Method 1694: Pharmaceuticals and

Personal Care Products in Water,

Soil, Sediment, and Biosolids by
HPLC/MS/MS

December 2007
December 2007 Method 1694

U.S. Environmental Protection Agency

Office of Water
Office of Science and Technology
Engineering and Analysis Division (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460

EPA-821-R-08-002
December 2007

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December 2007 Method 1694

Introduction
EPA Method 1694 determines pharmaceuticals and personal care products (PPCPs) in environmental
samples by high performance liquid chromatography combined with tandem mass spectrometry
(HPLC/MS/MS) using isotope dilution and internal standard quantitation techniques. This method has
been developed for use with aqueous, solid, and biosolids matrices.

Disclaimer
This method has been reviewed by the Engineering and Analytical Support Branch of the Engineering
and Analysis Division (EAD) in OST. The method is available for general use, but has not been
published in 40 CFR Part 136. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.

Contacts

Questions concerning this method or its application should be addressed to:

Brian Englert, Ph.D.

Environmental Scientist
Engineering & Analytical Support Branch
Engineering and Analysis Division (4303T)
Office of Science and Technology, Office of Water
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue NW
Washington, DC 20460
http://www.epa.gov/waterscience
ostcwamethods@epa.gov

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December 2007 Method 1694

Table of Contents
INTRODUCTION .................................................................................................................................................... III

DISCLAIMER .......................................................................................................................................................... III

1.0 SCOPE AND APPLICATION .....................................................................................................................1

2.0 SUMMARY OF METHOD ...........................................................................................................................2

3.0 DEFINITIONS AND UNITS OF MEASURE .............................................................................................3

4.0 INTERFERENCES .......................................................................................................................................3

5.0 SAFETY .........................................................................................................................................................5

6.0 EQUIPMENT AND SUPPLIES...................................................................................................................6

7.0 REAGENTS AND STANDARDS ..............................................................................................................10

8.0 SAMPLE COLLECTION, PRESERVATION, STORAGE, AND HOLDING TIMES ...........................14

9.0 QUALITY ASSURANCE/QUALITY CONTROL .....................................................................................15

10.0 CALIBRATION AND STANDARDIZATION............................................................................................19

11.0 SAMPLE PREPARATION .........................................................................................................................23

12.0 EXTRACTION AND CONCENTRATION ................................................................................................28

13.0 EXTRACT CLEANUP ................................................................................................................................32

14.0 LC/MS/MS ANALYSIS ..............................................................................................................................32

15.0 SYSTEM AND LABORATORY PERFORMANCE ...............................................................................33

16.0 QUALITATIVE DETERMINATION ..........................................................................................................34

17.0 QUANTITATIVE DETERMINATION .......................................................................................................35

18.0 ANALYSIS OF COMPLEX SAMPLES ...................................................................................................37

19.0 POLLUTION PREVENTION .....................................................................................................................38

20.0 WASTE MANAGEMENT ..........................................................................................................................39

21.0 METHOD PERFORMANCE .....................................................................................................................39

22.0 REFERENCES............................................................................................................................................39

23.0 TABLES AND FLOWCHART ...................................................................................................................42

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24.0 GLOSSARY ................................................................................................................................................69

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December 2007 Method 1694

Method 1694
Pharmaceuticals and Personal Care Products in Water, Soil,
Sediment, and Biosolids by HPLC/MS/MS

1.0 Scope and Application

1.1 Method 1694 is for determination of pharmaceuticals and personal care products (PPCPs)
in multi-media environmental samples by high performance liquid chromatography
combined with tandem mass spectrometry (HPLC/MS/MS).

1.2 This method was developed for use in Clean Water Act (CWA) programs; other
applications are possible. It is based on existing EPA methods (Reference 1) and
procedures developed at Axys Analytical Services (Reference 2) as well as previous work
on pharmaceuticals and personal care products (Reference 3).

1.3 The target analytes and their corresponding Chemical Abstracts Service Registry
Numbers (CASRNs) are listed in Table 1.

1.4 The detection limits and quantitation levels in this method are usually dependent on the
level of interferences rather than instrumental limitations. The method detection limits
(MDLs; 40 CFR 136, appendix B) and minimum levels of quantitation (MLs; 68 FR
11790) in Tables 3, 5, 7, and 9 are the levels at which the analytes can be determined in
the absence of interferences.

1.5 This method is restricted to use by or under the supervision of analysts experienced in
LC/MS/MS or under the close supervision of such qualified persons. Each laboratory
that uses this method must demonstrate the ability to generate acceptable results using the
procedure in Section 9.2.

1.6 This method is performance-based which means that you may modify the method to
improve performance (e.g., to overcome interferences or improve the accuracy or
precision of the results) provided that you meet all performance requirements in this
method. These requirements for establishing equivalency of a modification are in
Section 9.1.2. For Clean Water Act (CWA) uses, additional flexibility is described at 40
CFR 136.6. Modifications that are not within the scope of Part 136.6, or in Section 9 of
this method may require prior review and approval.

1.7 Some of the compounds in this method are controlled substances. Laboratories
performing this method should have all appropriate licenses and certifications and obtain
all needed standards and chemicals from licensed sources. For some of the compounds in
this method it may be necessary for laboratories to obtain a DEA license.

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December 2007 Method 1694

2.0 Summary of Method

The target analytes in this method are divided into four groups (1 through 4). Each group represents
an LC/MS/MS run, as detailed in Tables 2 to 9 in Section 23. Tables 2 and 3 are specific to Group
1. Tables 4 and 5 are specific to Group 2. Tables 6 and 7 are specific to Group 3. Tables 8 and 9
are specific to Group 4.

Groups 1, 2, and 3 are extracted under acidic (pH 2) conditions. Groups 1 and 2 are run in the
positive electrospray ionization (ESI+) mode and Group 3 is run in the negative electrospray
ionization (ESI-) mode. Group 4 is extracted under basic (pH 10) conditions and is run in the ESI+
mode. Group 3 is specific to the tetracyclines.

The general steps in this method are summarized in Section 2.1 to 2.7. A flow chart that
summarizes procedures for sample preparation, cleanup, and analysis is shown in Figure 1.

2.1 Aqueous samples absent visible particles and filtrate from samples with visible particles –
The pH of a 1-L sample aliquot is adjusted to 2 with acid. The pH of a second 1-L
aliquot of sample is adjusted with 10 with base. Stable, isotopically labeled analogs of
the analytes of interest are spiked into their respective acid or base fraction. The acid
fraction is stabilized with tetrasodium ethylenediamine-tetraacetate dihydrate
(NA4EDTA.2H2O•2H2O).

2.2 Solid and semi-solid samples, including biosolids and visible particles from aqueous
samples – A phosphate buffer and an ammonium hydroxide solution are used to adjust
the pH, respectively, of up to 1 g each of dry solids from a solid sample, or 1 g each of
dry solids filtered from an aqueous sample. The labeled compounds are spiked into their
respective acid and base fractions. The acid fraction is ultrasonically extracted three
times with a phosphate buffer/acetonitrile solution and the base fraction is ultrasonically
extracted three times with a ammonium hydroxide/acetonitrile solution. The solutions
are concentrated to remove the acetonitrile and diluted with reagent water. The acid
fraction is stabilized with NA4EDTA.2H2O•2H2O.

2.3 Sample cleanup – The acid and base fraction solutions are separately cleaned up using
solid-phase extraction (SPE) with hydrophilic-lipophillic balance (HLB) cartridges.
After cleanup, the fractions are exchanged to methanol, labeled injection internal
standards are added, and the final volume is adjusted to 4 mL with the LC elution solvent.

2.4 Determination by LC/MS/MS – The acid extract is analyzed in two positive electrospray
ionization (ESI+) LC/MS/MS runs and one negative electrospray ionization (ESI-) run,
each specific to a subset of the analytes of interest. The base extract is analyzed in a
single ESI+ run. The analytes are separated by the LC and detected by a tandem (1000
resolution) mass spectrometer. A daughter m/z for each compound is monitored
throughout a pre-determined retention time window.

