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86 / Anisyl Phenylacetate / Monographs FCC 9

Odor: Faint, honey-like, balsamic-rosy IDENTIFICATION

Solubility: Soluble in organic solvents and oils; insoluble in • INFRARED ABSORPTION, Spectrophotometric Identification
water; miscible with ethanol at room temperature Tests, Appendix IIIC
Boiling Point: ∼370° Reference standard: USP Anisyl Propionate RS
Function: Flavoring agent Sample and standard preparation: F
Acceptance criteria: The spectrum of the sample
IDENTIFICATION exhibits maxima at the same wavelengths as those in
• INFRARED ABSORPTION, Spectrophotometric Identification the spectrum of the Reference standard.
Tests, Appendix IIIC
Reference standard: USP Anisyl Phenylacetate RS ASSAY
Monographs

Sample and standard preparation: F • PROCEDURE: Proceed as directed under M-1b, Appendix
Acceptance criteria: The spectrum of the sample XI.
exhibits maxima at the same wavelengths as those in Reference standard: USP Anisyl Propionate RS
the spectrum of the Reference standard. Acceptance criteria: NLT 97%

ASSAY SPECIFIC TESTS

• PROCEDURE: Proceed as directed under M-1b, Appendix • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
XI. OILS), M-15, Appendix XI
Reference standard: USP Anisyl Phenylacetate RS Acceptance criteria: NMT 1.0
Acceptance criteria: NLT 97% • REFRACTIVE INDEX, Appendix IIB: At 20°
Acceptance criteria: 1.505–1.510
SPECIFIC TESTS • SPECIFIC GRAVITY: Determine at 25° by any reliable
• ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL method (see General Provisions).
OILS), M-15, Appendix XI Acceptance criteria: 1.070–1.086
Acceptance criteria: NMT 1.0
• REFRACTIVE INDEX, Appendix IIB: At 20°
Acceptance criteria: 1.553–1.563
• SPECIFIC GRAVITY: Determine at 25° by any reliable
Annatto Extracts
.

method (see General Provisions).

Acceptance criteria: 1.125–1.133 First Published: Prior to FCC 6

INS: 160b CAS: [1393-63-1]

UNII: 6PQP1V1B6O [annatto]
Anisyl Propionate
.

DESCRIPTION
First Published: Second Supplement, FCC 8 Annatto Extracts occur as dark red solutions, emulsions, or
suspensions in water or oil or as dark red powders. The
Anisyl Propanoate extract is prepared from annatto seeds, Bixa orellana L.
p-Methoxybenzyl Propionate (Fam. Bixaceae), using a food-grade extraction solvent.
4-Methoxybenzyl Propanoate Bixin is the principal pigment of oil-soluble Annatto
Extracts. Norbixin is the principal pigment of alkaline
water-soluble Annatto Extracts. Commercial preparations
are usually mixtures of bixin, norbixin, and other
carotenoids.
Function: Color
C11H14O3 Formula wt 194.23 Packaging and Storage: Store under refrigeration in full,
FEMA: 2102 well-closed containers that are made from steel or
CAS: [7549-33-9] aluminum and that are suitably lined.
UNII: IBJ5CR95MK [anisyl propionate]
IDENTIFICATION
DESCRIPTION • VISIBLE ABSORPTION SPECTRUM
Anisyl Propionate occurs as a colorless to slightly yellow Sample preparation
liquid. Oil-soluble extracts: Dilute a sample with acetone.
Odor: Sweet, fruity, floral, vanilla-like Water-soluble extracts: Dilute a sample with water.
Solubility: Soluble in organic solvents, oils; insoluble in Acceptance criteria
water, glycerol, propylene glycol; miscible with ethanol at Oil-soluble extracts: The solution exhibits absorbance
room temperature maxima at 439, 470, and 501 nm.
Boiling Point: ∼100 to 103° at 0.5 mm Hg
Function: Flavoring agent
FCC 9 Monographs / β-Apo-8’-Carotenal / 87

Water-soluble extracts: The solution exhibits Trichloroethylene and Dichloromethane: NMT

absorbance maxima at 451 to 455 nm and 480 to 0.003%, individually or in combination
484 nm.
• CARR-PRICE REACTION, Column Chromatography, Appendix SPECIFIC TESTS
IIA • COLOR INTENSITY
Column: Fill a 200- × 7-mm glass tube, stoppered with Analysis
glass wool, with alumina (80- to 200-mesh) slurried in Oil-soluble extracts: Transfer a sample, accurately
toluene so that the settled alumina fills about 2/3 of the weighed, into a solution of 1% glacial acetic acid in
tube. Use a rubber outlet tube and clamp to adjust the acetone, and dilute to a suitable volume (absorbance
flow rate to about 30 drops/min. of 0.5 to 1.0). Filter the sample to clarify it if

Monographs
Analysis necessary. Measure the absorbance at 454 nm, and
Oil-soluble extracts: Add to the top of the Column 3 calculate the color intensity by the formula:
mL of a solution containing sufficient sample, in
Result = A/(b × c)
toluene, to impart a color equivalent to a solution of
0.1% potassium dichromate. Elute with toluene until a
pale yellow fraction is washed from the column. Wash A = absorbance of the sample solution
the column with three 10-mL volumes of dry acetone b = pathlength of the cell (cm)
and follow with 5 mL of Carr-Price Reagent (see Test c = concentration of the sample solution (g/L)
Solutions (TS) and Other Reagents, Solutions and Water-soluble extracts: Proceed as directed for Oil-
Indicators), added to the top of the column. soluble extracts, but dissolve the sample in 0.1 M
Water-soluble extracts: Transfer 2 mL or 2 g of sample sodium hydroxide, and measure the absorbance at
into a 50-mL separatory funnel, and add sufficient 2 N 453 nm.
sulfuric acid to make the solution acidic to pH test Acceptance criteria: The sample meets the
paper (pH 1 to 2). Dissolve the red precipitate of representations of the vendor.
norbixin by mixing the solution with 50 mL of
toluene. Discard the water layer, and wash the toluene
phase with water until it no longer gives an acid
reaction. Remove any undissolved norbixin by β-Apo-8’-Carotenal
.

centrifugation or filtration, and dry the solution over

anhydrous sodium sulfate. Transfer 3 to 5 mL of the First Published: Prior to FCC 6
dry solution to the top of the Column and elute with
toluene, three 10-mL volumes of dry acetone, and 5 Apocarotenal
mL of Carr-Price Reagent (see Test Solutions (TS) and APO
Other Reagents, Solutions and Indicators,) added to the
top of the column.
Acceptance criteria
Oil-soluble extracts: The orange-red zone (bixin) at
the top of the column immediately turns blue-green.
Water-soluble extracts: The orange-red band C30H40O Formula wt 416.65
(norbixin) immediately turns blue-green. INS: 160e CAS: [1107-26-2]
UNII: V22N3E2U32 [8′-apo-β-carotenal]
IMPURITIES
Inorganic Impurities DESCRIPTION
• ARSENIC, Arsenic Limit Test, Appendix IIIB β-Apo-8’-Carotenal occurs as a fine, crystalline powder with
Sample solution: Prepare as directed for organic a dark, metallic sheen. It is freely soluble in chloroform and
compounds. sparingly soluble in acetone, but it is insoluble in water. It
Acceptance criteria: NMT 3 mg/kg melts at 136° to 140° with decomposition.
• LEAD, Lead Limit Test, Appendix IIIB Function: Color
Sample solution: Prepare as directed for organic Packaging and Storage: Store in tight, light-resistant
compounds. containers under inert gas.
Control: 10 µg Pb (10 mL of Diluted Standard Lead
Solution) IDENTIFICATION
Acceptance criteria: NMT 10 mg/kg • A. PROCEDURE
Organic Impurities Sample stock solution: Transfer 40 mg of sample into a
• RESIDUAL SOLVENTS, Appendix VIII 100-mL volumetric flask, add 10 mL of acid-free
Acceptance criteria chloroform to dissolve the sample, dilute to volume
Acetone: NMT 0.003% with cyclohexane, and mix. Pipet 2 mL of this solution
Hexanes: NMT 0.0025% into a 50-mL volumetric flask, dilute to volume with
Isopropyl alcohol: NMT 0.005% cyclohexane, and mix.
Methyl alcohol: NMT 0.005%
 88 / β-Apo-8’-Carotenal / Monographs FCC 9

Sample solution: Pipet 5 mL of Sample stock solution

ARA from Fungal (Mortierella alpina) Oil
.

into a 50-mL volumetric flask, dilute to volume with

cyclohexane, and mix. First Published: Third Supplement, FCC 7
Analysis: Determine the absorbance (A) of the Sample
solution at 488 nm and at 460 nm.
Arachidonic Acid-Rich Oil
Acceptance criteria: The ratio A488/A460 is between 0.77
ARA Fungal Oil
and 0.85.
ARA-Rich Oil
• B. PROCEDURE
ARA-Rich Single Cell Oil
Sample stock solution: Prepared as directed above
Mortierella alpina Oil
under Identification test A
Refined Arachidonic Acid-Rich Oil (RAO)
Monographs

Sample solution: Prepared as directed above under

Identification test A DESCRIPTION
Analysis: Determine the absorbance (A) of the Sample ARA from Fungal (Mortierella alpina) Oil occurs as a clear,
stock solution at 332 nm and of the Sample solution at yellow-colored oil providing a source of arachidonic acid
460 nm. (ARA, C20H32O2) (C20:4 n-6), an omega-6 long-chain
Acceptance criteria: The ratio A332/A460 is between 0.63 polyunsaturated fatty acid. It is obtained from fermentation
and 0.75. of the species of fungus Mortierella alpina usually followed
by solvent extraction. The oil may be winterized, bleached,
ASSAY and deodorized to substantially remove free fatty acids,
• PROCEDURE: [NOTE—Carry out all work in low-actinic
phospholipids, odor and flavor components, and other
glassware and in subdued light.]
material. Arachidonic acid is the main polyunsaturated fatty
Sample stock solution: Prepared as directed above
acid present; ARA content may be standardized with other
under Identification test A.
oils. Suitable antioxidants may be added.
Sample solution: Prepared as directed above under
Function: Source of ARA
Identification test A.
Packaging and Storage: Store in tight, light-resistant
Analysis: Using a suitable spectrophotometer and a 1-
containers. Avoid exposure to excessive heat.
cm cell, measure the absorbance at 460 nm of the
Sample solution; use cyclohexane for the blank. IDENTIFICATION
Calculate the weight (mg) of C30H40O in the sample • FATTY ACID COMPOSITION, Fatty Acid Composition
taken by the formula: (Saturated, cis-Monounsaturated, and cis-Polyunsaturated)
in Oils Containing Long Chain Polyunsaturated Fatty Acids,
Result = 25,000 × A/a
Appendix VII
Acceptance criteria: The retention times of the peaks of
A = absorbance of Sample solution B the arachidonic acid methyl ester from the Sample
a = absorptivity (264) of pure β-Apo-8’- Preparation correspond to those from the Standard
Carotenal Solution. The percentage of the fatty acids (calculated
Acceptance criteria: NLT 96.0% and NMT 101.0% of using the results from the corresponding methyl esters)
C30H40O from the Sample Preparation, determined as the
percentage of total fat, meet the requirements for each
IMPURITIES fatty acid indicated in the table below.
Inorganic Impurities
• ARSENIC, Arsenic Limit Test, Appendix IIIB Shorthand Lower Limit Upper Limit
Sample solution: Prepare as directed for organic Fatty Acid Notation (Area %) (Area %)
compounds. Myristic acid 14:0 0.1 0.5
Acceptance criteria: NMT 1 mg/kg Palmitic acid 16:0 4.3 8.1
• LEAD, Lead Limit Test, Appendix IIIB Palmitoleic acid 16:1 n-9 0 0.4
Sample solution: Prepare as directed for organic
Stearic acid 18:0 4.2 7.6
compounds.
Oleic acid 18:1 n-9 3.4 9.5
Control: 10 µg Pb (10 mL of Diluted Standard Lead
Solution) Linoleic acid 18:2 n-6 3.8 15.2
Acceptance criteria: NMT 10 mg/kg gamma-Linolenic
acid 18:3 n-6 1.7 2.7
SPECIFIC TESTS Arachidic acid 20:0 0.6 1.0
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
Dihomo-gamma-
Sample: 2 g linolenic acid 20:3 n-6 3.0 5.0
Acceptance criteria: NMT 0.2%
Arachidonic acid 20:4 n-6 38.0 48.5
Behenic acid 22:0 2.5 4.1
Lignoceric acid 24:0 7.8 12.6
FCC 9 Monographs / ARA from Fungal (Mortierella alpina) Oil / 89

ASSAY Standards and Technology (NIST) spectrometric

• ARA, Fatty Acid Composition (Saturated, cis- standard solutions].
Monounsaturated, and cis-Polyunsaturated) in Oils Calibration standard solutions: 2.0 µg/L, 5.0 µg/L,
Containing Long Chain Polyunsaturated Fatty Acids, 10.0 µg/L, 25.0 µg/L, and 50.0 µg/L in 2% nitric acid
Appendix VII from the Calibration standard stock solution
Analysis: Proceed as directed. Calculate the percentage 1% Palladium stock solution: Mix 1 g of ultrapure
of ARA (w/w, as a percentage of total fatty acids) in the palladium metal with 20 mL of water and 10 mL of
portion of the sample taken: nitric acid in a Teflon beaker, and warm the solution on
a hot plate to dissolve the palladium. Allow the
Result = (WFAMEx × FFAx)/ΣWTAG solution to cool to room temperature, transfer it into a

Monographs
100-mL volumetric flask, and dilute with deionized
[NOTE—Use the definitions provided for WFAMEx, FFAx, and
water to volume.
ΣWTAG in the method referenced, where x is
1% Magnesium nitrate stock solution: Mix 1 g of
arachidonic acid.]
ultrapure magnesium nitrate with 40 mL of water and
Acceptance criteria: NLT 38.0% arachidonic acid (ARA)
1 mL of nitric acid in a Teflon beaker, and warm the
IMPURITIES solution on a hot plate to dissolve. Allow the solution
Inorganic Impurities to cool to room temperature, transfer it into a 100-mL
• ARSENIC, Elemental Impurities by ICP, Appendix IIIC volumetric flask, and dilute with deionized water to
[NOTE—Alternatively, the arsenic content may be volume.
determined by the following method.] [NOTE—Because of the difficulty in preparing matrix
Apparatus modifier stock solutions with the required purity,
Sample digestion: Use a microwave oven1 equipped purchasing modifier stock solutions and using them
with advanced composite vessels with 100-mL Teflon to prepare modifier working solutions is
liners. Use rupture membranes to vent vessels should recommended.3]
the pressure exceed 125 psi. The vessels fit into a Modifier working solution: Transfer 3 mL of 1%
turntable, and each vessel can be vented into an Palladium stock solution and 2 mL of 1% Magnesium
overflow container. Equip the microwave oven with nitrate stock solution to a 10-mL volumetric flask, and
an exhaust tube to ventilate fumes. dilute with 2% nitric acid to volume. A volume of 5 µL
Sample analysis: Use a suitable graphite furnace provides 0.015 mg of palladium and 0.01 mg of
atomic absorption spectrophotometer (GFAAS) magnesium nitrate.
equipped with an autosampler, pyrolytically coated Sample solution: [CAUTION—Wear proper eye
graphite tubes, solid pyrolytic graphite platforms, and protection, protective clothing, and gloves during
an adequate means of background correction.2 An sample preparation. Closely follow the manufacturer’s
electrodeless discharge lamp serves as the source, safety instructions for use of the microwave digestion
argon as the purge gas, and air as the alternate gas. apparatus.] Transfer 500 mg of sample into a Teflon
Set up the instrument according to the digestion vessel liner. Prepare samples in duplicate. Add
manufacturer’s specifications, with consideration of 15 mL of nitric acid, and swirl gently. Cover the vessels
current good GFAAS practices. The instrument with lids, leaving the vent fitting off. Predigest
parameters are as follows: overnight under a hood. Place the rupture membrane
Wavelength: 193.7 nm in the vent fitting, and tighten the lid. Place all vessels
Lamp current: 300 (EDL) modulated on the microwave oven turntable. Connect the vent
Pyrolysis: 1000° tubes to the vent trap, and connect the pressure-
Atomization: 2400° sensing line to the appropriate vessel. Initiate a two-
Slit: 0.7 stage digestion procedure by heating the microwave at
Characteristic mass: 15 pg 15% power for 15 min followed by 25% power for 45
Glassware: Acid wash all glass, Teflon, and plastic min. Remove the turntable of vessels from the oven,
vessels by soaking them in a nitric acid bath containing and allow the vessels to cool to room temperature (a
a 4:1 solution of water and nitric acid. [CAUTION—Wear cool water bath may be used to speed the cooling
a full face shield, protective clothing, and gloves at all process). Vent the vessels when they reach room
times when working with acid baths.] After acid temperature. Remove the lids, and slowly add 2 mL of
soaking, rinse the acid-washed items in deionized 30% hydrogen peroxide to each. Allow the reactions to
water, dry them, and store them in clean, covered subside, and seal the vessels. Return the vessels on the
cabinets. turntable to the microwave oven, and heat for an
Calibration standard stock solution: 100 µg/L. Prepare additional 15 min at 30% power. Remove the vessels
from a suitable standard, which may be purchased from the oven, and allow them to cool to room
[accuracy certified against National Institute of temperature. Transfer the cooled digests into 25-mL
volumetric flasks, and dilute with deionized water to
1 CEM Model MDS-2100, or equivalent. volume.
2 This method was developed using a Perkin-Elmer Model 5100, HGA-600
furnace, and an AS-60 autosampler with Zeeman effect background 3 A palladium (0.3%) and magnesium nitrate (0.2%) solution may be

correction. purchased from High Purity Standards, or equivalent.

 90 / ARA from Fungal (Mortierella alpina) Oil / Monographs FCC 9

Analysis: The graphite furnace program is as follows: Sample digestion: Use a microwave oven1 equipped
1. Dry at 115° using a 1-s ramp, a 65-s hold, and a with advanced composite vessels with 100-mL Teflon
300-mL/min argon flow. liners. Use rupture membranes to vent vessels should
2. Char the sample at 1000° using a 1-s ramp, a 20-s the pressure exceed 125 psi. The vessels fit into a
hold, and a 300-mL/min air flow. turntable, and each vessel can be vented into an
3. Cool down and purge the air from the furnace for overflow container. Equip the microwave oven with
10 s using a 20° set temperature and a 300-mL/ an exhaust tube to ventilate fumes.
min argon flow. Sample analysis: See Apparatus in Lead Limit Test,
4. Atomize at 2400° using a 0-s ramp and a 5-s hold Atomic Absorption Spectrophotometric Graphite Furnace
with the argon flow stopped. Method, Method I, Appendix IIIB
Monographs

5. Clean out at 2600° with a 1-s ramp and a 5-s Calibration standard stock solution: 100 µg/L. Prepare
hold. from a suitable standard, which may be purchased
Use the autosampler to inject 20-µL aliquots of blanks, (accuracy certified against NIST spectrometric standard
Calibration standard solutions, Sample solution, and 5 µL solutions).
of Modifier working solution. Inject each solution in Calibration standard solutions: 2.0 µg/L, 5.0 µg/L,
duplicate, and average the results. Use peak area 10.0 µg/L, 25.0 µg/L, and 50.0 µg/L in 2% nitric acid,
measurement for all quantitations. After ensuring that from the Calibration standard stock solution
the furnace is clean by running a 5% nitric acid blank, 10% Ammonium dihydrogen phosphate stock
check the instrument’s sensitivity by running a 20-µL solution: Mix 10 g of ultrapure ammonium
aliquot of the 25.0-µg Calibration standard solution. dihydrogen phosphate with 40 mL of water and 1 mL
Compare the results obtained with the expected results of nitric acid to dissolve the phosphate. Dilute with
for the equipment used, and take the necessary steps deionized water to 100 mL.
to correct any problems. 1% Magnesium nitrate stock solution: Mix 1 g of
Calculate the characteristic mass. Record and track the ultrapure magnesium nitrate with 40 mL of water and
integrated absorbance and characteristic mass for 1 mL of nitric acid in a Teflon beaker, and warm on a
reference and quality assurance. hot plate to dissolve the solids. Allow the solution to
Inject each Calibration standard solution in duplicate. Use cool to room temperature, transfer it into a 100-mL
the algorithms provided in the instrument software to volumetric flask, and dilute with deionized water to
establish calibration curves. Recheck calibration volume.
periodically, and recalibrate if the recheck differs from [NOTE—Because of the difficulty in preparing matrix
the original calibration by more than 10%. modifier stock solutions with the required purity,
Inject the Sample solution in duplicate, and record the purchasing modifier stock solutions and using them
integrated absorbance. If the instrument response to prepare modifier working solutions is
exceeds that of the calibration curve, dilute with 5% recommended.4]
nitric acid to bring the sample’s response into the Modifier working solution: Transfer 4 mL of 10%
working range, and note the dilution factor (DF). All Ammonium dihydrogen phosphate stock solution and 2
sample analyses should be blank corrected using a mL of 1% Magnesium nitrate stock solution to a 10-mL
sample solution blank. volumetric flask, and dilute with 2% nitric acid to
If a computer-based instrument is used, the data output volume. A volume of 5 µL provides 0.2 mg of
is reported as µg/L. phosphate plus 0.01 mg of magnesium nitrate.
Calculate the concentration of arsenic, in µg/g Sample solution: Prepare as directed for Sample solution
(equivalent to mg/kg), in the original sample taken: in the test for Arsenic. [CAUTION—Wear proper eye
protection, protective clothing, and gloves during
Result = (C × DF × V)/W sample preparation. Closely follow the manufacturer’s
safety instructions for use of the microwave digestion
C = concentration of arsenic in the sample
apparatus.]
aliquot injected (µg/L)
Analysis: The graphite furnace program is as follows:
DF = dilution factor of the Sample solution
1. Dry at 120° using a 1-s ramp, a 55-s hold, and a
V = final volume of the Sample solution (L)
300-mL/min argon flow.
W = weight of the sample taken to prepare the
2. Char the sample at 850° using a 1-s ramp, a 30-s
Sample solution (g)
hold, and a 300-mL/min air flow.
[NOTE—To monitor recovery and ensure analytical
3. Cool down and purge the air from the furnace for
accuracy for proper quality assurance, analyze blanks,
10 s using a 20° set temperature and a 300-mL/
spiked blanks, and a spiked oil with each digestion
min argon flow.
set.]
4. Atomize at 2100° using a 0-s ramp and a 5-s hold
Acceptance criteria: NMT 0.1 mg/kg
with the argon flow stopped.
• CADMIUM, Elemental Impurities by ICP, Appendix IIIC
5. Clean out at 2600° with a 1-s ramp and a 5-s
Acceptance criteria: NMT 0.1 mg/kg
hold.
• LEAD, Elemental Impurities by ICP, Appendix IIIC
[NOTE—Alternatively, the lead content may be
determined by the following method.] 4An ammonium dihydrogen phosphate (4%) and magnesium nitrate (0.2%)
Apparatus solution may be purchased from High Purity Standards, or equivalent.
FCC 9 Monographs / ARA from Fungal (Mortierella alpina) Oil / 91

Use the autosampler to inject 20-µL aliquots of blanks, Wavelength: 253.6 nm

Calibration standard solutions, Sample solution, and 5 µL Slit: 0.7
of Modifier working solution. Inject each solution in Reagent setting: 5
duplicate, and average the results. Use peak-area Gas flow: 5–6 L/min
measurement for all quantitation. After ensuring that Reaction time: 0.5 min
the furnace is clean by running a 5% nitric acid blank, Glassware: Acid wash all glass, Teflon, and plastic
check instrument sensitivity by running an aliquot of vessels by soaking them in a nitric acid bath
the 25.0-µg Calibration standard solution. Compare the containing a 4:1 solution of water and nitric acid.
results obtained with the expected results for the [CAUTION—Wear a full face shield, protective clothing,
equipment used, and take the necessary steps to and gloves at all times when working with acid

Monographs
correct any problems. baths.] After acid soaking, rinse the acid-washed
Calculate the characteristic mass, and record and track items in deionized water, dry, and store them in
the integrated absorbance and characteristic mass for clean, covered cabinets.
reference and quality assurance. Calibration standard stock solution: 200 ng/g of
Inject each Calibration standard solution in duplicate. Use mercury. Prepare from a suitable standard, which
the algorithms provided in the instrument software to may be purchased (accuracy certified against NIST
establish calibration curves. Recheck the calibration spectrometric standard solutions).
periodically, and recalibrate if recheck differs from the Calibration standard solutions: 20 ng, 60 ng, 100
original calibration by more than 10%. ng, 200 ng, and 400 ng of mercury in 1 N
Inject the Sample solution in duplicate, and record the hydrochloric acid from the Calibration standard stock
integrated absorbance. If the instrument response solution
exceeds that of the calibration curve, dilute with 5% Reducing reagent: 5% Stannous chloride in 25%
nitric acid to bring the sample response into the hydrochloric acid (trace-metal grade). [NOTE—Prepare
working range, and note the dilution factor (DF). All daily.]
sample analyses should be blank corrected using a Sample solution: Prepare as directed for the Sample
sample solution blank. solution in the test for Arsenic. [CAUTION—Wear proper
If a computer-based instrument is used, the data output eye protection, protective clothing, and gloves during
is reported as µg/L. Calculate the concentration, in µg/ sample preparation. Closely follow the manufacturer’s
g (equivalent to mg/kg), of lead in the original sample: safety instructions for use of the microwave digestion
apparatus.]
Result = (C × DF × V)/W Analysis: Optimize the instrument settings for the
spectrophotometer as described in the instrument
C = concentration of lead in the sample aliquot
manual. The instrument parameters for cold vapor
injected (µg/L)
generation are as follows:
DF = dilution factor of the Sample solution
Wavelength: 253.6 nm
V = final volume of the Sample solution (L)
Slit: 0.70 nm
W = weight of the sample taken to prepare the
Reagent setting: 5
Sample solution (g)
Gas flow: 5–6 L/min
[NOTE—To monitor recovery and ensure analytical
Reaction time: 0.5 min
accuracy for proper quality assurance, analyze blanks,
Use a peak height integration method with a 40-s
spiked blanks, and a spiked oil with each digestion
integration time and a 20-s read delay in an
set.]
unheated absorption cell. Zero the instrument as
Acceptance criteria: NMT 0.1 mg/kg
follows: Place a Fleaker containing 50 mL of 1 N
• MERCURY
hydrochloric acid in the sample well of the hydride
Apparatus
generator. Press “start” on the vapor generator and
Sample digestion: Use a microwave oven1 equipped
“read” on the atomic absorption
with advanced composite vessels with 100-mL Teflon
spectrophotometer. The instrument will
liners. Use rupture membranes to vent vessels should
automatically flush the sample container with
the pressure exceed 125 psi. The vessels fit into a
nitrogen, dispense the designated amount of
turntable, and each vessel can be vented into an
reagent, stir the sample for a designated reaction
overflow container. Equip the microwave oven with
time, and purge the head volume again with
an exhaust tube to ventilate fumes.
nitrogen, sweeping any vapor into the quartz cell for
Sample analysis: Use a suitable atomic absorption
determination of absorption. The atomic absorption
spectrophotometer equipped with an atomic vapor
spectrophotometer will automatically zero on this
assembly.5 An electrodeless discharge lamp serves as
sample when “autozero” is selected from the
the source, with an inert gas such as argon or
calibration menu.
nitrogen as the purge gas. Set up the instrument
Generate a standard curve of concentration versus
according to the manufacturer’s specifications.
absorption by analyzing the five Calibration standard
Instrument parameters are as follows:
solutions prepared as described for daily standards
5 This method was developed using a Perkin-Elmer Model 5100 and IL 440 under Calibration standard solutions. Analyze each
Thermo Jarrell Ash atomic vapor assembly. solution in duplicate, generate the calibration curve,
 92 / ARA from Fungal (Mortierella alpina) Oil / Monographs FCC 9

and store, using procedures specific for the Syringe temperature: 60°
instrumentation. Carrier gas: Helium
Transfer an appropriate aliquot of Sample solution Flow rate: Optimize accordingly
(usually 2 mL) in a Fleaker containing 50 mL of 1 N Injection volume: 1000 µL
hydrochloric acid. Analyze solutions in duplicate Determination of calibration factors: Warm a 1000-µL
using the procedure specified in the instrument gas-tight syringe to 60°. Temper each Calibration
manual. Using the calibration algorithm provided in solution in a water bath maintained at 60° for exactly
the instrument software, calculate and report the 30 min, then, without removing the vial from the bath,
mercury concentration in ng of mercury in the use the gas-tight syringe and withdraw 1000 µL of the
aliquot analyzed. headspace above the oil. Inject immediately into the
Monographs

Calculate the level of mercury, as µg/g (equivalent to gas chromatograph, record the chromatograms from
mg/kg), in the original sample: each Calibration solution, and determine the peak areas.
For each of the Calibration solutions containing n-hexane
Result = (A × DF)/(W × 1000) (not including the blank solution) calculate the
calibration factor, F:
A = amount of mercury in the aliquot analyzed
(ng) F = (CS × AI)/[(AH − AB − AI) × CI]
DF = dilution factor (final volume of Sample
solution/volume taken for analysis) CS = concentration of n-hexane in the Calibration
W = weight of the sample taken to prepare the solution of interest (mg/kg)
Sample solution (g) AI = peak area corresponding to the Internal
[NOTE—To monitor recovery and ensure analytical standard in the chromatogram of the
accuracy for proper quality assurance, analyze Calibration solution
blanks, spiked blanks, and a spiked oil with each AH = total peak area of solvent hydrocarbons in
digestion set.] the chromatogram of the Calibration
Acceptance criteria: NMT 0.1 mg/kg solution, including the area of the Internal
Organic Impurities standard, not including peaks due to the
• HEXANE RESIDUES6 oxidation products
Vegetable oil: Use solvent-free vegetable oil that is AB = peak area of the solvent hydrocarbons
similar in nature to the sample. Deodorization of the oil present in the blank solution, minus the
in the laboratory may be used to reduce the amount of peak area of the Internal standard
extraction solvent present in the oil. CI = quantity of the Internal standard added to
Internal standard: n-Heptane the Calibration solution, in mg/kg of oil
Calibration solutions: Prepare a series of solutions by (680 mg)
adding 0 µL, 20 µL, 40 µL, 60 µL, 80 µL, and 100 µL [NOTE—Calculate calibration factors to three decimal
of n-hexane, separately, to a series of vials, each points. The mean calibration factor will be used in
containing 25 g of Vegetable oil. Close the vials, then the Analysis.]
mechanically shake them vigorously for 1 h at room Analysis: Warm a 1000-µL gas-tight syringe to 60°.
temperature. After shaking the vials, add 5 µL of the Temper the Sample solution in a water bath maintained
Internal standard to each vial, using a syringe. [NOTE— at 60° for exactly 30 min, then, without removing the
The vial with 0 µL of n-hexane added is the blank.] vial from the bath, use the gas-tight syringe and
Sample solution: Weigh 25.00 g of the sample into a withdraw 1000 µL of the headspace above the oil.
septum vial. Close the vial and, using a syringe, add 25 Inject immediately into the gas chromatograph, record
µL of the Internal standard to the sample. Shake the the chromatogram, and determine the peak areas.
vial vigorously for about 1 min before proceeding with Determine the residual solvent content, in mg/kg of
the Analysis. hexane:
Chromatographic system, Appendix IIA
Mode: Headspace gas chromatography Result = (AH − AI) × F × CI × (1/AI)
Detector: Flame ionization
AH = total peak area of solvent hydrocarbons in
Column: 30-m × 0.3-mm fused silica or glass capillary
the chromatogram of the Sample solution,
column coated with methyl polysiloxane (0.2-µm
including the area of the Internal standard
thickness)7
(do not include peaks due to oxidation
Temperature
products)
Oven: 40°
AI = peak area corresponding to the Internal
Injection port: 120°
standard in the chromatogram of the
Detector: 120°
Sample solution
Headspace sampling conditions
F = mean calibration factor determined above
Sample heating temperature: 60°
CI = quantity of the Internal standard added to
Sample heating time: 30 min
the Sample solution, in mg/kg of sample
6 Adapted from AOCS Method Ca 3b-87 (1997). The original method is (680 mg)
available from the American Oil Chemists’ Society (AOCS) at www.aocs.org. Acceptance criteria: NMT 1.0 mg/kg
7 HP-1 (Agilent Technologies), or equivalent.
 FCC 9 Monographs / L-Arginine / 93

SPECIFIC TESTS ASSAY

• ACID VALUE (FATS AND RELATED SUBSTANCES), Appendix VII • TOTAL CARBOHYDRATES
Acceptance criteria: NMT 1.0 Analysis: Determine the difference between 100% and
• ANISIDINE VALUE, Appendix VII the sum of the percent Ash (Total), Loss on Drying, and
Acceptance criteria: NMT 20 Protein.
• FREE FATTY ACIDS (AS OLEIC ACID), Appendix VII Acceptance criteria: NLT 80% (as arabinogalactan)
Analysis: Use 28.2 for the equivalence factor (e) in the
formula given in the procedure. IMPURITIES
Acceptance criteria: NMT 0.2% Inorganic Impurities
• PEROXIDE VALUE, Appendix VII • LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric

Monographs
Acceptance criteria: NMT 2.0 mEq/kg Graphite Furnace, Method I, Appendix IIIB
• UNSAPONIFIABLE MATTER, Method II, Appendix VII Acceptance criteria: NMT 0.1 mg/kg
Acceptance criteria: NMT 3.0%
SPECIFIC TESTS
OTHER REQUIREMENTS • ASH (TOTAL), Appendix IIC
• LABELING: Label to indicate the content of arachidonic Acceptance criteria: 10.0%
acid in mg/g (%). Indicate the name of any added • INSOLUBLE MATTER
antioxidant and the presence of any other oil(s) used to Sample: 5 g
standardize the arachidonic acid content. Analysis: Dissolve the Sample in about 100 mL of water
contained in a 250-mL Erlenmeyer flask, add 10 mL of
2.7 N hydrochloric acid, and boil gently for 15 min.
Filter the hot solution by suction through a tared
filtering crucible and wash the residue thoroughly with
Arabinogalactan
.

hot water. Dry the residue at 105° for 2 h, and weigh.

First Published: Prior to FCC 6 Acceptance criteria: NMT 0.1%
• LOSS ON DRYING, Appendix IIC: 105° for 5 h
Larch Fiber Acceptance criteria: NMT 8%
Larch Gum • PROTEIN, Nitrogen Determination, Method I, Appendix IIIC
INS: 409 CAS: [9036-66-2] Sample: 3.5 g
Analysis: Transfer the Sample into a 500-mL Kjeldahl
DESCRIPTION flask. Multiply the percentage of nitrogen determined
Arabinogalactan occurs as a white to yellow-white, coarse or
by 6.25.
fine powder. It is the dried water extract from the wood of
Acceptance criteria: NMT 1.0%
the larch trees Larix occidentalis and Larix laricina (Fam.
• STARCH
Pinaceae). It is a highly branched polysaccharide that has a
Sample solution: 100 mg/mL
molecular weight of 15,000 to 60,000 daltons and is
Analysis: Add a few drops of iodine TS to the Sample
composed of galactose units and arabinose units in the
solution.
approximate ratio of 6 : 1. It is freely dispersible in hot or
Acceptance criteria: No blue or red color appears.
cold water. It is insoluble in alcohol.
Function: Dietary fiber; humectant; stabilizer
Packaging and Storage: Store in well-closed containers.
L-Arginine
.

IDENTIFICATION
• A. PROCEDURE First Published: Prior to FCC 6
Sample: 20 g Last Revision: FCC 9
Sample solution: Add the Sample to 20 mL of water
and stir until completely dissolved. Pour the solution L-2-Amino-5-guanidinovaleric Acid
into a 500 mL beaker and add 100 mL of water.
Analysis: Transfer 7 mL of the Sample solution into a
250-mL beaker and add 0.2 mL of diluted lead
subacetate TS. [NOTE—Retain the remaining Sample
solution for Identification test B (below).]
Acceptance criteria: No precipitate forms. C6H14N4O2 Formula wt 174.20
• B. PROCEDURE CAS: [74-79-3]
Sample solution: Retained Sample solution from UNII: 94ZLA3W45F [arginine]
Identification test A (above)
Analysis: To the Sample solution, add 280 mL of 95% DESCRIPTION
ethyl alcohol. L-Arginine occurs as white crystals or as a white crystalline
Acceptance criteria: A precipitate forms. powder. It is soluble in water; sparingly soluble in alcohol;
insoluble in ether. It is strongly alkaline, and its water
solutions absorb carbon dioxide from the air.
Function: Nutrient
 94 / L-Arginine / Monographs FCC 9

Packaging and Storage: Store in well-closed, light-

L-Arginine Monohydrochloride
.

resistant containers.
First Published: Prior to FCC 6
IDENTIFICATION
Last Revision: FCC 6
• INFRARED ABSORPTION, Spectrophotometric Identification
Tests, Appendix IIIC
Reference standard: USP L-Arginine RS L-2-Amino-5-guanidinovaleric Acid Monohydrochloride
Sample and Standard preparation: K
Acceptance criteria: The spectrum of the sample
exhibits maxima at the same wavelengths as those in
Monographs

the spectrum of the Reference standard.