2.5 An individual compound is identified by comparing the LC retention time and presence
of the daughter m/z with the corresponding retention time and daughter m/z of an
authentic standard.

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December 2007 Method 1694

2.6 Quantitative analysis is performed in one of two ways, using selected ion current profile
(SICP) areas:

2.6.1 For a compound for which a labeled analog is available, the concentration is
determined using the isotope dilution technique and a multipoint calibration of all
the target analytes. Isotope dilution provides automatic correction of the target
analyte concentrations.

2.6.2 For a compound for which a labeled analog is not available, the concentration is
determined using the internal standard technique and a multipoint calibration of
all the target analytes. The labeled compounds are used to recovery correct
results of those analytes quantitated by the internal standard technique.

2.6.3 Additional labeled compounds may be incorporated into this method, at the
user’s discretion to determine the concentration of the native compound using the
isotope dilution technique provided that all performance requirements in this
method are met. Requirements for establishing equivalency are given in Section
9.1.2, and additionally for CWA uses, at 40 CFR 136.6.

2.7 The quality of the analysis is assured through reproducible calibration and testing of the
extraction, cleanup, and LC/MS/MS systems.

3.0 Definitions and Units of Measure

Definitions and units of measure are given in the glossary at the end of this method.

4.0 Interferences
4.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts,
elevated baselines, matrix enhancement or matrix suppression causing misinterpretation
of chromatograms. Specific selection of reagents and purification of solvents by
distillation in all-glass systems may be required. Where possible, reagents are cleaned by
extraction or solvent rinse.

4.2 Proper cleaning of glassware is extremely important, because glassware may not only
contaminate the samples but may also remove the analytes of interest by adsorption on
the glass surface.

4.2.1 Glassware should be rinsed with solvent and washed with a detergent solution as
soon after use as is practical. Sonication of glassware containing a detergent
solution for approximately 30 seconds may aid in cleaning. Glassware with
removable parts, particularly separatory funnels with fluoropolymer stopcocks,
must be disassembled prior to detergent washing.

4.2.2 After detergent washing, glassware should be rinsed immediately, first with
methanol, then with hot tap water. The tap water rinse is followed by another

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December 2007 Method 1694

methanol rinse, then acetone, and then methylene chloride.

4.2.3 Baking of glassware in a kiln or other high temperature furnace (300 – 500 ΕC)
may be useful after particularly dirty samples are encountered. The kiln or
furnace should be vented to prevent laboratory contamination by vapors. Baking
should be minimized, as repeated baking of glassware may cause active sites on
the glass surface that may irreversibly adsorb the compounds of interest.
Volumetric ware should not be baked at high temperature.

4.2.4 After drying and cooling, glassware should be sealed and stored in a clean
environment to prevent any accumulation of dust or other contaminants. Store
inverted or capped with solvent rinsed aluminum foil.

4.3 All materials used in the analysis must be demonstrated to be free from interferences by
running reference matrix method blanks (Section 9.5) initially and with each sample
batch (samples started through the extraction process on a given 12-hour shift, to a
maximum of 20 samples).

4.3.1 The reference matrix must simulate, as closely as possible, the sample matrix
under test. Ideally, the reference matrix should not contain the analytes of
interest in detectable amounts, but should contain potential interferents in the
concentrations expected to be found in the samples to be analyzed.

4.3.2 When a reference matrix that simulates the sample matrix under test is not
available, reagent water (Section 7.6.1) can be used to simulate water samples;
playground sand (Section 7.6.2) can be used to simulate soils; and peat moss
(Section 7.6.3) can be used to simulate biosolids.

4.4 Interferences co-extracted from samples will vary considerably from source to source,
depending on the diversity of the site being sampled. Interfering compounds may be
present at concentrations several orders of magnitude higher than the analytes of interest.
Because low levels of PPCPs are measured by this method, elimination of interferences is
essential. The cleanup steps given in Section 13 can be used to reduce or eliminate these
interferences and thereby permit reliable determination of the PPCPs at the levels shown
in Tables 3, 5, 7, and 9.

4.5 It may be useful to number reusable glassware is to associate that glassware with the
processing of a particular sample. This will assist the laboratory in tracking possible
sources of contamination for individual samples, identifying glassware associated with
highly contaminated samples that may require extra cleaning, and determining when
glassware should be discarded.

4.6 Contamination from personal care products used by laboratory staff that are also target
analytes is possible. Target analytes also include commonly used medications.
Therefore, it is important to take precautions to avoid contamination of the samples, for
example wearing of protective gloves and clothing (see Section 5).

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December 2007 Method 1694

5.0 Safety
The target analytes in this method have many beneficial uses as pharmaceuticals or over-the­
counter products. While their safety is less of a concern than for many environmental
contaminants, laboratory staff should avoid direct contact with samples and pure standards.
General guidelines are provided below.

5.1 The toxicity or carcinogenicity of each chemical used in this method has not been
precisely determined; however, each compound should be treated as a potential health
hazard. Pure standards of the compounds should be handled only by highly trained
personnel thoroughly familiar with handling and cautionary procedures and the
associated risks. It is recommended that the laboratory purchase dilute standard solutions
of the analytes in this method. However, if primary solutions are prepared, they should
be prepared in a hood, and a NIOSH/MESA approved toxic gas respirator may be
necessary when high concentrations are handled

5.2 This method does not address all safety issues associated with its use. The laboratory is
responsible for maintaining a current awareness file of OSHA regulations regarding the
safe handling of the chemicals specified in this method. A reference file of material
safety data sheets (MSDSs) should also be made available to all personnel involved in
these analyses. It is also suggested that the laboratory perform personal hygiene
monitoring of each analyst who uses this method and that the results of this monitoring
be made available to the analyst. Additional information on laboratory safety can be
found in References 4 – 7. The references and bibliography at the end of Reference 6 are
particularly comprehensive in dealing with the general subject of laboratory safety.

5.3 The pure PPCPs and samples suspected to contain high concentrations of these
compounds should be handled with care.

5.3.1 Facility – When finely divided samples (dusts, soils, dry chemicals) are handled,
all operations (including removal of samples from sample containers, weighing,
transferring, and mixing) should be performed in a glove box demonstrated to be
leak tight or in a fume hood demonstrated to have adequate air flow. Gross
losses to the laboratory ventilation system must not be allowed. Handling of the
dilute solutions normally used in analytical and animal work presents no
inhalation hazards except in the case of an accident.

5.3.2 Protective equipment – Disposable plastic gloves (Latex or non-Latex (such as

nitrile)), apron or lab coat, safety glasses or mask, and a glove box or fume hood
should be used. During analytical operations that may give rise to aerosols or
dusts, personnel should wear respirators equipped with activated carbon filters.
Eye protection (preferably full face shields) should be worn while working with
exposed samples or pure analytical standards. Latex or non-Latex (such as
nitrile) gloves are commonly used to reduce exposure of the hands.

5.3.3 Training – Workers must be trained in the proper method of removing

contaminated gloves and clothing without contacting the exterior surfaces.

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December 2007 Method 1694

5.3.4 Personal hygiene – Hands and forearms should be washed thoroughly after each
operation involving high concentrations of the analytes of interest, and before
breaks (coffee, lunch, and shift).

5.3.5 Confinement – Isolated work areas posted with signs, segregated glassware and
tools, and plastic absorbent paper on bench tops will aid in confining
contamination.

5.3.6 Waste handling – Good technique includes minimizing contaminated waste.

Plastic bag liners should be used in waste cans. Janitors and other personnel
should be trained in the safe handling of waste. See Section 20 for additional
information on waste handling and disposal.