ASSAY C6H14N4O2 · HCl Formula wt 210.66

• PROCEDURE CAS: [1119-34-12]
Sample: 200 mg UNII: F7LTH1E20Y [arginine hydrochloride]
Analysis: Dissolve the Sample in 3 mL of formic acid and
50 mL of glacial acetic acid. Add 2 drops of crystal DESCRIPTION
violet TS, and titrate with 0.1 N perchloric acid to a L-Arginine Monohydrochloride occurs as a white or nearly
green endpoint or until the blue color disappears white crystalline powder. It is soluble in water, slightly
completely. Each mL of 0.1 N perchloric acid consumed soluble in hot alcohol, and insoluble in ether. It is acidic
in the assay is equivalent to 8.710 mg of C6H14N4O2. and melts with decomposition at about 235°.
[CAUTION—Handle perchloric acid in an appropriate Function: Nutrient
fume hood.] Packaging and Storage: Store in well-closed, light-
Acceptance criteria: 98.5%–101.5% of C6H14N4O2, resistant containers.
calculated on the dried basis
IDENTIFICATION
IMPURITIES • INFRARED ABSORPTION, Spectrophotometric Identification
Inorganic Impurities Tests, Appendix IIIC
• LEAD, Lead Limit Test, Appendix IIIB Reference standard: USP Arginine Hydrochloride RS
Sample solution: Prepare as directed for organic Sample and Standard preparation: K
compounds. Acceptance criteria: The spectrum of the sample
Control: 5 µg Pb (5 mL of Diluted Standard Lead exhibits maxima at the same wavelengths as those in
Solution) the spectrum of the Reference standard.
Acceptance criteria: NMT 5 mg/kg
ASSAY
SPECIFIC TESTS • PROCEDURE
Sample: 100 mg, previously dried
Add the following: Analysis: Dissolve the Sample in 2 mL of formic acid,
• INSOLUBLE FOREIGN MATTER IN AMINO ACIDS, Appendix
▲ add exactly 15.0 mL of 0.1 N perchloric acid, and heat
IIC on a water bath for 30 min. [CAUTION—Handle
Acceptance criteria: No fibers longer than 5 mm are perchloric acid in an appropriate fume hood.] After
present, and the total weight of any foreign matter cooling, add 45 mL of glacial acetic acid, and titrate
present is NMT 5 mg/100 g of the sample.▲ FCC9 the excess perchloric acid with 0.1 N sodium acetate,
• LOSS ON DRYING, Appendix IIC: 105° for 3 h determining the endpoint potentiometrically. Perform a
Acceptance criteria: NMT 1.0% blank determination (see General Provisions), and make
• OPTICAL (SPECIFIC) ROTATION, Appendix IIB any necessary correction. Each mL of 0.1 N perchloric
Sample: 8 g, previously dried acid is equivalent to 10.53 mg of C6H14N4O2 · HCl.
Analysis: Dissolve the Sample in sufficient 6 N Acceptance criteria: NLT 98.5% and NMT 101.5% of
hydrochloric acid to make 100 mL. C6H14N4O2 · HCl on the dried basis
Acceptance criteria
[α]D20 between +26.0° and +27.9°, on the dried basis; or
IMPURITIES
Inorganic Impurities
[α]D25 between +25.8° and +27.7°, on the dried basis
• LEAD, Lead Limit Test, Appendix IIIB
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
Sample solution: Prepare as directed for organic
Sample: 1 g
compounds.
Acceptance criteria: NMT 0.2%
Control: 5 µg Pb (5 mL of Diluted Standard Lead
Solution)
Acceptance criteria: NMT 5 mg/kg

SPECIFIC TESTS
• LOSS ON DRYING, Appendix IIC: 105° for 3 h
Acceptance criteria: NMT 0.3%
 FCC 9 Monographs / Ascorbyl Palmitate / 95

• OPTICAL (SPECIFIC) ROTATION, Appendix IIB Acceptance criteria: NLT 99.0% and NMT 100.5% of
Sample: 8 g, previously dried C6H8O6
Analysis: Dissolve the Sample in sufficient 6 N
hydrochloric acid to make 100 mL. IMPURITIES
Acceptance criteria Inorganic Impurities
[α]D20 between +21.3° and +23.5°, on the dried basis; or • LEAD, Lead Limit Test, Flame Atomic Absorption
[α]D25 between +21.3° and +23.4°, on the dried basis. Spectrophotometric Method, Appendix IIIB
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC Sample: 10 g
Sample: 1 g Acceptance criteria: NMT 2 mg/kg
Acceptance criteria: NMT 0.1%
SPECIFIC TESTS

Monographs
• OPTICAL (SPECIFIC) ROTATION, Appendix IIB
Sample solution: 1 g in 10 mL of carbon dioxide-free
water
Ascorbic Acid
.

Acceptance criteria: [α]D25 between +20.5° and +21.5°

First Published: Prior to FCC 6 • RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
Sample: 2 g
Vitamin C Acceptance criteria: NMT 0.1%
L-Ascorbic Acid

Ascorbyl Palmitate
.

First Published: Prior to FCC 6

Last Revision: First Supplement, FCC 7
C6H8O6 Formula wt 176.13
INS: 300 CAS: [50-81-7] Palmitoyl L-Ascorbic Acid
UNII: PQ6CK8PD0R [ascorbic acid]

DESCRIPTION
Ascorbic Acid occurs as white or slightly yellow crystals or as
powder. It melts at about 190°. It gradually darkens on
exposure to light, is reasonably stable in air when dry, but
C22H38O7 Formula wt 414.54
rapidly deteriorates in solution in the presence of air. One
INS: 304 CAS: [137-66-6]
g is soluble in about 3 mL of water and in about 30 mL of
UNII: QN83US2B0N [ascorbyl palmitate]
alcohol. It is insoluble in chloroform and in ether.
Function: Antioxidant; meat-curing aid; nutrient DESCRIPTION
Packaging and Storage: Store in tight, light-resistant Ascorbyl Palmitate occurs as a white or yellow-white
containers. powder. It is very slightly soluble in water and in vegetable
IDENTIFICATION oils. One gram dissolves in about 4.5 mL of alcohol.
• A. PROCEDURE Function: Antioxidant
Sample solution: 20 mg/mL Packaging and Storage: Store in tightly closed
Acceptance criteria: The Sample solution slowly reduces containers, preferably in a cool, dry place.
alkaline cupric tartrate TS at 25°, but more readily upon IDENTIFICATION
heating. • A. INFRARED ABSORPTION, Spectrophotometric Identification
• B. INFRARED ABSORPTION, Spectrophotometric Identification Tests, Appendix IIIC
Tests, Appendix IIIC Reference standard: USP Ascorbyl Palmitate RS
Reference standard: USP Ascorbic Acid RS Sample and standard preparation: K
Sample and Standard preparation: K Acceptance criteria: The spectrum of the sample
Acceptance criteria: The spectrum of the sample exhibits maxima at the same wavelengths as those in
exhibits maxima at the same wavelengths as those in the spectrum of the Reference standard.
the spectrum of the Reference Standard. • B. PROCEDURE
ASSAY Sample solution: 100 mg/mL in alcohol
• PROCEDURE Acceptance criteria: The Sample solution decolorizes
Sample: 400 mg dichlorophenol–indophenol TS.
Analysis: Dissolve the Sample in a mixture of 100 mL of ASSAY
water, recently boiled and cooled, and 25 mL of 2 N • PROCEDURE
sulfuric acid. Titrate the solution immediately with 0.1 Sample: 300 mg
N iodine, adding starch TS near the endpoint. Each mL Analysis: Dissolve the Sample in 50 mL of alcohol in a
of 0.1 N iodine is equivalent to 8.806 mg of C6H8O6. 250-mL Erlenmeyer flask. Add 30 mL of water and
 96 / Ascorbyl Palmitate / Monographs FCC 9

immediately titrate with 0.1 N iodine to a yellow color alcohol and in ether. Its solutions are acid to litmus. It
that persists for at least 30 s. Each mL of 0.1 N iodine is melts at about 234°.
equivalent to 20.73 mg of C22H38O7. Function: Nutrient
Acceptance criteria: NLT 95.0% of C22H38O7, calculated Packaging and Storage: Store in well-closed, light-
on the dried basis resistant containers.

IMPURITIES IDENTIFICATION
Inorganic Impurities • INFRARED SPECTRA, Spectrophotometric Identification Tests,
• LEAD, Lead Limit Test, Flame Atomic Absorption Appendix IIIC
Spectrophotometric Method, Appendix IIIB Sample preparation: Mineral oil mull
Monographs

Sample: 10 g Acceptance criteria: The spectrum of the sample

Acceptance criteria: NMT 2 mg/kg exhibits relative maxima at the same wavelengths as
those of the spectrum below.
SPECIFIC TESTS
• LOSS ON DRYING, Appendix IIC: Vacuum oven at 56° to ASSAY
60° for 1 h • PROCEDURE
Acceptance criteria: NMT 2% Sample: 130 mg, previously dried
• MELTING RANGE OR TEMPERATURE DETERMINATION, Appendix Analysis: Dissolve the Sample in 3 mL of formic acid and
IIB 50 mL of glacial acetic acid, and titrate with 0.1 N
Analysis: Determine as directed in Procedure for Class Ia perchloric acid, determining the endpoint
Acceptance criteria: Between 107° and 117° potentiometrically. [CAUTION—Handle perchloric acid in
• OPTICAL (SPECIFIC) ROTATION, Appendix IIB an appropriate fume hood.] Perform a blank
Sample solution: 1 g in 10 mL of methanol determination (see General Provisions), and make any
Acceptance criteria: [α]D25 between +21° and +24°, necessary correction. Each mL of 0.1 N perchloric acid
calculated on the dried basis is equivalent to 13.21 mg of C4H8N2O3.
• RESIDUE ON IGNITION (SULFATED ASH), Method I, Appendix Acceptance criteria: NLT 98.0% and NMT 101.5% of
IIC C4H8N2O3, on the dried basis
Sample: 2 g
Acceptance criteria: NMT 0.1% IMPURITIES
Inorganic Impurities
• LEAD, Lead Limit Test, Appendix IIIB
Sample solution: Prepare as directed for organic
compounds.
L-Asparagine
.

Control: 5 µg Pb (5 mL of Diluted Standard Lead

First Published: Prior to FCC 6 Solution)
Acceptance criteria: NMT 5 mg/kg
L-α-Aminosuccinamic Acid
SPECIFIC TESTS
• LOSS ON DRYING, Appendix IIC: 130° for 3 h
Acceptance criteria: Between 11.5% and 12.5%
• OPTICAL (SPECIFIC) ROTATION, Appendix IIB
Sample solution: 10 g of a previously dried sample in
C4H8N2O3 Formula wt, anhydrous 132.12 sufficient 6 N hydrochloric acid to make 100 mL
C4H8N2O3 · H2O Formula wt, monohydrate 150.13 Acceptance criteria
CAS: anhydrous [70-47-3] [α]D20 between +33.0° and +36.5°, on the dried basis
CAS: monohydrate [5794-13-8] • RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
UNII: 7NG0A2TUHQ [asparagine anhydrous] Sample: 1g
Acceptance criteria: NMT 0.1%
DESCRIPTION
L-Asparagine occurs as white crystals or as a crystalline
powder. It is soluble in water and practically insoluble in
 FCC 9 Monographs / Aspartame / 97

Monographs
L-Asparagine (Mineral Oil Mull)

Acceptance criteria: The spectrum of the sample

Aspartame
.

exhibits maxima at the same wavelengths as those in

First Published: Prior to FCC 6 the spectrum of the Reference standard.

ASSAY
N-L-α-Aspartyl-L-phenylalanine 1-Methyl Ester • PROCEDURE
APM Sample: 300 mg
Analysis: Transfer the Sample to a 150-mL beaker,
dissolve in 1.5 mL of 96% formic acid, and add 60 mL
of glacial acetic acid. Add crystal violet TS, and titrate
immediately with 0.1 N perchloric acid to a green
endpoint. [CAUTION—Handle perchloric acid in an
appropriate fume hood.] [NOTE—Use 0.1 N perchloric
C14H18N2O5 Formula wt 294.31 acid previously standardized to a green endpoint. A
INS: 951 CAS: [22839-47-0] blank titration exceeding 0.1 mL may be due to
UNII: Z0H242BBR1 [aspartame] excessive water content and may cause loss of visual
endpoint sensitivity.] Perform a blank determination
DESCRIPTION (see General Provisions) and make any necessary
Aspartame occurs as a white, crystalline powder. It is correction. Each mL of 0.1 N perchloric acid is
sparingly soluble in water and slightly soluble in alcohol. equivalent to 29.43 mg of C14H18N2O5.
The pH of a 0.8% solution is between about 4.5 and 6.0. Acceptance criteria: NLT 98.0% and NMT 102.0% of
Function: Sweetener; sugar substitute; flavor enhancer C14H18N2O5, calculated on the dried basis
Packaging and Storage: Store in well-closed containers
in a cool, dry place. IMPURITIES
Inorganic Impurities
IDENTIFICATION • LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric
• INFRARED ABSORPTION, Spectrophotometric Identification Graphite Furnace Method, Method II, Appendix IIIB
Tests, Appendix IIIC Sample: 1 g
Reference standard: USP Aspartame RS Acceptance criteria: NMT 1 mg/kg
Sample and Standard preparation: K
 98 / Aspartame / Monographs FCC 9

Organic Impurities Use phosphoric acid to adjust the pH to 4.3, add 180
• 5-BENZYL-3,6-DIOXO-2-PIPERAZINEACETIC ACID mL of methanol, and mix.
Mobile phase: Dissolve 5.6 g of potassium phosphate Diluent: Add 200 mL of methanol to 1800 mL of
monobasic in 820 mL of water contained in a 1-L flask. water, and mix.
Use phosphoric acid to adjust the pH to 4.3, add 180 Standard stock solution: Dissolve 25 mg USP 5-Benzyl-
mL of methanol, and mix. 3,6-dioxo-2-piperazineacetic Acid RS in 10 mL of
Diluent: Add 200 mL of methanol to 1800 mL of methanol and dilute to 100 mL with water.
water, and mix. Standard solution: Pipet 15 mL of the Standard stock
Standard stock solution: Dissolve 25 mg USP 5-Benzyl- solution into a 50-mL volumetric flask, dilute to volume
3,6-dioxo-2-piperazineacetic Acid RS in 10 mL of with Diluent, and mix. [NOTE—This solution should be
Monographs

methanol and dilute to 100 mL with water. freshly prepared on the day of use.]
Standard solution: Pipet 15 mL of the Standard stock Sample solution: 5 mg/mL in Diluent. [NOTE—This
solution into a 50-mL volumetric flask, dilute to volume solution should be freshly prepared on the day of use.]
with Diluent, and mix. [NOTE—This solution should be Related substances solution: Pipet 2 mL of the Sample
freshly prepared on the day of use.] solution from the test for 5-Benzyl-3,6-dioxo-2-
Sample solution: 5 mg/mL in Diluent. [NOTE—This piperazineacetic Acid into a 100-mL volumetric flask,
solution should be freshly prepared on the day of use. dilute to volume with the Diluent, and mix.
Save the unused portion of this solution for use in the Chromatographic system, Appendix IIA
test for Related Impurities (below).] Mode: High-performance liquid chromatography
Chromatographic system, Appendix IIA Detector: UV 210 nm
Mode: High-performance liquid chromatography Column: 250-mm × 4.6-mm (id) or equivalent,
Detector: UV 210 nm packed with octadecyl silanized silica (10-µm Partisil
Column: 250-mm × 4.6-mm (id) or equivalent, ODS-3, or equivalent)
packed with octadecyl silanized silica (10-µm Partisil Column temperature: 40°
ODS-3, or equivalent) Flow rate: About 2 mL/min
Column temperature: 40° Injection volume: 20 µL
Flow rate: About 2 mL/min System suitability
Injection volume: 20 µL Sample: Standard solution
System suitability Suitability requirement: The area responses for
Sample: Standard solution three replicate injections show a relative standard
Suitability requirement: The area responses for deviation of NMT 2.0%.
three replicate injections show a relative standard Analysis: Separately inject the Related substances
deviation of NMT 2.0%. solution and the Sample solution into the
Analysis: Separately inject the Standard solution and the chromatograph, and record the chromatograms for a
Sample solution into the chromatograph, record the time equal to twice the retention time of aspartame.
chromatograms, and measure all the peak area Acceptance criteria: In the chromatogram obtained
responses. [NOTE—The approximate retention times of from the Sample solution, the sum of the responses of
5-benzyl-3,6-dioxo-2-piperazineacetic acid and all secondary peaks, other than those for 5-benzyl-3,6-
aspartame are 4 min and 11 min, respectively.] dioxo-2-piperazineacetic acid, is not more than the
Calculate the percentage of 5-benzyl-3,6-dioxo-2- response of the aspartame peak obtained in the
piperazineacetic acid in the sample taken by the chromatogram from the Related substances solution.
formula: (NMT 2.0%)

Result = (rU/rS) × (CS/CU) × 100 SPECIFIC TESTS

• LOSS ON DRYING, Appendix IIC: 105° for 4 h
Acceptance criteria: NMT 4.5%
rU = peak area response of 5-benzyl-3,6-dioxo-2- • OPTICAL (SPECIFIC) ROTATION, Appendix IIB
piperazineacetic acid in the Sample solution Sample solution: Dissolve 4 g of sample in sufficient
rS = peak area response of 5-benzyl-3,6-dioxo-2- 15 N formic acid to make 100 mL.
piperazineacetic acid in the Standard Analysis: Make the determination within 30 min of
solution preparation of the Sample solution.
CS = concentration (mg/mL) of 5-benzyl-3,6- Acceptance criteria: [α]D20 between +14.5° and +16.5°,
dioxo-2-piperazineacetic acid in the calculated on the dried basis
Standard solution • RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
CU = concentration (mg/mL) of the Sample Sample: 1 g
solution Acceptance criteria: NMT 0.2%
Acceptance criteria: NMT 1.5%
• OTHER RELATED IMPURITIES
Mobile phase: Dissolve 5.6 g of potassium phosphate
monobasic in 820 mL of water contained in a 1-L flask.
 FCC 9 Monographs / Aspartame–Acesulfame Salt / 99

VB = volume of titrant used for the blank (mL)

Aspartame–Acesulfame Salt
.

N = exact normality of the tetrabutylammonium

First Published: Prior to FCC 6 hydroxide used
Last Revision: FCC 6 Mr1 = formula weight of acesulfame, 163
Mr2 = formula weight of aspartame, 294
W = weight of the sample taken (g)
APM-Ace
F = factor, 10
[2-carboxy-β-(N-(b-methoxycarbonyl-2
Acceptance criteria
phenyl)ethylcarbamoyl)] ethanaminium 6-methyl-4-oxo-1,
Aspartame: NLT 63.0% and NMT 66.0%, calculated
2,3-oxathiazin-3-ide-2,2-dioxide
on the dried basis
L-Phenylalanine, L-α-aspartyl-2-methyl ester compound with

Monographs
Acesulfame: NLT 34.0% and NMT 37.0%, calculated
6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide (1:1)
as acid formed on the dried basis

IMPURITIES
Inorganic Impurities
• LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric
Graphite Furnace Method, Method II, Appendix IIIB
Acceptance criteria: NMT 1 mg/kg
C18H23O9N3S Formula wt 457.45 • POTASSIUM
INS: 962 CAS: [106372-55-8] Standard stock solution: 19.07 µg/mL potassium
UNII: IFE6C6BS24 [aspartame acesulfame] chloride (previously dried at 105° for 2 h), made to
1000 mL. This solution contains 10 µg/mL of
DESCRIPTION potassium.
Aspartame–Acesulfame Salt occurs as a white, crystalline Standard solutions: Pipet 10.0-, 15.0-, and 20.0-mL
powder. It is sparingly soluble in water and slightly soluble aliquots of the Standard stock solution into separate
in alcohol. 100-mL volumetric flasks; add 2.0 mL of a 200 mg/mL
Function: Sweetener solution of sodium chloride and 1.0 mL of hydrochloric
Packaging and Storage: Store in well-closed containers acid to each flask, dilute to volume, and mix. These
in a cool, dry place. Standard solutions contain, respectively, 1.0, 1.5, and
2.0 µg/mL of potassium.
IDENTIFICATION Sample solution: Transfer 3.0 g of sample into a 500-
• INFRARED ABSORPTION, Spectrophotometric Identification mL volumetric flask, dilute to volume, and mix. Transfer
Tests, Appendix IIIC 10 mL of this solution into a 100-mL volumetric flask
Reference standard: USP Aspartame–Acesulfame RS and add 2.0 mL of a 200 mg/mL sodium chloride
Sample and Standard preparation: K solution and 1.0 mL of hydrochloric acid, dilute to
Acceptance criteria: The spectrum of the sample volume, and mix. Filter the solution.
exhibits maxima at the same wavelengths as those in Analysis: Using a suitable atomic absorption
the spectrum of the Reference standard. spectrophotometer equipped with a potassium hollow-
cathode lamp and an air–acetylene flame with water as
ASSAY the blank, concomitantly determine the absorbances of
• PROCEDURE the Standard solutions and the Sample solution at the
Sample: 0.100 to 0.150 g potassium emission line of 766.5 nm. Plot the
Analysis: [NOTE—Use a combination pH-electrode for all absorbance of the Standard solutions versus
titrations.] Dissolve the Sample in 50 mL of ethanol. concentration, in µg/mL, of potassium and draw the
Under a flow of nitrogen, titrate the solution with straight line best fitting the plotted points. From the
standardized 0.1 N tetrabutylammonium hydroxide in graph, determine the potassium concentration, C, in
methanol or 2-propanol. Determine the volume of µg/mL, in the Sample solution. Calculate the percent
titrant needed to reach the first equivalence point and potassium in the sample by the formula:
the second equivalence point. Perform a blank titration
with 50 mL of ethanol. Calculate the percentages of Result = 500C/W
acesulfame and aspartame, respectively, taken by the
formulas:
C = potassium concentration in the Sample
Result = [(V1 − VB) × N × Mr1]/(W × F) solution, calculated from the standard curve
(µg/mL)
Result = [(V2 − VB) × N × Mr2]/(W × F) W = weight of sample taken to prepare the
Sample solution (mg)
Acceptance criteria: NMT 0.5%
V1 = volume of titrant used to reach the first Organic Impurities
equivalence point for the Sample (mL) • 5-BENZYL-3,6-DIOXO-2-PIPERAZINEACETIC ACID
V2 = volume of titrant used to reach the second Mobile phase: Dissolve 5.6 g of potassium phosphate
equivalence point for the Sample (mL) monobasic in 820 mL of water contained in a 1-L flask.
 100 / Aspartame–Acesulfame Salt / Monographs FCC 9

Use phosphoric acid to adjust the pH to 4.3, add 180 Acid into a 100-mL volumetric flask, dilute to volume
mL of methanol, and mix. Filter through a 0.45-µm with the Diluent, and mix.
disk, and degas. Analysis: Separately inject equal 20-µL portions of the
Diluent: Add 200 mL of methanol to 1800 mL of Standard solution and the Sample solution into the
water, and mix. chromatograph, and record the chromatograms for a
Standard stock solution: Dissolve 25 mg of USP 5- time equal to twice the retention time of aspartame.
Benzyl-3,6-dioxo-2-piperazineacetic Acid RS in 10 mL Acceptance criteria: In the chromatogram obtained
of methanol contained in a 100-mL volumetric flask, from the Sample solution, the sum of the responses of
and dilute to volume with water. all secondary peaks, other than those for 5-benzyl-3,6-
Standard solution: Pipet 15 mL of the Standard stock dioxo-2-piperazineacetic acid and acesulfame, is not
Monographs

solution into a 50-mL volumetric flask, dilute to volume more than the response of the aspartame peak
with Diluent, and mix. [NOTE—This solution should be obtained in the chromatogram from the Standard
freshly prepared on the day of use.] solution. (NMT 1.0%)
Sample solution: 5 mg/mL in Diluent. [NOTE—This
solution should be freshly prepared on the day of use. SPECIFIC TESTS
Save the unused portion of this solution for use in the • LOSS ON DRYING, Appendix IIC: 105° for 4 h
test for Related Impurities (below).] Acceptance criteria: NMT 0.5%
Chromatographic system, Appendix IIA • OPTICAL (SPECIFIC) ROTATION, Appendix IIB
Mode: High-performance liquid chromatography Sample: 6.2 g, previously dried
Detector: UV 210 nm Analysis: Dissolve the Sample in sufficient 15 N formic
Column: 250-mm × 4.6-mm (id) or equivalent, acid, made to 100 mL. Make the determination within
packed with octadecyl silanized silica (10-µm Partisil 30 min of preparation of the Sample. Divide the
ODS-3, or equivalent) calculated specific rotation by 0.646 to correct for the
Column temperature: 40° aspartame content in aspartame–acesulfame salt.
Flow rate: About 2 mL/min Acceptance criteria: [α]D20 between +14.5° and +16.5°,
Injection volume: 20 µL on the dried basis
System suitability
Sample: Standard solution
Suitability requirement: The area responses for
DL-Aspartic Acid
.

replicate injections show a relative standard

deviation of NMT 2.0%. First Published: Prior to FCC 6
Analysis: Separately inject the Standard solution and the
Sample solution into the chromatograph, record the DL-Aminosuccinic Acid
chromatograms, and measure all the peak area
responses. [NOTE—The approximate retention times of
5-benzyl-3,6-dioxo-2-piperazineacetic acid and
aspartame are 4 min and 11 min, respectively.]
Calculate the percentage of 5-benzyl-3,6-dioxo-2-
piperazineacetic acid in the sample taken by the C4H7NO4 Formula wt 133.10
formula: CAS: [617-45-8]
UNII: 28XF4669EP [aspartic acid, dl-]
Result = (rU/rS) × (CS/CU) × 100
DESCRIPTION
DL-Aspartic Acid occurs as colorless or white crystals. It is
rU = peak area response of 5-benzyl-3,6-dioxo-2-
piperazineacetic acid in the Sample solution slightly soluble in water, but insoluble in alcohol and in
rS = peak area response of 5-benzyl-3,6-dioxo-2- ether. It is optically inactive and melts with decomposition
piperazineacetic acid in the Standard at about 280°.
solution Function: Nutrient
CS = concentration of 5-benzyl-3,6-dioxo-2- Packaging and Storage: Store in well-closed, light-
piperazineacetic acid in the Standard resistant containers.
solution (mg/mL) IDENTIFICATION
CU = concentration of the Sample solution (mg/ • INFRARED SPECTRA, Spectrophotometric Identification Tests,
mL) Appendix IIIC
Acceptance criteria: NMT 0.5% Sample preparation: Mineral oil mull
• RELATED IMPURITIES Acceptance criteria: The spectrum of the sample
[NOTE—Determine as directed in the test for 5-Benzyl-3, exhibits relative maxima at the same wavelengths as
6-dioxo-2-piperazineacetic Acid (above), using the those of the spectrum below.
following Standard solution and Analysis.]
Standard solution: Pipet 1.5 mL of the Sample solution
from the test for 5-Benzyl-3,6-dioxo-2-piperazineacetic
 FCC 9 Monographs / L-Aspartic Acid / 101

ASSAY IMPURITIES
• PROCEDURE Inorganic Impurities
Sample: 200 mg • LEAD, Lead Limit Test, Appendix IIIB: Using a Sample
Analysis: Dissolve the Sample in 3 mL of formic acid and Solution prepared as directed for organic compounds
50 mL of glacial acetic acid. Add 2 drops of crystal Control: 5 µg Pb (5 mL of Diluted Standard Lead
violet TS and titrate with 0.1 N perchloric acid to a Solution)
green endpoint or until the blue color disappears Acceptance criteria: NMT 5 mg/kg
completely. [CAUTION—Handle perchloric acid in an
appropriate fume hood.] Perform a blank determination SPECIFIC TESTS
(see General Provisions) and make any necessary • LOSS ON DRYING, Appendix IIC: 105° for 3 h

Monographs
correction. Each mL of 0.1 N perchloric acid is Acceptance criteria: NMT 0.3%
equivalent to 13.31 mg of C4H7NO4. • RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
Acceptance criteria: NLT 98.5% and NMT 101.5% of Sample: 1 g
C4H7NO4, calculated on the dried basis Acceptance criteria: NMT 0.1%

DL-Aspartic Acid (Mineral Oil Mull)

DESCRIPTION
L-Aspartic Acid
.

L-Aspartic Acid occurs as white crystals or as a crystalline

First Published: Prior to FCC 6 powder. It is slightly soluble in water, but insoluble in
Last Revision: FCC 6 alcohol and in ether. It melts at about 270°.
Function: Nutrient
L-Aminosuccinic Acid Packaging and Storage: Store in well-closed, light-
resistant containers.

IDENTIFICATION
• INFRARED ABSORPTION, Spectrophotometric Identification
Tests, Appendix IIIC
Reference standard: USP Aspartic Acid RS
C4H7NO4 Formula wt 133.10 Sample and Standard preparation: K
CAS: [56-84-8]
UNII: 30KYC7MIAI [aspartic acid]
 102 / L-Aspartic Acid / Monographs FCC 9

Acceptance criteria: The spectrum of the sample diesters. Esterified astaxanthin is the primary carotenoid
exhibits maxima at the same wavelengths as those in present and the approximate astaxanthin composition is:
the spectrum of the Reference standard. 75% monoester, 20% diester, and 5% free form
astaxanthin. Astaxanthin Esters from Haematococcus
ASSAY pluvialis is soluble in n-hexane, acetone, and ether; partially
• PROCEDURE soluble in alcohol; practically insoluble in water and hot
Sample: 200 mg water. Suitable antioxidants may be added.
Analysis: Dissolve the Sample in 3 mL of formic acid and Function: Source of astaxanthin
50 mL of glacial acetic acid. Add 2 drops of crystal Packaging and Storage: Store in tight, light-resistant
violet TS, and titrate with 0.1 N perchloric acid to a containers in a cool place.
Monographs

green endpoint or until the blue color disappears

completely. [CAUTION—Handle perchloric acid in an IDENTIFICATION
appropriate fume hood.] Perform a blank determination • EPA CONTENT, Fatty Acid Composition (Saturated, cis-
(see General Provisions), and make any necessary Monounsaturated, and cis-Polyunsaturated) in Oils
correction. Each mL of 0.1 N perchloric acid is Containing Long Chain Polyunsaturated Fatty Acids,
equivalent to 13.31 mg of C4H7NO4. Appendix VII
Acceptance criteria: NLT 98.5% and NMT 101.5% of Analysis: Proceed as directed, then calculate the amount
C4H7NO4, calculated on the dried basis of EPA (eicosapentaenoic acid; C20:5 n-3) present as
the percentage of total fatty acids.
IMPURITIES Acceptance criteria: NMT 1.0%
Inorganic Impurities • THIN-LAYER CHROMATOGRAPHY, Appendix IIA
• LEAD, Lead Limit Test, Appendix IIIB Sample solution: 10 mg/mL in acetone
Sample solution: Prepare as directed for organic Standard solution: 10 mg/mL of USP Astaxanthin Esters
compounds. from Haematococcus pluvialis RS in acetone
Control: 5 µg Pb (5 mL of Diluted Standard Lead Adsorbent: 0.25-mm layer of chromatographic silica
Solution) gel. [NOTE—Dry silica gel at 110° for 1 h before use.]
Acceptance criteria: NMT 5 mg/kg Developing solvent system: Hexane and acetone
[70:30]
SPECIFIC TESTS
Application volume: 5 µL
• LOSS ON DRYING, Appendix IIC: 105° for 3 h
Analysis: Develop the chromatogram in the Developing
Acceptance criteria: NMT 0.25%
solvent system until the solvent front has moved about
• OPTICAL (SPECIFIC) ROTATION, Appendix IIB
three-fourths of the length of the plate. Remove the
Sample: 8 g, previously dried
plate from the chamber and dry in a current of air.
Analysis: Dissolve the Sample in sufficient 6 N
Acceptance criteria: The principal spots obtained from
hydrochloric acid to make 100 mL.
the Sample solution correspond in color, size, and RF
Acceptance criteria: [α]D20 between +24.5° and +26.0°,
value to those obtained from the Standard solution.
on the dried basis
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC ASSAY
Sample: 1 g
Acceptance criteria: NMT 0.1% Change to read:
• ASTAXANTHIN (TOTAL) [NOTE—Astaxanthin measured by
this method is total astaxanthin, including free
astaxanthin and both mono- and diesters.]
Astaxanthin Esters from Haematococcus
.

Buffer solution: Dissolve 6.06 g of

pluvialis tris(hydroxymethyl)aminomethane in 750 mL of water,
First Published: Third Supplement, FCC 7 adjust with 1 M hydrochloric acid to a pH of 7.0, and
dilute with water to 1000 mL.
Solution A: 4 U/mL of cholesterol esterase2 in Buffer
Astaxanthin1
solution. [NOTE—Prepare fresh daily.]
Astaxanthin Esters
Internal standard solution: 37.5 µg/mL of USP
Astaxanthin Fatty Acid Esters
Apocarotenal RS in acetone
(3S,3’S)-3,3’-dihydroxy-β,β-carotene-4,4’-dione
Standard stock solution: Transfer 30 mg of USP
DESCRIPTION Astaxanthin Esters from Haematococcus pluvialis RS to a
Astaxanthin Esters from Haematococcus pluvialis occurs as a 100-mL volumetric flask. Dissolve in 30 mL of acetone,
dark red, viscous oil. It is the product of the fermentation shake by mechanical means, and dilute with acetone to
of Haematococcus pluvialis, extracted with either super volume.
critical CO2 or acetone. It is a complex mixture, primarily Standard solution: Combine 2.0 mL of the Standard
composed of lipids, with astaxanthin esterified with stock solution and 1.0 mL of the Internal standard
common edible fatty acids to form both mono- and solution in a glass centrifuge tube. Add 3.0 mL of
2 Use Wako Pure Chemicals catalog no. 037-11221, available from

1 Commercial products marketed as astaxanthin are actually often mixtures of www.wakousa.com; Sigma catalog no. C9281, available from
free and esterified astaxanthin or primarily esterified astaxanthin. www.sigmaaldrich.com; or equivalent.
FCC 9 Monographs / Astaxanthin Esters / 103

Solution A to the tube, and mix gently by inversion. Phosphoric

t-Butyl- acid, 1%
Place the tube in a block heater set to 37° and allow
Time Methanol methylether aqueous
the reaction to continue for 45 min, gently and slowly (min) (%) (%) (%)
inverting the tube every 10 min. After 45 min, add 1 g
27 16 80 4
of sodium sulfate decahydrate and 2 mL of petroleum
27.1 81 15 4
ether to the tube. Vortex the tube for 30 s, then
centrifuge at 3000 rpm for 3 min. Carefully transfer the 35 81 15 4
petroleum ether layer to a 10-mL glass centrifuge tube
containing 1 g of sodium sulfate anhydrate. Be careful Analysis: Separately inject the Standard solution and the
to avoid pipetting the intermediate emulsive layer.
•Sample solutions•(ERR 1-Jan-2014) into the chromatograph.