5.4 Biosolids samples may contain high concentrations of biohazards, and must
be handled with gloves and opened in a hood or biological safety cabinet to
prevent exposure. Laboratory staff should know and observe the safety
procedures required in a microbiology laboratory that handles pathogenic
organisms when handling biosolids samples.

6.0 Equipment and Supplies

Note: Brand names, suppliers, and part numbers are cited for illustration purposes only. No
endorsement is implied. Equivalent performance may be achieved using equipment and materials
other than those specified here. Demonstration of equivalent performance that meets the
requirements of this method is the responsibility of the laboratory.

6.1 Sample bottles and caps

6.1.1 Liquid samples (waters, sludges and similar materials containing 5 percent
solids or less) – Sample bottle, amber glass, 1 L minimum, with screw cap.

6.1.2 Solid samples (soil, sediment, sludge, filter cake, compost, and similar
materials that contain more than 5 percent solids) – Sample bottle, wide mouth,
amber glass, 500-mL minimum.

6.1.3 If amber bottles are not available, samples must be protected from light.

6.1.4 Bottle caps – Threaded to fit sample bottles. Caps must be lined with
fluoropolymer.

6.1.5 Cleaning – Bottles are washed with detergent and water, then solvent rinsed
before use. Liners are washed with detergent and water and rinsed with reagent
water before use.

6.2 Compositing equipment – Automatic or manual compositing system incorporating glass

containers cleaned per bottle cleaning procedure above. Only glass or fluoropolymer
tubing must be used. If the sampler uses a peristaltic pump, a minimum length of
compressible silicone rubber tubing may be used in the pump only. Before use, the tubing

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December 2007 Method 1694

must be thoroughly rinsed with methanol, followed by repeated rinsing with reagent water
to minimize sample contamination. An integrating flow meter is used to collect
proportional composite samples.

6.3 Equipment for sample preparation

6.3.1 Laboratory fume hood of sufficient size to contain the sample preparation
equipment listed below.

6.3.2 Glove box (optional)

6.3.3 Tissue homogenizer – VirTis Model 45 Macro homogenizer (American Scientific

Products H-3515, or equivalent) with stainless steel Macro-shaft and Turbo-shear
blade.

6.3.4 Vortex mixer

6.3.5 Ultrasonic mixer

6.3.6 Oven – Capable of maintaining a temperature of 110∀5 °C

6.3.7 Desiccator

6.3.8 Balance, analytical – Capable of weighing 0.1 mg

6.3.9 Balance, top loading – Capable of weighing 10 mg

6.4 Apparatus for measuring pH

6.4.1 pH meter, with combination glass electrode

6.4.2 pH paper, wide range (Hydrion Papers, or equivalent)

6.5 Apparatus for ultrasonic and solid-phase extraction

6.5.1 Sonic disrupter – 375 watt with pulsing capability and ½ or ¾ in. disrupter horn
(Ultrasonics, Inc., Model 375, or equivalent)

6.5.2 Sonabox (or equivalent), for use with disrupter.

6.5.3 Vac-Elute Manifold (Analytichem International, or equivalent)

6.5.4 Vacuum trap: Made from 500-mL sidearm flask fitted with single-hole rubber
stopper and glass tubing.

6.5.5 Vacuum source – Capable of maintaining 25 in. Hg, equipped with shutoff valve
and vacuum gauge.

6.5.6 Rack for holding 50-mL volumetric flasks in the manifold.

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December 2007 Method 1694

6.5.7 SPE cartridge – Hydrophilic-Lipophilic-Balance (HLB) 60 mg, Waters Oasis, 20

cc/1 g LP, 60 μm, or equivalent, calibrated per the procedure in Section 10.6.

6.6 Filtration apparatus

6.6.1 Vacuum filtration apparatus – 1-L , including glass funnel, frit support, clamp,
adapter, stopper, filtration flask, and vacuum tubing. For wastewater samples,
the apparatus should accept 90- or 144-mm disks.

6.6.2 Glass-fiber filter – Whatman GMF 150 (or equivalent), 1 micron pore size, to fit
the vacuum filtration apparatus.

6.6.3 Pressure filtration apparatus – Millipore YT30 142 HW, or equivalent.

6.6.4 Whatman GF/A (1.6 μm), or equivalent, differing diameters, to fit the pressure
filtration apparatus.

6.6.5 Millipore, 0.2 μm, or equivalent to fit the pressure filtration apparatus.

6.7 Centrifuge – Capable of rotating 500-mL centrifuge bottles or 50-mL centrifuge tubes at
5,000 rpm minimum, equipped with 500-mL centrifuge bottles (glass or polypropylene
bottles) with screw-caps, and 50-mL centrifuge tubes with screw-caps, to fit centrifuge.

6.8 Pipet apparatus and pipets

6.8.1 Pipetter – variable volume

6.8.2 Pipet tips, disposable polypropylene, sizes from 1-10 μL to 5 mL

6.8.3 Disposable, Pasteur, 150-mm long x 5-mm ID (Fisher Scientific 13-678-6A, or

equivalent)

6.8.4 Disposable, serological, 50-mL (8- to 10- mm ID)

6.9 Rotary evaporator – Buchi/Brinkman-American Scientific No. E5045-10, or equivalent,

equipped with a variable temperature water bath and a vacuum source with shutoff valve at
the evaporator and vacuum gauge. A recirculating water pump and chiller are
recommended, as use of tap water for cooling the evaporator wastes large volumes of water
and can lead to inconsistent performance as water temperatures and pressures vary.

6.9.1 Round-bottom flask – 100-mL and 500-mL or larger, with ground-glass fitting
compatible with the rotary evaporator

6.9.2 Boiling chips

6.9.2.1 Glass or silicon carbide – Approximately 10/40 mesh, extracted with

methylene chloride and baked at 450 ΕC for one hour minimum

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December 2007 Method 1694

6.9.2.2 Fluoropolymer (optional) – Extracted with methylene chloride

6.10 Water bath – Heated, with concentric ring cover, capable of maintaining a temperature
within ∀ 2 ΕC, installed in a fume hood.

6.11 Nitrogen evaporation apparatus – Equipped with water bath controlled in the range of 30 –
60 ΕC (N-Evap, Organomation Associates, Inc., South Berlin, MA, or equivalent), installed
in a fume hood.

6.12 Amber glass vials, 2- to 5-mL with fluoropolymer-lined screw-cap

6.13 Clear glass vials, 0.3-mL, conical, with fluoropolymer-lined screw or crimp cap

6.14 HPLC/MS/MS System

6.14.1 HPLC system with high pressure inlet, multi-segment gradient capability, and
post-column pump for admission of calibrant. The system must be able to
produce the LC separations for the analytical runs detailed in Tables 3, 5, 7, and
9 under the instrument conditions detailed in Tables 2, 4, 6, and 8, and must meet
other HPLC requirements in this method (Waters 2690, 2795, or equivalent).

6.14.2 LC columns

6.14.2.1 C18 – 10.0 cm, 2.1 mm i.d., 3.5 :m particle size (Waters Xtera C18MS,
or equivalent)

6.14.2.2 Hydrophilic – 10 cm. 2.1 mm i.d., 3.0 μm particle size (Waters Atlantis
HILIC, or equivalent)

6.14.2.3 Alternative columns other than described above have not been tested and
are not allowed for this method. EPA may establish criteria for
equivalency in later versions of this method.

6.14.3 MS/MS system

6.14.3.1 Tandem MS with the necessary pumps, collision cell, makeup gases,
high vacuum system, and capability for positive and negative ion
electrospray ionization (ESI) of the effluent from the HPLC. (Waters
Quattro Ultima triple quadrupole MS, or equivalent). The system must
be able to produce parent-daughter transitions for the groups of
compounds in the acid and base fractions of the PPCPs for the
analytical runs detailed in Tables 3, 5, 7, and 9.

6.14.3.2 Instrument control and data system – Interfaced to the HPLC and
MS/MS to control the LC gradient and other LC and MS/MS operating
conditions, and to acquire, store, and reduce LC/MS/MS data. The data
system must be able to identify a compound by retention time and
parent-daughter m/zs, and quantify the compound using linear or

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December 2007 Method 1694

quadratic multi-point relative responses and response factors by isotope

dilution and internal standard techniques.

6.15 Miscellaneous labware – Beakers, 400- to 500-mL; Erlenmeyer flasks; volumetric flasks;
pipets; syringes; stainless steel spatulas; etc.

7.0 Reagents and Standards

Note: All reagents are ACS Reagent Grade unless specified otherwise.

7.1 pH adjustment and solution stabilization

7.1.1 Potassium hydroxide – Dissolve 20 g reagent grade KOH in 100 mL reagent water.

7.1.2 Sulfuric acid – Reagent grade (specific gravity 1.84)

7.1.3 Hydrochloric acid – Reagent grade, 6N

7.1.4 Phosphoric acid (H3PO4) – Reagent grade (85%), Fisher, or equivalent

7.1.5 Sodium chloride – Reagent grade, prepare at 5% (w/v) solution in reagent water

7.1.6 Ammonium hydroxide (NH4OH) – Reagent grade, Anachemia, or equivalent

7.1.7 Sodium dihydrogen phosphate monohydrate – Reagent grade, J.T. Baker, or

equivalent

7.1.8 Oxalic acid, anhydrous

7.2 Prepurified nitrogen

7.3 Solvents, reagents, and solutions

7.3.1 Acetic acid, acetone, acetonitrile ammonium acetate, formic acid, methanol,
methylene chloride, HPLC water, ammonium formate.

7.3.2 Solvents and purchased solutions should be lot-certified to be free of interferences.

If necessary, solvents should be analyzed by this method to demonstrate that they
are interference free.

7.4 Buffer and elution solutions

7.4.1 Phosphate buffer (sodium phosphate monohydrate/phosphoric acid) – 0.14 M

NaH2PO4.H2O /85% H3PO4 (1.93 g NaH2PO4.H2O in 99 mL of reagent water + 1
mL of 85% H3PO4)

7.4.2 Tetrasodium ethylenediamine tetraacetate hydrate (Na4EDTA•2H2O ~+99.5%

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December 2007 Method 1694

titration), Sigma, used as received

7.4.3 Formic acid solutions – Alfa Aesar, >99 percent purity

7.4.3.1 2% v/v in methanol

7.4.3.2 0.1% v/v in methanol

7.4.3.3 Formic acid/ammonium formate (0.1%) in water – dissolve 4 mL of

formic acid and 4 g of ammonium formate in 4.0 L of HPLC water.
Mix thoroughly and sonicate for 5 min.

7.4.3.4 Formic acid (0.1%) in methanol:water (75:25) – add 4 mL of formic acid

to 3.0 L methanol premixed with 1.0 L HPLC-grade water. Mix
thoroughly and sonicate for 5 min.

7.4.4 Acetonitrile:methanol (1:1) – mix 500 mL methanol and 500 mL of acetonitrile.

Sonicate for 5 min.

7.4.5 Oxalic acid solution (5 mM) – dissolve 0.45 g anhydrous oxalic acid in 1.0 L of
HPLC water. Mix thoroughly and sonicate for 5 min.

7.4.6 Oxalic acid/acetonitrile/methanol (5 mM) – dissolve 0.45 g anhydrous oxalic acid

in 500 mL acetonitrile premixed with 500 mL methanol. Mix thoroughly and
sonicate for 5 min.

7.4.7 Acetonitrile/water (90%) – Add 400 mL HPLC-grade water to 3600 mL of

acetonitrile. Mix thoroughly and sonicate for 5 min.

7.4.8 Ammonium acetate/acetic acid, 1 mM (0.1%) in water – Add 4 g NH4OAC and 4

mL acetic acid to 4.0 L of HPLC-grade water. Mix thoroughly and sonicate for 5
min.

7.5 Sodium iodide/cesium iodide mass calibration solution – 2 mg/mL NaI and 50 μg/mL CsI
in (1:1) isopropyl alcohol:water (Waters 700000889, or equivalent) or other based on
manufacture’s specifications.

7.6 Reference matrices – Matrices in which the PPCPs and interfering compounds are not
detected by this method

7.6.1 Reagent water – Bottled water purchased locally, or prepared by passage through
activated carbon

7.6.2 High-solids reference matrix – Playground sand or similar material.

7.6.2.1 Playground sand is used to simulate the base fraction of solids in this
method, including biosolids (see Section 7.6.3.1 for simulation of the
biosolids acid fraction) – Place 1 g of sand in a 50-mL centrifuge tube.
Add 15 mL of reagent water and adjust the pH to 10 ± 0.5 with NH4OH.

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Add 20 mL of acetonitrile and sonicate for 20 minutes. Discard the

aqueous phase.

7.6.2.2 Extract with a second 20-mL portion of acetonitrile. Decant and discard
the acetonitrile. The sand is now ready for spiking (Section 11.5.4).

7.6.3 Biosolids (sludge) reference matrix – Dry peat moss, purchase from local garden
center. Note: Store peat moss in closed container to prevent further drying. Sand
may be used for the acid fraction if QC acceptance criteria (Section 9) are met.

7.6.3.1 Peat moss is used to simulate the acid fraction of biosolids in this
method (see Section 7.6.2.1 for information on the biosolids base
fraction) – Place 1 g of peat moss in a 50-mL centrifuge tube. Add 15
mL of phosphate buffer (Section 7.4.1) and vortex to mix. Extract with
20 mL of acetonitrile and discard the aqueous phase.

7.6.3.2 Extract with a second 20-mL portion of acetonitrile. Decant and discard
the acetonitrile. The peat moss is now ready for spiking (Section
11.4.3).

7.6.4 Other matrices – Other reference matrices of interest may be used if the results
from the tests given in Section 9.2 demonstrate acceptable performance. Ideally,
the matrix should be free of the analytes of interest, but in no case must the
background level of the analytes in the reference matrix exceed the minimum
levels in Tables 3, 5, 7, and 9. If low background levels of the analytes of interest
are present in the reference matrix, the spike level of the analytes used in Section
9.2 should be increased to provide a spike-to-background ratio of approximately 5
(Reference 8).

7.7 Standard solutions – Prepare from materials of known purity and composition or purchase
as solutions or mixtures with certification to their purity, concentration, and authenticity. If
the chemical purity is 98 % or greater, the weight may be used without correction to
calculate the concentration of the standard. Observe the safety precautions in Section 5.

7.7.1 Preparation and storage of solutions - For preparation of stock solutions from neat
materials, dissolve an appropriate amount of assayed reference material in solvent.
For example, weigh 10 to 20 mg of Ampicillin to three significant figures in a 10­
mL ground-glass-stoppered volumetric flask and fill to the mark with methanol.
After the compound is completely dissolved, transfer the solution to a clean 15-mL
vial with fluoropolymer-lined cap. When not being used, store standard solutions in
the dark at less than -10 ºC in screw-capped vials with fluoropolymer-lined caps or
under a non-reactive gas (e.g., nitrogen) in a flame-sealed glass ampul. Place a
mark on the vial or ampul at the level of the solution so that solvent loss by
evaporation can be detected. Replace the solution if solvent loss has occurred.

7.7.2 Native (unlabeled; authentic) compound spiking solution – Separately prepare

Group 1 to Group 4 native compounds at the concentrations shown in column 3 of
Table 10 in methanol, or purchase prepared solutions. If additional native
compounds are to be determined, include these compounds in this stock solution.

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December 2007 Method 1694

Stock solutions should be prepared at a frequency necessary to preclude

degradation from affecting the analysis. For example, it may be necessary to
prepare the tetracycline compounds weekly if concentrations drop more than 30 %
of their original concentration. Stock solutions should also be checked for signs of
degradation prior to preparation of calibration or performance test standards.