Monographs
Evaporate the petroleum ether layer using a vacuum or Record the chromatograms, and identify the peaks by
a stream of inert gas at room temperature, add 3 mL of comparison to the Reference Chromatograms supplied
acetone, sonicate, and filter the mixture. The filtered with the USP Apocarotenal RS (internal standard) and
solution is the Standard solution. with the USP Astaxanthin Esters from Haematococcus
Sample stock solution: Warm a quantity of the sample pluvialis RS. [NOTE—The approximate retention times for
in a water bath at 50°–60° for 30 min. Shake the 13-cis-astaxanthin, trans-astaxanthin, 9-cis-astaxanthin,
sample well at 10-min intervals. After 30 min, transfer and the internal standard apocarotenal (trans-beta-apo-
30 mg of the sample to a 100-mL volumetric flask. 8’-carotenal) are 9 min, 10 min, 14 min, and 17 min,
Dissolve in 30 mL of acetone, shake by mechanical respectively.]
means, and dilute with acetone to volume. [NOTE— For the Standard solution and the •Sample solutions,•(ERR 1-
Prepare in triplicate.] Jan-2014) separately calculate the ratios of the peak

Sample solution: •[NOTE—Prepare one Sample solution responses of total astaxanthin to the internal standard
as follows from each of the three Sample stock obtained from the individual analysis:
solutions.]•(ERR 1-Jan-2014) Combine 2.0 mL of the Sample
Result = (F1P13-cis + Ptrans + F2P9-cis)/PIS
stock solution and 1.0 mL of the Internal standard
solution in a glass centrifuge tube. Add 3.0 mL of F1 = relative response coefficient of 13-cis-
Solution A to the tube, and mix gently by inversion. astaxanthin to trans-astaxanthin (1.3)
Place the tube in a block heater set to 37° and allow P13- = peak response for 13-cis-astaxanthin
the reaction to continue for 45 min, gently and slowly cis obtained from the chromatogram
inverting the tube every 10 min. After 45 min, add 1 g Ptrans = peak response for trans-astaxanthin obtained
of sodium sulfate decahydrate and 2 mL of petroleum from the chromatogram
ether to the tube. Vortex the tube for 30 s, then F2 = relative response coefficient of 9-cis-
centrifuge at 3000 rpm for 3 min. Carefully transfer the astaxanthin to trans-astaxanthin (1.1)
petroleum ether layer to a 10-mL glass centrifuge tube P9- = peak response for 9-cis-astaxanthin obtained
containing 1 g of sodium sulfate anhydrate. Be careful cis from the chromatogram
to avoid pipetting the intermediate emulsive layer. PIS = peak response for the internal standard,
Evaporate the petroleum ether layer using a vacuum or apocarotenal
a stream of inert gas at room temperature, add 3 mL of Calculate the percentage of astaxanthin (w/w) in the
acetone, sonicate, and filter the mixture. The filtered portion of the sample taken •(as the average from the
solution is the Sample solution. three Sample solutions):•(ERR 1-Jan-2014)
Chromatographic system, Appendix IIA
Mode: High-performance liquid chromatography Result = (RU/RS) × (CS/CU) × 100
Detector: 474 nm
Column: 4.6-mm × 250-mm column with a C30 silane RU = ratio of peak responses of total astaxanthin
bonded stationary phase on fully porous spherical to the internal standard obtained from the
silica, 5-µm in diameter3 Sample solution
Column temperature: 25° RS = ratio of peak responses of total astaxanthin
Flow rate: 1.0 mL/min to the internal standard obtained from the
Injection size: 20 µL Standard solution
Mobile phase: See the gradient table below. CS = concentration of astaxanthin in the Standard
solution (mg/mL)
Phosphoric CU = concentration of the Sample solution (mg/
t-Butyl- acid, 1% mL)
Time Methanol methylether aqueous Acceptance criteria: 5.0%–15.0%
(min) (%) (%) (%)
0 81 15 4 IMPURITIES
15 66 30 4
Inorganic Impurities
• ARSENIC, Elemental Impurities by ICP, Appendix IIIC
23 16 80 4
[NOTE—Alternatively, the arsenic content may be
determined by the following method.]
3 YMC-Carotenoid S 5-µm column, available at www.ymc.co.jp/en, or Apparatus
equivalent.
 104 / Astaxanthin Esters / Monographs FCC 9

Sample digestion: Use a microwave oven4 equipped recommended. A palladium (0.3%) and magnesium
with advanced composite vessels with 100-mL Teflon nitrate (0.2%) solution may be purchased from High
liners. Use rupture membranes to vent vessels should Purity Standards, or equivalent.]
the pressure exceed 125 psi. The vessels fit into a Modifier working solution: Transfer 3 mL of 1%
turntable, and each vessel can be vented into an Palladium stock solution and 2 mL of 1% Magnesium
overflow container. Equip the microwave oven with nitrate stock solution to a 10-mL volumetric flask, and
an exhaust tube to ventilate fumes. dilute with 2% nitric acid to volume. A volume of 5 µL
Sample analysis: Use a suitable graphite furnace provides 0.015 mg of palladium and 0.01 mg of
atomic absorption spectrophotometer (GFAAS) magnesium nitrate.
equipped with an autosampler, pyrolytically coated Sample solution: [CAUTION—Wear proper eye
Monographs

graphite tubes, solid pyrolytic graphite platforms, and protection and protective clothing and gloves during
an adequate means of background correction.5 An sample preparation. Closely follow the manufacturer’s
electrodeless discharge lamp serves as the source, safety instructions for use of the microwave digestion
argon as the purge gas, and air as the alternate gas. apparatus.]
Set up the instrument according to manufacturer’s Transfer 500 mg of the sample into a Teflon digestion
specifications, with consideration of current good vessel liner. Prepare samples in duplicate. Add 15 mL
GFAAS practices. The instrument parameters are as of nitric acid, and swirl gently. Cover the vessels with
follows: lids, leaving the vent fitting off. Predigest overnight
Wavelength: 193.7 nm under a hood. Place the rupture membrane in the vent
Lamp current: 300 (EDL) modulated fitting, and tighten the lid. Place all vessels on the
Pyrolysis: 1000° microwave oven turntable. Connect the vent tubes to
Atomization: 2400° the vent trap, and connect the pressure-sensing line to
Slit: 0.7 the appropriate vessel. Initiate a two-stage digestion
Characteristic mass: 15 pg procedure by heating the microwave at 15% power for
Glassware: Acid wash all glass, Teflon, and plastic 15 min followed by 25% power for 45 min. Remove
vessels by soaking them in a nitric acid bath containing the turntable of vessels from the oven, and allow the
a 4:1 solution of water and nitric acid. [CAUTION—Wear vessels to cool to room temperature (a cool water bath
a full face shield and protective clothing and gloves at may be used to speed the cooling process). Vent the
all times when working with acid baths.] After acid vessels when they reach room temperature. Remove
soaking, rinse acid-washed items in deionized water, the lids, and slowly add 2 mL of 30% hydrogen
dry them, and store them in clean, covered cabinets. peroxide to each. Allow the reactions to subside, and
Calibration standard stock solution: 100 µg/L seal the vessels. Return the vessels on the turntable to
Prepare from a suitable standard, which may be the microwave oven, and heat for an additional 15 min
purchased [accuracy certified against National Institute at 30% power. Remove the vessels from the oven, and
of Standards and Technology (NIST) spectrometric allow them to cool to room temperature. Transfer the
standard solutions]. cooled digests into 25-mL volumetric flasks, and dilute
Calibration standard solutions: 2.0 µg/L, 5.0 µg/L, with deionized water to volume.
10.0 µg/L, 25.0 µg/L, and 50.0 µg/L in 2% nitric acid Analysis: The graphite furnace program is as follows:
from the Calibration standard stock solution 1. Dry at 115° using a 1-s ramp, a 65-s hold, and a
1% Palladium stock solution: Mix 1 g of ultrapure 300-mL/min argon flow.
palladium metal, with 20 mL of water and 10 mL of 2. Char the sample at 1000° using a 1-s ramp, a 20-s
nitric acid in a Teflon beaker, and warm the solution on hold, and a 300-mL/min air flow.
a hot plate to dissolve the palladium. Allow the 3. Cool down and purge the air from the furnace for
solution to cool to room temperature, transfer it into a 10 s using a 20° set temperature and a 300-mL/
100-mL volumetric flask, and dilute with deionized min argon flow.
water to volume. 4. Atomize at 2400° using a 0-s ramp and a 5-s hold
1% Magnesium nitrate stock solution: Mix 1 g of with the argon flow stopped.
ultrapure magnesium nitrate, with 40 mL of water and 5. Clean out at 2600° with a 1-s ramp and a 5-s
1 mL of nitric acid in a Teflon beaker, and warm the hold.
solution on a hot plate to dissolve. Allow the solution Use the autosampler to inject 20-µL aliquots of blanks,
to cool to room temperature, transfer it into a 100-mL Calibration standard solutions, and Sample solutions and
volumetric flask, and dilute with deionized water to 5 µL of Modifier working solution. Inject each solution in
volume. duplicate, and average the results. Use peak area
[NOTE—Because of the difficulty in preparing matrix measurement for all quantitations. After ensuring that
modifier stock solutions with the required purity, the furnace is clean by running a 5% nitric acid blank,
purchasing modifier stock solutions and using them check the instrument’s sensitivity by running a 20-µL
to prepare working modifier solutions is aliquot of the 25-µg Calibration standard solution.
Compare the results obtained with the expected results
4 CEM Model MDS-2100, or equivalent. for the equipment used, and take the necessary steps
5 This method was developed using a Perkin-Elmer Model 5100, HGA-600 to correct any problems.
furnace, and an AS-60 autosampler with Zeeman effect background
correction.
FCC 9 Monographs / Astaxanthin Esters / 105

Calculate the characteristic mass. Record and track the of nitric acid to dissolve the phosphate. Dilute with
integrated absorbance and characteristic mass for deionized water to 100 mL.
reference and quality assurance. 1% Magnesium nitrate stock solution: Mix 1 g of
Inject each Calibration standard solution in duplicate. Use ultrapure magnesium nitrate, with 40 mL of water and
the algorithms provided in the instrument software to 1 mL of nitric acid in a Teflon beaker, and warm on a
establish calibration curves. Recheck calibration hot plate to dissolve the solids. Allow the solution to
periodically, and recalibrate if the recheck differs from cool to room temperature, transfer it into a 100-mL
the original calibration by more than 10%. volumetric flask, and dilute with deionized water to
Inject the Sample solution in duplicate, and record the volume.
integrated absorbance. If the instrument response [NOTE—Because of the difficulty in preparing matrix

Monographs
exceeds that of the calibration curve, dilute with 5% modifier stock solutions with the required purity,
nitric acid to bring the sample’s response into the purchasing modifier stock solutions and using them
working range, and note the dilution factor (DF). All to prepare working solutions is recommended. An
sample analyses should be blank corrected using a ammonium dihydrogen phosphate (4%) and
sample solution blank. magnesium nitrate (0.2%) solution may be purchased
If a computer-based instrument is used, the data output from High Purity Standards, or equivalent.]
is reported as µg/L. Calculate the concentration of Modifier working solution: Transfer 4 mL of 10%
arsenic, in µg/g (equivalent to mg/kg), in the original Ammonium dihydrogen phosphate stock solution and 2
sample taken: mL of 1% Magnesium nitrate stock solution to a 10-mL
volumetric flask, and dilute with 2% nitric acid to
Result = (C × DF × V)/W volume. A volume of 5 µL provides 0.2 mg of
phosphate plus 0.01 mg of magnesium nitrate.
C = concentration of arsenic in the sample
Sample solution: Prepare as directed for the Sample
aliquot injected (µg/L)
solution in the Arsenic test.
DF = dilution factor of the Sample solution
[CAUTION—Wear proper eye protection and protective
V = final volume of the Sample solution (L)
clothing and gloves during sample preparation.
W = weight of the sample taken to prepare the
Closely follow the manufacturer’s safety instructions
Sample solution (g)
for use of the microwave digestion apparatus.]
[NOTE—To monitor recovery and ensure analytical
Analysis: The graphite furnace program is as follows:
accuracy for proper quality assurance, analyze blanks,
1. Dry at 120° using a 1-s ramp, a 55-s hold, and a
spiked blanks, and a spiked oil with each digestion
300-mL/min argon flow.
set.]
2. Char the sample at 850° using a 1-s ramp, a 30-s
Acceptance criteria: NMT 2.0 mg/kg
hold, and a 300-mL/min air flow.
• CADMIUM, Elemental Impurities by ICP, Appendix IIIC
3. Cool down and purge the air from the furnace for
Acceptance criteria: NMT 1.0 mg/kg
10 s using a 20° set temperature and a 300-mL/
• LEAD, Elemental Impurities by ICP, Appendix IIIC
min argon flow.
[NOTE—Alternatively, the lead content may be
4. Atomize at 2100° using a 0-s ramp and a 5-s hold
determined by the following method.]
with the argon flow stopped.
Apparatus 5. Clean out at 2600° with a 1-s ramp and a 5-s
Sample digestion: Use a microwave oven4 equipped hold.
with advanced composite vessels with 100-mL Teflon Use the autosampler to inject 20-µL aliquots of blanks,
liners. Use rupture membranes to vent vessels should Calibration standard solutions, Sample solutions, and 5
the pressure exceed 125 psi. The vessels fit into a µL of Modifier working solution. Inject each solution in
turntable, and each vessel can be vented into an duplicate, and average the results. Use peak-area
overflow container. Equip the microwave oven with measurement for all quantitation. After ensuring that
an exhaust tube to ventilate fumes. the furnace is clean by running a 5% nitric acid blank,
Sample analysis: See Apparatus in Lead Limit Test, check instrument sensitivity by running an aliquot of
Atomic Absorption Spectrophotometric Graphite Furnace the 25-µg Calibration standard solution. Compare the
Method, Method I, Appendix IIIB. results obtained with the expected results for the
Calibration standard stock solution: 100 µg/L equipment used, and take the necessary steps to
Prepare from a suitable standard, which may be correct any problems.
purchased [accuracy certified against National Institute Calculate the characteristic mass, and record and track
of Standards and Technology (NIST) spectrometric the integrated absorbance and characteristic mass for
standard solutions]. reference and quality assurance.
Calibration standard solutions: 2.0 µg/L, 5.0 µg/L, Inject each Calibration standard solution in duplicate. Use
10.0 µg/L, 25.0 µg/L, and 50.0 µg/L in 2% nitric acid the algorithms provided in the instrument software to
from the Calibration standard stock solution establish calibration curves. Recheck the calibration
10% Ammonium dihydrogen phosphate stock periodically and recalibrate if recheck differs from the
solution: Mix 10 g of ultrapure ammonium original calibration by more than 10%.
dihydrogen phosphate, with 40 mL of water and 1 mL Inject the Sample solution in duplicate, and record the
integrated absorbance. If the instrument response
 106 / Astaxanthin Esters / Monographs FCC 9

exceeds that of the calibration curve, dilute with 5% hydrochloric acid from the Calibration standard stock
nitric acid to bring the sample response into the solution
working range, and note the dilution factor (DF). All Reducing reagent: 5% stannous chloride in 25%
sample analyses should be blank corrected using a hydrochloric acid (trace-metal grade). [NOTE—Prepare
sample solution blank. daily.]
If a computer-based instrument is used, the data output Sample solution: Prepare as directed for the Sample
is reported as micrograms per liter. solution in the Arsenic test.
Calculate the concentration, in µg/g (equivalent to mg/ [CAUTION—Wear proper eye protection and
kg), of lead in the original sample taken: protective clothing and gloves during sample
preparation. Closely follow the manufacturer’s
Monographs

Result = (C × DF × V)/W safety instructions for use of the microwave

digestion apparatus.]
C = concentration of lead in the sample aliquot
Analysis: Optimize the instrument settings for the
injected (µg/L)
spectrophotometer as described in the instrument
DF = dilution factor of the Sample solution
manual. The instrument parameters for cold vapor
V = final volume of the Sample solution (L)
generation are as follows:
W = weight of the sample taken to prepare the
Wavelength: 253.6 nm
Sample solution (g)
Slit: 0.70 nm
[NOTE—To monitor recovery and ensure analytical
Reagent setting: 5
accuracy for proper quality assurance, analyze blanks,
Gas flow: 5–6 L/min
spiked blanks, and a spiked oil with each digestion
Reaction time: 0.5 min
set.]
Use a peak height integration method with a 40-s
Acceptance criteria: NMT 1.0 mg/kg
integration time and a 20-s read delay in an
• MERCURY
unheated absorption cell. Zero the instrument as
Apparatus
follows: Place a Fleaker containing 50 mL of 1 N
Sample digestion: Use a microwave oven (CEM
hydrochloric acid in the sample well of the hydride
Model MDS-2100, or equivalent) equipped with
generator. Press “start” on the vapor generator and
advanced composite vessels with 100-mL Teflon
“read” on the atomic absorption
liners. Use rupture membranes to vent vessels should
spectrophotometer. The instrument will
the pressure exceed 125 psi. The vessels fit into a
automatically flush the sample container with
turntable, and each vessel can be vented into an
nitrogen, dispense the designated amount of
overflow container. Equip the microwave oven with
reagent, stir the sample for a designated reaction
an exhaust tube to ventilate fumes.
time, and purge the head volume again with
Sample analysis: Use a suitable atomic absorption
nitrogen, sweeping any vapor into the quartz cell for
spectrophotometer equipped with an atomic vapor
determination of absorption. The atomic absorption
assembly. This method was developed using a Perkin-
spectrophotometer will automatically zero on this
Elmer Model 5100 and IL 440 Thermo Jarrell Ash
sample when “autozero” is selected from the
atomic vapor assembly. An electrodeless discharge
calibration menu.
lamp serves as the source, with an inert gas such as
Generate a standard curve of concentration versus
argon or nitrogen as the purge gas. Set up the
absorption by analyzing the five Calibration standard
instrument according to manufacturer’s specifications.
solutions prepared as described for daily standards
Instrument parameters are as follows:
under Calibration standard solutions. Analyze each
Wavelength: 253.6 nm
solution in duplicate, generate the calibration curve,
Slit: 0.7
and store, using procedures specific for the
Reagent setting: 5
instrumentation.
Gas flow: 5–6 L/min
Transfer an appropriate aliquot of the Sample solution
Reaction time: 0.5 min
(usually 2 mL) in a Fleaker containing 50 mL of 1 N
Glassware: Acid wash all glass, Teflon, and plastic
hydrochloric acid. Analyze solutions in duplicate
vessels by soaking them in a nitric acid bath
using the procedure specified in the instrument
containing a 4:1 solution of water and nitric acid.
manual. Using the calibration algorithm provided in
[CAUTION—Wear a full face shield and protective
the instrument software, calculate and report the
clothing and gloves at all times when working with
mercury concentration in nanograms of mercury in
acid baths.] After acid soaking, rinse acid-washed
the aliquot analyzed.
items in deionized water, dry, and store them in
Calculate the level of mercury as µg/g (equivalent to
clean, covered cabinets.
mg/kg), in the original sample taken:
Calibration standard stock solution: 200 ng/g of
mercury. Prepare from a suitable standard, which Result = (A × DF)/(W × 1000)
may be purchased [accuracy certified against National
Institute of Standards and Technology (NIST) A = amount of mercury in the aliquot analyzed
spectrometric standard solutions]. (ng)
Calibration standard solutions: 20 ng, 60 ng, 100 DF = dilution factor (final volume of Sample
ng, 200 ng, and 400 ng of mercury in 1 N solution/volume taken for analysis)
FCC 9 Monographs / Azodicarbonamide / 107

W = weight of the sample taken to prepare the

Azodicarbonamide
.

Sample solution (g)

[NOTE—To monitor recovery and ensure analytical First Published: Prior to FCC 6
accuracy for proper quality assurance, analyze
blanks, spiked blanks, and a spiked oil with each
Azodicarboxylic Acid Diamide
digestion set.]
Acceptance criteria: NMT 1.0 mg/kg
Organic Impurities
• PHEOPHORBIDE CONTENT
Solution A: 50 mg/mL of sodium sulfate

Monographs
Solution B: Saturated solution of sodium sulfate C2H4N4O2 Formula wt 116.08
Sample stock solution: Transfer 100 mg of the sample INS: 927a CAS: [123-77-3]
to a 10-mL test tube, add 10 mL of acetone, and UNII: 56Z28B9C8O [azodicarbonamide]
dissolve with sonication. Quantitatively transfer this
solution to a separatory funnel, rinsing the test tube DESCRIPTION
three times with 10-mL portions of acetone and adding Azodicarbonamide occurs as a yellow to orange-red,
the rinsings to the funnel. Add 30 mL of ethyl ether to crystalline powder. It is practically insoluble in water and in
the separatory funnel, followed by 50 mL of Solution A. most organic solvents. It is slightly soluble in dimethyl
Mix the contents of the separatory funnel by shaking sulfoxide. It melts above 180° with decomposition.
gently, then draw off and discard the lower layer. Function: Maturing agent for flour
Repeat washing with Solution A three times. Dehydrate Packaging and Storage: Store in well-closed, light-
the remaining extract with sodium sulfate anhydrate, resistant containers.
then transfer the extract to a 50-mL volumetric flask.
Dilute with ethyl ether to volume. IDENTIFICATION
Sample solution: Transfer 20 mL of the Sample stock • ULTRAVIOLET ABSORPTION
solution to a small beaker. Add 20 mL of 17% Sample solution: 35 µg/mL
hydrochloric acid, and mix the solution vigorously. Acceptance criteria: The Sample solution exhibits an
Transfer the hydrochloric acid layer to a separatory ultraviolet absorption maximum at about 245 nm.
funnel and repeat the extraction with a second 10-mL
portion of 17% hydrochloric acid, adding the ASSAY
hydrochloric acid layer to the separatory funnel. Add • PROCEDURE
150 mL of Solution B, 20 mL of ethyl ether, and mix Sample: 225 mg, previously dried
the contents of the separatory funnel by shaking. Analysis: Transfer the Sample into a 250-mL glass-
Transfer the ethyl ether layer to a 20-mL volumetric stoppered iodine flask. Add about 23 mL of dimethyl
flask, and dilute with ethyl ether to volume. sulfoxide to the flask, washing any adhered sample
Analysis: Using a suitable spectrophotometer, down with the solvent, then stopper the flask and place
determine the absorbance of the Sample solution at about 2 mL of the solvent in the cup or lip of the flask.
667 nm in a 1-cm cuvette, using ethyl ether as the Swirl occasionally, until dissolution of the Sample is
blank. If necessary, the Sample solution may be further complete, and then loosen the stopper to drain the
diluted with ethyl ether to obtain an absorbance within remainder of solvent into the flask and to rinse down
the linear operating range of the instrument. any dissolved sample into the solution. Add 5.0 g of
Calculate the percentage of pheophorbide in the potassium iodide followed by 15 mL of water, then
portion of the sample taken: immediately pipet 10 mL of 0.5 N hydrochloric acid
into the flask, and rapidly stopper. Swirl until the
Result = AU/(CU × E) × 100 potassium iodide dissolves, and allow to stand for 20 to
25 min protected from light. Titrate the liberated iodine
AU = absorbance of the Sample solution with 0.1 N sodium thiosulfate to the disappearance of
CU = concentration of the Sample solution (mg/ the yellow color. Titrate with additional thiosulfate if
mL) any yellow color appears within 15 min. Perform a
E = absorption constant for 1 mg/mL blank determination (see General Provisions) on a
pheophorbide in ethyl ether at 667 nm in a solution consisting of 25 mL of dimethyl sulfoxide, 5.0
1-cm cuvette (70.2 mL/mg−1cm−1) g of potassium iodide, 15 mL of water, and 5 mL of 0.5
Acceptance criteria: NMT 0.02% N hydrochloric acid, and make any necessary
correction. Each mL of 0.1 N sodium thiosulfate is
SPECIFIC TESTS equivalent to 5.804 mg of C2H4N4O2.
• WATER, Water Determination, Appendix IIB
Acceptance criteria: NLT 98.6% and NMT 100.5% of
Acceptance criteria: NMT 1.0%
C2H4N4O2, on the dried basis
OTHER REQUIREMENTS
• LABELING Label to indicate the name of any added
antioxidant.
 108 / Azodicarbonamide / Monographs FCC 9

IMPURITIES
Azorubine1
.

Inorganic Impurities
• LEAD, Lead Limit Test, Appendix IIIB First Published: FCC 8
Sample solution: Prepare as directed for organic
compounds. Carmoisine
Control: 5 µg Pb (5 mL of Diluted Standard Lead CI Food Red 3
Solution) CI No. 14720
Acceptance criteria: NMT 5 mg/kg Class: Mono-Azo
SPECIFIC TESTS Disodium 4-hydroxy-3-(4-sulfonato-1-naphthylazo)-1-
naphthalenesulfonate
Monographs

• LOSS ON DRYING, Appendix IIC: Vacuum oven at 50° for 2

h
Acceptance criteria: NMT 0.5%
• NITROGEN, Appendix IIIC
Sample: 50 mg
Analysis: Transfer the Sample into a 100-mL Kjeldahl
flask, add 3 mL of concentrated hydriodic acid solution
(57% freshly assayed), and digest the mixture with
gentle heating for 1.25 h, adding sufficient water, when C20H12N2Na2O7S2 Formula wt 502.44
necessary, to maintain the original volume. Increase the INS: 122 CAS: [3567-69-9]
heat at the end of the digestion period, and continue UNII: DR4641L47F [azorubine]
heating until the volume is reduced by about one-half.
Cool to room temperature, add 1.5 g of potassium DESCRIPTION
sulfate, 3 mL of water, and 4.5 mL of sulfuric acid, and Azorubine occurs as red powder or granules. It is principally
heat until iodine fumes no longer evolve. Allow the the disodium salt of 4-hydroxy-3-(4-sulfonato-1-
mixture to cool, wash down the sides of the flask with naphthylazo)-1-naphthalenesulfonate and subsidiary
water, heat until charring occurs, and again cool to coloring matters, with sodium chloride and/or sodium
room temperature. Add 40 mg of mercuric oxide to the sulfate as the principal uncolored components. It is soluble
charred material, heat until the color of the solution is in water and sparingly soluble in ethanol.
pale yellow, then cool, wash down the sides of the flask Function: Color
with a few mL of water, and digest the mixture for an Packaging and Storage: Store in well-closed containers.
additional 3 h. Cool the digest, add 20 mL of
ammonia-free water, 16 mL of a 50% sodium IDENTIFICATION
hydroxide solution, and 5 mL of a 44% sodium • VISIBLE ABSORPTION SPECTRUM
thiosulfate solution. Immediately connect the flask to a Sample solution: Dissolve a sample in water, and dilute
distillation apparatus and distill, collecting the distillate appropriately.
in 10 mL of a 4% boric acid solution. Add a few drops Analysis: Measure the absorption spectrum of the
of methyl red-methylene blue TS to the distillate, and Sample solution, using a suitable UV-visible
titrate with 0.05 N sulfuric acid. Perform a blank spectrophotometer.
determination (see General Provisions), and make any Acceptance criteria: The Sample solution exhibits a
necessary correction. Each mL of 0.05 N sulfuric acid is wavelength maximum at 516 nm.
equivalent to 0.7004 mg of nitrogen. ASSAY
Acceptance criteria: Between 47.2% and 48.7% • TOTAL COLOR, Color Determination, Methods I and II,
• PH, pH Determination, Appendix IIB Appendix IIIC: Both methods must be used.
Sample preparation: 2 g in 100 mL of water, agitated Method I: (Spectrophotometric)
with a power stirrer for 5 min Sample solution: 10 mg/mL
Acceptance criteria: NLT 5.0 Analysis: Determine as directed at 516 nm, using
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC 0.051 L/(mg · cm) for the absorptivity (a) for
Sample: 1.5 g Azorubine.
Acceptance criteria: NMT 0.15% Method II: (TiCl3 Titration)
Sample: 0.5–0.6 g
Analysis: Determine as directed, except under
Procedure, use 15 g of Sodium Bitartrate instead of
21–22 g, and use 150 mL of water instead of 275 mL.
For the calculation, use 7.962 as the stoichiometric
factor (FS) for the disodium salt of Azorubine.
Acceptance criteria: The average of the results obtained
from Method I and Method II is NLT 85% total coloring
matters.
1 Azorubine is approved for use in some countries but banned in others, such

as the United States.

FCC 9 Monographs / Azorubine / 109

IMPURITIES CU = concentration of sample in the Sample

Inorganic Impurities solution (mg/mL)
• LEAD, Lead Limit Test, Appendix IIIB F = mg-to-µg conversion factor, 1000
Sample solution: Prepare as directed for organic Acceptance criteria: 4-amino-1-naphthalenesulfonic
compounds. acid and 4-hydroxy-1-naphthalenesulfonic acid: NMT
Control: 2 µg Pb (2 mL of Diluted Standard Lead 0.5% combined
Solution)
Acceptance criteria: NMT 2 mg/kg SPECIFIC TESTS
Organic Impurities • COMBINED TESTS
• UNCOMBINED INTERMEDIATES AND PRODUCTS OF SIDE Tests

Monographs
REACTIONS • LOSS ON DRYING (VOLATILE MATTER), Color Determination,
Solution A: 0.2 N ammonium acetate Appendix IIIC
Solution B: Methanol • CHLORIDE, Sodium Chloride, Color Determination,
Mobile phase: Exponential gradient program from Appendix IIIC
(99% A and 1% B) to (0% A and 100% B) at a rate of • SULFATES (AS SODIUM SALTS), Sodium Sulfate, Color
2% per min, followed by 6 min of 100% B to wash the Determination, Appendix IIIC
column, and (0% A and 100% B) to (99% A and 1% Acceptance criteria: NMT 15%, combined as the sum
B) in 14 min to return to the initial gradient of all three tests
composition and equilibrate column. • ETHER EXTRACTS, Color Determination, Appendix IIIC
Standard solution: 25 µg/mL of 4-amino-1- Acceptance criteria: NMT 0.2%
naphthalenesulfonic acid and 25 µg/mL of 4-hydroxy- • SUBSIDIARY COLORING MATTERS
1-naphthalenesulfonic acid in 0.02 M ammonium [NOTE—In this method, subsidiary coloring matters are
acetate separated from the main coloring matter of Azorubine
Sample solution: 5 mg/mL in 0.02 M ammonium by ascending paper chromatography (see Paper
acetate Chromatography, Appendix IIA), and extracted
Chromatographic system, Appendix IIA separately from the chromatographic paper. The
Mode: High-performance liquid chromatography absorbance of each extract is measured at the
Detector: UV wavelength of maximum absorption for Azorubine (516
Column nm) by visible spectrophotometry. Because it is
Guard column: 15-mm × 4.6-mm; 5-µm C18 impractical to identify each subsidiary coloring matter
column using this procedure, and because the subsidiary
Analytical column: 25-cm × 4.6-mm; 5-µm C18 coloring matters are usually minor components of food
column colors, the method assumes that the maximum
Column temperature: Ambient absorbance of each subsidiary coloring matter is the
Flow rate: 1.0 mL/min same as that of the total coloring matters. The
Injection volume: 20 µL subsidiary coloring matters content is calculated by
Analysis: Separately inject equal volumes of the adding together the absorbances of the extracts in
Standard solution and Sample solution into the conjunction with the total coloring matters content of
chromatograph, and measure the responses for the the sample.]
major peaks on the resulting chromatograms. Chromatographic apparatus: The chromatography tank
Calculate the percentage of both impurities (4-amino-1- (Figures 1 and 2) is composed of a glass tank (A) and
naphthalenesulfonic acid and 4-hydroxy-1- cover (B); frame to support chromatography paper (C);
naphthalenesulfonic acid) in the portion of the sample solvent tray (D); wire secondary frame (E) for
taken: supporting “drapes” of the filter paper; and 20-cm ×
20-cm chromatography grade paper.2 Mark out the
Result = (rU/rS) × (CS/CU) × F × 100 chromatography paper as shown in Figure 3.

rU = peak area for analyte in the Sample solution

rS = peak area for analyte in the Standard solution
CS = concentration of analyte in the Standard
solution (µg/mL) 2 Whatman No 1, or equivalent.
 110 / Azorubine / Monographs FCC 9
Monographs

Figure 1. Assembly of the Chromatographic Apparatus

Figure 2. Components of the Chromatographic Apparatus

Figure 3. Method for Marking the Chromatographic Paper

FCC 9 Monographs / Azorubine / 111

Chromatographic solvent: Prepare a mixture of 2- nm, and correct the absorbances of the colored extracts
butanone, acetone, water, and saturated aqueous with the blank values.
solution of ammonium hydroxide (specific gravity of Calculate the percentage of subsidiary coloring matter in
0.880), (700:300:300:2). Shake for 2 min, allow the the portion of the sample taken:
layers to separate, and use the upper layer.
Sample solution: 10 mg/mL sample Result = 0.01 × D × [(Aa + Ab + Ac ...An)/As] × 100
Standard solution: 0.1 mg/mL sample, prepared by
0.01 = dilution factor for the Standard solution
diluting the Sample solution
D = total coloring matter content of the sample,

Monographs
Application volume: 0.10 mL
determined from the Total Color test above
Analysis: NLT 2 h before analysis, arrange the filter-
and expressed as a decimal
paper drapes in the glass tank, and pour sufficient
As = the absorbance from the Standard solution
Chromatographic solvent over the drapes and into the
bottom of the tank to cover the bottom of the tank to Aa + Ab + Ac ...An = the sum of the absorbances of the
a depth of 1 cm. Place the solvent tray in position, and subsidiary coloring matters from the Sample solution,
fit the cover to the tank. Using a microsyringe capable corrected for the blank values
of delivering 0.1 mL with a tolerance of ±0.002 mL,
apply to separate chromatography sheets 0.1-mL Acceptance criteria: NMT 1%
aliquots of the Sample solution and Standard solution, as • UNSULFONATED PRIMARY AROMATIC AMINES
uniformly as possible within the confines of the 18-cm [NOTE—Under the conditions of this test, unsulfonated
× 7-mm rectangle, holding the nozzle of the primary aromatic amines are extracted into toluene
microsyringe steadily in contact with the paper. Allow from an alkaline solution of the sample, reextracted into
the papers to dry at room temperature for 1–2 h or at acid, then determined spectrophotometrically after
50° in a drying cabinet for 5 min followed by 15 min at diazotization and coupling.]
room temperature. Mount the dried sheets, together R salt solution: 0.05 N 2-naphthol-3,6-disulfonic acid,
with two plain sheets to act as blanks on the disodium salt
supporting frame. [NOTE—If required, several dried Sodium carbonate solution: 2 N sodium carbonate
sheets may be developed simultaneously.] Standard stock solution: Weigh 0.100 g of redistilled
Pour sufficient Chromatographic solvent into the solvent aniline into a small beaker, and transfer to a 100-mL
tray to bring the surface of the solvent about 1 cm volumetric flask, rinsing the beaker several times with
below the base line of the chromatography sheets. The water. Add 30 mL of 3 N hydrochloric acid, and dilute
volume necessary will depend on the dimensions of the with water at room temperature to the mark. Dilute
apparatus and should be predetermined. Put the 10.0 mL of this solution to 100 mL with water, and mix
supporting frame into position, and replace the cover. well; 1 mL of this solution is equivalent to 0.0001 g of
Allow the solvent front to ascend approximately 17 cm aniline. [NOTE—Prepare the Standard stock solution
above base line, then remove the supporting frame and fresh.]
transfer it to a drying cabinet at 50–60° for 10–15 min. Standard solutions: Separately dilute 5-mL, 10-mL, 15-
Remove the sheets from the frame. mL, 20-mL, and 25-mL aliquots of the Standard stock
For the Sample solution sheets, cut each subsidiary band solution with 1 N hydrochloric acid to 100 mL.
from each chromatography sheet as a strip, and cut an Standard blank solution: In a 25-mL volumetric flask
equivalent strip from the corresponding position of the mix 10.0 mL of 1 N hydrochloric acid, 10.0 mL of the
plain (blank) sheet. For the Standard solution sheet, cut Sodium carbonate solution, and 2.0 mL of the R salt
the entire band from the sheet, and cut an equivalent solution, and dilute with water to volume.
strip from the corresponding position of the plain Sample solution: Add 2.0 g of the sample to a
(blank) sheet. Place each strip, subdivided into a separatory funnel containing 100 mL of water; rinse
suitable number of approximately equal portions, in a down the sides of the funnel with 50 mL of water,
separate test tube. Add 5.0 mL of a mixture of water swirling to dissolve the sample; and add 5 mL of 1 N
and acetone (1:1 by volume) to each test tube, swirl for sodium hydroxide. Extract with two 50-mL portions of
2–3 min, add 15.0 mL of 0.05 N sodium hydrogen toluene, and wash the combined toluene extracts with
carbonate solution, and shake the tube to ensure 10-mL portions of 0.1 N sodium hydroxide to remove
mixing. traces of color. Extract the washed toluene with three
Filter the colored extracts and the blanks through 9-cm 10-mL portions of 3 N hydrochloric acid, and dilute the
coarse-porosity filter papers into clean test tubes, and combined extract with water to 100 mL.
determine the absorbances of the colored extracts at Sample blank solution: In a 25-mL volumetric flask mix
516 nm, using a suitable spectrophotometer with 40- 2.0 mL of R salt solution, 10 mL of Sodium carbonate
mm closed cells against a filtered mixture of 5.0 mL of solution, and 10.0 mL of the Sample solution, and dilute
water and acetone (1:1 by vol) and 15.0 mL of 0.05 N with water to volume.
sodium hydrogen carbonate solution. Measure the Analysis: Pipet 10-mL aliquots of each of the Standard
absorbances of the extracts of the blank strips at 516 solutions and the Sample solution into separate clean,
dry test tubes. Cool the tubes for 10 min by immersion
 112 / Azorubine / Monographs FCC 9

in a beaker of ice water, and add 1 mL of 50% spectrophotometer with 40-mm cells, against the
potassium bromide solution and 0.05 mL of 0.5 N Sample blank solution. From the standard curve,
sodium nitrite solution. Mix and allow the tubes to determine the weight (g) of aniline in each 100 mL of
stand for 10 min in the ice water bath while the aniline the Sample solution.
is diazotized. Into each of five 25-mL volumetric flasks, Calculate the percentage of unsulfonated primary
measure 1 mL of the R salt solution and 10 mL of the aromatic amine (as aniline) in the portion of the sample
Sodium carbonate solution. Separately pour each taken:
diazotized aniline solution into a 25-mL volumetric flask
containing R salt solution and Sodium carbonate solution; Result = WA/W × 100
rinse each test tube with a small volume of water to
WA = weight of aniline in the Sample solution,
Monographs

allow for a quantitative transfer. Dilute with water to

calculated from the standard curve (g/100
the mark, stopper the flasks, mix the contents well, and
mL)
allow them to stand for 15 min in the dark.
W = weight of sample used to prepare the Sample
Measure the absorbance of each of the solutions
solution (g)
containing the coupled Standard solutions at 510 nm,
Acceptance criteria: NMT 0.01%, calculated as aniline
using a suitable spectrophotometer with 40-mm cells,
• WATER-INSOLUBLE MATTER, Color Determination, Appendix
against the Standard blank solution. Plot a standard
IIIC
curve relating absorbance to weight (g) of aniline in
Acceptance criteria: NMT 0.2%
each 100 mL of the Standard solutions.
Measure the absorbance of the solutions containing the
coupled Sample solution at 510 nm, using a suitable
 FCC 9 Monographs / Basil Oil, Comoros Type / 113

Acceptance criteria: The spectrum of the sample

Balsam Peru Oil
.

exhibits relative maxima at the same wavelengths as

First Published: Prior to FCC 6 those of the spectrum below.
Last Revision: Third Supplement, FCC 7 SPECIFIC TESTS
• ACID VALUE (ESSENTIAL OILS AND FLAVORS), Appendix VI
CAS: [8007-00-9] Acceptance criteria: 30–60
UNII: DIK0395679 [balsam peru oil] • ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
IIB: Use a 100-mm tube.
DESCRIPTION Acceptance criteria: Between −1° and +2°
Balsam Peru Oil occurs as a yellow to pale brown, slightly

Monographs
• ESTERS, Ester Value, Appendix VI
viscous liquid having a sweet, balsamic odor. It is obtained
Sample: 1 g
by extraction or distillation of Peruvian Balsam obtained
Acceptance criteria: 200–225
from Myroxylon pereirae Royle Klotzsche (Fam.
• REFRACTIVE INDEX, Appendix IIB
Leguminosae). Occasionally, crystals may occur within the
[NOTE—Use an Abbé or other refractometer of equal or
liquid. It is soluble in most fixed oils, and is soluble, with
greater accuracy.]
turbidity, in mineral oil. It is partly soluble in propylene
Acceptance criteria: 1.567–1.579 at 20°
glycol, but it is practically insoluble in glycerin.
• SOLUBILITY IN ALCOHOL, Appendix VI
Function: Flavoring agent
Acceptance criteria: One mL of the sample dissolves in
Packaging and Storage: Store in a cool place protected
0.5 mL of 90% alcohol, and remains in solution on
from light in full, tight containers that are made from steel
further dilution to 10 mL.
or aluminum and that are suitably lined.
• SPECIFIC GRAVITY: Determine by any reliable method (see
IDENTIFICATION General Provisions).
• INFRARED SPECTRA, Spectrophotometric Identification Tests, Acceptance criteria: 1.110–1.120
Appendix IIIC

Balsam Peru Oil

DESCRIPTION
Basil Oil, Comoros Type
.