7.7.3 Labeled compound spiking solution – Prepare Group 1 to Group 4 labeled

compounds at the concentrations shown in column 3 of Table 10 in methanol, or
purchase prepared solutions. If additional labeled compounds are to be used,
include these compounds in this solution. Note: The Group 2, acid extracted
positive ESI (tetracyclines) contains the same labeled compounds as for Group 1
and 3, acid extracted positive and negative ESI, yet the only labeled compounds
used in determination of the Group 2 are Thiabendazole-d6 and 13C3-Atrazine.
This minimizes the work required to prepare solutions. Some of those surrogates
are used to quantify the Group 1 and 2 and some Group 3 in separate runs of the
same extract. This is not a requirement.

7.7.4 Labeled injection internal standard spiking solutions – For the labeled injection
internal standards for Groups 1 and 2, prepare 13C-Atrazine in methanol at the
concentration shown column 3 of Table 10. For the labeled injection internal
standard for Group 3, prepare 13C6-2,4,5-Trichlorophenoxyacetic acid (TCPAA) in
methanol at the concentration shown in column 3 of Table 10. For the labeled
injection internal standards for Group 4, prepare 13C3-Atrazine and Continine-d3 in
methanol at the concentrations shown in column 3 of Table 10. If additional
labeled injection internal standards are to be used, include these compounds in
these solutions.

7.7.5 Calibration standards – Combine and dilute the solutions in Sections 7.7.1 and
7.7.2 to produce the calibration solutions in Table 11 or purchase prepared
standards for the CS-1 to CS-5 set of calibration solutions. These solutions permit
the relative response (labeled to native) and response factor to be determined as a
function of concentration. The CS-3 standard is used for calibration verification
(VER).

7.8 QC Check Sample – A QC Check Sample should be obtained from a source independent of
the calibration standards. Ideally, this check sample would be a Standard Reference
Material (SRM) from the National Institute of Standards and Technology (NIST)
containing the compounds of interest in known concentrations in a sample matrix similar to
the matrix of interest. If no SRM is available, a certified reference material (CRM) may be
used or a QC check sample may be prepared from materials from a source or lot of
standards separate from those used for calibration and spiked into a clean reference matrix.

7.9 Stability of solutions – standard solutions used for quantitative purposes (Sections 7.7.2 -
7.7.5) should be assayed periodically (e.g., every 6 months) against SRMs from NIST (if
available), or against certified reference materials from a source that will attest to the
authenticity and concentration, to assure that the composition and concentrations have not
changed.

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December 2007 Method 1694

8.0 Sample Collection, Preservation, Storage, and Holding Times

8.1 Collect samples in amber glass containers following conventional sampling practices
(Reference 9).

8.2 Aqueous samples

8.2.1 Samples that flow freely are collected as grab samples or in refrigerated bottles
using automatic sampling equipment. Collect 1-L each for the acid and base
fractions (2 L total). If high concentrations of the analytes of interest are expected,
collect two smaller volumes (e.g., 100 mL each) in addition to the 1-L samples.
Do not rinse the bottle with sample before collection.

8.2.2 If residual chlorine is present, add 80 mg sodium thiosulfate per liter of water. Any
method suitable for field use may be employed to test for residual chlorine.
Ascorbic acid has also been used by a number of other groups as a preservative for
a number of pharmaceuticals however it has not been tested for all of the
pharmaceuticals covered under this method (Reference 10).

8.2.3 Maintain aqueous samples in the dark at <6 ΕC from the time of collection until
receipt at the laboratory (see 40 CFR 136.6(e), Table II). If the sample will be
frozen, allow room for expansion.

8.3 Solid, mixed-phase, and semi-solid samples, including biosolids

8.3.1 Collect samples as grab samples using wide-mouth jars. Collect a sufficient
amount of wet material to produce a minimum of 10 g of solids.

8.3.2 Maintain solid, semi-solid, and mixed-phase samples in the dark at <6 ΕC from the
time of collection until receipt at the laboratory. Store solid, semi-solid, and
mixed-phase samples in the dark at less than -10 ΕC.

8.4 Store sample extracts in the dark at less than -10 ΕC until analyzed. Analyze extracts within
40 days of extraction.

8.5 Holding times

EPA has not conducted formal holding time studies for these analytes to date. Use
the information below as guidance. Exceeding these default holding times does not
invalidate the sample results.

8.5.1 Aqueous samples – Anecdotal evidence suggests that some may degrade rapidly in
aqueous samples. Therefore, begin sample extraction within 7 days of collection
(within 48 hours is strongly encouraged). Extracts should be analyzed within 40
days of extraction. Freezing of aqueous samples is encouraged to minimize
degradation, in which case, samples should be extracted within 48 hours of
removal from the freezer.

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December 2007 Method 1694

8.5.2 Biosolid, solid, mixed-phase, and semi-solid samples – Anecdotal evidence

suggests that some may degrade rapidly in these samples. Therefore, begin sample
extraction within 7 days of collection (within 48 hours is strongly encouraged).
Extracts should be analyzed within 40 days of extraction. Freezing of biosolids,
mixed phase and semisolid samples is encouraged to minimize degradation, in
which case, samples should be extracted within 48 hours of removal from the
freezer..

8.5.3 If extraction within 48 hours is not practical, samples should be frozen to increase
the holding time to seven days.

8.5.4 If the sample will not be extracted within 48 hours of collection, the laboratory
should adjust the pH of aqueous samples to 5.0 to 9.0 with a sodium hydroxide or
sulfuric acid solution. Record the volume of acid or base used. If aqueous samples
are stored frozen, extraction should begin within 48 hours of removal from the
freezer.

9.0 Quality Assurance/Quality Control

9.1 Each laboratory that uses this method is required to operate a formal quality assurance
program (Reference 11). The minimum requirements of this program consist of an initial
demonstration of laboratory capability, analysis of samples spiked with labeled compounds
to evaluate and document data quality, and analysis of standards and blanks as tests of
continued performance. Laboratory performance is compared to established performance
criteria to determine if the results of analyses meet the performance characteristics of the
method.

If the method is to be applied to sample matrix other than water (e.g., soil, sediment, filter
cake, compost) the most appropriate alternate reference matrix (Sections 7.6.1 – 7.6.4) is
substituted for the reagent water matrix (Section 7.6.1) in all performance tests.

9.1.1 The laboratory must make an initial demonstration of the ability to generate
acceptable precision and recovery with this method. This demonstration is given in
Section 9.2.

9.1.2 In recognition of advances that are occurring in analytical technology, and to

overcome matrix interferences, the laboratory is permitted certain options to
improve separations or lower the costs of measurements. These options include
alternate extraction, concentration, and cleanup procedures, and changes in
columns and detectors (see also 40 CFR 136.6). Alternate determinative
techniques, such as the substitution of spectroscopic or immunoassay techniques,
and changes that degrade method performance, are not allowed. If an analytical
technique other than the techniques specified in this method is used, that technique
must have a specificity equal to or greater than the specificity of the techniques in
this method for the analytes of interest.

9.1.2.1 Each time a modification is made to this method, the laboratory is

15
December 2007 Method 1694

required to repeat the procedure in Section 9.2. If the detection limit of

the method will be affected by the change, the laboratory is required to
demonstrate that the MDLs (40 CFR Part 136, Appendix B) are lower
than one-third the regulatory compliance level or the MDLs in this
method, whichever are greater. If calibration will be affected by the
change, the instrument must be recalibrated per Section 10. Once the
modification is demonstrated to produce results equivalent or superior to
results produced by this method as written, that modification may be
used routinely thereafter, so long as the other requirements in this
method are met (e.g., labeled compound recovery).