Basil Oil, Comoros Type occurs as a light yellow liquid with

First Published: Prior to FCC 6 a spicy odor. It is obtained by steam distillation of the
flowering tops or the entire plant of Ocimum basilicum L.
Basil Oil Exotic (Fam. Lamiaceae). It may be distinguished from other
Basil Oil, Réunion Type types, such as basil oil, European type, by its
camphoraceous odor and physicochemical constants. It is
UNII: Z129UMU8LE [basil oil]
 114 / Basil Oil, Comoros Type / Monographs FCC 9

soluble in most fixed oils and, with turbidity, in mineral oil. Analysis: Calculate the Ester value after acetylation by the
One mL is soluble in 20 mL of propylene glycol with slight formula:
haziness, but it is insoluble in glycerin.
Function: Flavoring agent Result = A × 28.05/B
Packaging and Storage: Store in a cool place protected
from light in full, tight containers that are made from steel
A = amount (mL) of 0.5 N alcoholic potassium
or aluminum and that are suitably lined.
hydroxide consumed in the saponification
IDENTIFICATION B = weight (g) of acetylated sample oil taken
• INFRARED SPECTRA, Spectrophotometric Identification Tests, Acceptance criteria: Between 25 and 45
• REFRACTIVE INDEX, Appendix IIB
Monographs

Appendix IIIC
Acceptance criteria: The spectrum of the sample [NOTE—Use an Abbé or other refractometer of equal or
exhibits relative maxima at the same wavelengths as greater accuracy.]
those of the spectrum below. Acceptance criteria: Between 1.512 and 1.520 at 20°
• SAPONIFICATION VALUE, Esters, Appendix VI
SPECIFIC TESTS Sample: 5 g
• ACID VALUE (ESSENTIAL OILS AND FLAVORS), Appendix VI Acceptance criteria: Between 4 and 10
Acceptance criteria: NMT 1.0 • SOLUBILITY IN ALCOHOL, Appendix VI
• ANGULAR ROTATION, Optical (Specific) Rotation, Appendix Acceptance criteria: One mL of the sample dissolves in
IIB: Use a 100 mm tube. 4 mL of 80% alcohol.
Acceptance criteria: Between −2° and +2° • SPECIFIC GRAVITY: Determine by any reliable method (see
• ESTER VALUE AFTER ACETYLATION, Linalool Determination, General Provisions).
Appendix VI Acceptance criteria: Between 0.952 and 0.973
Sample: 2.5 g of the dry acetylated oil

Basil Oil, Comoros Type

DESCRIPTION
Basil Oil, European Type
.

Basil Oil, European Type occurs as a pale yellow to yellow

First Published: Prior to FCC 6 liquid with a floral-spicy odor. It is obtained by the steam
distillation of the flowering tops or the entire plant of
Basil Oil, Italian Type Ocimum basilicum L. It may be distinguished from other
Sweet Basil Oil types, such as basil oil, Comoros type, or basil oil, Réunion
CAS: [8015-73-4] type, by its more floral odor and its physicochemical
UNII: Z129UMU8LE [basil oil] constants. It is soluble in most fixed oils and, with
turbidity, in mineral oil. One mL is soluble in 20 mL of
 FCC 9 Monographs / Bay Oil / 115

propylene glycol with slight haziness, but it is insoluble in Sample: 2.5 g of the dry acetylated oil
glycerin. Analysis: Calculate the Ester value after acetylation by the
Function: Flavoring agent formula:
Packaging and Storage: Store in a cool place protected
from light in full, tight containers that are made from steel Result = A × 28.05/B
or aluminum and that are suitably lined.

IDENTIFICATION A = amount (mL) of 0.5 N alcoholic potassium

• INFRARED SPECTRA, Spectrophotometric Identification Tests, hydroxide consumed in the saponification
Appendix IIIC B = weight (g) of acetylated sample oil taken
Acceptance criteria: Between 140 and 180

Monographs
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as • REFRACTIVE INDEX, Appendix IIB
those of the spectrum below. [NOTE—Use an Abbé or other refractometer of equal or
greater accuracy.]
SPECIFIC TESTS Acceptance criteria: Between 1.483 and 1.493 at 20°
• ACID VALUE (ESSENTIAL OILS AND FLAVORS), Appendix VI • SOLUBILITY IN ALCOHOL, Appendix VI
Acceptance criteria: NMT 2.5 Analysis: Use 4 mL of 80% alcohol.
• ANGULAR ROTATION, Optical (Specific) Rotation, Appendix Acceptance criteria: Passes test
IIB: Use a 100-mm tube. • SPECIFIC GRAVITY: Determine by any reliable method (see
Acceptance criteria: Between −5° and −15° General Provisions).
• ESTER VALUE AFTER ACETYLATION, Linalool Determination, Acceptance criteria: Between 0.900 and 0.920
Appendix VI

Basil Oil, European Type

Kostel (Fam. Myrtaceae). It is soluble in alcohol and in

Bay Oil
.

glacial acetic acid. Its solutions in alcohol are acid to

First Published: Prior to FCC 6 litmus.
Function: Flavoring agent
Packaging and Storage: Store in full, tight containers in
Myrcia Oil
a cool place protected from light.
UNII: 3T5GC5CQ33 [bay oil (pimenta racemosa)]
IDENTIFICATION
DESCRIPTION • A. INFRARED SPECTRA, Spectrophotometric Identification
Bay Oil occurs as a yellow or brown-yellow liquid with a Tests, Appendix IIIC
pleasant, aromatic odor and a pungent, spicy taste. It is
the volatile oil distilled from the leaves of Pimenta acris
 116 / Bay Oil / Monographs FCC 9

Acceptance criteria: The spectrum of the sample SPECIFIC TESTS

exhibits relative maxima at the same wavelengths as • ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
those of the spectrum below. IIB: Use a 100-mm tube.
• B. PROCEDURE Acceptance criteria: Levorotatory (not more
Sample: 1 mL levorotatory than −3°)
Analysis: Shake the Sample with 20 mL of hot water, • REFRACTIVE INDEX, Appendix IIB
and filter. Test the filtrate with litmus, and then add 1 [NOTE—Use an Abbé or other refractometer of equal or
drop of ferric chloride TS. greater accuracy.]
Acceptance criteria: The filtrate is acid to litmus and, Acceptance criteria: Between 1.507 and 1.516 at 20°
after addition of ferric chloride TS, yields only a • SPECIFIC GRAVITY: Determine by any reliable method (see
Monographs

transient gray-green, not a blue or purple, color. General Provisions).

Acceptance criteria: Between 0.950 and 0.990
ASSAY
• PHENOLS, Appendix VI
Acceptance criteria: NLT 50% and NMT 65%, by
volume, of phenols.

Bay Oil

in water and sparingly soluble in cold alcohol. Boiling

Beeswax, White
.

alcohol dissolves cerotic acid and part of the myricin. It is

First Published: Prior to FCC 6 completely soluble in chloroform, in ether, and in fixed
and volatile oils. It is partly soluble in cold carbon disulfide
and is completely soluble in it at temperatures of 30° or
White Wax
above.
INS: 901 Function: Surface-finishing (glazing) agent; release agent;
UNII: 7G1J5DA97F [white wax] raw material for flavoring agent
Packaging and Storage: Store in well-closed containers.
DESCRIPTION
Beeswax, White, occurs as a yellow-white solid, somewhat IMPURITIES
translucent in thin layers, with a faint, characteristic odor, Inorganic Impurities
free from rancidity. It is the bleached, purified wax from • LEAD, Lead Limit Test, Appendix IIIB
the honeycomb of the bee Apis mellifera L. (Fam. Apidae), Sample solution: Prepare as directed for organic
and it consists primarily of myricyl palmitate (myricin), compounds.
cerotic acid and ester, and some high-carbon paraffins. Its Control: 5 µg Pb (5 mL of Diluted Standard Lead
specific gravity is about 0.95. Beeswax, White, is insoluble Solution)
FCC 9 Monographs / Beeswax, Yellow / 117

Acceptance criteria: NMT 5 mg/kg 30 mL of the Saponifying solution, attach a reflux

Organic Impurities condenser to the flask, and heat the mixture gently on
• CARNAUBA WAX a steam bath for 2 h. At the end of this period, remove
Sample: 100 mg the reflux condenser, insert a thermometer into the
Analysis: Place the Sample in a test tube, and add 20 solution, and place the flask in an 80° water bath.
mL of n-butanol. Immerse the test tube in boiling Rotate the flask while both the bath and the solution
water, and shake the mixture gently until dissolution is cool to 65°.
complete. Transfer the test tube into a beaker of water Acceptance criteria: The solution shows no cloudiness
at 60°, and allow it to cool to room temperature. A or globule formation before the solution reaches 65°.
loose mass of fine, needlelike crystals separate from a

Monographs
clear mother liquor.
Acceptance criteria: Under the microscope, the crystals
Beeswax, Yellow
.

appear as loose needles or stellate clusters, and no

amorphous masses are observed, indicating the
First Published: Prior to FCC 6
absence of carnauba wax.
• FATS, JAPAN WAX, ROSIN, AND SOAP
Sample: 1 g Yellow Wax
INS: 901 CAS: [8012-89-3]
Analysis: Boil the Sample for 30 min with 35 ml of a
143 mg/mL sodium hydroxide solution, maintaining UNII: 2ZA36H0S2V [yellow wax]
the volume by the occasional addition of water. Cool
DESCRIPTION
the mixture. The wax separates and the liquid remains
Beeswax, Yellow, occurs as a yellow to gray-brown solid
clear. Filter the cold mixture and acidify the filtrate
with an agreeable, honey odor. It is the purified wax from
with hydrochloric acid.
the honeycomb of the bee Apis mellifera L. (Fam. Apidae),
Acceptance criteria: No precipitate forms.
and it consists primarily of myricyl palmitate (myricin),
SPECIFIC TESTS cerotic acid and ester, and some high-carbon paraffins. It is
• ACID VALUE (FATS AND RELATED SUBSTANCES), Appendix VII somewhat brittle when cold, and presents a dull, granular,
Sample: 3 g noncrystalline fracture when broken. It becomes pliable at
Analysis: Warm the Sample, in a 200-mL flask with 25 a temperature of about 35°. Its specific gravity is about
mL of absolute alcohol, previously neutralized to 0.95. Beeswax, Yellow, is insoluble in water and sparingly
phenolphthalein with potassium hydroxide, until the soluble in cold alcohol. Boiling alcohol dissolves cerotic
sample is melted. Shake the mixture and add 1 mL of acid and part of the myricin. It is completely soluble in
phenolphthalein TS. Titrate the warm solution with 0.5 chloroform, in ether, and in fixed and volatile oils. It is
N alcoholic potassium hydroxide to a permanent, faint partly soluble in cold carbon disulfide and completely
pink color. [NOTE—Save this solution for the Ester Value soluble in it at temperatures of 30° or above.
test.] Function: Candy glaze and polish; raw material for
Acceptance criteria: Between 17 and 24 flavoring agent
• ESTER VALUE Packaging and Storage: Store in well-closed containers.
Sample: Solution resulting from the determination of
IMPURITIES
Acid Value (above).
Inorganic Impurities
Analysis: Add 25.0 mL of 0.5 N alcoholic potassium
• LEAD, Lead Limit Test, Appendix IIIB
hydroxide and 50 mL of alcohol to Sample solution,
Sample solution: Prepare as directed for organic
heat the mixture under a reflux condenser for 4 h, and
compounds.
titrate the excess alkali with 0.5 N hydrochloric acid.
Control: 5 µg Pb (5 mL of Diluted Standard Lead
Perform a residual blank titration, and calculate the
Solution)
Ester Value as the number of mg of potassium
Acceptance criteria: NMT 5 mg/kg
hydroxide required for each g of the sample taken for
Organic Impurities
the test.
• CARNAUBA WAX
Acceptance criteria: Between 72 and 79
Sample: 100 mg
• MELTING RANGE OR TEMPERATURE DETERMINATION, Procedure
Analysis: Place the Sample in a test tube, and add 20
for Class II, Appendix IIB
mL of n-butanol. Immerse the test tube in boiling
Acceptance criteria: Between 62° and 65°
water, and shake the mixture gently until dissolution is
• SAPONIFICATION CLOUD TEST
complete. Transfer the test tube into a beaker of water
Saponifying solution: Dissolve 40 g of potassium
at 60°, and allow it to cool to room temperature. A
hydroxide in about 900 mL of aldehyde-free alcohol
loose mass of fine, needlelike crystals separate from a
maintained at a temperature of 15° until solution is
clear mother liquor.
complete. Warm to room temperature, and add
Acceptance criteria: Under the microscope, the crystals
sufficient aldehyde-free alcohol to make 1000 mL.
appear as loose needles or stellate clusters, and no
Sample: 3.00 g
amorphous masses are observed, indicating the
Analysis: Transfer the Sample into a round-bottom, 100-
absence of carnauba wax.
mL boiling flask provided with a ground-glass joint, add
 118 / Beeswax, Yellow / Monographs FCC 9

• FATS, JAPAN WAX, ROSIN, AND SOAP

Bentonite
.

Sample: 1 g
Analysis: Boil the Sample for 30 min with 35 ml of a First Published: Prior to FCC 6
143 mg/mL sodium hydroxide solution, maintaining Last Revision: Third Supplement, FCC 7
the volume by the occasional addition of water. Cool
the mixture. The wax separates and the liquid remains
Smectite
clear. Filter the cold mixture and acidify the filtrate
Aluminum Silicate
with hydrochloric acid. INS: 558 CAS: [1302-78-9]
Acceptance criteria: No precipitate forms.
UNII: A3N5ZCN45C [bentonite]
Monographs

SPECIFIC TESTS
• ACID VALUE (FATS AND RELATED SUBSTANCES), Appendix VII
DESCRIPTION
Bentonite occurs commercially as powders ranging in colors
Sample: 3 g
and tints from off white to pale brown to gray depending
Analysis: Warm the Sample, in a 200-mL flask with 25
on the cations present in natural deposits. It comprises
mL of absolute alcohol, previously neutralized to
natural smectite clays consisting primarily of colloidal
phenolphthalein with potassium hydroxide, until the
hydrated aluminum silicates of the montmorillonite or
sample is melted. Shake the mixture and add 1 mL of
hectorite type of minerals with varying quantities of
phenolphthalein TS. Titrate the warm solution with 0.5
alkalies, alkaline earths, and iron. It is insoluble in water, in
N alcoholic potassium hydroxide to a permanent, faint
alcohol, in dilute acids, and in alkalies. The pH of a 2%
pink color. [NOTE—Save this solution for the Ester Value
suspension of Bentonite is typically in the range of
test]
4.5–10.5.
Acceptance criteria: Between 18 and 24
Function: Clarifying, filter agent
• ESTER VALUE
Packaging and Storage: Store in tight containers.
Sample: Solution resulting from the determination of
Acid Value (above) IDENTIFICATION
Analysis: Add 25.0 mL of 0.5 N alcoholic potassium • A. X-RAY DIFFRACTION
hydroxide and 50 mL of alcohol to Sample solution, Sample preparation A: With intense agitation, add 2 g
heat the mixture under a reflux condenser for 4 h, and of sample, in small portions, to 100 mL of water. Allow
titrate the excess alkali with 0.5 N hydrochloric acid. the mixture to stand for 12 h to ensure complete
Perform a residual blank titration, and calculate the hydration. Place 2 mL of the mixture so obtained on a
Ester Value as the number of mg of potassium suitable glass slide, and allow it to air dry at room
hydroxide required for each g of the sample taken for temperature to produce an oriented film. Place the slide
the test. in a vacuum desiccator over a free surface of ethylene
Acceptance criteria: Between 72 and 77 glycol. Evacuate the desiccator, and close the stopcock
• MELTING RANGE OR TEMPERATURE DETERMINATION, Procedure so that ethylene glycol saturates the desiccator
for Class II, Appendix IIB chamber. Allow the slide to stand for 12 h.
Acceptance criteria: Between 62° and 65° Sample preparation B: Prepare a random powder
• SAPONIFICATION CLOUD TEST specimen of the sample.
Saponifying solution: Dissolve 40 g of potassium Analysis: Record the X-ray diffraction pattern using a
hydroxide in about 900 mL of aldehyde-free alcohol copper source, and calculate the d values. [NOTE—For
maintained at a temperature of 15° until solution is Sample preparation B, determine the d value between
complete. Warm to room temperature, and add the range of 1.48 and 1.54 Å.]
sufficient aldehyde-free alcohol to make 1000 mL. Acceptance criteria: For Sample preparation A, the
Sample: 3.00 g largest peak corresponds to a d value between 15.0 and
Analysis: Transfer the Sample into a round-bottom, 100- 17.2 Å. For Sample preparation B, the peak is between
mL boiling flask provided with a ground-glass joint, add 1.492 and 1.504 Å or between 1.510 and 1.540 Å.
30 mL of the Saponifying solution, attach a reflux • B. PROCEDURE
condenser to the flask, and heat the mixture gently on Sample: 0.5 g
a steam bath for 2 h. At the end of this period, remove Analysis: Add 1 g of potassium nitrate and 3 g of
the reflux condenser, insert a thermometer into the anhydrous sodium carbonate to the Sample contained
solution, and place the flask in an 80° water bath. in a metal crucible, heat until the mixture has melted,
Rotate the flask while both the bath and the solution and allow it to cool. Add 20 mL of boiling water to the
cool to 65°. residue, mix, filter, and wash the residue with 50 mL of
Acceptance criteria: The solution shows no cloudiness water. Add 1 mL of hydrochloric acid and 5 mL of
or globule formation before the solution reaches 65°. water to the residue, and filter. Add 1 mL of 10 N
sodium hydroxide to the filtrate, filter, and add 3 mL of
2 M ammonium chloride.
Acceptance criteria: A gelatinous, white precipitate
forms.
FCC 9 Monographs / Benzaldehyde / 119

IMPURITIES dispersed. Dry at 100°–105°, and weigh. The difference

Inorganic Impurities in weight corresponds to the measure of coarse
• ARSENIC, Arsenic Limit Test, Appendix IIIB particles.
Sample solution: Transfer 8.0 g of dried sample into a Acceptance criteria: NMT 0.5% of sample is retained
250-mL beaker containing 100 mL of 1:25 hydrochloric on a 75-µm sieve
acid, mix, cover with a watch glass, and boil gently, • GEL FORMATION
stirring occasionally, for 15 min without allowing Sample: 6 g
excessive foaming. Filter the hot supernatant liquid Analysis: Mix the Sample with 300 mg of magnesium
through a rapid-flow filter paper into a 200-mL oxide. Add the mixture, in several divided portions, to
volumetric flask, and wash the filter with four 25-mL 200 mL of water contained in a blender jar with an

Monographs
portions of hot 4% hydrochloric acid, collecting the approximately 500-mL capacity. Blend thoroughly for 5
washings in the volumetric flask. Cool the combined min at high speed, transfer 100 mL of the mixture into
filtrates to room temperature, add 4% hydrochloric a 100-mL graduated cylinder, and leave undisturbed for
acid to volume, and mix. 24 h.
Control: 5 µg As (5 mL of Standard Arsenic Solution) Acceptance criteria: NMT 2 mL of supernatant appears
Analysis: Proceed as directed using a 25-mL aliquot of on the surface.
the Sample solution. • LOSS ON DRYING, Appendix IIC (105° for 2 h)
Acceptance criteria: NMT 5 mg/kg Acceptance criteria: NMT 12.0%
• LEAD • MICROBIAL LIMITS
[NOTE—The Standard solution and the Sample solution [NOTE—Current methods for the following tests may be
may be modified, if necessary, to obtain solutions of found by accessing the Food and Drug Administration’s
suitable concentrations, adaptable to the linear or Bacteriological Analytical Manual (BAM) online at
working range of the spectrophotometer used.] www.fda.gov/Food/default.htm.]
Sample preparation: Transfer 3.75 g of dried sample Acceptance criteria
into a 250-mL beaker containing 100 mL of 1:25 Aerobic plate count: NMT 1000 cfu/g
hydrochloric acid, stir, cover with a watch glass, and E. coli: Negative in 25 g
boil for 15 min. Cool to room temperature, and allow
the insoluble matter to settle. Decant the supernatant
through a rapid-flow filter paper into a 400-mL beaker.
Benzaldehyde
.

Wash the filter with four 25-mL portions of hot water,

collecting the filtrate in the 400-mL beaker. First Published: Prior to FCC 6
Concentrate the combined extracts by gentle boiling Last Revision: FCC 7
to approximately 20 mL. If a precipitate forms, add
2–3 drops of nitric acid, heat to boiling, and cool to
room temperature. Filter the concentrated extracts
through a rapid-flow filter paper into a 50-mL
volumetric flask. Transfer the remaining contents of the
400-mL beaker through the filter paper and into the
flask with water. Dilute with water to volume, and mix.
Standard solution: 3 µg/mL Pb: from Lead Nitrate Stock C7H6O Formula wt 106.12
Solution, Lead Limit Test, Flame Atomic Absorption FEMA: 2127
Method, Appendix IIIB. [NOTE—Prepare this solution on UNII: TA269SD04T [benzaldehyde]
the day of use.]
Analysis: Using a suitable atomic absorption DESCRIPTION
spectrophotometer equipped with a lead hollow- Benzaldehyde occurs as a colorless liquid with a burning
cathode lamp, deuterium arc background correction, a taste. It may contain a suitable antioxidant.
single-slot burner, and using an oxidizing air–acetylene Odor: Bitter almond oil
flame, determine the absorbances of the Sample Solubility: Slightly soluble in water; miscible in alcohol,
preparation and the Standard solution at 284 nm. ether, most fixed oils, volatile oils
Acceptance criteria: The absorbance of the Sample Boiling Point: ∼178°
preparation is not greater than that of the Standard Function: Flavoring agent
solution (NMT 4 mg/kg). IDENTIFICATION
SPECIFIC TESTS • INFRARED ABSORPTION, Spectrophotometric Identification
• COARSE PARTICLES Tests, Appendix IIIC
Sample preparation: 20 g in 100 mL of water, mixed Reference standard: USP Benzaldehyde RS
for 15 min at NLT 5000 rpm Sample and standard preparation: F
Analysis: Transfer the Sample preparation to a wet sieve Acceptance criteria: The spectrum of the sample
of nominal mesh aperture (75 µm), previously dried at exhibits maxima at the same wavelengths as those in
100°–105° and weighed, and wash with three 500-mL the spectrum of the Reference standard.
volumes of water, ensuring that any agglomerates are
 120 / Benzaldehyde / Monographs FCC 9

ASSAY C10H12O3 Formula wt 180.20

• PROCEDURE: Proceed as directed under M-1b, Appendix FEMA: 2129
XI. UNII: DTD5NUE52Q [benzaldehyde glyceryl acetal]
Acceptance criteria: NLT 98.0% of C7H6O
DESCRIPTION
SPECIFIC TESTS Benzaldehyde Glyceryl Acetal occurs as a colorless to pale
• REFRACTIVE INDEX, Appendix II: At 20° yellow liquid.
Acceptance criteria: Between 1.544 and 1.547 Odor: Mild almond
• SPECIFIC GRAVITY: Determine at 25° by any reliable Boiling Point: ∼185°
method (see General Provisions). Solubility in Alcohol, Appendix VI: One mL dissolves in 1
Monographs

Acceptance criteria: Between 1.041 and 1.046 mL of 95% alcohol.

Function: Flavoring agent
OTHER REQUIREMENTS
• CHLORINATED COMPOUNDS, Appendix VI IDENTIFICATION
Acceptance criteria: Passes test • INFRARED SPECTRA, Spectrophotometric Identification Tests,
• HYDROCYANIC ACID, M-8, Appendix XI Appendix IIIC
Acceptance criteria: Passes test Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

Benzaldehyde Glyceryl Acetal ASSAY

.

• PROCEDURE: Proceed as directed under M-1a, Appendix

First Published: Prior to FCC 6 XI.
Acceptance criteria: NLT 95.0% of C10H12O3 (sum of
Mixture of 1,2- and 1,3-Benzaldehyde Cyclic Acetals of four isomers; each isomer between 5% and 40%)
Glycerin
SPECIFIC TESTS
• ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
OILS), M-15, Appendix XI
Acceptance criteria: NMT 2.0
• REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.535 and 1.541
• SPECIFIC GRAVITY: Determine at 25° by any reliable
method (see General Provisions).
Acceptance criteria: Between 1.181 and 1.191

Benzaldehyde Glyceryl Acetal

 FCC 9 Monographs / 1,2-Benzodihydropyrone / 121

Benzaldehyde Propylene Glycol Acetal Benzenethiol

. .

First Published: Second Supplement, FCC 8 First Published: Second Supplement, FCC 8

4-Methyl-2-phenyl-m-dioxolane Phenyl Mercaptan

4-Methyl-2-phenyl-1,3-dioxolane Thiophenol

Monographs
C6H6S Formula wt 110.17
C10H12O2 Formula wt 164.2 FEMA: 3616
FEMA: 2130 CAS: [108-98-5]
CAS: [2568-25-4] UNII: 7K011JR4T0 [benzenethiol]
UNII: ELQ3FTL5B1 [benzaldehyde propylene glycol acetal]
DESCRIPTION
DESCRIPTION Benzenethiol occurs as a colorless to pale yellow liquid.
Benzaldehyde Propylene Glycol Acetal occurs as a colorless Odor: Penetrating, garlic-like
liquid. Solubility: Soluble in oils; slightly soluble in alcohol and
Odor: Mild, almond-like odor ether; insoluble in water
Solubility: Slightly soluble in water; soluble in oils; miscible Boiling Point: ∼169° (46.4° at 10 mm Hg)
in ethanol at room temperature Function: Flavoring agent
Boiling Point: ∼83°–85° at 4 mm Hg
Function: Flavoring agent IDENTIFICATION
• INFRARED ABSORPTION, Spectrophotometric Identification
IDENTIFICATION Tests, Appendix IIIC
• INFRARED ABSORPTION, Spectrophotometric Identification Reference standard: USP Benzenethiol RS
Tests, Appendix IIIC Sample and standard preparation: F
Reference standard: USP Benzaldehyde Propylene Acceptance criteria: The spectrum of the sample
Glycol Acetal RS exhibits maxima at the same wavelengths as those in
Sample and standard preparation: F the spectrum of the Reference standard.
Acceptance criteria: The spectrum of the sample
exhibits maxima at the same wavelengths as those in ASSAY
the spectrum of the Reference standard. • PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
ASSAY Reference standard: USP Benzenethiol RS
• PROCEDURE: Proceed as directed under M-1b, Appendix Acceptance criteria: NLT 97%
XI.
Reference standard: USP Benzaldehyde Propylene SPECIFIC TESTS
Glycol Acetal RS • REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: NLT 95% Acceptance criteria: 1.589–1.593
• SPECIFIC GRAVITY: Determine at 25° by any reliable
SPECIFIC TESTS method (see General Provisions).
• ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL Acceptance criteria: 1.073–1.080
OILS), M-15, Appendix XI
Acceptance criteria: NMT 1.0
• REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: 1.506–1.516 1,2-Benzodihydropyrone
.

• SPECIFIC GRAVITY: Determine at 25° by any reliable

method (see General Provisions). First Published: Prior to FCC 6
Acceptance criteria: 1.061–1.071
Dihydrocoumarin

C9H8O2 Formula wt 148.16

FEMA: 2381
UNII: NM5K1Y1BT2 [benzodihydropyrone]
 122 / 1,2-Benzodihydropyrone / Monographs FCC 9

DESCRIPTION ASSAY
1,2-Benzodihydropyrone occurs as a colorless to pale yellow • PROCEDURE: Proceed as directed under M-1b, Appendix
liquid. XI.
Odor: Coconut Acceptance criteria: NLT 99.0% of C9H8O2
Boiling Point: ∼272°
Solubility in Alcohol, Appendix VI: One mL dissolves in 1 SPECIFIC TESTS
mL of 95% alcohol. • REFRACTIVE INDEX, Appendix II: At 20°
Function: Flavoring agent Acceptance criteria: Between 1.555 and 1.559
• SPECIFIC GRAVITY: Determine at 25° by any reliable
IDENTIFICATION method (see General Provisions).
Monographs

• INFRARED SPECTRA, Spectrophotometric Identification Tests, Acceptance criteria: Between 1.186 and 1.192
Appendix IIIC
Acceptance criteria: The spectrum of the sample OTHER REQUIREMENTS
exhibits relative maxima at the same wavelengths as • SOLIDIFICATION POINT, Appendix IIB
those of the spectrum below. Acceptance criteria: NLT 22°

1,2-Benzodihydropyrone

chloroform, and in 3 mL of ether. It is soluble in fixed and

Benzoic Acid
.

in volatile oils and is sparingly soluble in hexane.

First Published: Prior to FCC 6 Function: Preservative; antimicrobial agent
Packaging and Storage: Store in well-closed containers.

IDENTIFICATION
• PROCEDURE
Sample solution: 1 g in a 20:1 (v/v) mixture of water
and 1 N sodium hydroxide
Analysis: Filter the Sample solution and add about 1 mL
C7H6O2 Formula wt 122.12 of ferric chloride TS.
INS: 210 CAS: [65-85-0] Acceptance criteria: A buff-colored precipitate forms.
UNII: 8SKN0B0MIM [benzoic acid]
ASSAY
DESCRIPTION • PROCEDURE
Benzoic Acid occurs as white crystals, scales, or needles. It Sample: 500 mg
begins to sublime at about 100° and is volatile with steam. Analysis: Dissolve the Sample in 25 mL of 50% alcohol,
One gram is soluble in 275 mL of water at 25°, in 20 mL previously neutralized with 0.1 N sodium hydroxide,
of boiling water, in 3 mL of alcohol, in 5 mL of add phenolphthalein TS, and titrate with 0.1 N sodium
 FCC 9 Monographs / Benzophenone / 123

hydroxide. Each mL of 0.1 N sodium hydroxide is DESCRIPTION

equivalent to 12.21 mg of C7H6O2. Benzophenone occurs as a white rhombic crystal or flaky
Acceptance criteria: NLT 99.5% and NMT 100.5% of solid.
C7H6O2, calculated on the anhydrous basis Odor: Delicate, persistent, rose
Solubility: Soluble in most fixed oils; slightly soluble in
IMPURITIES propylene glycol; insoluble or practically insoluble in
Inorganic Impurities glycerin
• LEAD, Lead Limit Test, Flame Atomic Absorption Boiling Point: ∼305°
Spectrophotometric Method, Appendix IIIB Solubility in Alcohol, Appendix VI: One g dissolves in 10
Sample: 10 g mL of 80% alcohol.

Monographs
Acceptance criteria: NMT 2.0 mg/kg Function: Flavoring agent
• WATER, Water Determination, Method I, Appendix IIB
[NOTE—Use methanol:pyridine (1:2) as the solvent.] IDENTIFICATION
Acceptance criteria: NMT 0.7% • INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
SPECIFIC TESTS Acceptance criteria: The spectrum of the sample
• READILY CARBONIZABLE SUBSTANCES, Appendix IIB exhibits relative maxima at the same wavelengths as
Sample solution: Dissolve 500 mg of sample in 5 mL of those of the spectrum below.
95% sulfuric acid.
Acceptance criteria: The color resulting from treatment ASSAY
of the Sample solution is no darker than Matching Fluid • PROCEDURE: Proceed as directed under M-1a, Appendix
Q. XI.
• READILY OXIDIZABLE SUBSTANCES Acceptance criteria: NLT 98.0% of C13H10O
Sample: 1 g
Analysis: Add 0.1 N potassium permanganate, drop OTHER REQUIREMENTS
wise, to a mixture of 100 mL of water and 1.5 mL of • CHLORINATED COMPOUNDS, Appendix VI
sulfuric acid heated to 100°, until a pink color persists Acceptance criteria: Passes test
for 30 s. Dissolve the Sample in the hot solution. Titrate • LEAD, M-9, Appendix XI
with 0.1 N potassium permanganate to a pink color Acceptance criteria: NMT 10 mg/kg
that persists for 15 s.
Acceptance criteria: The volume of 0.1 N potassium
permanganate consumed in the titration does not
exceed 0.5 mL.
• RESIDUE ON IGNITION (SULFATED ASH), Method I, Appendix
IIC
Sample: 2 g
Acceptance criteria: NMT 0.05%
• SOLIDIFICATION POINT, Appendix IIB
Acceptance criteria: Between 121° and 123°

Benzophenone
.

First Published: Prior to FCC 6

Benzoylbenzene
Diphenyl Ketone

C13H10O Formula wt 182.22

FEMA: 2134
UNII: 701M4TTV9O [benzophenone]
 124 / Benzophenone / Monographs FCC 9

• SOLIDIFICATION POINT, Appendix IIB

Acceptance criteria: NLT 47°
Monographs

Benzophenone

gradually to boiling, and continue boiling for 15 min.

Benzoyl Peroxide
.

Cool, dilute to 200 mL with water, and make the

First Published: Prior to FCC 6 solution strongly acid with 0.5 N hydrochloric acid.
Extract with ether, dry the extract with anhydrous
sodium sulfate, and then evaporate to dryness on a
steam bath.
Acceptance criteria: The residue of benzoic acid so
obtained melts between 121° and 123°.

ASSAY
C14H10O4 Formula wt 242.23 • PROCEDURE
INS: 928 CAS: [94-36-0] Sample: 250 mg
UNII: W9WZN9A0GM [benzoyl peroxide] Analysis: Dissolve the Sample in 15 mL of acetone
contained in a 100-mL glass-stoppered bottle and add
DESCRIPTION 3 mL of a 500 mg/mL solution of potassium iodide.
Benzoyl Peroxide occurs as a colorless, crystalline solid. It is Swirl for 1 min and immediately titrate with 0.1 N
insoluble in water, slightly soluble in alcohol, and soluble in sodium thiosulfate (without the addition of starch TS).
chloroform and in ether. It melts between 103° and 106° Each mL of 0.1 N sodium thiosulfate is equivalent to
with decomposition. 12.11 mg of C14H10O4.
Function: Bleaching agent Acceptance criteria: NLT 96.0% of C14H10O4
Packaging and Storage: Store in the original container
and observe the safety precautions printed on the label. IMPURITIES
[CAUTION—Benzoyl Peroxide, especially in the dry form, is a Inorganic Impurities
dangerous, highly reactive oxidizing material and has been • LEAD, Lead Limit Test, Appendix IIIB
known to explode spontaneously. Observe safety Sample solution: Prepare as directed for organic
precautions printed on the label of the container.] compounds from the residue of a mixture prepared by
mixing 1 g of sample with 10 mL of 1 N sodium
IDENTIFICATION hydroxide, slowly evaporating to dryness on a steam
• MELTING RANGE OR TEMPERATURE DETERMINATION, Appendix bath, and cooling.
IIB Control: 4 µg Pb (4 mL of Diluted Standard Lead
Sample preparation: Add 50 mL of 0.5 N alcoholic Solution)
potassium hydroxide to 500 mg of sample, heat Acceptance criteria: NMT 4 mg/kg
 FCC 9 Monographs / Benzyl Acetate / 125

Acceptance criteria: The spectrum of the sample

Benzyl Acetate
.

exhibits relative maxima at the same wavelengths as

First Published: Prior to FCC 6 those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
Acceptance criteria: NLT 98.0% of C9H10O2

SPECIFIC TESTS
• ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL

Monographs
C9H10O2 Formula wt 150.18
FEMA: 2135 OILS), M-15, Appendix XI [NOTE—Use phenol red TS as
UNII: 0ECG3V79ZJ [benzyl acetate] the indicator.]
Acceptance criteria: NMT 1.0
DESCRIPTION • REFRACTIVE INDEX, Appendix II: At 20°
Benzyl Acetate occurs as a colorless liquid. Acceptance criteria: Between 1.501 and 1.504
Odor: Sweet, floral, fruity • SPECIFIC GRAVITY: Determine at 25° by any reliable
Solubility: Soluble in alcohol, most fixed oils, propylene method (see General Provisions).
glycol; insoluble or practically insoluble in glycerin, water Acceptance criteria: Between 1.052 and 1.056
Boiling Point: ∼214°
Solubility in Alcohol, Appendix VI: One mL dissolves in 5 OTHER REQUIREMENTS
mL of 60% alcohol. • CHLORINATED COMPOUNDS, Appendix VI
Function: Flavoring agent Acceptance criteria: Passes test

IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC

Benzyl Acetate
 126 / Benzyl Acetoacetate / Monographs FCC 9

C7H8O Formula wt 108.14

Benzyl Acetoacetate
.

FEMA: 2137
First Published: Third Supplement, FCC 8 UNII: LKG8494WBH [benzyl alcohol]

DESCRIPTION
Benzyl 3-oxobutanoate
Benzyl Alcohol occurs as a colorless liquid with a sharp
Benzyl acetylacetate
burning taste.
Benzyl beta-ketobutyrate
Odor: Faint, aromatic
Solubility: Miscible in alcohol, chloroform, ether; 1 mL
dissolves in 30 mL of water
Monographs

Boiling Point: ∼206° (decomp)

Function: Flavoring agent

C11H12O3 Formula wt 192.21 IDENTIFICATION

FEMA: 2136 • INFRARED ABSORPTION, Spectrophotometric Identification
CAS: [5396-89-4] Tests, Appendix IIIC
UNII: F9S0XGV18X [benzyl acetoacetate] Reference standard: USP Benzyl Alcohol RS
Sample and Standard preparation: F
DESCRIPTION Acceptance criteria: The spectrum of the sample
Benzyl Acetoacetate occurs as a colorless oily liquid. exhibits maxima at the same wavelengths as those in
Odor: Balsamic, herbaceous, and fruity the spectrum of the Reference standard.
Solubility: Soluble in organic solvents, oils; miscible with
ethanol at room temperature; insoluble in water ASSAY
Boiling Point: ∼249° • PROCEDURE: Proceed as directed under M-1a, Appendix XI
Function: Flavoring agent Acceptance criteria: NLT 99.0% of C7H8O

IDENTIFICATION SPECIFIC TESTS

• INFRARED ABSORPTION, Spectrophotometric Identification • REFRACTIVE INDEX, Appendix II: At 20°
Tests, Appendix IIIC Acceptance criteria: Between 1.539 and 1.541
Reference standard: USP Benzyl Acetoacetate RS • SPECIFIC GRAVITY: Determine at 25° by any reliable
Sample and standard preparation: F method (see General Provisions).
Acceptance criteria: The spectrum of the sample Acceptance criteria: Between 1.042 and 1.047
exhibits maxima at the same wavelengths as those in
the spectrum of the Reference standard. OTHER REQUIREMENTS
• ALDEHYDES, M-1b, Appendix XI
ASSAY Acceptance criteria: NMT 0.2%
• PROCEDURE: Proceed as directed under M-1b, Appendix • CHLORINATED COMPOUNDS, Appendix VI
XI. Acceptance criteria: Passes test
Reference standard: USP Benzyl Acetoacetate RS • DISTILLATION RANGE, Appendix IIB
Acceptance criteria: NLT 98% of benzyl acetoacetate Acceptance criteria: NLT 95% distills between 202.5°
and 206.5°
SPECIFIC TESTS
• ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
OILS), M-15, Appendix XI
Acceptance criteria: NMT 3.0
Benzyl Benzoate
.

• REFRACTIVE INDEX, Appendix IIB: At 20°

Acceptance criteria: 1.498–1.520 First Published: Prior to FCC 6
• SPECIFIC GRAVITY: Determine at 25° by any reliable Last Revision: FCC 6
method (see General Provisions).
Acceptance criteria: 1.112–1.120

Benzyl Alcohol
.

First Published: Prior to FCC 6 C14H12O2 Formula wt 212.25

Last Revision: FCC 6 FEMA: 2138
UNII: N863NB338G [benzyl benzoate]
Phenyl Carbinol DESCRIPTION
Benzyl Benzoate is a colorless, oily liquid.
Odor: Slight, balsamic
 FCC 9 Monographs / Benzyl Butyrate / 127

Solubility: Miscible in alcohol, chloroform, ether; insoluble IDENTIFICATION

or practically insoluble in glycerin, water • INFRARED ABSORPTION, Spectrophotometric Identification
Boiling Point: ∼323° Tests, Appendix IIIC
Function: Flavoring agent Reference standard: USP 3-Benzyl-4-Heptanone RS
Sample and standard preparation: F
IDENTIFICATION Acceptance criteria: The spectrum of the sample
• INFRARED ABSORPTION, Spectrophotometric Identification exhibits maxima at the same wavelengths as those in
Tests, Appendix IIIC the spectrum of the Reference standard.
Reference standard: USP Benzyl Benzoate RS
Sample and Standard preparation: F ASSAY

Monographs
Acceptance criteria: The spectrum of the sample • PROCEDURE: Proceed as directed under M-1b, Appendix
exhibits maxima at the same wavelengths as those in XI.
the spectrum of the Reference standard. Reference standard: USP 3-Benzyl-4-Heptanone RS
Acceptance criteria: NLT 99% of 3-benzyl-4-heptanone
ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix SPECIFIC TESTS
XI. • REFRACTIVE INDEX, Appendix IIB: At 20°
Acceptance criteria: NLT 99.0% of C14H12O2. Acceptance criteria: 1.490–1.495
• SPECIFIC GRAVITY: Determine at 25° by any reliable
SPECIFIC TESTS method (see General Provisions).
• ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL Acceptance criteria: 0.931–0.937
OILS), M-15, Appendix XI
Acceptance criteria: NMT 1.0
• REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.568 and 1.570
Benzyl Butyrate
.

• SPECIFIC GRAVITY, Determine at 25° by any reliable

method (see General Provisions). First Published: Prior to FCC 6
Acceptance criteria: Between 1.116 and 1.120
Benzyl n-Butyrate
OTHER REQUIREMENTS
• CHLORINATED COMPOUNDS, Appendix VI
Acceptance criteria: Passes test
• SOLIDIFICATION POINT, Appendix IIB
Acceptance criteria: NLT 18°
C11H14O2 Formula wt 178.23
FEMA: 2140
UNII: 84L0NDE31F [benzyl butyrate]
3-Benzyl-4-Heptanone
.

First Published: Second Supplement, FCC 8 DESCRIPTION

Benzyl Butyrate occurs as a colorless liquid.
Benzyl Dipropyl Ketone Odor: Floral, fruity, plum
Morellone Solubility: Soluble in alcohol, most fixed oils; insoluble or
1-Phenyl-2-Ethyl-3-Hexanone practically insoluble in glycerin, propylene glycol, water
Boiling Point: ∼239°
Solubility in Alcohol, Appendix VI: One mL dissolves in 2
mL of 80% alcohol.
Function: Flavoring agent

IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
C14H20O Formula wt 204.31 Appendix IIIC
FEMA: 2146 Acceptance criteria: The spectrum of the sample
CAS: [7492-37-7] exhibits relative maxima at the same wavelengths as
UNII: 64UYT55005 [3-benzyl-4-heptanone] those of the spectrum below.

DESCRIPTION ASSAY
3-Benzyl-4-Heptanone occurs as a colorless, oily liquid. • PROCEDURE: Proceed as directed under M-1b, Appendix
Odor: Fruity, berry, woody, raisin XI.
Solubility: Soluble in organic solvents, oils; miscible with Acceptance criteria: NLT 98.0% of C11H14O2
ethanol at room temperature; insoluble in water
Boiling Point: ∼158°–160° (10 mm Hg) SPECIFIC TESTS
Function: Flavoring agent • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
OILS), M-15, Appendix XI
 128 / Benzyl Butyrate / Monographs FCC 9

Acceptance criteria: NMT 1.0 • SPECIFIC GRAVITY: Determine at 25° by any reliable
• REFRACTIVE INDEX, Appendix II: At 20° method (see General Provisions).
Acceptance criteria: Between 1.492 and 1.496 Acceptance criteria: Between 1.006 and 1.009
Monographs

Benzyl Butyrate

Function: Flavoring agent

Benzyl Cinnamate
.

IDENTIFICATION
First Published: Prior to FCC 6
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
C16H14O2 Formula wt 238.29 • PROCEDURE: Proceed as directed under M-1b, Appendix
FEMA: 2142 XI.
UNII: V67O3RO97U [benzyl cinnamate] Acceptance criteria: NLT 98.0% of C16H14O2

DESCRIPTION SPECIFIC TESTS

Benzyl Cinnamate occurs as a white to pale yellow solid. • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
Odor: Sweet, balsamic OILS), M-15, Appendix XI
Solubility: Soluble in most fixed oils; insoluble or Acceptance criteria: NMT 1.0
practically insoluble in glycerin, propylene glycol OTHER REQUIREMENTS
Boiling Point: ∼195° (5 mm Hg) • CHLORINATED COMPOUNDS, Appendix VI
SOLUBILITY IN ALCOHOL, Appendix VI: One g dissolves in 8 Acceptance criteria: Passes test
mL of 90% alcohol.
 FCC 9 Monographs / Benzyl Formate / 129

• SOLIDIFICATION POINT, Appendix IIB

Acceptance criteria: Between 33.0° and 35.0°

Monographs
Benzyl Cinnamate

Reference standard: USP Benzyl Disulfide RS

Benzyl Disulfide
.

Sample and standard preparation: K

First Published: Third Supplement, FCC 8 Acceptance criteria: The spectrum of the sample
exhibits maxima at the same wavelengths as those in
the spectrum of the Reference standard.
alpha-(Benzyldithio)toluene
BDS ASSAY
Di(phenylmethyl)disulfide • PROCEDURE: Proceed as directed in M-1b, Appendix XI.
Reference standard: USP Benzyl Disulfide RS
Acceptance criteria: NLT 98% of benzyl disulfide

OTHER REQUIREMENTS
• MELTING RANGE OR TEMPERATURE DETERMINATION, Appendix
IIB
C14H14S2 Formula wt 246.39
Acceptance criteria: 71°–74°
FEMA: 3617
CAS: [150-60-7]
UNII: BG7680605N [benzyl disulfide]

Benzyl Formate
.

DESCRIPTION
Benzyl Disulfide occurs as pale yellowish leafy crystals or First Published: Prior to FCC 6
leaflets.
Odor: Burnt caramellic
Solubility: Soluble in hot alcohol and ether; slightly soluble
or insoluble in water
Boiling Point: >270° (with decomposition)
Function: Flavoring agent

IDENTIFICATION C8H8O2 Formula wt 136.15

• INFRARED ABSORPTION, Spectrophotometric Identification FEMA: 2145
Tests, Appendix IIIC UNII: 79GJF97O0Y [benzyl formate]
 130 / Benzyl Formate / Monographs FCC 9

DESCRIPTION ASSAY
Benzyl Formate occurs as a colorless to pale yellow liquid. • PROCEDURE: Proceed as directed under M-1b, Appendix
Odor: Sweet, balsamic, floral XI.
Boiling Point: ∼203° Acceptance criteria: NLT 95.0% of C8H8O2
SOLUBILITY IN ALCOHOL, Appendix VI: One mL dissolves in 1
mL of 95% alcohol. SPECIFIC TESTS
Function: Flavoring agent • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
OILS), M-15, Appendix XI
IDENTIFICATION Acceptance criteria: NMT 3.0
• INFRARED SPECTRA, Spectrophotometric Identification Tests, • REFRACTIVE INDEX, Appendix II: At 20°
Monographs

Appendix IIIC Acceptance criteria: Between 1.508 and 1.515

Acceptance criteria: The spectrum of the sample • SPECIFIC GRAVITY: Determine at 25° by any reliable
exhibits relative maxima at the same wavelengths as method (see General Provisions).
those of the spectrum below. Acceptance criteria: Between 1.082 and 1.092

Benzyl Formate

Solubility: Soluble in alcohol, most fixed oils; slightly

Benzyl Isobutyrate
.

soluble in propylene glycol; insoluble or practically

First Published: Prior to FCC 6 insoluble in glycerin
Boiling Point: ∼229°
SOLUBILITY IN ALCOHOL, Appendix VI: One mL dissolves in 6
Benzyl 2-Methyl Propionate
mL of 70% alcohol.
Function: Flavoring agent

IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
C11H14O2 Formula wt 178.23 Acceptance criteria: The spectrum of the sample
FEMA: 2141 exhibits relative maxima at the same wavelengths as
UNII: P98PE45V9M [benzyl isobutyrate] those of the spectrum below.

DESCRIPTION ASSAY
Benzyl Isobutyrate occurs as a colorless liquid. • PROCEDURE: Proceed as directed under M-1b, Appendix
Odor: Floral, fruity, jasmine XI.
Acceptance criteria: NLT 97.0% of C11H14O2
 FCC 9 Monographs / Benzyl Isovalerate / 131

SPECIFIC TESTS • REFRACTIVE INDEX, Appendix II: At 20°

• ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL Acceptance criteria: Between 1.488 and 1.492
OILS), M-15, Appendix XI • SPECIFIC GRAVITY: Determine at 25° by any reliable
Acceptance criteria: NMT 1.0 method (see General Provisions).
Acceptance criteria: Between 1.000 and 1.005

Monographs
Benzyl Isobutyrate

Function: Flavoring agent

Benzyl Isovalerate
.

IDENTIFICATION
First Published: Prior to FCC 6
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Benzyl 3-Methyl Butyrate Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
C12H16O2 Formula wt 192.26 XI.
FEMA: 2152 Acceptance criteria: NLT 98.0% of C12H16O2 (one
UNII: 87UKH01DMA [benzyl isovalerate] isomer)

DESCRIPTION SPECIFIC TESTS

Benzyl Isovalerate occurs as a colorless liquid. • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
Odor: Fruity, herbaceous, apple OILS), M-15, Appendix XI
Solubility: Soluble in alcohol, most fixed oils; slightly Acceptance criteria: NMT 1.0
soluble in propylene glycol; insoluble or practically • REFRACTIVE INDEX, Appendix II: At 20°
insoluble in glycerin, water Acceptance criteria: Between 1.486 and 1.490
Boiling Point: ∼246° • SPECIFIC GRAVITY: Determine at 25° by any reliable
Solubility in Alcohol, Appendix VI: One mL dissolves in 3 method (see General Provisions).
mL of 80% alcohol, and remains in solution on dilution. Acceptance criteria: Between 0.983 and 0.989
 132 / Benzyl Isovalerate / Monographs FCC 9
Monographs

Benzyl Isovalerate

Function: Flavoring agent

Benzyl Mercaptan
.

IDENTIFICATION
First Published: Third Supplement, FCC 8
• INFRARED ABSORPTION, Spectrophotometric Identification
Tests, Appendix IIIC
Benzenemethanethiol Reference standard: USP Benzyl Mercaptan RS
Benzylthiol Sample and standard preparation: F
alpha-Mercaptoluene Acceptance criteria: The spectrum of the sample
Thiobenzyl alcohol exhibits maxima at the same wavelengths as those in
the spectrum of the Reference Standard.

ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
C7H8S Formula wt 124.2 XI.
FEMA: 2147 Reference standard: USP Benzyl Mercaptan RS
CAS: [100-53-8] Acceptance criteria: NLT 98% of benzyl mercaptan
UNII: OS34A21OBZ [benzyl mercaptan]
SPECIFIC TESTS
DESCRIPTION • REFRACTIVE INDEX, Appendix IIB: At 20°
Benzyl Mercaptan occurs as a colorless or pale-straw-colored Acceptance criteria: 1.573–1.578
mobile liquid. • SPECIFIC GRAVITY: Determine at 25° by any reliable
Odor: Powerful, roasted, burnt beef method (see General Provisions).
Solubility: Soluble in oils; insoluble in water Acceptance criteria: 1.050–1.058
Boiling Point: ∼194° to 195°
Solubility in Alcohol, Appendix VI: One mL dissolves in 1
mL of 95% alcohol.
 FCC 9 Monographs / Benzyl Phenylacetate / 133

Benzyl Methyl Sulfide Benzyl Phenylacetate

. .

First Published: Third Supplement, FCC 8 First Published: Prior to FCC 6

alpha-(Methylthio)toluene
Methyl Benzyl Sulfide
Methylthiomethylbenzene

Monographs
C15H14O2 Formula wt 226.27
FEMA: 2149
C8H10S Formula wt 138.28 UNII: A7LDA0CIWF [benzyl phenylacetate]
FEMA: 3597
CAS: [766-92-7] DESCRIPTION
UNII: Y3900RBK5 [benzyl methyl sulfide] Benzyl Phenylacetate occurs as a colorless liquid.
Odor: Sweet, floral, honey undertone
Solubility: Miscible in alcohol, chloroform, ether
DESCRIPTION Boiling Point: ∼317°
Benzyl Methyl Sulfide occurs as a colorless liquid. Solubility in Alcohol, Appendix VI: One mL dissolves in 3
Odor: Powerful, roasted, burnt beef mL of 90% alcohol to give a clear solution.
Solubility: Soluble in fats; slightly soluble in water Function: Flavoring agent
Boiling Point: ~197°; ~133° (4 mm Hg); ~87° to 88° (11
mm Hg) IDENTIFICATION
Function: Flavoring agent • INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
IDENTIFICATION Acceptance criteria: The spectrum of the sample
• INFRARED ABSORPTION, Spectrophotometric Identification exhibits relative maxima at the same wavelengths as
Tests, Appendix IIIC those of the spectrum below.
Reference standard: USP Benzyl Methyl Sulfide RS
Sample and standard preparation: F ASSAY
Acceptance criteria: The spectrum of the sample • PROCEDURE: Proceed as directed under M-1b, Appendix
exhibits maxima at the same wavelengths as those in XI.
the spectrum of the Reference standard. Acceptance criteria: NLT 98.0% of C15H14O2

ASSAY SPECIFIC TESTS

• PROCEDURE: Proceed as directed under M-1b, Appendix • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
XI. OILS), M-15, Appendix XI
Reference standard: USP Benzyl Methyl Sulfide RS Acceptance criteria: NMT 1.0
Acceptance criteria: NLT 98% of benzyl methyl sulfide • REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.553 and 1.558
SPECIFIC TESTS • SPECIFIC GRAVITY: Determine at 25° by any reliable
• REFRACTIVE INDEX, Appendix IIB: At 20° method (see General Provisions).
Acceptance criteria: 1.563–1.573 Acceptance criteria: Between 1.095 and 1.099
• SPECIFIC GRAVITY: Determine at 25° by any reliable
method (see General Provisions).
Acceptance criteria: 1.015–1.020
 134 / Benzyl Phenylacetate / Monographs FCC 9
Monographs

Benzyl Phenylacetate

Function: Flavoring agent

Benzyl Propionate
.

IDENTIFICATION
First Published: Prior to FCC 6
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Benzyl Propanoate Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
C10H12O2 Formula wt 164.20 XI.
FEMA: 2150 Acceptance criteria: NLT 98.0% of C10H12O2
UNII: 307DN1208L [benzyl propionate]
SPECIFIC TESTS
DESCRIPTION • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
Benzyl Propionate occurs as a colorless liquid. OILS), M-15, Appendix XI
Odor: Sweet, floral, fruity Acceptance criteria: NMT 1.0
Solubility: Soluble in alcohol, most fixed oils; slightly • REFRACTIVE INDEX, Appendix II: At 20°
soluble in propylene glycol; insoluble or practically Acceptance criteria: Between 1.496 and 1.500
insoluble in glycerin, water • SPECIFIC GRAVITY: Determine at 25° by any reliable
Boiling Point: ∼222° method (see General Provisions).
Solubility in Alcohol, Appendix VI: One mL dissolves in 3 Acceptance criteria: Between 1.028 and 1.032
mL of 70% alcohol, and remains in solution on dilution to
10 mL.
 FCC 9 Monographs / Benzyl Salicylate / 135

Monographs
Benzyl Propionate

IDENTIFICATION
Benzyl Salicylate
.

• INFRARED SPECTRA, Spectrophotometric Identification Tests,

First Published: Prior to FCC 6 Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
C14H12O3 Formula wt 228.25 Acceptance criteria: NLT 98.0% of C14H12O3
FEMA: 2151
UNII: WAO5MNK9TU [benzyl salicylate] SPECIFIC TESTS
• ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
DESCRIPTION OILS), M-15, Appendix XI
Benzyl Salicylate occurs as an almost colorless liquid. [NOTE—Use phenol red TS as the indicator.]
Odor: Faint, sweet Acceptance criteria: NMT 1.0
Solubility: Soluble in most fixed oils; insoluble or • REFRACTIVE INDEX, Appendix II: At 20°
practically insoluble in glycerin, propylene glycol Acceptance criteria: Between 1.573 and 1.582
Boiling Point: ∼300° • SPECIFIC GRAVITY: Determine at 25° by any reliable
Solubility in Alcohol, Appendix VI: One mL dissolves in 5 method (see General Provisions).
mL of 95% alcohol. Acceptance criteria: Between 1.176 and 1.180
Function: Flavoring agent
 136 / Benzyl Salicylate / Monographs FCC 9

OTHER REQUIREMENTS
• SOLIDIFICATION POINT, Appendix IIB
Acceptance criteria: NLT 23.5°
Monographs

Benzyl Salicylate

ASSAY
Bergamot Oil, Coldpressed
.

• ESTERS, Ester Determination, Appendix VI

First Published: Prior to FCC 6 Sample: 2 g
Last Revision: Second Supplement, FCC 7 Analysis: Heat the mixture for 30 min on a steam bath,
rather than for 1 h. Use 98.15 as the equivalence factor
FEMA: 2153 (e) in the calculation.
CAS: [8007-75-8] Acceptance criteria: NLT 36.0% of esters, calculated as
UNII: 39W1PKE3JI [bergamot oil] linalyl acetate (C12H20O2)

DESCRIPTION SPECIFIC TESTS

Bergamot Oil, Coldpressed occurs as a green to yellow- • ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
green or yellow-brown liquid with a fragrant, sweet-fruity IIB: Use a 100-mm tube.
odor. It is a volatile oil obtained by pressing, without the Acceptance criteria: Between +12° and +30°
aid of heat, the fresh peel of the fruit of Citrus bergamia • REFRACTIVE INDEX, Appendix IIB
Risso et Poiteau (Fam. Rutaceae). It is miscible with alcohol [NOTE—Use an Abbé or other refractometer of equal or
and with glacial acetic acid. It is soluble in most fixed oils, greater accuracy.]
but is insoluble in glycerin and in propylene glycol. It may Acceptance criteria: Between 1.465 and 1.468 at 20°
contain a suitable antioxidant. • RESIDUE ON EVAPORATION, Appendix VI: Heat sample for
Function: Flavoring agent 5 h.
Packaging and Storage: Store in a cool place protected Acceptance criteria: NMT 6.0%
from light in full, tight containers that are made from steel • SOLUBILITY IN ALCOHOL, Appendix VI
or aluminum and that are suitably lined. Acceptance criteria: One mL of the sample dissolves in
2 mL of 90% alcohol.
IDENTIFICATION • SPECIFIC GRAVITY: Determine by any reliable method (see
• INFRARED SPECTRA, Spectrophotometric Identification Tests, General Provisions).
Appendix IIIC Acceptance criteria: 0.871–0.879
Acceptance criteria: The spectrum of the sample • ULTRAVIOLET ABSORBANCE, Ultraviolet Absorbance of Citrus
exhibits relative maxima at the same wavelengths as Oils, Appendix VI
those of the spectrum below. Sample: 50 mg
 FCC 9 Monographs / Beta Glucan from Baker’s Yeast / 137

Acceptance criteria: The absorbance difference is NLT

0.32. [NOTE—The absorbance maximum occurs at
315 ± 3 nm.]

Monographs
Bergamot Oil, Coldpressed

Packaging and Storage: Store in closed, sealed packages

Beta Glucan from Baker’s Yeast
.

in a dry controlled environment (21° and 50% RH).

(Saccharomyces cerevisiae)
IDENTIFICATION
First Published: Third Supplement, FCC 7
• 1HNMR SPECTROSCOPY, Nuclear Magnetic Resonance
Spectroscopy, Appendix IIC
Baker’s Yeast Beta Glucan Reference standard solution: Dissolve 10 mg of USP
(1-3), (1-6)-β-d-glucan, Poly-(1-6)-β-d-glucopyranosyl-(1,3)- Beta Glucan RS in 0.6 mL of dimethyl sulfoxide–d6 at
β-d glucopyranose 100° for 1 h. After incubation time, add 0.1 mL of D2O,
mix the solution, and transfer to an NMR tube.
DESCRIPTION
Sample solution: Dissolve 10 mg of sample in 0.6 mL
Beta Glucan from Baker’s Yeast (Saccharomyces cerevisiae) is
of dimethyl sulfoxide–d6 at 100° for 1 h. After
a light beige to tan fine powder. This ingredient is the
incubation time, add 0.1 mL of D2O, mix the solution,
result of the fermentation of food-grade baker’s yeast
and transfer to an NMR tube.
(Saccharomyces cerevisiae) and later lysis through a thermal
Analysis: Collect 1HNMR spectra at 80°, and compare
process. The cell wall component is separated from the
individual resonances from the Sample solution to those
yeast extract using centrifugation. Then, the cell wall
from the Reference standard solution. The major signals
isolate undergoes a caustic treatment to strip the
associated with this ingredient are shown in the table
mannosylated cell wall proteins that are linked to the cell
below. The relative area of the resonance corresponding
wall and to remove the residual cellular lipids. After that,
to the 1H signal of (1,6) linked beta glucan with respect
the isolate undergoes an acid treatment, which results in
to the 1H signal of (1,3) linked beta glucan is also
the removal of most of the chitin. Lastly, the yeast wall
measured and compared to the range shown in the
slurry undergoes flash sterilization, followed by pH
table.
adjustment steps, which results in the final dry product. It
is comprised mainly of β-(1,3)/(1,6) branched glucan 1 HNMR Major Signals USP Beta Glucan RS
polymers, and trace amounts of protein and lipid. Small
H-1 (1,3-glucan) 4.52, d, J = 7.5 Hz, 1H
amounts of β-(1,6)-glucan and chitin are also expected to
be present in the final product. H-2, 4, and 5 (1,3-) 3.27–3.33, m, 3H
Function: Nutrient H-3 and 6b (1,3-) 3.45–3.48, m, 2H
H-6a (1,3-) 3.71, d, J = 11 Hz, 1H
 138 / Beta Glucan from Baker’s Yeast / Monographs FCC 9

1 HNMR Major Signals USP Beta Glucan RS glucosidase4 with Buffer solution A in a 100-mL
H-1 (1,6-glucan) 4.27, d, J = 7.7 Hz, 1H volumetric flask. A pre-mix of the enzymes may be used
Relative % of 1,6 10%–18% as an alternative5. Mix well by inverting at least 10
times. [NOTE—Store on ice during the procedure, and
Integrate the area under the peaks five times for each for use in a same-day assay. Unused Polishing enzyme
sample, and average. Integration values are then used in mix can be refrozen once at NMT −15° with an
the equation below for the peaks at 4.52 and 4.27 ppm expiration date of 2 years.]
to determine the relative percentage of (1,6) linked Glucose oxidase/peroxidase buffer6: In a 1-L
glucan in the sample. volumetric flask add 45.287 g of potassium phosphate
dibasic, 30.382 g of p-hydroxybenzoic acid, and 4 g of
Monographs

Result = {A/(A + B)} × 100 sodium azide. Carefully add 800 mL of water. Mix with
a stir bar and mild heat until fully dissolved. Transfer
A = integration values of H-1 from (1,6) glucan
the contents to a large beaker. Adjust to a pH of 7.4
B = integration values of H-1 from (1,3) glucan
with 2 M KOH solution. Transfer the solution back to
Acceptance criteria: The spectrum obtained for the
the 1-L volumetric flask, and fill with water to volume.
Sample solution exhibits a chemical shift pattern with
Mix by inverting at least six times. [NOTE—Store buffer
signal locations and intensities that match those
in an amber bottle with an expiration date of 3 years at
obtained from the preparation of the Reference standard
4°.]
solution. Also, the relative percentage of (1,6) linked
Glucose oxidase/peroxidase reagent: Dissolve 50 mL
glucan is 10%–18% of the total linkages.
of Glucose oxidase/peroxidase buffer in water to a total
ASSAY volume of 1 L. In this entire volume, dissolve the
• PROCEDURE contents of the Glucose Determination Reagent7.
Buffer solution A (sodium acetate buffer, pH 5, 200 [NOTE—Store reagent in an amber bottle, and label
mM): Add 11.6 mL of glacial acetic acid to with an expiration date of 3 months at a temperature
approximately 900 mL of water with stirring. Adjust to between 2° and 8° or 1 year at NMT −17°. Minimize
a pH of 5 using sodium hydroxide solution (4 M). time spent at room temperature.]
Transfer to a 1-L volumetric flask, and adjust to volume. [NOTE—Prepare the Sample solution and Standard solution
Buffer solution B (sodium acetate buffer, pH 3.8, 1.2 in triplicate. It is critical for the success of the assay that
M): Add 69.6 mL of glacial acetic acid to the sample is well dispersed.]
approximately 800 mL of water with stirring. Adjust to Sample solution: Accurately weigh 15–20 mg of sample
a pH of 3.8 using sodium hydroxide solution (4 M). into a 16-mm × 100-mm glass vial. Place the vial in an
Transfer to a 1-L volumetric flask, and adjust to volume. ice bath. Add 0.4 mL of cold KOH solution (2 M) all in
10 X TES buffer (10 X (hydroxymethyl)aminomethane one aliquot while vortexing to disperse the powder.
(TRIS)/EDTA/Saline): Dissolve 12.12 g of TRIS, 11.69 Return the vial to the ice bath. Continue cycling
g of NaCl, and 4.16 g of EDTA tetrasodium dihydrate through vortexing and placing the vials in the ice bath
salt in approximately 900 mL of purified water with as much as possible for 20 min. The mixture should
stirring. Adjust to a pH of 7.5 with concentrated HCl or turn into a homogenous, translucent dispersion.
4 M NaOH. Transfer the solution to a 1-L volumetric Standard solution: Repeat the Sample solution steps
flask, and dilute with water to volume. [NOTE—Buffer with USP Beta Glucan RS (15–20 mg).
can be stored for 1 year at 2°–8°.] Lyticase digestion: Upon removal of all vials containing
Lyticase solution (10 U/µL in 1 X TES buffer): Prepare Sample solution or Standard solution from the ice bath,
the required volume of lyticase from Arthrobacter luteus1 add 1.6 mL of Buffer solution B and 600 µL of Lyticase
at a concentration of 10 U/µL by dissolving the quantity solution to each vial. Incubate the mixture at 50° for
stated by the manufacturer (U/mg) in a solution 12–18 h, and cool to room temperature.
containing 10% 10 X TES buffer (v/v). [NOTE—Unused (1,6)-Glucanase digestion: After cooling of all vials,
solution can be stored at NMT −15° with an expiration remove a 130-µL aliquot of each vial and digest further
date of 1 year. Every time a different lot of lyticase is by adding 25 µL of KOH solution (2 M) and 300 µL of
used, the concentration of lyticase solution required (1,6)-Glucanase solution. Incubate vials at 80° for 15
needs to be qualified.] min, and cool to room temperature.
(1,6)-Glucanase solution: Dissolve lyophilized (1,6)- Beta glucanase/glucosidase digestion: After cooling of
glucanase2 in Buffer solution A in amounts that yield all vials, add 390 µL of the Polishing enzyme mix to each
1U/300 µL solution. [NOTE—Solids may not fully vial, and incubate the vials at 40° for 1 h. Cool them to
dissolve. So, this solution should be handled as a room temperature, centrifuge, and transfer the 50-µL
homogeneous suspension. Solution is stable for at least aliquots (in duplicate) to new vials.
60 days at NMT −15°.] Enzyme blank solution: Prepare enzyme blanks in
Polishing enzyme mix: For 100 mL total volume, mix triplicate by combining all the reagents used during the
2000 U of exo-beta-glucanase3 and 400 U of beta- 4 200 U/bottle, Megazyme, or equivalent.
5 E-EXBGOS, Megazyme, or equivalent.
6 This buffer is also available as Bottle #3 of the K-YBGL kit (Megazyme), or

1 Lyticase from Arthrobacter luteus, Sigma L4025, or equivalent. Bottle #1 of the GOPOD kit (Megazyme).
2 Commercially available as Pustulanase, Cel136, Prokazyme, or equivalent. 7 Bottle #4 of K-YBGL kit, or Bottle #2 of GOPOD kit, Megazyme, or

3 E-EXBGL 200 U/mL, 200 U/bottle, Megazyme, or equivalent. equivalent.

FCC 9 Monographs / Beta Glucan from Baker’s Yeast / 139

digestion steps except the Sample solution or Standard 1630 U/mL of amyloglucosidase and 500 U/mL of
solution. invertase.
Analysis: Dilute the 50-µL aliquots obtained after the Analysis: Weigh 100 mg of sample in triplicate into
Beta glucanase/glucosidase digestion with 50 µL of water, individual 16-mm × 150-mm glass screw cap vials.
and then add 3 mL of Glucose oxidase/peroxidase Place the vials in an ice bath, and add to each vial 2
reagent. Incubate the vials for 20 min at 40°. Using a mL of cold 2 M KOH (in one aliquot) while vortexing
suitable spectrophotometer, determine the absorbance to disperse the powder. Return the vial to the ice bath.
of each vial with Sample solution or Standard solution at Continue cycling through vortexing and placing vials in
510 nm against the Enzyme blank solution. Prepare a the ice bath as much as possible for 20 min. The
standard curve using the absorbance of similarly treated mixture should turn into a homogenous, translucent

Monographs
series of glucose standards (0 mg/mL, 0.1 mg/mL, 0.25 dispersion. Add 8 mL of Buffer solution B. Vortex
mg/mL, 0.5 mg/mL, and 1.0 mg/mL). From the slope thoroughly, and immediately add 200 µL of
of the standard curve and the absorbance of the Amyloglucosidase/invertase solution and vortex again.
digested Sample solution and Standard solution, Incubate the mixture at 40° for 30–35 min. Cool to
determine the concentration of liberated glucose in the room temperature. Vortex again, transfer to a suitable
cuvette (C), in mg/mL: centrifuge tube, and centrifuge until a clear
supernatant is obtained. Transfer duplicate 50-µL
C = (AbsS − AbsB)/slope aliquots of supernatant into new vials, and proceed
with the Analysis as outlined in the Assay section.
AbsS = average absorbance of sample or USP Beta
Acceptance criteria: NMT 1.0%
Glucan RS
• MANNOSE
AbsB = average absorbance of Enzyme blank solution
Mobile phase A: 100% Purified Water
Calculate the percentage of beta glucan as glucose in
Mobile phase B: 956 mM NaOH
the sample:
Internal standard solution: Dissolve USP Inositol RS, or
Result = 100 × C/{[(WTS/F1) × (F2/F3)]/2} equivalent, in water (0.8 mg/mL).
Sample solution: Weigh 2.0–4.0 mg of sample in
C = concentration of liberated glucose in the duplicate into vials with stir bars. Add 500 µL of pure
cuvette (mg/mL) trifluoroacetic acid (TFA), and allow the mixture to
WTS = original weight of the sample or USP Beta form a uniform dispersion by stirring for 1 h at room
Glucan RS (mg) temperature. Incubate in an 80° water bath for 2 h
F1 = total volume in the vial during Lyticase with stirring, and then cool to room temperature. Add
digestion, 2.6 mL 100 µL of Internal standard solution to each vial, and
F2 = volume of the sample or USP Beta Glucan RS incubate with stirring in a boiling water bath for 15
transferred to a new vial during (1,6)- min. Cool again to room temperature, then add 1.07
glucanase digestion, 0.130 mL mL of water to each vial, and incubate with stirring in
F3 = total volume during Beta glucanase/ a boiling water bath for 1 h. Cool the solutions to
glucosidase digestion, 0.845 mL room temperature, and dry overnight on a SpeedVac,
Acceptance criteria: NLT 70% beta glucan as glucose, or equivalent, at low heat with the cryopumping
calculated on the dried basis system off. Dissolve the dried preparation in 2.5 mL of
deionized water, and filter through a 0.2-µm PTFE
IMPURITIES syringe filter. Dilute with an equal volume of water
Inorganic Impurities before injection.
• ARSENIC, Elemental Impurities by ICP, Method I: ICP-OES, Standard solutions: Dissolve USP Dextrose RS or
Appendix IIIC equivalent, and USP Mannose RS or equivalent, in
Acceptance criteria: NMT 0.5 ppm water, aliquot them in duplicate as shown in the table
• CADMIUM, Elemental Impurities by ICP, Method I: ICP-OES, below, and freeze dry them. Continue preparation as
Appendix IIIC directed in the Sample solution, beginning with “Add
Acceptance criteria: NMT 0.5 ppm 500 µL of pure trifluoroacetic acid”.
• LEAD, Elemental Impurities by ICP, Method I: ICP-OES,
Appendix IIIC Standard µL/vial µL/vial
Acceptance criteria: NMT 0.5 ppm Identification of 4.0 µg/mL of 80 µg/mL
• MERCURY, Elemental Impurities by ICP, Method I: ICP-OES, Number Glucose Mannose
Appendix IIIC 0 0 0
Acceptance criteria: NMT 0.1 ppm 1 100 25
Organic Impurities
2 200 50
• GLYCOGEN
3 300 100
Amyloglucosidase/invertase solution8: Dissolve
amyloglucosidase and invertase in 20 mL of glycerol 4 500 200
solution (50% v/v) to obtain a solution containing 5 1000 400
8Alternatively, Bottle #2 of K-YBGL kit (Megazyme, or equivalent) could be
used directly.
 140 / Beta Glucan from Baker’s Yeast / Monographs FCC 9

Chromatographic system, Appendix IIA SPECIFIC TESTS

Mode: High-performance liquid chromatography • LOSS ON DRYING, Appendix IIC: 105°, 3 h
Column: Strong anion-exchange column 4.0-mm × Sample: 0.9–1.2 g
250-mm (CarboPac MA-1, with CarboPac MA-1 4.0- Acceptance criteria: NMT 8.0%
mm × 50-mm guard column, Dionex, or equivalent) • RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
Column temperature: 30° Sample: 2.0 g
Flow rate: 0.4 mL/min Acceptance criteria: NMT 2.5%
Injection volume: 10 µL
Gradient program: See the gradient table below.
Monographs

Time Mobile phase A Mobile phase B

Betaine
.