9.1.2.2 The laboratory is required to maintain records of modifications made to

this method. These records include the following, at a minimum:

9.1.2.2.1 The names, titles, addresses, and telephone numbers of the

analyst(s) that performed the analyses and modification, and
of the quality control officer that witnessed and will verify
the analyses and modifications.

9.1.2.2.2 A list of compounds (s) measured, by name and CAS

Registry number.

9.1.2.2.3 A narrative stating reason(s) for the modifications.

9.1.2.2.4 Results from all quality control (QC) tests comparing the
modified method to this method, including:

a) Calibration (Section 10).

b) Calibration verification (Section 15.2).
c) Initial precision and recovery (Section 9.2).
d) Labeled compound recovery (Section 9.3).
e) Analysis of blanks (Section 9.5).
f) Accuracy assessment (Section 9.4).

9.1.2.2.5 Data that will allow an independent reviewer to validate

each determination by tracing the instrument output (peak
height, area, or other signal) to the final result. These data
are to include:

a) Sample numbers and other identifiers.

b) Extraction dates.
c) Analysis dates and times.
d) Analysis sequence/run chronology.
e) Sample weight or volume (Section 11).
f) Sample or extract volume prior to each cleanup step
(Section 12).
g) Extract volume after each cleanup step (Section 12).
h) Final extract volume prior to injection (Section 12).
i) Injection volume (Sections 10.2.1 and 14.2).

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December 2007 Method 1694

j) Dilution data, differentiating between dilution of a

sample or extract (Section 17.5).
k) Instrument and operating conditions.
l) Column (dimensions, material, particle size, etc).
m) Operating conditions (flow rates, elution solvents,
gradient, flow rates).
n) Detector (type, operating conditions, etc).
o) Chromatograms, printer tapes, and other recordings of
raw data.
p) Quantitation reports, data system outputs, and other data
to link the raw data to the results reported.

9.1.3 Analyses of method blanks are required to demonstrate freedom from

contamination (Section 4.3). The procedures and criteria for analysis of a method
blank are given in Sections 9.5 and 15.4.

9.1.4 The laboratory must spike all samples with labeled compounds to monitor method
performance. This test is described in Section 9.3. When results of these spikes
indicate atypical method performance for samples, the samples are diluted to bring
method performance within acceptable limits. Procedures for dilution are given in
Section 17.5.

9.1.5 The laboratory must, on an ongoing basis, demonstrate through calibration

verification and the analysis of the ongoing precision and recovery standard (OPR)
and blanks that the analytical system is in control. These procedures are given in
Sections 15.1 through 15.4.

9.1.6 The laboratory should maintain records to define the quality of data generated.
Development of accuracy statements is described in Section 9.4.

9.2 Initial precision and recovery (IPR) – To establish the ability to generate acceptable
precision and recovery, the laboratory must perform the following operations.

9.2.1 For aqueous samples containing less than 1% solids, analyze four 1-L aliquots of
reagent water (7.6.1) each for the acid and base fractions according to the
procedures in Sections 11 through 18. For an alternate sample matrix, four aliquots
each for the acid and base fractions of the alternate reference matrix (Sections
7.6.2-7.6.4) are used. All sample processing steps that are to be used for
processing samples, including preparation (Section 11), extraction (Section 12),
and cleanup (Section 13), must be included in this test.

9.2.2 Using results of the set of four analyses, compute the average percent recovery (X)
of the concentration of each compound in each extract and the relative standard
deviation (RSD) of the concentration for each compound, by isotope dilution for
compounds with a labeled analog, and by internal standard for compounds without
a labeled analog and for the labeled compounds.

9.2.3 For each native and labeled compound, compare RSD and X with the
corresponding limits for initial precision and recovery in Table 12. If RSD and X

17
December 2007 Method 1694

for all compounds meet the acceptance criteria, system performance is acceptable
and analysis of blanks and samples may begin. If, however, any individual RSD
exceeds the precision limit or any individual X falls outside the range for recovery,
system performance is unacceptable for that compound. Correct the problem and
repeat the test (Section 9.2).

9.3 To assess method performance on the sample matrix, the laboratory must spike all samples
with the Labeled spiking solution (Section 7.7.3).

9.3.1 Analyze each sample according to the procedures in Sections 11 through 18.

9.3.2 Compute the percent recovery of the labeled compounds using the internal standard
method (Sections 10.4 and 7.2).

9.3.3 The recovery of each labeled compound must be within the limits in Table 12. If
the recovery of any compound falls outside of these limits, method performance is
unacceptable for that compound in that sample. Additional cleanup procedures
must then be employed to attempt to bring the recovery within the normal range. If
the recovery cannot be brought within the normal range after all cleanup
procedures have been employed, water samples are diluted and smaller amounts of
soils, sludges, sediments, and other matrices are analyzed per Section 18.

9.4 Recovery of labeled compounds from samples should be assessed and recorded.

9.4.1 After the analysis of 30 samples of a given matrix type (water, soil, sludge, pulp,
etc.) for which the labeled compounds pass the tests in Section 9.3, compute the
average percent recovery (R) and the standard deviation of the percent recovery
(SR) for the labeled compounds only. Express the assessment as a percent recovery
interval from R ! 2SR to R + 2SR for each matrix. For example, if R = 90% and SR
= 10% for 30 analyses of biosolids, the recovery interval is expressed as 70 to
110%.

9.4.2 Update the accuracy assessment for each labeled compound in each matrix on a
regular basis (e.g., after each 5-10 new measurements).

9.5 Method blanks – A reference matrix method blank is analyzed with each sample batch
(Section 4.3) to demonstrate freedom from contamination. The matrix for the method
blank must be similar to the sample matrix for the batch, e.g., a 1-L reagent water blank
(Section 7.6.1), high-solids reference matrix blank (Section 7.6.2), biosolids reference
matrix blank (Section 7.6.3) or alternate reference matrix blank (Section 7.6.4).

9.5.1 Process the method blank(s) along with the IPR or batch of samples according to
the procedures in Sections 11 through 18. Analyze the blank immediately after
analysis of the OPR (Section 15.4) to demonstrate freedom from contamination.

9.5.2 If any compound of interest (Table 1) is found in the blank at greater than the
minimum level (Tables 3, 5, 7, or 9) or one-third the regulatory compliance limit,
whichever is greater; or if any potentially interfering compound is found in the
blank above the minimum level for each native compound in Tables 3, 5, 7, or 9

18
December 2007 Method 1694

(assuming a response factor of 1 relative to the quantitation reference in Tables 3,

5, 7, or 9 for a potentially interfering compound; i.e., a compound not listed in this
method), analysis of samples must be halted until the sample batch is re-extracted
and the extracts re-analyzed, and the blank associated with the sample batch shows
no evidence of contamination at these levels. All samples must be associated with
an uncontaminated method blank before the results for those samples may be
reported or used for permitting or regulatory compliance purposes.

9.6 QC Check Sample – If available, analyze the QC Check Sample (Section 7.8) periodically
to assure the accuracy of calibration standards and the overall reliability of the analytical
process. It is suggested that the QC Check Sample be analyzed at least quarterly.

9.7 The specifications contained in this method can be met if the apparatus used is calibrated
properly and then maintained in a calibrated state. The standards used for calibration
(Section 10), calibration verification (Section 15.2), and for initial (Section 9.2) and
ongoing (Section 15.4) precision and recovery should be identical, so that the most precise
results will be obtained. A LCMSMS instrument will provide the most reproducible results
if dedicated to the settings and conditions required for determination of PPCPs by this
method.

9.8 Depending on specific program requirements, field replicates may be collected to determine
the precision of the sampling technique, and spiked samples may be required to determine
the accuracy of the analysis when the internal standard method is used.

10.0 Calibration and Standardization

10.1 Establish the LC/MS/MS operating conditions for the Group 1 through Group 4
compounds, as suggested in Tables 2, 4, 6, and 8, to meet the retention times in Tables 3, 5,
7, and 9, respectively. The LC conditions may be optimized for compound separation and
sensitivity. Once optimized, the same conditions must be used for the analysis of all
standards, blanks, IPR and OPR standards, and samples.

10.2 Retention time calibration for the native and labeled compounds

10.2.1 Inject the volume of CS-3 calibration standard (Section 7.7.5 and Table 11) listed
in Table 2, 4, 6, or 8, or other volume appropriate to system optimization.
Establish the beginning and ending retention times for the parent-daughter
descriptors in Tables 3, 5, 7, and 9. Descriptors other than those listed may be used
provided the MLs in those tables are met. Store the retention time (RT) for each
compound in the data system.

10.2.2 The absolute retention time of last-eluted compound in each of the four Groups
must be equal to or greater than its retention time in Tables 3, 5, 7, or 9; otherwise,
the LC operating conditions must be adjusted and this test repeated until this
minimum retention time criterion is met.

10.3 Mass spectrometer calibration and optimization

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December 2007 Method 1694

10.3.1 Mass calibration – The mass spectrometer must undergo mass calibration
according to manufacture’s specifications to ensure accurate assignments of m/z's
by the instrument. This mass calibration must be performed at least annually to
maintain instrument sensitivity and stability. It must be repeated after performing
major maintenance on the mass spectrometer.

In the absence of vendor-specific instructions and acceptance criteria, the following

procedure may be used.

10.3.1.1 Introduce the NaCsI calibration solution (Section 7.5) to the MS at the
flow rate necessary to produce a stable aerosol spray (e.g., 10 μL/min).

10.3.1.2 Scan the MS/MS over the mass range from 20 to 3000 Daltons. Adjust
the source parameters to optimize peak intensity and shape across the
mass range. The exact m/z's for NaCsI calibration are:

Calibration Masses (Daltons)

22.9898 1521.9321
132.9054 1671.8264
172.8840 1821.7206
322.7782 1971.6149
472.6725 2121.5091
622.5667 2271.4033
772.4610 2421.2976
922.3552 2571.1918
1072.2494 2721.0861
1222.1437 2870.9803
1372.0379

10.3.1.3 Mass calibration is judged on the basis of the presence or absence of the
exact calibration masses, e.g., a limit of the number of masses that are
“missed.” Absent vendor-specific instructions, all of the masses from
22.9898 to 1971.6149 must be present. If peaks above 1971 are missing
or not correctly identified, adjust the MS/MS and repeat the test. Only
after the MS/MS is properly calibrated may standards, blanks, and
samples be analyzed.

10.3.2 Mass spectrometer optimization – Prior to measurements of a given analyte

Group (Table 2, 4, 6, or 8), the mass spectrometer must be separately optimized
for that Group.

10.3.2.1 Using the post-column pump (Section 6.14.1), infuse the CS-3
calibration solution (Table 11 a, b, or c) for the Group of interest.

10.3.2.2 Optimize sensitivity to the daughter m/z's for the high mass compounds
in each Group (Table 3, 5, 7, or 9).

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December 2007 Method 1694

10.3.2.3 After MS calibration and optimization and LC/MS/MS calibration

(Sections 10.4 and 10.5), MS and LC/MS/MS conditions may not be
altered without verifying calibration (Section 15.2).

10.4 Calibration by isotope dilution − Isotope dilution is used for calibration of each native
compound for which a labeled analog is available. The reference compound for each
native compound is its labeled analog, as listed in Tables 3, 5, 7, and 9. A 5-point
calibration encompassing the concentration range is prepared for each native compound.
The calibration solutions are listed in Table 11.

10.4.1 To calibrate the analytical system by isotope dilution, inject calibration standards
CS-1 through CS-5 (Section 7.7.5 and Table 11). Use the volume shown in
identical to the volume chosen in Section 10.2.1, the procedure in Section 14, and
the optimized operating conditions from Sections 10.1 - 10.3.

10.4.2 For the compounds determined by isotope dilution, the relative response (RR)
(labeled to native) vs. concentration in the calibration solutions (Table 11) is
computed over the calibration range according to the procedures below.
Determine the response of each compound relative to its labeled analog using the
area responses of the daughter m/zs specified in Tables 3, 5, 7, and 9. Use the
labeled compounds listed in the tables as the quantitation reference and the
daughter m/zs of these labeled compounds for quantitation. The area of the
daughter m/z for the native compound is divided by the area of the daughter m/z
of the labeled quantitation reference compound.

Note: Other quantitation references and procedures may be used provided that the results produced are
as accurate as results produced by the quantitation references and procedures described in this method.

10.4.3 Calibrate the native compounds with a labeled analog using the following
equation:

A n Cl
RR =
Al Cn

Where:
An = The area of the daughter m/z for the native compound
Al = The area of the daughter m/z for the labeled compound.
Cl = The concentration of the labeled compound in the calibration
standard (Table 11) (ng/mL).
Cn = The concentration of the native compound in the calibration
standard (Table 11) (ng/mL).

10.4.4 Compute the average (mean) RR, and the standard deviation and relative
standard deviation (RSD) of the 5 RRs.

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December 2007 Method 1694

10.4.5 Linearity – If the RR for any compound is constant (less than 20% RSD), the
average RR may be used for that compound; otherwise, the complete calibration
curve for that compound must be used over the calibration range.

10.5 Calibration by internal standard – Internal standard calibration is applied to determination

of the native compounds for which a labeled compound is not available, and to
determination of the labeled compounds for performance tests and intra-laboratory
statistics (Sections 9.4 and 15.4.4). The reference compound for each native compound
is listed in Table 3, 5, 7, or 9. For the labeled compounds, calibration is performed at a
single concentration using data from the 5 points in the calibration (Section 10.4).

10.5.1 Response factors – Internal standard calibration requires the determination of

response factors (RF) defined by the following equation:

A n Cis
RF =
A is C n
Where:
An = The area of the daughter m/z for the native compound
Ais = The area of the daughter m/z for the internal standard.
Cis = The concentration of the internal standard (Table 11) (ng/mL).
Cn = The concentration of the native compound in the calibration
standard (Table 11) (ng/mL).

10.5.2 To calibrate the analytical system for compounds that do not have a labeled
analog, and for the labeled compounds, use the data from the 5-point calibration
(Section 10.4 and Table 11).

10.5.3 Compute and store the response factor (RF) for all native compounds that do not
have a labeled analog. Use the labeled compounds and daughter m/zs listed in
Tables 3, 5, 7, and 9 as the quantitation references.

10.5.4 Compute and store the response factor (RF) for the labeled compounds using the
labeled injection internal standard as the quantitation reference, as given in
Tables 3, 5, 7, and 9.

10.5.5 Linearity − If the RF for any native compound without a labeled analog or for
any labeled compound is constant (less than 35% RSD), the average RF may be
used for that compound; otherwise, the complete calibration curve for that
compound must be used over the calibration range.

10.6 SPE cartridge performance check

In order to be used for extraction of aqueous samples or cleanup of solid-sample extracts,

the performance of the HLB SPE cartridges must be checked at least once for each
manufacturer’s lot of cartridges. This performance check is accomplished by processing
a spiked reagent water sample through the extraction procedure is Section 12 and
analyzing the extract. Separate checks are performed for the acid and base fractions.
Labeled compounds are not added to these check samples before extraction because the

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December 2007 Method 1694

recovery correction inherent in isotope dilution will mask problems with the cartridges.
Cartridge performance is acceptable if the recoveries of the native analytes are within the
QC acceptance criteria for the OPR in Table 12. Perform this cartridge check as outlined
below. Note – This performance check is performed when a new lot number of cartridges
is purchased.