(min) (%) (%)

0 36.0 64.0
First Published: First Supplement, FCC 7
15.0 36.0 64.0
Trimethylglycine
35.0 (sample
injection) 59.4 40.6
2-Trimethylammonioacetate
TMG
80.0 59.4 40.6
Glycine betaine
FEMA: 4223
[NOTE—The run time typically required is 80 min.]
Detector mode: Integrated amperometry
Detector range: 3000 µC (may be modified if
needed)
Working electrode: Gold
Reference electrode: pH, Ag/AgCl C5H11O2N Formula wt, anhydrous 117.15
Electrochemical waveform: See the table below. C5H11O2N · H2O Formula wt, monohydrate 135.16
CAS: anhydrous [107-43-7]
Time Potential
monohydrate [590-47-6]
(s) (V) Integration UNII: 3SCV180C9W [betaine]
0.00 0.10 —
DESCRIPTION
0.20 0.10 Start
Betaine occurs as a white, very hygroscopic powder. It is
0.40 0.10 End recovered and purified from the aqueous liquor (molasses)
0.41 −2.00 — remaining from the production of sucrose from sugar
0.42 −2.00 — beets. It is very soluble in water, freely soluble in methanol,
0.43 0.60 —
and soluble in ethanol.
Function: Source of betaine, flavoring agent
0.44 −0.10 —
Packaging and Storage: Store in well-closed containers.
0.50 −0.10 —
IDENTIFICATION
Analysis: Separately inject equal volumes of the • INFRARED ABSORPTION, Spectrophotometric Identification
Standard solutions and Sample solution into the Tests, Appendix IIIC
chromatograph, and measure the responses for the Reference standard: USP Betaine RS
major peaks on the resulting chromatograms. For each Sample and standard preparation: K
injection, calculate the ratios of the area of the glucose Acceptance criteria: The spectrum of the sample
and mannose peaks to the area of the internal standard exhibits absorption bands at approximately the
inositol peak. Make two standard curves of the peak following wavelengths, which correspond in intensity
area ratio versus the concentration for the glucose and and wavelength to those in the spectrum of the
mannose standards, and calculate their linear Reference standard (s = strong, m = medium): 1415
regression. The glucose and mannose concentration of cm-1 (s), 1393 cm-1 (s), 1333 cm-1 (s), 932 cm-1 (s), 892
the sample is then calculated from their peak area ratio cm-1 (s), 625 cm-1 (s), and 604 cm-1 (m).
using the respective slope and intercept of the
standards regression lines. ASSAY
Calculate the percentage of mannose present in the • PROCEDURE
sample: [NOTE—Betaine is very hygroscopic and should be
handled accordingly for appropriate test procedures.]
Result = [CM/(CM + CG)] × 100 Mobile phase: 0.142 g/L anhydrous sodium sulfate.
Adjust with sodium hydroxide solution to a pH of 9.
CM = mannose concentration in the sample (µL/ Standard solution: 7.0 mg/mL USP Betaine RS, filtered
mL) through a 0.2-µm filter
CG = glucose concentration in the sample (µL/mL) Sample solution: 10 mg/mL, filtered through a 0.2-µm
Acceptance criteria: NMT 1.0% mannose, as a function filter
of total hexose recovered (glucose and mannose)
FCC 9 Monographs / BHA / 141

Chromatographic system, Appendix IIA suitable spectrophotometer. Calculate the color using
Mode: High-performance liquid chromatography the following formula:
Detector: Refractive index
Analytical column: 7.8-mm × 300-mm column packed Color = (AU × 10,000)/(CU × L)
with a strong cation-exchange sodium form resin1,
AU = absorbance of the Sample solution
and a 4.6-mm × 30-mm guard column2
CU = concentration of the Sample solution on the
Flow rate: 0.6 mL/min
anhydrous basis (g/100 mL)
Injection volume: 10 µL
L = pathlength (cm)
Elution: Isocratic
Acceptance criteria: NMT 20
Column temperature: 75°
• PH, pH Determination, Appendix IIB

Monographs
System suitability
Sample solution: 5 g/100 mL
Sample: Standard solution
Acceptance criteria: 5–7
Relative standard deviation: Peak heights NMT 1.0%
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
for replicate injections
Sample: 1 g
Analysis: Separately inject equal volumes of the Standard
Acceptance criteria: NMT 0.1%, calculated on the
solutions and Sample solution into the chromatograph,
anhydrous basis
record the chromatograms, and measure the peak
• WATER, Water Determination, Method I, Appendix IIB
responses. Calculate the percent betaine in the sample
Acceptance criteria
taken by the equation:
Anhydrous: NMT 2%
Result = (rU/rS) × (CS/CU) × 100 Monohydrate: NMT 15%

rU = peak response from the Sample solution OTHER REQUIREMENTS

rS = peak response from the Standard solution • LABELING: Indicate whether the material is anhydrous or
CS = concentration of the Standard solution (mg/ monohydrate.
mL)
CU = concentration of the Sample solution (mg/
mL)
BHA
.

Acceptance criteria: NLT 99.0% of betaine (C5H11O2N),

calculated on the anhydrous basis First Published: Prior to FCC 6
Last Revision: FCC 8
IMPURITIES
Inorganic Impurities
• ARSENIC, Arsenic Limit Test, Appendix IIIB Butylated Hydroxyanisole
Sample: 1 g
Acceptance criteria: NMT 1 mg/kg, calculated on the
anhydrous basis
• LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric
Graphite Furnace Method, Method I, Appendix IIIB
Sample: 5 g C11H16O2 Formula wt 180.25
Acceptance criteria: NMT 1 mg/kg, calculated on the INS: 320 CAS: [25013-16-5]
anhydrous basis UNII: REK4960K2U [butylated hydroxyanisole]
• SULFATE, Chloride and Sulfate Limit Tests, Sulfate Limit Test,
Appendix IIIB DESCRIPTION
Sample: Equivalent to 4.0 g on the anhydrous basis BHA occurs as a white or slightly yellow, waxy solid. It is
Control: 400 µg sulfate (40 mL of Standard Sulfate predominantly 3-tert-butyl-4-hydroxyanisole (3-BHA), with
Solution) varying amounts of 2-tert-butyl-4-hydroxyanisole (2-BHA).
Acceptance criteria: Any turbidity produced by the It melts between 48° and 63°. It is freely soluble in alcohol
Sample solution does not exceed that produced by the and in propylene glycol, and insoluble in water.
Control (NMT 0.01%). Function: Antioxidant
Packaging and Storage: Store in well-closed containers
SPECIFIC TESTS protected from light and heat.
• COLOR
Sample: Equivalent to 5 g on the anhydrous basis IDENTIFICATION
Sample solution: Transfer the Sample into a 100-mL • A. PROCEDURE
volumetric flask, and dissolve in and dilute with water Sample solution: 100 µg/mL in 72% alcohol
to volume. Pass through a 0.45-µm filter. Analysis: Add 2 mL of sodium borate TS and 1 mL of a
Analysis: Measure the absorbance of the Sample solution 100 µg/mL solution of 2,6-dichloroquinone chlorimide
in a suitable cuvette at 420 nm against water using a in absolute alcohol to 5 mL of the Sample solution and
mix.
Acceptance criteria: A blue color appears.
1 Transgenomic Coregel 87N, or equivalent
2 Bio-Rad 125-0508, or equivalent
 142 / BHA / Monographs FCC 9

• B. PROCEDURE Sample: 10 g
Acceptance criteria: The retention times of 3-tert-butyl- Acceptance criteria: NMT 0.05%
4-hydroxyanisole and 2-tert-butyl-4-hydroxyanisole from
the Sample solution correspond to those from the
Standard solution, as obtained in the Assay.
BHT
.

ASSAY
• PROCEDURE First Published: Prior to FCC 6
Solution A: 5% Acetic acid
Mobile phase: Acetonitrile and Solution A (45:55) Butylated Hydroxytoluene
Monographs

Standard solution: 90 µg/mL of USP 3-tert-Butyl-4- 2,6-Di-tert-butyl-p-cresol

hydroxyanisole RS and 10 µg/mL of USP 2-tert-Butyl-4-
hydroxyanisole RS in Mobile phase
Sample solution: 100 µg/mL in Mobile phase
Chromatographic system, Appendix IIA
Mode: HPLC
Detector: UV 290 nm
Column: 4.6-mm × 75-mm; packed with 3.5-µm
octadecylsilane chemically bonded to porous silica or C15H24O Formula wt 220.35
ceramic micro-particles packing1 INS: 321 CAS: 128-37-0
Column temperature: 30° UNII: 1P9D0Z171K [butylated hydroxytoluene]
Flow rate: 1.2 mL/min
Injection size: 20 µL
DESCRIPTION
BHT occurs as a white, crystalline solid. It is freely soluble in
System suitability
alcohol, and insoluble in water and in propylene glycol.
Sample: Standard solution
Function: Antioxidant
[NOTE—The retention times of 3-tert-butyl-4-
Packaging and Storage: Store in well-closed containers.
hydroxyanisole and 2-tert-butyl-4-hydroxyanisole are
about 4.2 and 4.6 min, respectively.] IDENTIFICATION
Suitability requirements • PROCEDURE
Resolution: NLT 1.5 between the 3-tert-butyl-4- Sample solution: 100 µg/mL in methanol
hydroxyanisole isomer and 2-tert-butyl-4- Analysis: Dissolve 200 mg of 3,3’-dimethoxy-
hydroxyanisole isomer peaks benzidine dihydrochloride in a mixture of 40 mL of
Tailing factor: NMT 1.5 methanol and 60 mL of 1 N hydrochloric acid. Add 5
Relative standard deviation: NMT 2.0% for the 3- mL of the resulting dianisidine solution to 10 mL of
tert-butyl-4-hydroxyanisole isomer and 2-tert-butyl-4- water and 2 mL of a 30 mg/mL solution of sodium
hydroxyanisole isomer peaks nitrite. Add this solution to 10 mL of the Sample
Analysis: Separately inject equal volumes of the Standard solution. An orange-red color appears within 3 min. Add
solution and Sample solution into the chromatograph, 5 mL of chloroform, and shake.
and measure the responses for the major peaks on the Acceptance criteria: The chloroform layer exhibits a
resulting chromatograms. purple or magenta color that fades when exposed to
Calculate the percentage of each isomer (3-tert-butyl-4- light.
hydroxyanisole and 2-tert-butyl-4-hydroxyanisole) in the
portion of the sample taken: ASSAY
• SOLIDIFICATION POINT, Appendix IIB
Result = (rU/rS) × (CS/CU) × 100 Acceptance criteria: NLT 69.2° (indicating a purity of
NLT 99.0% C15H24O).
rU = peak area response for the analyte in the
Sample solution SPECIFIC TESTS
rS = peak area response for the analyte in the • RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
Standard solution Sample: 50 g
CS = concentration of the analyte in the Standard Analysis: Transfer the Sample into a tared crucible, ignite
solution (µg/mL) until thoroughly charred, and cool. Moisten the ash
CU = concentration of the sample in the Sample with 1 mL of sulfuric acid, and complete the ignition by
solution (µg/mL) heating for 15-min periods at 800° ± 25° to constant
Calculate the percentage of C11H16O2 in the sample taken weight.
by adding the percentages of the two isomers. Acceptance criteria: NMT 0.002%
Acceptance criteria: NLT 98.5% C11H16O2

SPECIFIC TESTS
• RESIDUE ON IGNITION (SULFATED ASH), Method I, Appendix
IIC
1 Symmetry C18 (Waters), or equivalent.
 FCC 9 Monographs / Biphenyl / 143

Acceptance criteria: Between 229° and 232°, with

Biotin
.

decomposition
First Published: Prior to FCC 6 • OPTICAL (SPECIFIC) ROTATION, Appendix IIB
Sample solution: 20 mg/mL in 0.1 N sodium hydroxide
Acceptance criteria: [α]D20 between +89° and +93°
cis-Hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-valeric
Acid
d-Biotin

Biphenyl
.

First Published: Third Supplement, FCC 8

Monographs
Bibenzene
C10H16N2O3S Formula wt 244.31 Diphenyl
CAS: [58-85-5] Phenylbenzene
UNII: 6SO6U10H04 [biotin] 1,1’-Biphenyl
Lemonene
DESCRIPTION
Biotin occurs as a practically white, crystalline powder. It is
stable to air and heat. One g dissolves in about 5000 mL
of water at 25° and in about 1300 mL of alcohol; it is
more soluble in hot water and in dilute alkali, and it is
insoluble in other common organic solvents.
Function: Nutrient C12H10 Formula wt 154.21
Packaging and Storage: Store in tight containers. FEMA: 3129
CAS: [92-52-4]
IDENTIFICATION UNII: 2L9GJK6MGN [biphenyl]
• A. INFRARED ABSORPTION, Spectrophotometric Identification
Tests, Appendix IIIC DESCRIPTION
Reference standard: USP Biotin RS Biphenyl occurs as a white to light brown leaflet solid.
Sample and Standard preparation: K Odor: Pungent green aroma, rose-like upon dilution
Acceptance criteria: The spectrum of the sample Solubility: Soluble in ethanol; insoluble in water
exhibits maxima at the same wavelengths as those in Boiling Point: ∼254°
the spectrum of the Reference standard. Function: Flavoring agent
• B. PROCEDURE
IDENTIFICATION
Sample solution: Saturated solution in warm water
• INFRARED ABSORPTION, Spectrophotometric Identification
Acceptance criteria: The Sample solution decolorizes
Tests, Appendix IIIC
bromine TS, added dropwise.
Reference standard: USP Biphenyl RS
ASSAY Sample and standard preparation: M
• PROCEDURE Acceptance criteria: The spectrum of the sample
Sample: 500 mg exhibits maxima at the same wavelengths as those in
Analysis: Mix the Sample with 100 mL of water, add the spectrum of the Reference standard.
phenolphthalein TS and, while heating and stirring
ASSAY
continuously, slowly titrate the suspension with 0.1 N
• PROCEDURE: Proceed as directed under M-1b, Appendix
sodium hydroxide to a pink color. Each mL of 0.1 N
XI.
sodium hydroxide is equivalent to 24.43 mg of
Reference standard: USP Biphenyl RS
C10H16N2O3S.
Acceptance criteria: NLT 99% of biphenyl
Acceptance criteria: NLT 97.5% and NMT 100.5%
C10H16N2O3S SPECIFIC TESTS
• ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
IMPURITIES OILS), M-15, Appendix XI
Inorganic Impurities
Acceptance criteria: NMT 1.0
• LEAD, Lead Limit Test, Flame Atomic Absorption
• MELTING RANGE OR TEMPERATURE DETERMINATION, Appendix
Spectrophotometric Method, Appendix IIIB
IIB
Sample: 10 g
Acceptance criteria: 69°–71°
Acceptance criteria: NMT 2 mg/kg

SPECIFIC TESTS
• MELTING RANGE OR TEMPERATURE DETERMINATION, Appendix
IIB
 144 / Birch Tar Oil, Rectified / Monographs FCC 9

IDENTIFICATION
Birch Tar Oil, Rectified
.

• INFRARED SPECTRA, Spectrophotometric Identification Tests,

First Published: Prior to FCC 6 Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima (that may vary in intensity) at
UNII: 7JK2RXJ8G7 [betula pendula tar oil]
the same wavelengths as those of the spectrum below.
DESCRIPTION SPECIFIC TESTS
Birch Tar Oil, Rectified occurs as a clear, dark brown liquid
• SOLUBILITY IN ALCOHOL, Appendix VI
with a strong leather odor. It is the pyroligneous oil
Acceptance criteria: One mL of sample dissolves in 3
obtained by dry distillation of the bark and the wood of
Monographs

mL of absolute alcohol.
Betula pendula Roth and related species of Betula (Fam.
• SPECIFIC GRAVITY: Determine by any reliable method (see
Betulaceae) and rectified by steam distillation. It is soluble
General Provisions).
in most fixed oils, but it is insoluble in glycerin, in mineral
Acceptance criteria: Between 0.886 and 0.950
oil, and in propylene glycol.
Function: Flavoring agent
Packaging and Storage: Store in a cool place protected
from light in full, tight containers that are made from steel
or aluminum and that are suitably lined.

Birch Tar Oil, Rectified

 FCC 9 Monographs / Black Pepper Oil / 145

Add the following: bisabolene may co-elute depending on the exact

analysis conditions used.]
Bisabolene
.

▲
SPECIFIC TESTS
First Published: FCC 9 • ACID VALUE, Flavor Chemicals (Other Than Essential Oils),
M-15, Appendix XI
Limene Acceptance criteria: NMT 1.0
(E)-1-Methyl-4-(6-methylhepta-2,5-dien-2-yl)cyclohex-1-ene • REFRACTIVE INDEX, Appendix IIB: At 20°
(alpha-bisabolene) Acceptance criteria: 1.493–1.497
(S)-1-Methyl-4-(6-methylhepta-1,5-dien-2-yl)cyclohex-1-ene • SPECIFIC GRAVITY: Determine at 25° by any reliable

Monographs
(beta-bisabolene) method (see General Provisions).
(Z)-1-Methyl-4-(6-methylhept-5-en-2-ylidene)cyclohex-1-ene Acceptance criteria: 0.850–0.858▲ FCC9
(gamma-bisabolene)

Black Pepper Oil

.

First Published: Prior to FCC 6

CAS: [8006-82-4]
UNII: U17J84S19Z [black pepper oil]

DESCRIPTION
Black Pepper Oil occurs as an almost colorless to slightly
green liquid with the characteristic odor of pepper and a
relatively mild taste. It is the volatile oil obtained by steam
C15H24 Formula wt 204.35 distillation from the dried, unripened fruit of the plant Piper
CAS: alpha-isomer [17627-44-0]
nigrum L. (Fam. Piperaceae). It is soluble in most fixed oils,
beta-isomer [495-61-4]
in mineral oil, and in propylene glycol. It is sparingly
gamma-isomer [495-62-5]
soluble in glycerin.
FEMA: 3331
Function: Flavoring agent
UNII: E6941S3U3Q [gamma-bisabolene] Packaging and Storage: Store in a cool place protected
from light in full, tight containers that are made from steel
DESCRIPTION
or aluminum and that are suitably lined.
Bisabolene occurs as a colorless, slightly viscous oil. It
typically contains a mixture of alpha-, beta-, and gamma- IDENTIFICATION
isomers of bisabolene. • INFRARED SPECTRA, Spectrophotometric Identification Tests,
Odor: Sweet-spicy-balsamic, woody, citrus, valencene, Appendix IIIC
fruity, tropical, terpy, green, and banana Acceptance criteria: The spectrum of the sample
Solubility: Soluble in oils; insoluble in water and ethanol exhibits relative maxima (that may vary in intensity) at
Boiling Point: ~262° (760 mm Hg) the same wavelengths as those of the spectrum below.
Function: Flavoring agent
SPECIFIC TESTS
IDENTIFICATION • ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
• INFRARED ABSORPTION, Spectrophotometric Identification IIB: Use a 100-mm tube.
Tests, Appendix IIIC Acceptance criteria: Between −1° and −23°
Reference standard: USP Bisabolene RS • REFRACTIVE INDEX, Appendix IIB
Sample and standard preparation: F [NOTE—Use an Abbé or other refractometer of equal or
Acceptance criteria: The spectrum of the sample greater accuracy.]
exhibits maxima at the same wavelengths as those in Acceptance criteria: Between 1.479 and 1.488 at 20°
the spectrum of the Reference standard. • SOLUBILITY IN ALCOHOL, Appendix VI
Acceptance criteria: One mL of sample dissolves in 3
ASSAY
mL of 95% alcohol.
• PROCEDURE: Proceed as directed under M-1b, Appendix XI.
• SPECIFIC GRAVITY: Determine by any reliable method (see
Reference standard: USP Bisabolene RS
General Provisions).
Acceptance criteria: NLT 97% of bisabolene (sum of all
Acceptance criteria: Between 0.864 and 0.884
isomers) [NOTE—Alpha-, beta-, and gamma-isomers of
 146 / Black Pepper Oil / Monographs FCC 9
Monographs

Black Pepper Oil

IDENTIFICATION
Bohenin
.

• FATTY ACID COMPOSITION, Appendix VII

First Published: Prior to FCC 6 Acceptance criteria: A sample exhibits the following
fatty acid composition profile:
1,3-Behenic-2-oleic Glyceride
Weight %
Fatty Acid (Range)
16:0 <1.5
18:0 <3.0
18:1 >25.0
20:0 <7.0
22:0 >58.0
24:0 <3.0
UNII: 1E62B2M7C5 [bohenin]

DESCRIPTION IMPURITIES
Bohenin occurs as a white to light tan, waxy solid. It is a Inorganic Impurities
triglyceride containing behenic acid at the 1- and 3- • LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric
positions and oleic acid at the 2-position. Behenic acid is a Method II, Appendix IIIB
saturated fatty acid that occurs naturally in peanuts, most Acceptance criteria: NMT 0.5 mg/kg
seed fats, animal milk fat, and marine oils. It is produced
by the interesterification of triolein and ethyl behenate in SPECIFIC TESTS
the presence of a suitable lipase enzyme preparation. It • ACID VALUE (FATS AND RELATED SUBSTANCES), Appendix VII
melts at approximately 52°. It is insoluble in water; soluble Acceptance criteria: NMT 0.3
in hexane, in chloroform, and in acetone; and slightly • DIGLYCERIDES AND TRIGLYCERIDES
soluble in hot ethanol. Mobile phase: Acetone, acetonitrile [80:20]
Function: Tempering aid and antibloom agent in the Sample preparation: Transfer 5 g of sample into a 50-
manufacture of chocolate and chocolate coatings mL beaker and warm at 80° to melt. Transfer 300 µL of
Packaging and Storage: Store in closed containers away melted sample into a 10-mL volumetric flask, add 9 mL
from excessive heat. of acetone, and swirl to dissolve. If crystals of bohenin
form, warm the flask to 40° in a water bath. After
complete dissolution, dilute to volume with acetone.
 FCC 9 Monographs / Bois de Rose Oil / 147

Chromatographic system, Appendix IIA the volatile oil obtained by steam distillation from the
Mode: High-performance liquid chromatography chipped wood of Aniba rosaeodora var. amazonica Ducke
[NOTE—Use a system equipped with an autosampler (Fam. Lauraceae). The oils from the coastal region of Brazil
injection unit, a mobile-phase degasser, a column and the Amazon valley tend to differ in odor and in
heating block or oven, and a computing integrator.] linalool content from that produced in the Loreto province
Detector: Differential refractometer of Peru. It is soluble in most fixed oils and in propylene
Column: Lichrosorb RP-18 250 mm × 4.5 mm (id) (GL glycol. It is soluble in mineral oil, occasionally with
Science, Inc., or equivalent) and YMC-Pack ODA-A A- turbidity, but it is only slightly soluble in glycerin.
303 250 mm × 4.5 mm (id) (YMC Company, Ltd., or Function: Flavoring agent
equivalent), connected in a series, or equivalent Packaging and Storage: Store in a cool place protected

Monographs
Column temperature: 50° from light in full, tight containers that are made from steel
Flow rate: 2 mL/min. [NOTE—The column should be or aluminum and that are suitably lined.
equilibrated using a rate of 0.9 mL/ min, until a stable
baseline is obtained.] IDENTIFICATION
Injection size: 30 µL • INFRARED SPECTRA, Spectrophotometric Identification Tests,
Analysis: Analyze duplicate aliquots of the Sample Appendix IIIC
preparation. Calculate the percent of diglycerides (%D) Acceptance criteria: The spectrum of the sample
in the Sample preparation by the following equation: exhibits relative maxima at the same wavelengths as
those of the spectrum below.
%D = 100(DG/SA)
ASSAY
• LINALOOL, Linalool Determination, Appendix VI
DG = total sum of the peak areas at retention Sample: 1.2 g of acetylated sample
times between 11 and 14 min Acceptance criteria: NLT 82.0% and NMT 92.0% of
SA = total sum of all peak areas total alcohols, calculated as linalool (C10H18O).
Calculate the percent of triglycerides (%T) in the Sample
preparation by the following equation: SPECIFIC TESTS
• ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
%T = 100 [(SA – DG) / SA] IIB: Use a 100-mm tube.
Acceptance criteria: Between −4° and +6°
Acceptance criteria • DISTILLATION RANGE, Appendix IIB: Use a 125-mL
Diglycerides: NMT 5.0% distillation flask.
Triglycerides: NLT 95.0% Sample: 50 mL, previously dried over anhydrous sodium
• IODINE VALUE, Appendix VII sulfate
Acceptance criteria: Between 24 and 30 Acceptance criteria: NLT 70% distills between 195° and
• PEROXIDE VALUE, Appendix VII 205°.
Acceptance criteria: NMT 0.3 mEq/kg • REFRACTIVE INDEX, Appendix IIB
• SAPONIFICATION VALUE, Appendix VII [NOTE—Use an Abbé or other refractometer of equal or
Acceptance criteria: Between 162 and 172 greater accuracy.]
Acceptance criteria: Between 1.462 and 1.470 at 20°
• SOLUBILITY IN ALCOHOL, Appendix VI
Acceptance criteria: One mL of sample dissolves in 6
Bois de Rose Oil
.

mL of 60% alcohol.
First Published: Prior to FCC 6 • SPECIFIC GRAVITY: Determine by any reliable method (see
General Provisions).
Acceptance criteria: Between 0.868 and 0.889
UNII: F2522O5L7B [rosewood oil]

DESCRIPTION
Bois de Rose Oil occurs as a colorless to pale yellow liquid
with a slightly camphoraceous, pleasant, floral odor. It is
 148 / Bois de Rose Oil / Monographs FCC 9
Monographs

Bois de Rose Oil

Solubility in Alcohol, Appendix VI: One g dissolves in 2

Borneol
.

mL of 95% ethanol.
First Published: Prior to FCC 6 Function: Flavoring agent

IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.
C10H18O Formula wt 154.25
ASSAY
FEMA: 2157
• PROCEDURE: Proceed as directed under M-1b, Appendix
UNII: M89NIB437X [borneol] XI.
Acceptance criteria: NLT 97.0% of C10H18O
DESCRIPTION
Borneol occurs as a white to off-white crystal. OTHER REQUIREMENTS
Odor: Piney, camphoraceous • MELTING RANGE OR TEMPERATURE DETERMINATION,
Solubility: Slightly soluble in propylene glycol; very slightly Appendix IIB
soluble in water; insoluble or practically insoluble in Acceptance criteria: NLT 202°
vegetable oils
Boiling Point: ∼210°
 FCC 9 Monographs / Bornyl Acetate / 149

Monographs
Borneol

Function: Flavoring agent

Bornyl Acetate
.

IDENTIFICATION
First Published: Prior to FCC 6
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
L-Bornyl Acetate Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
Acceptance criteria: NLT 98.0% of C12H20O2
C12H20O2 Formula wt 196.29
FEMA: 2159 SPECIFIC TESTS
UNII: 213431586X [bornyl acetate] • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
OILS), M-15, Appendix XI
DESCRIPTION Acceptance criteria: NMT 1.0
Bornyl Acetate occurs as a colorless liquid, semicrystalline • REFRACTIVE INDEX, Appendix II: At 20°
mass, or white crystalline solid. Acceptance criteria: Between 1.462 and 1.466
Odor: Sweet, herbaceous, piney • SPECIFIC GRAVITY: Determine at 25° by any reliable
Solubility: Soluble in alcohol, most fixed oils; slightly method (see General Provisions).
soluble in water; insoluble or practically insoluble in Acceptance criteria: Between 0.981 and 0.985
glycerin, propylene glycol
Boiling Point: ∼226° OTHER REQUIREMENTS
Solubility in Alcohol, Appendix VI: One mL dissolves in 3 • ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
mL of 70% alcohol, and remains in solution on dilution to IIB: Use a 100-mm tube.
10 mL. Acceptance criteria: Between −39.5° and −45.0°
 150 / Bornyl Acetate / Monographs FCC 9

• SOLIDIFICATION POINT, Appendix IIB

Acceptance criteria: NLT 25°
Monographs

Bornyl Acetate

Add the following: coloring matters together with sodium chloride and/or
sodium sulfate as the principal uncolored components.
Brilliant Black PN is typically the sodium salt, but may also
Brilliant Black PN1
.

▲
be the calcium or potassium salts. It is soluble in water;
First Published: FCC 9 sparingly soluble in ethanol.
Function: Color
CI Food Black 1 Packaging and Storage: Store in well-closed containers.
Black PN
Brilliant Black BN IDENTIFICATION
CI No. 28440 • VISIBLE ABSORPTION SPECTRUM
Class: Bis-Azo Sample solution: Dissolve a sample in water, and dilute
Tetrasodium 4-acetamido-5-hydroxy-6-({7-sulfonato-4-[(4- appropriately.
sulfonatophenyl)diazenyl]naphthalen-1- Analysis: Measure the absorption spectrum of the
yl}diazenyl)naphthalene-1,7-disulfonate Sample solution using a suitable UV-visible
spectrophotometer.
Acceptance criteria: The Sample solution exhibits a
wavelength maximum at about 570 nm.

ASSAY
• TOTAL COLOR, Color Determination, Methods I and II,
Appendix IIIC: [NOTE—Both methods must be used.]
Method I (Spectrophotometric)
Sample solution: 10 mg/mL
Analysis: Determine as directed at 570 nm using 0.053
L/(mg · cm) for the absorptivity (a) for Brilliant Black
C28H17N5Na4O14S4 Formula wt 867.69 PN.
CAS: [2519-30-4]
Method II (TiCl3 Titration)
INS: 151
Sample solution: 0.6–0.7 g
DESCRIPTION Analysis: Determine as directed, except under
Brilliant Black PN occurs as black powder or granules. It Procedure use 15 g of Sodium Bitartrate instead of
consists of tetrasodium salt of 4-acetamido-5-hydroxy-6- 21–22 g, and use 150 mL of water instead of 275 mL.
({7-sulfonato-4-[(4-sulfonatophenyl)diazenyl]naphthalen-1- For the calculation, use 9.208 as the stoichiometric
yl}diazenyl)naphthalene-1,7-disulfonate and subsidiary factor (FS) for the sodium salt of Brilliant Black PN.
Acceptance criteria: The average of results obtained
1 Brilliant Black PN is approved for use in some countries but banned in from Method I and Method II is NLT 80% total coloring
others, such as the United States. matters.
FCC 9 Monographs / Brilliant Black PN / 151

IMPURITIES Detector: UV-Vis

Inorganic Impurities Column: 25-cm × 4.6-mm C18 analytical column (5-
• LEAD, Lead Limit Test, Appendix IIIB µm), with a 15-mm × 4.6-mm C18 guard column (5-
Sample solution: Prepare as directed for organic µm)
compounds. Column temperature: Ambient
Control: 2 µg Pb (2 mL of Diluted Standard Lead Flow rate: 1.0 mL/min
Solution) Injection volume: 20 µL
Acceptance criteria: NMT 2 mg/kg Analysis: Separately inject equal volumes of the
Organic Impurities Standard solution and Sample solution into the
• UNCOMBINED INTERMEDIATES AND PRODUCTS OF SIDE chromatograph, and measure the responses for the

Monographs
REACTIONS major peaks on the resulting chromatograms. [NOTE—
Solution A: 0.2 N ammonium acetate For peak identification, solutions for each analyte can
Solution B: Methanol be prepared using the USP Reference Standards and
Mobile phase: See Table 1 for the gradient table. separately injected to establish relative retention times.]
Calculate the individual percentages for each of the five
Table 1 impurities (Color Related Compounds 001, 002, 003,
Solution A Solution B 004, and sulfanilic acid) in the sample taken:
Time (min) (%) (%) Comments
Result = (rU/rS) × (CS/CU) × (1/F) × 100
0 98 2 Analysis
49 0 100 Wash rU = peak area of analyte in the Sample solution
Return to rS = peak area of analyte in the Standard solution
initial CS = concentration of analyte in the Standard
gradient and solution (µg/mL)
column
55 0 100 equilibration
CU = concentration of the Sample solution (mg/
mL)
69 100 0
F = mg-to-µg conversion factor, 1000
Acceptance criteria: NMT 0.8%, combined
Sample solution: 5.0 mg/mL in 0.02 M ammonium
acetate SPECIFIC TESTS
Standard solution: 40 µg/mL each of USP Color Combined Tests
Related Compound 001 RS, USP Color Related Tests
Compound 002 RS, USP Color Related Compound 003 • Loss on Drying (Volatile Matter), Color Determination,
RS, USP Color Related Compound 004 RS, and USP Appendix IIIC
Sulfanilic Acid RS. See Table 2 for chemical information • Chloride, Sodium Chloride, Color Determination, Appendix
on all five impurities. IIIC
• Sulfates (as sodium salts), Sodium Sulfate, Color
Table 2
Determination, Appendix IIIC
Chemical Acceptance criteria: NMT 20%, combined as the sum of
USP RS Name Name Synonym CAS
all three tests
4-Acetamido-5- • ETHER EXTRACTS, Color Determination, Appendix IIIC
USP Color 4-Acetamido-5- hydroxy-1,7-
Acceptance criteria: NMT 0.2%
Related hydroxynaph- 134-34-9
Compound thalene-1,7- naphthalene- • SUBSIDIARY COLORING MATTERS
001 RS disulfonic acid disulfonic acid 134-34-9 [NOTE—In this method, subsidiary coloring matters are
USP Color 4-Amino-5- 4-Amino-5- separated from the main coloring matter of Brilliant
Related hydroxynaph- hydroxy-1,7- Black PN using ascending paper chromatography (see
Compound thalene-1,7- naphthalene- Paper Chromatography, Appendix IIA) and extracted
002 RS disulfonic acid disulfonic acid 130-23-4 separately from the chromatographic paper. The
4,4’-(Triaz-1- 4,4’- absorbance of each extract is measured at the
USP Color ene-1,3- Diazoamino- wavelength of maximum absorption for Brilliant Black
Related diyl)dibenze- di(benzene-
Compound nesulfonic sulfuric acid);
(570 nm) by visible spectrophotometry. The analysis is
003 RS acid DAADBSA 17596-06-4 carried out twice using two separate developing
USP Color
solvents, and results added together to determine total
Related 8-Aminonaph- 8-Amino-2- subsidiary coloring matters. Because it is impractical to
Compound thalene-2- naphthalene- identify each subsidiary coloring matter using this
004 RS sulfonic acid sulfonic acid 119-28-8 procedure, and because the subsidiary coloring matters
4-Aminobenze- are usually minor components of food colors, the
USP Sulfanilic nesulfonic method assumes that the maximum absorbance of each
Acid RS Sulfanilic acid acid 121-57-3
subsidiary coloring matter is the same as that of the
total coloring matters. The subsidiary color matters
Chromatographic system, Appendix IIA
content is calculated by adding together the
Mode: High-performance liquid chromatography
 152 / Brilliant Black PN / Monographs FCC 9

absorbances of the extracts in conjunction with the “drapes” of the filter paper; and 20-cm × 20-cm
total coloring matters content of the sample.] chromatography grade paper.2 Mark out the
Chromatographic apparatus: The chromatography tank chromatography paper as shown in Figure 3.
(Figures 1 and 2) is comprised of a glass tank (A) and
cover (B); frame to support chromatography paper (C);
solvent tray (D); secondary frame (E) for supporting 2 Whatman No 1, or equivalent.
Monographs

Figure 1. Assembly of the Chromatographic Apparatus

Figure 2. Components of the Chromatographic Apparatus

FCC 9 Monographs / Brilliant Black PN / 153

Monographs
Figure 3. Method for Marking the Chromatographic Paper

Chromatographic solvent 1: Prepare a mixture of Collect all Sample solution sheets, cut each subsidiary
water, 28%–30% ammonium hydroxide, and trisodium band from each chromatogram sheet as a strip, and cut
citrate (95 mL: 5 mL: 2 g). Shake for 2 min, allow the an equivalent strip from the corresponding position of
layers to separate, and use the upper layer as the the plain (blank) sheet. For the Standard solution sheet,
chromatographic solvent. cut the entire band from the sheet, and cut an
Chromatographic solvent 2: Prepare a mixture of 2- equivalent strip from the corresponding position of the
butanol, acetone, water, and 28%–30% ammonium plain (blank) sheet. Place each strip, subdivided into a
hydroxide, (700:300:300:2). Shake for 2 min, allow the suitable number of approximately equal portions, in a
layers to separate, and use the upper layer as the separate test tube. Add 5.0 mL of a mixture of water
chromatographic solvent. and acetone (1:1 by vol) to each test tube, swirl for 2–3
Sample solution: 10 mg/mL sample min, add 15.0 mL of 0.05 N sodium hydrogen
Standard solution: 0.4 mg/mL sample prepared by carbonate solution, and shake the tube to ensure
diluting the Sample solution mixing. Filter the colored extracts and blanks through
Application volume: 0.10 mL 9-cm coarse porosity filter papers into clean test tubes,
Analysis: NLT 2 h before analysis, arrange the filter- and determine the absorbances of the colored extracts
paper drapes in the glass tank, and pour sufficient at 570 nm using a suitable spectrophotometer with 40-
Chromatographic solvent 1 over the drapes and into the mm closed cells against a filtered mixture of 5.0 mL of
bottom of the tank to cover the bottom of the tank to a mixture of water and acetone (1:1 by vol) and 15.0
a depth of 1 cm. Place the solvent tray in position, and mL of the 0.05 N sodium hydrogen carbonate solution.
fit the cover to the tank. Using a microsyringe capable Measure the absorbances of the extracts of the blank
of delivering 0.1 mL with a tolerance of ±0.002 mL, strips at 570 nm, and correct the absorbances of the
apply to separate chromatography sheets 0.1-mL colored extracts with the blank values.
aliquots of the Sample solution and Standard solution, as Calculate the percentage of subsidiary coloring matter in
uniformly as possible within the confines of the 18-cm the portion of the sample taken using Chromatographic
× 7-mm rectangle, holding the nozzle of the solvent 1 (Result1):
microsyringe steadily in contact with the paper. Allow
the papers to dry at room temperature for 1–2 h or at Result1 = F × D [(Aa + Ab + Ac ... An)/AS] × 100
50° in a drying cabinet for 5 min followed by 15 min at
F = dilution factor for the Standard solution, 0.04
room temperature. Mount the dried sheets, together
D = total coloring matter content of the sample,
with two plain sheets to act as blanks on the
determined from the Total Color test above
supporting frame. [NOTE—If required, several dried
and expressed as a decimal
sheets may be developed simultaneously.]
AS = absorbance from the Standard solution
Pour sufficient Chromatographic solvent 1 into the solvent
tray to bring the surface of the solvent about 1 cm (Aa + Ab + Ac ... An) = sum of the absorbances of the
below the base line of the chromatography sheets. The subsidiary coloring matters from the Sample solution,
volume necessary will depend on the dimensions of the corrected for the blank values
apparatus and should be predetermined. Put the
supporting frame into position, and replace the cover. Repeat the above procedure, but using Chromatographic
Allow the solvent front to ascend approximately 17 cm solvent 2 instead of Chromatographic solvent 1. Add the
above baseline. Remove the supporting frame, and percentages of subsidiary coloring matters determined
transfer it to a drying cabinet at 50°–60° for 10–15 in this second analysis to Result 1 to calculate the total
min. Remove the sheets from the frame. subsidiary coloring matter.
Acceptance criteria: NMT 4%
 154 / Brilliant Black PN / Monographs FCC 9

• UNSULFONATED PRIMARY AROMATIC AMINES Measure the absorbance of the solutions containing the
[NOTE—Under the conditions of this test, unsulfonated coupled Sample solution at 510 nm using a suitable
primary aromatic amines are extracted into toluene spectrophotometer with 40-mm cells against the
from an alkaline solution of the sample, re-extracted Sample blank solution. From the standard curve,
into acid, and then determined spectrophotometrically determine the weight (g) of aniline in each 100 mL of
after diazotization and coupling.] the Sample solution.
R salt solution: 0.05 N 2-naphthol-3,6-disulfonic acid, Calculate the percentage of unsulfonated primary
disodium salt aromatic amine (as aniline) in the portion of the sample
Sodium carbonate solution: 2 N sodium carbonate taken:
Standard stock solution: Weigh 0.100 g of redistilled
Monographs

aniline into a small beaker, and transfer to a 100-mL Result = WA/W × 100
volumetric flask, rinsing the beaker several times with
WA = weight of aniline in the Sample solution
water. Add 30 mL of 3 N hydrochloric acid, and dilute
calculated from the standard curve (g/100
to the mark with water at room temperature. Dilute
mL)
10.0 mL of this solution with water to 100 mL, and mix
W = weight of the sample used to prepare the
well; 1 mL of this solution is equivalent to 0.0001 g of
Sample solution (g)
aniline. [NOTE—Prepare the Standard stock solution
Acceptance criteria: NMT 0.01%, calculated as aniline
fresh.]
• WATER-INSOLUBLE MATTER, Color Determination, Appendix
Standard solutions: Separately dilute 5-mL, 10-mL, 15-
IIIC
mL, 20-mL, and 25-mL aliquots of the Standard stock
Acceptance criteria: NMT 0.2%▲ FCC9
solution with 1 N hydrochloric acid to 100 mL.
Standard blank solution: In a 25-mL volumetric flask
mix 10.0 mL of 1 N hydrochloric acid, 10.0 mL of
Sodium carbonate solution, 2.0 mL of R salt solution, and
Brilliant Blue1
.

dilute with water to volume.

Sample solution: Add 2.0 g of the sample into a First Published: Prior to FCC 6
separatory funnel containing 100 mL of water, rinse Last Revision: Third Supplement, FCC 8
down the sides of the funnel with 50 mL of water,
swirling to dissolve the sample, and add 5 mL of 1 N Brilliant Blue FCF
sodium hydroxide. Extract with two 50-mL portions of CI 42090
toluene, and wash the combined toluene extracts with Class: Triphenylmethane
10-mL portions of 0.1 N sodium hydroxide to remove
traces of color. Extract the washed toluene with three
10-mL portions of 3 N hydrochloric acid, and dilute the
combined extract with water to 100 mL.
Sample blank solution: In a 25-mL volumetric flask mix
10.0 mL of the Sample solution, 10 mL of Sodium
carbonate solution, and 2.0 mL of R salt solution, and
dilute with water to volume.
Analysis: Pipet 10-mL aliquots of the Sample solution
C37H34N2O9S3Na2 Formula wt 792.86
and each of the Standard solutions into separate, clean INS: 133 CAS: [3844-45-9]
dry test tubes. Cool the tubes for 10 min by immersion
UNII: H3R47K3TBD [fd&c blue no. 1]
in a beaker of ice water, add 1 mL of 50% potassium
bromide solution and 0.05 mL of 0.5 N sodium nitrite DESCRIPTION
solution. Mix, and allow the tubes to stand for 10 min Brilliant Blue occurs as a dark purple to bronze powder or
in the ice water bath while the aniline is diazotized. granules. It is principally the disodium salt of ethyl[4-[p-
Into each of six 25-mL volumetric flasks, measure 1 mL [ethyl(m-sulfobenzyl)amino]-α-(o-sulfophenyl)benzylidene]-
of R salt solution and 10 mL of Sodium carbonate 2,5-cyclohexadien-1-ylidene](m-sulfobenzyl)ammonium
solution. Separately pour each diazotized aniline solution hydroxide inner salt. It dissolves in water to give a solution
into a 25-mL volumetric flask containing R salt solution green-blue at neutrality, green in weak acid, and yellow in
and Sodium carbonate solution; rinse each test tube with stronger acid. Addition of base to its neutral solution
a small volume of water to allow for a quantitative produces a violet color only on boiling. When dissolved in
transfer. Dilute to the mark with water, stopper the concentrated sulfuric acid, it yields a yellow solution that
flasks, mix the contents well, and allow them to stand
for 15 min in the dark. 1To be used or sold for use to color food that is marketed in the United
Measure the absorbance of each of the solutions States, this color additive must be from a batch that has been certified by the
containing the coupled Standard solutions at 510 nm U.S. Food and Drug Administration (FDA). If it is not from an FDA-certified
using a suitable spectrophotometer with 40-mm cells batch, it is not a permitted color additive for food use in the United States,
even if it is compositionally equivalent. The name FD&C Blue No. 1 can be
against the Standard blank solution. Plot a standard applied only to FDA-certified batches of this color additive. Brilliant Blue is a
curve relating absorbance to weight (g) of aniline in common name given to the uncertified colorant. See the monograph entitled
each 100 mL of the Standard solutions. FD&C Blue No. 1 for directions for producing an FDA-certified batch.
FCC 9 Monographs / Brilliant Blue / 155

turns green when diluted with water. It is slightly soluble in Acceptance criteria
ethanol. o-, m-, and p-Sulfobenzaldehydes: NMT 1.5%,
Function: Color combined
Packaging and Storage: Store in well-closed containers. N-Ethyl-N-(3-sulfobenzyl)-sulfanilic acid: NMT 0.3%

IDENTIFICATION SPECIFIC TESTS

• PROCEDURE • COMBINED TESTS
Sample solution: 10 µg/mL, freshly prepared Tests
Analysis: Adjust the pH of three aliquots of the Sample • LOSS ON DRYING (VOLATILE MATTER), Color Determination,
solution to pH 1, pH 7, and pH 13. Measure the Appendix IIIC

Monographs
absorbance intensities (A) and wavelength maxima of • CHLORIDE, Sodium Chloride, Color Determination,
these solutions with a suitable UV-visible Appendix IIIC
spectrophotometer. • SULFATES (AS SODIUM SALTS), Sodium Sulfate, Color
Acceptance criteria Determination, Appendix IIIC
pH 1: A = 0.95 at 629 nm and A = 0.2 at 410 nm Acceptance criteria: NMT 15.0% in combination
pH 7: A = 1.11 at 630 nm • ETHER EXTRACTS, Color Determination, Appendix IIIC
pH 13: A = 1.29 at 630 nm and A = 0.15 at 408 nm Acceptance criteria: NMT 0.2%
• LEUCO BASE, Color Determination, Appendix IIIC
ASSAY Sample solution: 120 µg/mL
• TOTAL COLOR, Color Determination, Methods I and II, Acceptance criteria: NMT 5.0%
Appendix IIIC: Both methods must be used. • SUBSIDIARY COLORS, Thin-Layer Chromatography, Appendix
Method I (Spectrophotometric) IIA
Sample: 50–75 mg Adsorbent: Silica Gel G
Analysis: Transfer the Sample into a 1-L volumetric Developing solvent system: Acetonitrile, isoamyl
flask; dissolve in and dilute with water to volume. alcohol, 2-butanone, water, and ammonium hydroxide
Determine as directed at 630 nm using 0.164 L/(mg · (10:10:3:1:1)
cm) for the absorptivity (a) for Brilliant Blue. Sample solution: Transfer 1 g of sample into a 100-mL
Method II (TiCl3 Titration) volumetric flask. Fill the flask about 3/4 full with water,
Sample: 0.5 g place it in the dark for 1 h, dilute with water to
Analysis: Determine as directed using 2.52 as the volume, and mix well.
stoichiometric factor (FS) for Brilliant Blue. Application volume: 0.1 mL
Acceptance criteria: The average of results obtained Analysis: Prepare a 20- × 20-cm glass plate coated with
from Methods I and II is NLT 85.0% coloring matters. a 0.25-mm layer of Adsorbent. Spot the Sample solution
3 cm from the bottom edge. Allow the plate to dry for
IMPURITIES
about 20 min in the dark, then develop with the
Inorganic Impurities
Developing solvent system in an unlined tank
• ARSENIC, Arsenic Limit Test, Appendix IIIB
equilibrated for at least 20 min before the plate is
Sample solution: Prepare as directed for organic
inserted. Allow the solvent front to reach within about
compounds.
3 cm of the top of the plate. Dry the developed plate
Acceptance criteria: NMT 3 mg/kg
in the dark. When the plate has dried, scrape off all the
• CHROMIUM, Color Determination, Appendix IIIC
colored bands above the Brilliant Blue, which remains
Acceptance criteria: NMT 0.005%
close to the origin, into a 30-mL beaker. Extract the
• LEAD, Lead Limit Test, Appendix IIIB
subsidiary colors with three 6-mL portions of 95%
Sample solution: Prepare as directed for organic
ethanol, or until no color remains on the gel by visual
compounds.
inspection. Record the volume of ethanol used and the
Control: 10 µg Pb (10 mL of Diluted Standard Lead
spectrum of the solution between 400 and 700 nm.
Solution)
Calculate the percent of subsidiary colors:
Acceptance criteria: NMT 10 mg/kg
• MANGANESE, Manganese Limit Test, Appendix IIIB Result = (A × V × 100)/(a × W × b)
Sample solution: 15 mg/mL
Acceptance criteria: NMT 0.01%
Organic Impurities A = absorbance at the wavelength maximum
• UNCOMBINED INTERMEDIATES AND PRODUCTS OF SIDE V = volume of the ethanol solution (mL)
REACTIONS, Color Determination, Method I, Appendix IIIC a = absorptivity, 0.126 L/(mg · cm)
Analysis: Calculate the concentrations of o-, m-, and p- W = weight of the sample taken to prepare the
sulfobenzaldehyde and N-ethyl-N-(3-sulfobenzyl)- Sample solution (mg)
sulfanilic acid using the following absorptivities: a = b = cell pathlength (cm)
0.0495 L/(mg · cm) at 246 nm (acid solution) for o-, Acceptance criteria
m-, and p-sulfobenzaldehyde, and a = 0.078 L/(mg · Ethyl[4-[p-[ethyl(p-sulfobenzyl)amino]-1-(o-
cm) at 277 nm (alkaline solution) for N-ethyl-N-(3- sulfophenyl)benzylidene]-2,5-cyclohexadien-1-
sulfobenzyl)-sulfanilic acid. ylidene](p-sulfobenzyl) ammonium hydroxide inner
salt, Isomeric disodium salts
 156 / Brilliant Blue / Monographs FCC 9

and • SPECIFIC GRAVITY: Determine by any reliable method (see

Ethyl[4-[p-[ethyl(o-sulfobenzyl)amino]-α-(o- General Provisions) at the temperature specified by the
sulfophenyl)benzylidene]-2,5-cyclohexadien-1- vendor.
ylidene](o-sulfobenzyl) ammonium hydroxide inner Acceptance criteria: Within the range specified by the
salt, Isomeric disodium salts: NMT 6.0%, combined vendor
• WATER-INSOLUBLE MATTER, Color Determination, Appendix
IIIC
Acceptance criteria: NMT 0.2%
Brown HT1
.

First Published: Third Supplement, FCC 7

Monographs

Brominated Vegetable Oil

.

Chocolate Brown HT
First Published: Prior to FCC 6 CI Food Brown 3
CI No. 20285
Class: Bis-azo
DESCRIPTION Disodium 4,4’-(2,4-dihydroxy-5-hydroxymethyl-1,3-
Brominated Vegetable Oil occurs as a pale yellow to dark phenylene-bisazo) di-1-naphthalene-sulfonate
brown, viscous, oily liquid. It is a bromine addition product
of vegetable oil or oils. It is soluble in chloroform, in ether,
in hexane, and in fixed oils, and is insoluble in water.
Function: Flavoring agent; beverage stabilizer
Packaging and Storage: Store in well-closed containers.

IDENTIFICATION
• PROCEDURE
Sample: 0.2 mL C27H18N4Na2O9S2 Formula wt 652.57
Analysis: Mix the Sample with 1 g of anhydrous sodium INS: 155 CAS: [4553-89-3]
carbonate in a suitable crucible, cover the mixture with
an additional 1 g of sodium carbonate; compact the DESCRIPTION
mixture by gentle tapping; and heat the crucible over Brown HT occurs as a brown powder or granules. It is
an open flame until the crucible turns red. Cool the principally the disodium salt of 4,4’-(2,4-dihydroxy-5-
crucible and its contents, dissolve the residue in 20 mL hydroxymethyl-1,3-phenylene-bisazo) di-1-naphthalene-
of hot water, and filter. Add 1.7 N nitric acid to the sulfonate and subsidiary coloring matters with sodium
filtrate until effervescence ceases, then add 1 mL of chloride and/or sodium sulfate as the principal uncolored
silver nitrate TS. A curdy, yellow precipitate forms. components. It is soluble in water and insoluble in ethanol.
Acceptance criteria: The yellow precipitate is insoluble Function: Color
in nitric acid but soluble in an excess of ammonia TS, Packaging and Storage: Store in well-closed containers.
stronger (stronger ammonia water).
IDENTIFICATION
SPECIFIC TESTS • VISIBLE ABSORPTION SPECTRUM
• FREE BROMINE Sample solution: Dissolve a sample in water and adjust
Sample: 1 g the pH to 7.
Analysis: Dissolve the Sample in 20 mL of acetone, add Analysis: Measure the absorption spectrum of the
1 g of sodium iodide, and allow the mixture to stand in Sample solution using a suitable UV-visible
a stoppered flask in the dark for 30 min; shake the flask spectrophotometer.
occasionally. Add 25 mL of water and 1 mL of starch Acceptance criteria: The Sample solution exhibits a
TS. wavelength maximum at 460 nm.
Acceptance criteria: No blue color appears.
• FREE FATTY ACIDS (AS OLEIC ACID), Appendix VII
ASSAY
• TOTAL COLOR, Color Determination, Method I, Appendix IIIC
Sample: See Table in Appendix VII
Diluent: Phosphate buffer, pH 7, prepared by
Analysis: Titrate with the appropriate normality of
combining 50 mL of 0.2 M potassium dihydrogen
sodium hydroxide solution, shaking the flask vigorously,
phosphate and 29.54 mL of 0.2 M sodium hydroxide,
to the first permanent pink color of the same intensity
and diluting with water to 200 mL
as that of the neutralized alcohol (if the sample color
Sample stock solution: 250 mg/L in diluent
interferes, titrate to a pH of 8.5, determined with a
Sample solution: 10 mg/L prepared by diluting the
suitable instrument). Use 28.2 as the equivalence factor
Sample stock solution with diluent
(e) in the calculation for oleic acid.
Analysis: Determine the absorbance of the Sample
Acceptance criteria: NMT 2.5%
solution (instead of the directed sample preparation) at
• IODINE VALUE, Appendix VII
Acceptance criteria: NMT 16
1 Brown HT is approved for use in some countries but banned in others, such

as the United States.

FCC 9 Monographs / Brown HT / 157

460 nm. Calculate the total color as directed using CU = concentration of sample in the Sample
0.0403 L/(mg · cm) for the absorptivity (a) for Brown solution (mg/mL)
HT. 1000 = mg-to-µg conversion factor
Acceptance criteria: NLT 70.0% total coloring matters Acceptance criteria: NMT 0.7%

IMPURITIES SPECIFIC TESTS

Inorganic Impurities • COMBINED TESTS
• LEAD, Lead Limit Test, Appendix IIIB Tests
Sample solution: Prepare as directed for organic • LOSS ON DRYING (VOLATILE MATTER), Color Determination,
compounds. Appendix IIIC

Monographs
Control: 2 µg Pb (2 mL of Diluted Standard Lead • CHLORIDE, Sodium Chloride, Color Determination,
Solution) Appendix IIIC
Acceptance criteria: NMT 2 mg/kg • SULFATES (AS SODIUM SALTS), Sodium Sulfate, Color
Organic Impurities Determination, Appendix IIIC
• 4-AMINONAPHTHALENE-1-SULFONIC ACID Acceptance criteria: NMT 30%, combined as the sum
Solution A: 0.2 N ammonium acetate of all three tests
Solution B: Methanol • ETHER EXTRACTS, Color Determination, Appendix IIIC
Mobile phase: Exponential gradient program from Acceptance criteria: NMT 0.2%
(99% A and 1% B) to (0% A and 100% B) at a rate of • SUBSIDIARY COLORING MATTERS
2% per min [NOTE—In this method, subsidiary coloring matters are
Sample solution: 5 mg/mL in 0.02 M ammonium separated from the main coloring matter of Brown HT
acetate by ascending paper chromatography (see Paper
Standard solution: 35 µg/mL of 4-aminonaphthalene- Chromatography, Appendix IIA), and extracted
1-sulfonic acid separately from the chromatographic paper. The
Chromatographic system, Appendix IIA absorbance of each extract is measured at the
Mode: High-performance liquid chromatography wavelength of maximum absorption for Brown HT (460
Detector: UV nm) by visible spectrophotometry. Because it is
Column: 25-cm × 4.6-mm C18 analytical column (5- impractical to identify each subsidiary coloring matter
µm), with a 15-mm × 4.6-mm C18 guard column (5- using this procedure, and because the subsidiary
µm) coloring matters are usually minor components of food
Column temperature: Ambient colors, the method assumes that the maximum
Flow rate: 1.0 mL/min absorbance of each subsidiary coloring matter is the
Injection volume: 20 µL same as that of the total coloring matters. The
Analysis: Separately inject equal volumes of the subsidiary coloring matters content is calculated by
Standard solution and Sample solution into the adding together the absorbances of the extracts in
chromatograph, and measure the responses for the conjunction with the total coloring matters content of
major peaks on the resulting chromatograms. the sample.]
Calculate the percentage of 4-aminonaphthalene-1- Chromatographic apparatus: The chromatography tank
sulfonic acid in the sample taken: (Figures 1 and 2) is comprised of a glass tank (A) and
cover (B); frame to support chromatography paper (C);
Result = (rU/rS) × (CS/CU) × 1000 × 100 solvent tray (D); secondary frame (E) for supporting
“drapes” of the filter paper; and 20-cm × 20-cm
rU = peak area for 4-aminonaphthalene-1-sulfonic
chromatography grade paper2. Mark out the
acid in the Sample solution
chromatography paper as shown in Figure 3.
rS = peak area for 4-aminonaphthalene-1-sulfonic
acid in the Standard solution
CS = concentration of 4-aminonaphthalene-1-
sulfonic acid in the Standard solution (µg/
mL) 2 Whatman No 1, or equivalent.
 158 / Brown HT / Monographs FCC 9
Monographs

Figure 1. Assembly of the Chromatographic Apparatus.

Figure 2. Components of the Chromatographic Apparatus.

Figure 3. Method for Marking the Chromatographic Paper.

FCC 9 Monographs / Brown HT / 159

Chromatographic solvent: Prepare a mixture of n- and acetone (1:1 by vol), 14.9 mL of the 0.05 N
butanol, glacial acetic acid, and water (4:1:5). Shake for sodium hydrogen carbonate solution, and 0.1 mL of
2 min, allow the layers to separate, and use the upper water.
layer as the chromatographic solvent. Calculate the percentage of subsidiary coloring matter
Sample solution: 10 mg/mL sample in the portion of the sample taken:
Standard solution: Dilute 1.0 mL of the Sample solution
with water to 100 mL, and mix. Transfer 0.1 mL of this Result = 0.01 × D × [(AA + AB + AC ... AN)/AS] × 100
solution to a test tube. Add 5.0 mL of a water and
0.01 = dilution factor for the Standard solution

Monographs
acetone mixture (1:1 v/v), and then add 14.9 mL of
D = total coloring matter content of the sample,
0.05 N sodium carbonate solution, and shake the tube
determined from the Total Color test above,
to ensure mixing.
expressed as a decimal
Application volume: 0.10 mL
AS = absorbance from the Standard solution
Analysis: NLT 2 h before analysis, arrange the filter-
corrected for the blank
paper drapes in the glass tank and pour a sufficient
amount of the Chromatographic solvent over the drapes AA + AB + AC ... AN = sum of the absorbances of the
and into the bottom of the tank to cover the bottom of subsidiary coloring matters from the Sample solution,
the tank to a depth of 1 cm. Place the solvent tray in corrected for the blank values
position and fit the cover to the tank. Using a
microsyringe capable of delivering 0.1 mL with a Acceptance criteria: NMT 10%
tolerance of ±0.002 mL, apply a 0.1-mL aliquot of the • UNSULFONATED PRIMARY AROMATIC AMINES
Sample solution to a chromatograph sheet as uniformly [NOTE—Under the conditions of this test, unsulfonated
as possible within the confines of the 18-cm × 7-mm primary aromatic amines are extracted into toluene
rectangle, holding the nozzle of the microsyringe from an alkaline solution of the sample, re-extracted
steadily in contact with the paper. Allow the papers to into acid, and then determined spectrophotometrically
dry at room temperature for 1–2 h or at 50° in a drying after diazotization and coupling.]
cabinet for 5 min followed by 15 min at room R salt solution: 0.05 N 2-naphthol-3,6-disulfonic acid,
temperature. Mount the dried sheet together with a disodium salt
plain sheet to act as a blank on the supporting frame. Sodium carbonate solution: 2 N sodium carbonate
[NOTE—If required, several dried sheets may be Standard stock solution: Weigh 0.100 g of redistilled
developed simultaneously.] Pour a sufficient amount of aniline into a small beaker and transfer to a 100-mL
the Chromatographic solvent into the solvent tray to volumetric flask, rinsing the beaker several times with
bring the surface of the solvent about 1 cm below the water. Add 30 mL of 3 N hydrochloric acid, and dilute
base line of the chromatography sheets. The volume to the mark with water at room temperature. Dilute
necessary will depend on the dimensions of the 10.0 mL of this solution with water to 100 mL, and mix
apparatus and should be predetermined. Put the well; 1 mL of this solution is equivalent to 0.0001 g of
supporting frame into position and replace the cover. aniline. [NOTE—Prepare the Standard stock solution
Allow the system to develop for approximately 14 h, fresh.]
then remove the supporting frame and transfer it to a Standard solutions: Separately dilute 5-mL, 10-mL, 15-
drying cabinet at 50–60° for 10–15 min. Remove the mL, 20-mL, and 25-mL aliquots of the Standard stock
sheets from the frame. Cut each subsidiary band from solution with 1 N hydrochloric acid to 100 mL.
each chromatogram sheet as a strip, and cut an Standard blank solution: In a 25-mL volumetric flask
equivalent strip from the corresponding position of the mix 10.0 mL of 1 N hydrochloric acid, 10.0 mL of
plain (blank) sheet. Add 5.0 mL of water and acetone Sodium carbonate solution, 2.0 mL of R salt solution, and
(1:1 by vol) to each test tube, swirl for 2–3 min, add dilute with water to volume.
15.0 mL of 0.05 N sodium hydrogen carbonate Sample solution: Add 2.0 g of the sample into a
solution, and shake the tube to ensure mixing. Filter the separatory funnel containing 100 mL of water, rinse
colored extracts and blanks through 9-cm coarse down the sides of the funnel with 50 mL of water,
porosity filter papers into clean test tubes and swirling to dissolve the sample, and add 5 mL of 1 N
determine the absorbances of the colored extracts at sodium hydroxide. Extract with two 50-mL portions of
460 nm using a suitable spectrophotometer with 40- toluene, and wash the combined toluene extracts with
mm closed cells against a filtered mixture of 5.0 mL of 10-mL portions of 0.1 N sodium hydroxide to remove
water and acetone (1:1 by vol) and 15.0 mL of the traces of color. Extract the washed toluene with three
0.05 N sodium hydrogen carbonate solution. Measure 10-mL portions of 3 N hydrochloric acid, and dilute the
the absorbances of the extracts of the blank strips at combined extract with water to 100 mL.
460 nm, and correct the absorbances of the colored Sample blank solution: In a 25-mL volumetric flask mix
extracts with the blank values. 10.0 mL of the Sample solution, 10 mL of Sodium
Measure the absorbance of the Standard solution at 460 carbonate solution, and 2.0 mL of R salt solution, and
nm using a suitable spectrophotometer with 40-mm dilute with water to volume.
closed cells against a filtered mixture of 5.0 mL of water
 160 / Brown HT / Monographs FCC 9

Analysis: Pipet 10-mL aliquots of the Sample solution regulator (if required), and shortstop. It also occurs as a
and each of the Standard solutions into separate, clean solid rubber produced by the solution copolymerization of
dry test tubes. Cool the tubes for 10 min by immersion butadiene and styrene in a hexane solution, using butyl
in a beaker of ice water, add 1 mL of 50% potassium lithium as a catalyst. Solvents and volatiles are removed by
bromide solution and 0.05 mL of 0.5 N sodium nitrite processing with hot water or by drum drying.
solution. Mix, and allow the tubes to stand for 10 min The latex, which has a pH between 9.5 and 11.0 and a
in the ice water bath while the aniline is diazotized. solids content between 26% and 63%, is coagulated with
Into each of five 25-mL volumetric flasks, measure 1 mL or without other food-grade ingredients in a heated kettle.
of R salt solution and 10 mL of Sodium carbonate The coagulated mass is squeezed to drain off sera, and the
solution. Separately pour each diazotized aniline solution coagulum is washed with hot water (with or without
Monographs

into a 25-mL volumetric flask containing R salt solution alkali), and it is rinsed with water until the batch is neutral.
and Sodium carbonate solution, and rinse each test tube Finally, the coagulum is dried to remove residual volatiles.
with a small volume of water to allow for a quantitative When butadiene-styrene rubber is purchased in the latex
transfer. Dilute to the mark with water, stopper the form, it must be washed by the preceding or an equivalent
flasks, mix the contents well, and allow them to stand procedure. In the case of the solvent-polymerized product,
for 15 min in the dark. solvent and volatiles are removed by processing in hot
Measure the absorbance of each of the solutions water or by drum drying.
containing the coupled Standard solutions at 510 nm Both of the solid forms are supplied by the manufacturer
using a suitable spectrophotometer with 40-mm cells either as a slab or as a uniform, free-flowing crumb and
against the Standard blank solution. Plot a standard may contain a suitable food-grade antioxidant. The crumb
curve relating absorbance to weight (g) of aniline in form, in addition, may contain a suitable food-grade
each 100 mL of the Standard solutions. partitioning agent.
Measure the absorbance of the solutions containing the Function: Masticatory substance in chewing gum base
coupled Sample solution at 510 nm using a suitable Packaging and Storage: Store in well-closed containers.
spectrophotometer with 40-mm cells against the
[NOTE—The following requirements apply to the solid
Sample blank solution. From the standard curve,
rubber as supplied by the manufacturer or to the washed
determine the weight (g) of aniline in each 100 mL of
and dried coagulum obtained from the latex as described
the Sample solution.
above.]
Calculate the percentage of unsulfonated primary
aromatic amine (as aniline) in the portion of the sample IDENTIFICATION
taken: • INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Result = WA/W × 100
Sample preparation (latex): Dry a sample at 105° for 4
WA = weight of aniline in the Sample solution h, then dissolve it in hot toluene and evaporate the
calculated from the standard curve (g/100 solution on a potassium bromide plate.
mL) Sample preparation (solid): Dissolve a sample in hot
W = weight of sample used to prepare the Sample toluene and evaporate the solution on a potassium
solution (g) bromide plate.
Acceptance criteria: NMT 0.01%, calculated as aniline Acceptance criteria: The spectrum of the sample
• WATER-INSOLUBLE MATTER, Color Determination, Appendix exhibits relative maxima at the same wavelengths as
IIIC those of the appropriate spectrum below.
Acceptance criteria: NMT 0.2%
IMPURITIES
Inorganic Impurities
• CADMIUM, Cadmium Limit Test, Appendix IIIB
[NOTE—Alternatively, the cadmium content may be
Butadiene-Styrene Rubber
.

determined by the following method]

First Published: Prior to FCC 6 [NOTE—For this assay, use reagent-grade chemicals with
the lowest practicable Sb, As, Bi, Cd, Cu, Pb, Hg, Ag,
and Sn levels, and use only high-purity water and
gases. Rinse all glass- and plastic-ware twice with 10%
nitric acid and twice with 10% hydrochloric acid, and
then rinse thoroughly with High-purity water.]
High-purity water: Obtain from a mixed-bed strong-
acid, strong-base ion exchange apparatus capable of
producing water of more than 15-megohm resistivity.
DESCRIPTION 10% nitric acid solution: 1:10 (v/v) nitric acid in High-
Butadiene-Styrene Rubber occurs as a synthetic liquid latex purity water
or solid rubber produced by the emulsion polymerization 10% hydrochloric acid solution: 1:10 (v/v)
of butadiene and styrene, using fatty acid soaps as hydrochloric acid in High-purity water
emulsifiers, and a suitable catalyst, molecular weight
FCC 9 Monographs / Butadiene-Styrene Rubber / 161

Cadmium stock solution: Use any commercially the instrument manufacturer. Select analytical
available NIST traceable 1000 ppm (1000 mg/kg) wavelengths to yield adequate sensitivity and freedom
plasma-grade standard stock solution of cadmium. from interference.
Cadmium calibration standards: Tare three clean, dry Analyze the Calibration blank solution. Results for
4-oz polyethylene bottles (or equivalent). Add cadmium should indicate a concentration of less than
approximately 50 g of High-purity water to each. Slowly 0.01 mg/kg. If the results are NLT 0.01 mg/kg, repeat
add 28 ± 1 g of concentrated nitric acid, mix the analysis. In the event that reanalysis is unsuccessful,
thoroughly, slowly add 12 ± 1 g of concentrated take steps consistent with the manufacturer’s
hydrochloric acid, and mix thoroughly again. Using a recommendations to identify and remediate the
precision micropipet, add 10, 50, and 500 µL, sources of contamination or interference. Do not

Monographs
respectively, of Cadmium stock solution to one of each proceed with the analysis until the sources of
of the bottles. Dilute each solution to 100.0 ± 0.1 g contamination or interference have been identified and
with High-purity water, and mix thoroughly to obtain corrected.
calibration standards with 0.1, 0.5, and 5.0 mg/kg, Subsequently analyze all three Cadmium calibration
respectively. standards, from lowest concentration to highest. Results
• CALIBRATION BLANK SOLUTION: Prepare one solution as for each of the Cadmium calibration standards should
directed under Cadmium calibration standards, omitting indicate concentrations of 100 ± 5 mg/kg, 500 ± 25 mg
the addition of Cadmium stock solution. /kg, and 5000 ± 250 mg/kg, respectively. If the results
are not as indicated, repeat the analysis. In the event
[NOTE—For the solutions listed below, use a Parr Closed
that reanalysis is unsuccessful, take steps consistent
Digestion Vessel (catalog number 4748) with
with the manufacturer’s recommendations to identify
polyethylene vessel liners. Any equivalent apparatus
and remediate the sources of contamination or
may be used if the predigestion fortification recoveries
interference. Do not proceed with the analysis until the
are within the specifications noted below.]
sources of contamination or interference have been
Fortification solution: Use any commercially available
identified and addressed. After successful calibration of
NIST traceable 1000 ppm (1000 mg/kg) plasma-grade
the instrument for cadmium, reanalyze the Calibration
standard stock solution of cadmium.
blank solution to demonstrate that there is no carryover
Sample digestion preparation: Weigh a representative
of the cadmium.
sample on a balance with 0.1-mg precision (see
Next, analyze the Sample digestion preparation, Digestion
Apparatus for Tests and Assays, Weights and Balances,
blank preparation (2 replicates), and the Digestion
Appendix I). Transfer the sample to a digestion vessel
fortification preparation in groups of no more than 10.
that has been cleaned according to the manufacturer’s
[NOTE—At a minimum, each group should contain a
specifications. Slowly add 5.0 mL of concentrated nitric
digestion blank, a prepared digestion sample, a second
acid to the digestion vessel, seal, and heat the vessel for
replicate of the prepared digestion sample, and that
8 to 16 h at 210° ± 5°. Allow the vessel to cool to
same digestion sample prepared as a fortification
room temperature, and quantitatively transfer its
sample.] Analyze the Calibration blank solution followed
contents into a clean, dry, tared 1-oz polyethylene
by any of the Cadmium calibration standards between
bottle. Slowly add concentrated hydrochloric acid to
each group of ten samples.
achieve a final concentration of 10% (w/w), and dilute
To determine the calibration curve, aspirate the
to an appropriate final mass with High-purity water.
Cadmium calibration standards and the Calibration blank
Digestion blank preparation: Prepare as directed under
solution. If possible, use the calibration function
Sample digestion preparation, omitting the addition of
incorporated in the ICP-AES instrument’s software or
the sample.
firmware. If necessary, plot instrument response versus
Digestion fortification preparation: Prepare as directed
concentration of cadmium. Fit this line with a linear
under Sample digestion preparation, except immediately
equation of the form y = mx + b, in which y is
before heating the sample, add 25 µL of the Fortification
instrument response, m is the slope of the best-fit line,
solution. Determine the recovery of the Digestion
x is concentration, and b is the y intercept of the best-
fortification preparation by analyzing the Sample
fit line. The correlation coefficient for the best-fit line
digestion preparation for cadmium. Calculate the percent
should be ≥ 0.99. Concentrations of cadmium in the
recovery for cadmium by subtracting the unfortified
Calibration blanks, Cadmium calibration standards,
assay result from the fortified assay result and
Digestion blank preparations, Sample digestion
multiplying the difference, in mg/kg, by 100. The
preparations, and Digestion fortification preparations can
fortification level for cadmium is 1 mg/kg. Acceptable
be directly read from the ICP-AES when using its
recoveries are in the 85% to 110% range.
software or firmware, or they can be calculated from
Analysis: Use an Inductively Coupled Plasma Atomic
the best-fit equation.
Emission Spectrometer (ICP-AES), or equivalent
Acceptance criteria: NMT 1 mg/kg
instrumentation with similar capabilities. Follow the
• LEAD, Appendix IIIB
instrument manufacturer’s instructions for setting
Lead stock solution: Use any commercially available
instrument parameters for assay of cadmium. Select
NIST traceable 1000 mg/kg plasma-grade standard
appropriate background correction points for the
stock solution of lead.
cadmium analyte according to the recommendations of
 162 / Butadiene-Styrene Rubber / Monographs FCC 9

Lead calibration standards: Tare three clean, dry 4-oz hydrochloric acid, and mix thoroughly again. Using a
polyethylene bottles (or equivalent). Add approximately precision micropipet, add 10, 50, and 500 µL,
50 g of High-purity water to each. Slowly add 28 ± 1 g respectively, of Mercury stock solution to one of each of
of concentrated nitric acid, mix thoroughly, slowly add the bottles. Dilute each solution to 100.0 ± 0.1 g with
12 ± 1 g of concentrated hydrochloric acid, and mix High-purity water, and mix thoroughly to obtain
thoroughly again. Using a precision micropipet, add calibration standards with 0.1, 0.5, and 5.0 mg/kg,
10, 50, and 500 µL, respectively, of Lead stock solution respectively.
to one of each of the bottles. Dilute each solution to Analysis: Determine as directed in the alternate
100.0 ± 0.1 g with High-purity water, and mix procedure for Cadmium (above) using the Mercury stock
thoroughly to obtain calibration standards with 0.1, solution and Mercury calibration standards in place of
Monographs

0.5, and 5.0 mg/kg, respectively. the Cadmium stock solution and Cadmium calibration
Analysis: Determine as directed in the alternate standards. Substitute the word “mercury” for
procedure for Cadmium (above) using the Lead stock “cadmium” throughout the test.
solution and Lead calibration standards in place of the Acceptance criteria: NMT 3 mg/kg
Cadmium stock solution and Cadmium calibration Organic Impurities
standards. Substitute the word “lead” for “cadmium” • QUINONES, Appendix IV
throughout the test. Acceptance criteria: NMT 0.002%
Acceptance criteria: NMT 3 mg/kg • RESIDUAL HEXANE
• LITHIUM [NOTE—The isooctane (also called 2,2,4-
Standard solution: Transfer 399.3 mg of reagent-grade trimethylpentane) used in this test should be of
lithium carbonate to a 1000-mL volumetric flask, chromatographic-grade quality.]
dissolve in a minimal amount of 1:1 hydrochloric Internal standard stock solution: 3.0 mg/mL of
acid:water, dilute to volume with water, and mix. n-nonane, in isooctane
Transfer 10.0 mL of this solution to a 100-mL Internal standard solution: 6 µg/mL of n-nonane in
volumetric flask, dilute to volume with water, and mix. isooctane: made from Internal standard stock solution
Finally, transfer 10.0 mL of this solution to a second Standard stock solution: 30 µg/mL of n-hexane in
100-mL volumetric flask, add 1.0 mL of hydrochloric isooctane
acid, dilute to volume with water, and mix. This Standard solution: Pipet 10.0 mL of Standard stock
solution contains 75 µg of lithium per 100 mL. solution and 10.0 mL of Internal standard stock solution
Sample solution: Weigh 1 g of a solid-rubber sample, into a 50-mL volumetric flask, dilute to volume with
wrap it tightly in ashless filter paper, and place it in a isooctane, and mix.
tared platinum crucible. Heat in an oven at 100° for 15 Sample preparation: Transfer 1.5 g of a solid-rubber
min, and then transfer to a muffle furnace sample into a 4-oz bottle and pipet 25.0 mL of the
programmed to reach 500° within 1 to 3 h after Internal standard solution into the bottle. Stopper the
introduction of the sample. Remove the crucible from bottle, and shake mechanically overnight to dissolve
the furnace 15 to 20 min after 500° has been reached, the rubber. Add 50 mL of methanol to precipitate the
and cool in a desiccator. Quantitatively transfer the polymer, and shake vigorously for 15 min. Allow the
contents of the crucible to a 100-mL volumetric flask, mixture to settle, and decant the liquid phase into a
using 1 mL of hydrochloric acid and water, dilute to 250-mL separatory funnel. Wash the polymer with 25
volume with water, and mix. mL of methanol, and add the wash to the separatory
Analysis: Use a suitable atomic absorption funnel. Add 50 to 75 mL of cold water to the
spectrophotometer equipped with a lithium hollow- separatory funnel, and shake vigorously for 1 min,
cathode lamp, capable of measuring the radiation venting periodically to release any pressure. Allow the
absorbed by lithium in the 670-nm spectral band. phases to separate, drain off the bottom (aqueous)
Following the manufacturer’s instructions for operating phase, and rewash the isooctane phase with a second
the instrument, aspirate a suitable portion of the 50-mL portion of cold water. Shake again, allow to
Standard solution through the flame. In a similar separate, and drain off the bottom layer. Transfer 10
manner, aspirate a suitable portion of the Sample mL of the isooctane phase to a 20-mL vial for the
solution. analysis.
Acceptance criteria: Any absorbance produced by the Chromatographic system, Appendix IIA
Sample solution should not exceed that produced by Mode: Gas chromatography
the Standard solution. (NMT 0.0075%) Detector: Flame-ionization
• MERCURY, Appendix IIIB Column: 3-m × 3-mm stainless steel column, or
Mercury stock solution: Use any commercially available equivalent, packed with 60-to 80-mesh Chromosorb
NIST traceable 1000 ppm (1000 mg/kg) plasma-grade P containing 15% didecyl phthalate and capable of
standard stock solution of mercury. separating hexane, isooctane, and n-nonane, or
Mercury calibration standards: Tare three clean, dry 4- equivalent
oz polyethylene bottles (or equivalent). Add Temperature
approximately 50 g of High-purity water to each. Slowly Column: 120° (isothermal)
add 28 ± 1 g of concentrated nitric acid, mix Injection port: 240°
thoroughly, slowly add 12 ± 1 g of concentrated Detector: 250°
FCC 9 Monographs / Butadiene-Styrene Rubber / 163

Carrier gas: Helium n-nonane areas) produced by the Sample preparation

Flow rate: 30 mL/min. does not exceed that produced by the Standard
Injection volume: 5 µL solution. (NMT 0.01%)
Analysis: Separately inject the Standard solution and the • RESIDUAL STYRENE, Appendix IV
Sample preparation into the chromatograph (duplicate Acceptance criteria: NMT 0.003%
injections for each) and record the resulting
chromatograms. Measure the areas under the hexane SPECIFIC TESTS
and n-nonane peaks on the resulting chromatograms. • BOUND STYRENE, Appendix IV
Acceptance criteria: The peak area ratio of hexane to Acceptance criteria: Between 1.0% and 50.0%
n-nonane (i.e., sum of hexane areas divided by sum of

Monographs
Butadiene-Styrene 50/50 Rubber (Solid)

Butadiene-Styrene 75/25 Rubber (Emulsion-Polymerized Latex)

 164 / Butadiene-Styrene Rubber / Monographs FCC 9
Monographs

Butadiene-Styrene 75/25 Rubber (Emulsion-Polymerized Solid)

Butadiene-Styrene 50/50 Rubber (Latex)

DESCRIPTION
Butan-3-one-2-yl Butanoate
.

Butan-3-one-2-yl Butanoate occurs as a white to slightly

First Published: Prior to FCC 6 yellow liquid.
Odor: Sweet, red berry character
Solubility: Soluble in alcohol, propylene glycol, most fixed
oils; insoluble or practically insoluble in water
Function: Flavoring agent

IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
C8H14O3 Formula wt 158.20
Appendix IIIC
FEMA: 3332
UNII: SEN8DCI58L [butan-3-one-2-yl butanoate]
 FCC 9 Monographs / Butane / 165

Acceptance criteria: The spectrum of the sample SPECIFIC TESTS

exhibits relative maxima at the same wavelengths as • REFRACTIVE INDEX, Appendix II: At 20°
those of the spectrum below. Acceptance criteria: Between 1.408 and 1.429
• SPECIFIC GRAVITY: Determine at 25° by any reliable
ASSAY method (see General Provisions).
• PROCEDURE: Proceed as directed under M-1a, Appendix Acceptance criteria: Between 0.972 and 0.992
XI.
Acceptance criteria: NLT 98.0% of C8H14O3

Monographs
Butan-3-one-2-yl Butanoate

[NOTE—For obtaining a test sample of gas, use a stainless

Butane
.

steel specimen cylinder equipped with a stainless steel

First Published: Prior to FCC 6 valve and having a capacity of not less than 200 mL and
Last Revision: FCC 6 a pressure rating of 240 psi or more. Dry the cylinder
with the valve open at 110° for 2 h, and evacuate the hot
cylinder to less than 1 mm Hg. Close the valve, and cool
and weigh the cylinder. Tightly connect one end of a
charging line to the sample cylinder, and loosely connect
the other end to the specimen cylinder. Carefully open
n-Butane the sample cylinder, and allow the sample gas to flush
C4H10 Formula wt 58.12 out the charging line through the loose connection. Avoid
INS: 943a CAS: [106-97-8] excessive flushing that causes moisture to freeze in the
UNII: 6LV4FOR43R [butane] charging line and connections. Tighten the fitting on the
specimen cylinder, and open its valve, allowing the
DESCRIPTION sample gas to flow into the evacuated cylinder. Continue
Butane occurs as a colorless, flammable gas. One volume of until the desired amount of sample gas is obtained, then
water dissolves 0.15 volume; 1 volume of alcohol dissolves close the sample cylinder valve, and finally, close the
18 volumes; 1 volume of ether dissolves 25 volumes, at specimen cylinder valve. Weigh the charged specimen
17° and 770 mm Hg. Its boiling temperature is −0.5°. cylinder again, and calculate the sample gas weight.]
Function: Propellant; aerating agent [CAUTION—Do not overload the specimen cylinder.]
Packaging and Storage: Store in tight cylinders
protected from heat. IDENTIFICATION
[CAUTION—Butane is highly flammable and explosive. • INFRARED ABSORPTION SPECTRUM
Observe precautions and perform sampling and analytical Acceptance criteria: The spectrum of a sample exhibits
operations in a well-ventilated fume hood.] absorptions, among others, at approximately 3.4 µm
(vs), 6.8 µm (s), 7.2 µm (m), and 10.4 µm (m).
 166 / Butane / Monographs FCC 9

• VAPOR PRESSURE through the gas dispersion tube at a rate of about 100
Analysis: Determine the vapor pressure of the sample mL/min; if necessary, heat the sampling cylinder gently
gas at 21° by means of a suitable pressure gauge. to maintain this flow rate.
Acceptance criteria: Approximately 213 kPa absolute Acceptance criteria: NMT 10 mg/kg
(31 psia) Organic Impurities
• HIGH-BOILING RESIDUE
ASSAY Analysis: Prepare a cooling coil from copper tubing
• PROCEDURE (about 6.1 m × 6 mm (od)) to fit into a suitable
Chromatographic system, Appendix IIA vacuum-jacketed flask. Immerse the cooling coil in a
Mode: Gas chromatography mixture of Dry Ice and acetone in a vacuum-jacketed
Monographs

Detector: Thermal conductivity flask, and connect one end of the tubing to the sample
Column: 6-m × 3-mm aluminum column, or cylinder (see Note above on sampling). Carefully open
equivalent, packed with 10 weight percent the sample cylinder valve, flush the cooling coil with
tetraethylene glycol dimethyl ether liquid phase on a about 50 mL of the liquified sample, and discard this
support of crushed firebrick (GasChrom R, or portion of liquid. Continue delivering liquid from the
equivalent), which has been calcined or burned with a cooling coil, and collect it in a previously chilled 1000-
clay binder above 900° and silanized, or equivalent mL sedimentation cone until the cone is filled to the
Column temperature: 33° 1000-mL mark (approximately 600 g). Using a warm
Carrier gas: Helium water bath maintained at about 40° to reduce
Flow rate: 50 mL/min evaporating time, allow the liquid to evaporate. When
Injection volume: 2 µL all of the liquid has evaporated, rinse the
System suitability sedimentation cone with two 50-mL portions of
Sample: Sample gas pentane, and combine the rinsings in a tared 150-mL
Suitability requirement: The peak responses obtained evaporating dish. Transfer 100 mL of the pentane
for the sample gas in the chromatograms from solvent to a second, tared 150-mL evaporating dish,
duplicate determinations agree within 1%. place both evaporating dishes on a water bath,
Analysis: Connect one sample cylinder to the evaporate to dryness, and heat the dishes in an oven at
chromatograph through a suitable sampling valve and a 100° for 60 min. Cool the dishes in a desiccator, and
flow control valve downstream from the sampling valve. weigh them. Repeat the heating for 15-min periods
Flush the liquid sample through the sampling valve, until successive weighings are within 0.1 mg. [NOTE—
taking care to avoid trapping gas or air in the valve. Retain the residue for the test for Acidity of Residue
Inject a sample and record the chromatogram. (below).] The weight of the residue obtained from the
Calculate the purity of the sample using the following sample is the difference between the weights of the
formula: residues in the two evaporating dishes. Calculate the
mg/kg of high-boiling residue based on a sample
Result = 100S/Σs
weight of 600 g.
Acceptance criteria: NMT 5 mg/kg
S = sample peak response • SULFUR COMPOUNDS
Σs = sum of all of the peak responses in the Analysis: Carefully open the container valve to produce
chromatogram a moderate flow of gas. Do not direct the gas stream
Acceptance criteria: NLT 97.0% of C4H10 toward the face, but deflect a portion of the stream
toward the nose.
IMPURITIES Acceptance criteria: The gas is free from the
Inorganic Impurities characteristic odor of sulfur compounds.
• WATER, Water Determination, Appendix IIB
Sample: 100 g (see Note above on sampling) SPECIFIC TESTS
Analysis: Proceed as directed using the following • ACIDITY OF RESIDUE
modifications: (a) Provide the closed-system titrating Sample: The residue obtained in the test for High-Boiling
vessel with an opening and pass through it a coarse- Residue (above)
porosity gas dispersion tube connected to a sampling Analysis: Add 10 mL of water to the Sample, mix by
cylinder. (b) Dilute the reagent with anhydrous swirling for about 30 s, add 2 drops of methyl orange
methanol to give a water equivalence factor of TS, insert the stopper in the tube, and shake the tube
between 0.2 and 1.0 mg/mL; age this diluted solution vigorously.
for not less than 16h before standardization. (c) Acceptance criteria: No pink or red color appears in the
Introduce the gas Sample into the titration vessel aqueous layer.
 FCC 9 Monographs / 2-Butanone / 167

FEMA: 2170
2,3-Butanedithiol
.

UNII: 6PT9KLV9IO [methyl ethyl ketone]

First Published: Third Supplement, FCC 8
DESCRIPTION
2-Butanone occurs as a colorless, mobile liquid.
2,3-Dimercaptobutane
Odor: Ethereal, nauseating
Solubility: One mL dissolves in 4 mL of water; miscible in
alcohol, ether, and most fixed oils
Boiling Point: ∼78.6° to 80°
Function: Flavoring agent

Monographs
C4H10S2 Formula wt 122.25 IDENTIFICATION
FEMA: 3477 • INFRARED SPECTRA, Spectrophotometric Identification Tests,
CAS: [4532-64-3] Appendix IIIC
UNII: 69G298U39K [2,3-butanedithiol] Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
DESCRIPTION those of the spectrum below.
2,3-Butanedithiol occurs as a colorless liquid.
Odor: Sulfureous, meaty, and alliaceous with a coffee ASSAY
nuance • PROCEDURE: Proceed as directed under M-1b, Appendix
Solubility: Insoluble in water; miscible with fat XI.
Boiling Point: ∼69° to 70° (28 mm Hg) Acceptance criteria: NLT 99.5% of C4H8O
Function: Flavoring agent
SPECIFIC TESTS
IDENTIFICATION • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
• INFRARED ABSORPTION, Spectrophotometric Identification OILS), M-15, Appendix XI
Tests, Appendix IIIC Acceptance criteria: NMT 2.0
Reference standard: USP 2,3-Butanedithiol RS • REFRACTIVE INDEX, Appendix II: At 20°
Sample and standard preparation: F Acceptance criteria: Between 1.375 and 1.384
Acceptance criteria: The spectrum of the sample • SPECIFIC GRAVITY: Determine at 25° by any reliable
exhibits maxima at the same wavelengths as those in method (see General Provisions).
the spectrum of the Reference standard. Acceptance criteria: Between 0.801 and 0.803
ASSAY OTHER REQUIREMENTS
• PROCEDURE: Proceed as directed under M-1b, Appendix • DISTILLATION RANGE, Appendix IIB
XI. Acceptance criteria: Within 1.5°
Reference standard: USP 2,3-Butanedithiol RS • WATER, Water Determination, Method I, Appendix IIB:
Acceptance criteria: NLT 99% of 2,3-butanedithiol [NOTE—Use freshly distilled pyridine as solvent.]
Acceptance criteria: NMT 0.2%
SPECIFIC TESTS
• REFRACTIVE INDEX, Appendix IIB: At 20°
Acceptance criteria: 1.517–1.527
• SPECIFIC GRAVITY: Determine at 20° by any reliable
method (see General Provisions).
Acceptance criteria: 0.995–0.997

2-Butanone
.

First Published: Prior to FCC 6

Methyl Ethyl Ketone

C4H8O Formula wt 72.11

 168 / 2-Butanone / Monographs FCC 9
Monographs

2-Butanone

Function: Flavoring agent

2-sec-Butyl Cyclohexanone
.

IDENTIFICATION
First Published: Prior to FCC 6
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Freskomenthe Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
C10H18O Formula wt 154.25 XI.
FEMA: 3261 Acceptance criteria: NLT 97.0% of C10H18O (sum of two
UNII: 5WA6R1KL5J [2-sec-butyl cyclohexanone] isomers)

DESCRIPTION SPECIFIC TESTS

2-sec-Butyl Cyclohexanone occurs as a colorless to pale • REFRACTIVE INDEX, Appendix II: At 20°
yellow liquid. Acceptance criteria: Between 1.456 and 1.462
Odor: Camphoraceous • SPECIFIC GRAVITY: Determine at 25° by any reliable
Solubility: Soluble in propylene glycol, vegetable oils; method (see General Provisions).
insoluble or practically insoluble in water Acceptance criteria: Between 0.910 and 0.915
Boiling Point: ∼76° (8 mm Hg)
Solubility in Alcohol, Appendix VI: One mL dissolves in 1
mL of 95% ethanol.
 FCC 9 Monographs / Butyl 2-Methyl Butyrate / 169

Monographs
2-sec-Butyl Cyclohexanone

Function: Flavoring agent

Butyl 2-Methyl Butyrate
.

IDENTIFICATION
First Published: Prior to FCC 6
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
C9H18O2 Formula wt 158.24 • PROCEDURE: Proceed as directed under M-1b, Appendix
FEMA: 3393 XI.
UNII: W313K2V7PG [butyl 2-methyl butyrate] Acceptance criteria: NLT 98.0% of C9H18O2

DESCRIPTION SPECIFIC TESTS

Butyl 2-Methyl Butyrate occurs as a colorless to pale yellow • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
liquid. OILS), M-15, Appendix XI
Odor: Fruity Acceptance criteria: NMT 1.0
Solubility: Soluble in propylene glycol, vegetable oils; • REFRACTIVE INDEX, Appendix II: At 20°
insoluble or practically insoluble in water Acceptance criteria: Between 1.407 and 1.413
Boiling Point: ∼173° (730 mm Hg) • SPECIFIC GRAVITY: Determine at 25° by any reliable
Solubility in Alcohol, Appendix VI: One mL dissolves in 1 method (see General Provisions).
mL of 95% ethanol. Acceptance criteria: Between 0.858 and 0.863
 170 / Butyl 2-Methyl Butyrate / Monographs FCC 9
Monographs

Butyl 2-Methyl Butyrate

ASSAY
Butyl Acetate
.

• PROCEDURE: Proceed as directed under M-1b, Appendix

First Published: Prior to FCC 6 XI.
Last Revision: FCC 7 Acceptance criteria: NLT 98.0% of C6H12O2

SPECIFIC TESTS
n-Butyl Acetate • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
OILS), M-15, Appendix XI
Acceptance criteria: NMT 2.0
• REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.393 and 1.396
C6H12O2 Formula wt 116.16 • SPECIFIC GRAVITY: Determine at 25° by any reliable
FEMA: 2174 method (see General Provisions).
UNII: 464P5N1905 [butyl acetate] Acceptance criteria: Between 0.876 and 0.880

DESCRIPTION OTHER REQUIREMENTS

Butyl Acetate occurs as a colorless, mobile liquid. • DISTILLATION RANGE, Appendix IIB
Odor: Strong, fruity Acceptance criteria: Between 120° and 128°
Solubility: Miscible in alcohol, ether, propylene glycol; one
mL in 145 mL water
Boiling Point: ∼126°
Butyl Alcohol
.

Function: Flavoring agent

IDENTIFICATION First Published: Prior to FCC 6

• INFRARED ABSORPTION, Spectrophotometric Identification
Tests, Appendix IIIC 1-Butanol
Reference standard: USP Butyl Acetate RS
Sample and standard preparation: F
Acceptance criteria: The spectrum of the sample
exhibits maxima at the same wavelengths as those in C4H10O Formula wt 74.12
the spectrum of the Reference standard. FEMA: 2178
UNII: 8PJ61P6TS3 [butyl alcohol]
 FCC 9 Monographs / Butyl Butyrate / 171

DESCRIPTION Acceptance criteria: NLT 99.5% of C4H10O

Butyl Alcohol occurs as a colorless, mobile liquid.
Odor: Vinous SPECIFIC TESTS
Solubility: Miscible in alcohol, ether; 1 mL dissolves in 15 • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
mL water OILS), M-15, Appendix XI
Boiling Point: ∼117.7° Acceptance criteria: NMT 2.0
Function: Flavoring agent • REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.397 and 1.402
IDENTIFICATION • SPECIFIC GRAVITY: Determine at 25° by any reliable
• INFRARED SPECTRA, Spectrophotometric Identification Tests, method (see General Provisions).

Monographs
Appendix IIIC Acceptance criteria: Between 0.807 and 0.809
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as OTHER REQUIREMENTS
those of the spectrum below. • BUTYL ETHER, M-1b, Appendix XI
Acceptance criteria: NMT 0.15%
ASSAY • DISTILLATION RANGE, Appendix IIB
• PROCEDURE: Proceed as directed under M-1b, Appendix Acceptance criteria: NMT 1.5° (between beginning and
XI. end)

Butyl Alcohol

Boiling Point: ∼165°

Butyl Butyrate
.

Function: Flavoring agent

First Published: Prior to FCC 6
IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
n-Butyl n-Butyrate Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

C8H16O2 Formula wt 144.21 ASSAY

FEMA: 2186 • PROCEDURE: Proceed as directed under M-1b, Appendix
UNII: 1BHV00T1M4 [butyl butyrate] XI.
Acceptance criteria: NLT 98.0% of C8H16O2
DESCRIPTION
Butyl Butyrate occurs as a colorless liquid. SPECIFIC TESTS
Odor: Fruity, pineapple on dilution • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
Solubility: Slightly soluble in propylene glycol, water; OILS), M-15, Appendix XI
miscible in alcohol, ether, most vegetable oils; 1 mL Acceptance criteria: NMT 1.0
dissolves in 3 mL 70% alcohol • REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.405 and 1.407
 172 / Butyl Butyrate / Monographs FCC 9

• SPECIFIC GRAVITY: Determine at 25° by any reliable Acceptance criteria: Between 0.867 and 0.871
method (see General Provisions).
Monographs

Butyl Butyrate

Function: Flavoring agent

Butyl Butyryllactate
.

IDENTIFICATION
First Published: Prior to FCC 6
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Butyl Ester Acceptance criteria: The spectrum of the sample
Butyrate exhibits relative maxima at the same wavelengths as
Butyryllactic Acid those of the spectrum below.
Lactic Acid
ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
Acceptance criteria: NLT 95.0% of C11H20O4

C11H20O4 Formula wt 216.28 SPECIFIC TESTS

FEMA: 2190 • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
UNII: OCR0ONT89C [butyl butyryllactate] OILS), M-15, Appendix XI
Acceptance criteria: NMT 1.0
DESCRIPTION • REFRACTIVE INDEX, Appendix II: At 20°
Butyl Butyryllactate occurs as a colorless liquid. Acceptance criteria: Between 1.420 and 1.423
Odor: Mild, buttery, cream • SPECIFIC GRAVITY: Determine at 25° by any reliable
Solubility: Soluble in propylene glycol; miscible in alcohol, method (see General Provisions).
most fixed oils; insoluble or practically insoluble in water Acceptance criteria: Between 0.970 and 0.974
Solubility in Alcohol, Appendix VI: One mL dissolves in 3
mL of 70% alcohol.
 FCC 9 Monographs / Butyl Heptanoate / 173

Monographs
Butyl Butyryllactate

Acceptance criteria: The spectrum of the sample

Butyl Cinnamate
.

exhibits maxima at the same wavelengths as those in

First Published: Third Supplement, FCC 8 the spectrum of the Reference standard.

ASSAY
Butyl 3-Phenylpropenoate • PROCEDURE: Proceed as directed under M-1b, Appendix
Butyl beta-Phenylacrylate XI.
Cinnamic Acid, Butyl Ester Acceptance criteria: NLT 98% of butyl cinnamate and
Cinnamic Acid n-Butyl Ester its isomers
n-Butyl Cinnamate
SPECIFIC TESTS
• REFRACTIVE INDEX, Appendix IIB: At 20°
Acceptance criteria: 1.539–1.545
• SPECIFIC GRAVITY: Determine at 25° by any reliable
method (see General Provisions).
C13H16O2 Formula wt 204.27 Acceptance criteria: 1.008–1.014
FEMA: 2192
CAS: [538-65-8]

DESCRIPTION Butyl Heptanoate

.

Butyl Cinnamate occurs as a colorless, oily, somewhat

viscous liquid. First Published: Third Supplement, FCC 8
Odor: Sweet, oily, balsamic, fruity aroma
Solubility: Slightly soluble to insoluble in water; miscible Butyl Heptylate
with ethanol Butyl Oenanthate
Boiling Point: ~287° Heptanoic Acid, Butyl Ester
Function: Flavoring agent

IDENTIFICATION
• INFRARED ABSORPTION, Spectrophotometric Identification
Tests, Appendix IIIC C11H22O2 Formula wt 186.29
Reference standard: USP Butyl Cinnamate RS FEMA: 2199
Sample and standard preparation: F CAS: [5454-28-4]
 174 / Butyl Heptanoate / Monographs FCC 9

DESCRIPTION UNII: PW9UJ5C0F3 [butyl isobutyrate]

Butyl Heptanoate occurs as a colorless liquid.
Odor: Herbaceous; slightly fruity aroma DESCRIPTION
Solubility: Soluble in most organic solvents; slightly Butyl Isobutyrate occurs as a colorless liquid.
soluble in water Odor: Fresh, fruity, apple-pineapple
Boiling Point: ~226.2° Solubility: Miscible in alcohol, ether, most fixed oils;
Function: Flavoring agent insoluble or practically insoluble in glycerin, propylene
glycol, water
IDENTIFICATION Boiling Point: ∼166°
• INFRARED ABSORPTION, Spectrophotometric Identification Solubility in Alcohol, Appendix VI: One mL dissolves in 7
Monographs

Tests, Appendix IIIC mL of 60% alcohol.

Reference standard: USP Butyl Heptanoate RS Function: Flavoring agent
Sample and standard preparation: F
Acceptance criteria: The spectrum of the sample IDENTIFICATION
exhibits maxima at the same wavelengths as those in • INFRARED SPECTRA, Spectrophotometric Identification Tests,
the spectrum of the Reference standard. Appendix IIIC
Acceptance criteria: The spectrum of the sample
ASSAY exhibits relative maxima at the same wavelengths as
• PROCEDURE: Proceed as directed in M-1b, Appendix XI. those of the spectrum below.
Reference standard: USP Butyl Heptanoate RS
Acceptance criteria: NLT 98% ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
SPECIFIC TESTS XI.
• REFRACTIVE INDEX, Appendix IIB: At 20° Acceptance criteria: NLT 97.0% of C8H16O2 (one
Acceptance criteria: 1.419–1.423 isomer)
• SPECIFIC GRAVITY: Determine at 25° by any reliable
method (see General Provisions). SPECIFIC TESTS
Acceptance criteria: 0.866 (±0.002) • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
OILS), M-15, Appendix XI
Acceptance criteria: NMT 1.0
• REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.401 and 1.404
Butyl Isobutyrate
.

• SPECIFIC GRAVITY: Determine at 25° by any reliable

First Published: Prior to FCC 6 method (see General Provisions).
Acceptance criteria: Between 0.859 and 0.864

C8H16O2 Formula wt 144.21

FEMA: 2188
 FCC 9 Monographs / Butyl Isovalerate / 175

Monographs
Butyl Isobutyrate

IDENTIFICATION
Butyl Isovalerate
.

• INFRARED SPECTRA, Spectrophotometric Identification Tests,

First Published: Prior to FCC 6 Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix XI
C9H18O2 Formula wt 158.24 Acceptance criteria: NLT 97.0% of C9H18O2 (one
FEMA: 2218 isomer)
UNII: 4UX6V9QM2J [butyl isovalerate]
SPECIFIC TESTS
DESCRIPTION • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
Butyl Isovalerate occurs as a colorless to pale yellow liquid. OILS), M-15, Appendix XI
Odor: Fruity Acceptance criteria: NMT 1.0
Solubility: Soluble in alcohol, most fixed oils; insoluble or • REFRACTIVE INDEX, Appendix II: At 20°
practically insoluble in propylene glycol, water Acceptance criteria: Between 1.407 and 1.411
Boiling Point: ∼175° • SPECIFIC GRAVITY: Determine at 25° by any reliable
Solubility in Alcohol, Appendix VI: One mL dissolves in 1 method (see General Provisions).
mL of 95% alcohol. Acceptance criteria: Between 0.856 and 0.859
Function: Flavoring agent
 176 / Butyl Isovalerate / Monographs FCC 9
Monographs

Butyl Isovalerate

IDENTIFICATION
Butyl Phenylacetate
.

• INFRARED SPECTRA, Spectrophotometric Identification Tests,

First Published: Prior to FCC 6 Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1a, Appendix
C12H16O2 Formula wt 196.26 XI.
FEMA: 2209 Acceptance criteria: NLT 98.0% of C12H16O2
UNII: TR6RZ109SI [butyl phenylacetate]
SPECIFIC TESTS
DESCRIPTION • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
Butyl Phenylacetate occurs as a colorless to pale yellow OILS), M-15, Appendix XI
liquid. Acceptance criteria: NMT 1.0
Odor: Honey, rose • REFRACTIVE INDEX, Appendix II: At 20°
Boiling Point: ∼260° Acceptance criteria: Between 1.488 and 1.492
Solubility in Alcohol, Appendix VI: One mL dissolving in • SPECIFIC GRAVITY: Determine at 25° by any reliable
1 mL of 95% alcohol. method (see General Provisions).
Function: Flavoring agent Acceptance criteria: Between 0.990 and 0.997
 FCC 9 Monographs / Butyl Stearate / 177

Monographs
Butyl Phenylacetate

Acceptance criteria: The spectrum of the sample

Butyl Stearate
.

exhibits maxima at the same wavelengths as those in

First Published: Prior to FCC 6 the spectrum of the Reference standard.▲ FCC9
Last Revision: FCC 9 Add the following:
• ▲ B. GC PEAK IDENTITY
Butyl Octadecanoate
Acceptance criteria: The retention time of the major
peaks of the Sample solution corresponds to that of
Standard solution A, as obtained in the Assay.▲ FCC9

ASSAY
C22H44O2 Formula wt 340.59
FEMA: 2214 Add the following:
UNII: 6Y0AI5605C [butyl stearate] • ▲ PROCEDURE
Standard solution A: 2.0 mg/mL USP Butyl Stearate RS
DESCRIPTION in chloroform
Butyl Stearate occurs as a colorless, waxy solid. Standard solution B: 0.2 mg/mL in USP Butyl Palmitate
Odor: Odorless to faintly fatty RS chloroform
Solubility: Soluble in alcohol, most fixed oils; insoluble or System suitability solution: 1.4 µg/mL USP Butyl
practically insoluble in propylene glycol, water Stearate RS and 1.0 µg/mL USP Butyl Palmitate RS in
Boiling Point: ∼223° chloroform, prepared from Standard solution A and
Solubility in Alcohol, Appendix VI: One mL dissolves in 6 Standard solution B
mL of 95% alcohol. Sample solution: 2.0 mg/mL in chloroform
Function: Flavoring agent Chromatographic system, Appendix IIA
Mode: Gas chromatography
IDENTIFICATION Detector: Flame ionization
Add the following: Column: 30-m × 0.32-mm (id) capillary column;
bonded with a 0.25-µm layer of 5% phenyl–95%
• ▲ A. INFRARED ABSORPTION, Spectrophotometric Identification methylpolysiloxane1
Tests, Appendix IIIC
Reference standard: USP Butyl Stearate RS
Sample and standard preparation: F 1 DB-5 from J & W Scientific, or equivalent.
 178 / Butyl Stearate / Monographs FCC 9

Temperatures • SAPONIFICATION VALUE, Appendix VII

Injection port: 280° Acceptance criteria: 65–180
Detector: 280°
Column: See Table 1.

Butylated Hydroxymethylphenol
.

Table 1
Hold Time at
First Published: Prior to FCC 6
Initial Temperature Final Final
Temperature Ramp Temperature Temperature
(°) (°/min) (°) (min)
Monographs

60 — 60 2
60 4 250 20.5

Carrier gas: Helium

Flow rate: 1.4 mL/min
Injection volume: 1 µL
Split ratio: 5:1 C15H24O2 Formula wt 236.35
System suitability UNII: 46ND6GQI48 [butylated hydroxymethylphenol]
Samples: Standard solution A and System suitability
solution
DESCRIPTION
Butylated Hydroxymethylphenol occurs as a nearly white,
[NOTE—The approximate relative retention times for
crystalline solid. It is freely soluble in alcohol, and insoluble
butyl palmitate and butyl stearate are 0.91 and 1.00,
in water and in propylene glycol.
respectively.]
Function: Antioxidant
Suitability requirement 1: The resolution, R,
Packaging and Storage: Store in well-closed containers.
between butyl palmitate and butyl stearate is NLT
20 for the System suitability solution. IDENTIFICATION
Suitability requirement 2: The relative standard • PROCEDURE
deviation, RSD, for the butyl stearate peak is NMT Analysis: Butylated Hydroxymethylphenol may be
3.0% for Standard solution A. identified by its solidification point, as determined in
Analysis: Inject Standard solution A, Standard solution B, the Assay (below).
and the Sample solution into the chromatograph. Acceptance criteria: Passes test
Identify and record the butyl palmitate and butyl
stearate peaks in the Sample solution based on those in ASSAY
Standard solution A and Standard solution B. Calculate • SOLIDIFICATION POINT, Appendix IIB
the percentage of butyl palmitate and butyl stearate in Acceptance criteria: The solidification point is NLT 140°
the sample taken: (indicating a purity of NLT 98.0%, by weight of
C15H24O2).
Result = (rU/rS) × (CS/CU) × 100

rU = peak response of butyl palmitate or butyl

stearate in the chromatogram of the
1,3-Butylene Glycol
.

Sample solution
rS = peak response of butyl palmitate in the First Published: Prior to FCC 6
chromatogram of Standard solution B or
peak response of butyl stearate in the Butane-1,3-diol
chromatogram of Standard solution A
CS = concentration of USP Butyl Palmitate RS in
Standard solution B (mg/mL) or
concentration of USP Butyl Stearate RS (mg
/mL) in Standard solution A C4H10O2 Formula wt 90.12
CU = concentration of the Sample solution (mg/ CAS: [107-88-0]
mL) UNII: 3XUS85K0RA [butylene glycol]
Acceptance criteria
Butyl stearate: NLT 90.0% DESCRIPTION
Butyl stearate and butyl palmitate: NLT 96.0%▲ FCC9 1,3-Butylene Glycol occurs as a clear, colorless, hygroscopic,
viscous liquid. It is miscible with water, with acetone, and
OTHER REQUIREMENTS with ether in all proportions, but is immiscible with fixed
• IODINE VALUE, Appendix VII oils. It dissolves most essential oils and synthetic flavoring
Acceptance criteria: NMT 1 substances.
• MELTING RANGE OR TEMPERATURE DETERMINATION, Appendix Function: Solvent for flavoring agents
IIB Packaging and Storage: Store in well-closed containers.
Acceptance criteria: 17°–21°
 FCC 9 Monographs / Butyric Acid / 179

ASSAY Reference standard: USP Butyraldehyde RS

• PROCEDURE Standard and sample preparation: F
Acetylating reagent: Prepare within one week of use, Acceptance criteria: The spectrum of the sample
by mixing 3.4 mL of water and 130 mL of acetic exhibits maxima at the same wavelengths as those in
anhydride with 1000 mL of anhydrous pyridine. the spectrum of the Reference standard.
Sample: 1 g
Analysis: Pipet 20 mL of Acetylating reagent into a 250- ASSAY
mL iodine flask and add the Sample. Attach a dry reflux • PROCEDURE: Proceed as directed under M-2c, Appendix XI.
condenser to the flask, and reflux for 1 h. Allow the Acceptance criteria: NLT 98.0% of C4H8O
flask to cool to room temperature, then rinse the SPECIFIC TESTS

Monographs
condenser with 50 mL of chilled (10°) carbon dioxide- • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
free water, allowing the water to drain into the flask. OILS), M-15, Appendix XI: Use methyl red TS as the
Stopper the flask, cool to below 20°, and add indicator.
phenolphthalein TS. Titrate with 0.5 N sodium Acceptance criteria: NMT 5.0
hydroxide, swirling the contents of the flask • REFRACTIVE INDEX, Appendix II: At 20°
continuously during the titration. Perform a blank Acceptance criteria: Between 1.381 and 1.387
determination (see General Provisions), and make any • SPECIFIC GRAVITY: Determine at 25° by any reliable
necessary correction. Each mL of 0.5 N sodium method (see General Provisions).
hydroxide is equivalent to 22.53 mg of C4H10O2. Acceptance criteria: Between 0.797 and 0.802
Acceptance criteria: NLT 99.0% of C4H10O2
OTHER REQUIREMENTS
IMPURITIES • DISTILLATION RANGE, Appendix IIB
Inorganic Impurities Acceptance criteria: Between 72° and 80° (first 95%)
• LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric • para-BUTYRALDEHYDE, M-1b, Appendix XI
Graphite Furnace Method, Method I, Appendix IIIB Acceptance criteria: NMT 2.5%
Acceptance criteria: NMT 2 mg/kg • WATER, Water Determination, Method I, Appendix IIB
SPECIFIC TESTS Acceptance criteria: NMT 0.5%
• DISTILLATION RANGE, Appendix IIB
Acceptance criteria: Between 200° and 215°
• SPECIFIC GRAVITY : Determine by any reliable method (see
Butyric Acid
.

General Provisions).
Acceptance criteria: Between 1.004 and 1.006 at 20° First Published: Prior to FCC 6

Butyraldehyde
.

First Published: Prior to FCC 6

Last Revision: Third Supplement, FCC 8 C4H8O2 Formula wt 88.11
FEMA: 2221
Butyl Aldehyde UNII: 40UIR9Q29H [butyric acid]

DESCRIPTION
Butyric Acid occurs as a colorless liquid.
Odor: Strong, rancid, buttery
Solubility: Miscible in alcohol, most fixed oils, propylene
C4H8O Formula wt 72.11
glycol, water
FEMA: 2219
Boiling Point: ∼164°
UNII: H21352682A [butyraldehyde]
Function: Flavoring agent
DESCRIPTION IDENTIFICATION
Butyraldehyde occurs as a colorless, mobile liquid. It may • INFRARED SPECTRA, Spectrophotometric Identification Tests,
contain a suitable antioxidant. Appendix IIIC
Odor: Pungent, nutty Acceptance criteria: The spectrum of the sample
Solubility: 1 mL dissolves in 15 mL of water; miscible in exhibits relative maxima at the same wavelengths as
alcohol, ether those of the spectrum below.
Boiling Point: ∼74.8°
Function: Flavoring agent ASSAY
• PROCEDURE: Proceed as directed under M-3a, Appendix
IDENTIFICATION XI.
• INFRARED ABSORPTION, Spectrophotometric Identification Acceptance criteria: NLT 99.0% of C4H8O2
Tests, Appendix IIIC
 180 / Butyric Acid / Monographs FCC 9

SPECIFIC TESTS Acceptance criteria: Between 0.953 and 0.957

• REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.397 and 1.399 OTHER REQUIREMENTS
• SPECIFIC GRAVITY: Determine at 25° by any reliable • REDUCING SUBSTANCES, M-14, Appendix XI
method (see General Provisions). Acceptance criteria: Passes test
Monographs

Butyric Acid

IDENTIFICATION
γ-Butyrolactone
.

• INFRARED SPECTRA, Spectrophotometric Identification Tests,

First Published: Prior to FCC 6 Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1a, Appendix
C4H6O2 Formula wt 86.09 XI.
FEMA: 3291 Acceptance criteria: NLT 98.0% of C4H6O2
UNII: OL659KIY4X [butyrolactone]
SPECIFIC TESTS
DESCRIPTION • REFRACTIVE INDEX, Appendix II: At 20°
γ-Butyrolactone occurs as a colorless to slightly yellow liquid. Acceptance criteria: Between 1.430 and 1.440
Odor: Faint, sweet, caramel • SPECIFIC GRAVITY: Determine at 25° by any reliable
Solubility: Soluble in water; miscible in alcohol method (see General Provisions).
Boiling Point: ∼204° Acceptance criteria: Between 1.120 and 1.130
Function: Flavoring agent
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FCC 9 Monographs / γ-Butyrolactone / 181

Monographs
γ-Butyrolactone