10.6.1 Acid fraction – Acidify a 1.0-L aliquot of reagent water to pH 2.0 ± 0.5. Add 500
mg Na4EDTA (Section 7.4.2) and spike with the Group 1, 2, and 3 native
compounds (Section 7.7.2 and Table 10). Do not spike the labeled compounds.
Process the solution through the SPE HLB procedure for the acid fraction in
Section 12. After processing, spike the solution with the Group 1, 2, and 3
labeled compounds (Section 7.7.3 and Table 10) and complete the analysis per
Sections 12 - 15. Recovery of the native compounds must be within the QC
acceptance crieria for the OPR in Table 12. If the compounds are not recovered
in this range, adjust the elution volumes or reject the cartridge batch.

10.6.2 Base fraction – Adjust the pH a 1.0-L aliquot of reagent water to pH 10.0 ± 0.5
and spike with the Group 4 native compounds (Section 7.7.2 and Table 10). Do
not spike the labeled compounds. Process the solution through the SPE HLB
procedure for the base fraction in Section 12. After processing, spike the extract
with the Group 4 labeled compounds (Section 7.7.3 and Table 10) and complete
the analysis per Sections 12 - 15. Recovery of the native compounds must be
within the QC acceptance criteria for the OPR in Table 12. If the compounds are
not recovered in this range, adjust the elution volumes or reject the cartridge
batch.

11.0 Sample Preparation

Sample preparation involves modifying the physical form of the sample so that the analytes can
be extracted efficiently. In general, the samples must be in a liquid form or in the form of finely
divided solids in order for efficient extraction to take place. Table 13 lists the phases and
suggested quantities for extraction of various sample matrices. For samples known or expected to
contain high levels of the analytes, the smallest sample size representative of the entire sample
should be used (see Section 18).

Biosolids and solid samples are prepared per Section 11.4, extracted per Sections 12.3 and 12.4,
and cleaned up using SPE HLB cleanup in Sections 12.1 and 12.2.

Aqueous samples - Because the analytes may be bound to suspended particles, the preparation of
aqueous samples is depends on the presence of visible particles. Aqueous samples absent visible
particles are prepared per Section 11.3 and processed using SPE HLB cleanup in Sections 12.1
and 12.2.

Aqueous samples with visible particles - If visible particles can be seen in aqueous samples they
should be filtered and the solids and aqueous portions of these samples should be extracted and
combined prior to clean up as follows. Filtration of particles - assemble a clean filtration
apparatus (Section 6.6). Apply vacuum to the apparatus, and pour the entire contents of the

23
December 2007 Method 1694

sample bottle through the filter, swirling the sample remaining in the bottle to suspend any
particles. Rinse the sample bottle twice with approximately 5 mL portions of reagent water to
transfer any remaining particles onto the filter. Rinse any particles off the sides of the filtration
apparatus with small quantities of reagent water. Weigh the empty sample bottle to ±1 g.
Determine the weight of the sample by difference. Save the bottle for further use. Prepare and
extract the filtrate using the procedure in Section 11.3. Prepare and extract the filter containing
the particles using the same procedure for biosolids and solid samples in Section 11.4, Sections
12.3 and 12.4, and Sections 12.1 and 12.2. These extracts should be combined prior to analysis
(Section 14) or results of separate analysis combined. It should be noted that the judgment of the
analyst must be used to determine the need to analyze samples with visible particles that compose
less than 1 % of the sample weight per Section 11.1.

Procedures for grinding, homogenization, and blending of various sample phases are given in
Section 11.5.

Note: Each sample batch (Section 4.3) is accompanied by a blank and an OPR. If the acid fraction
(Groups 1, 2, and 3) only is to be analyzed then 1 acid blank and OPR must be used. If both the acid
(Groups 1, 2, and 3) and base (Group 4) fractions are to be analyzed, 1 acid blank and OPR as well as 1
base blank and OPR must accompany the batch. If the base fraction (Group 4) only is to be analyzed, a
base blank and OPR must accompany the base batch.

11.1 Determination of solids content

The solids content of the bulk sample is determined from a subsample that is used only for the
solids determination. Separate procedures are used for the solids determination, based on the
sample matrix, as described below.

11.1.1 Aqueous liquids and multi-phase samples consisting of mainly an aqueous phase.

11.1.1.1 Dry a GF/A filter (Section 6.6.4) and weigh to three significant figures.
Mix the bulk sample in the original container (e.g., cap the bottle and
shake) and take a 10.0 ∀ 0.2 mL aliquot. Filter that aliquot through the
filter. Dry the filter in an oven for a minimum of 12 hours at 110 ∀ 5
ΕC and cool in a dessicator.

11.1.1.2 Weigh the filter and calculate percent solids as follows:

Weight of sample aliquot after drying (g) - weight of filter (g)

% Solids = x 100
10 g

11.1.2 Non-aqueous liquids, solids, semi-solid samples, and multi-phase samples in

which the main phase is not aqueous

11.1.2.1 Weigh 5 to 10 g of the bulk sample to three significant figures in a

tared beaker, weighing pan, or other suitable container. Dry for a
minimum of 12 hours at 110 ∀ 5 ΕC, and cool in a dessicator.

24
December 2007 Method 1694

11.1.2.2 Weigh the dried aliquot and calculate percent solids as follows:

Weight of sample aliquot after drying (g)

% Solids = x 100
Weight of sample aliquot before drying (g)
11.2 Estimation of particle size

Extraction of a sample matrix is affected by the size of particles in the sample. Ideally,
the particles should be 1 mm or less. The particle size can be estimated using the sample
aliquot filtered or dried in Sections 11.1.1 or 11.1.2. Spread the aliquot on a piece of
filter paper or aluminum foil in a fume hood or glove box. Visually estimate the size of
the particles in the sample. If the size of the largest particles is greater than 1 mm, use
one of the procedures in Section 11.5 to reduce the particle size to 1 mm or less prior to
extraction. If the largest particles are 1 mm or less, proceed with sample preparation,
using the procedures in Section 11.4

11.3 Preparation of aqueous samples absent visible particles and corresponding QC samples.

Two separate sample aliquots are required to analyze all of the target analytes in this
procedure: one aliquot is adjusted to pH 2 ± 0.5 (Section 11.3.3.1) and the other aliquot
is adjusted to pH 10 ± 0.5 (Section 11.3.4.1). Following this pH adjustment, both
aliquots are filtered separately, and the two filtrates are extracted using the SPE HLB
cartridge per Section 12.

11.3.1 Mark the original level of the sample on each of the two sample bottles.
Designate one bottle for the acid fraction and the other for the base fraction.
Weigh each sample plus bottle to the nearest 1 g. If only one sample bottle was
provided, and both the acid and base fractions are to be analyzed, split the sample
in half and place each new aliquot in a separate clean container.

11.3.2 For each sample batch (Section 4.3) to be extracted during the same 12-hour
shift, transfer four 1-L aliquots of reagent water to clean sample bottles or flasks.
Two of these aliquots will serve as method blanks (one for the acid fraction and
one for the base fraction) and the other two aliquots will be used to prepare the
OPR samples (one acid and one base). (If both acid and base fractions are not
required, prepare only the reference matrix aliquots appropriate for the fraction of
interest.)

11.3.3 Acid fraction - typically 500 mL to 1 L

11.3.3.1 Acidify the filtrate for the acid fraction to pH 2.0 ± 0.5 with HCl
while swirling or stirring the water. Re-adjust the pH as necessary to
achieve pH 2.0 ± 0.5. Maintain the pH above 1.95 to preclude
deuterium-hydrogen exchange on the deuterium-labeled compounds.

11.3.3.2 Spike the acid fraction (Group 1, 2, and 3) native compounds

(Section 7.7.2 and Table 10) into the reagent water aliquot that will
serve as the acid fraction OPR. Acidify the OPR aliquot and the

25
December 2007 Method 1694

blank aliquot in the same manner as the acid fraction of the field
sample (11.3.3.1).

11.3.3.3 Spike the acid fraction (Group 1, 2, and 3) labeled compounds

(Section 7.7.3 and Table 10) into the acid fractions of the samples
and QC aliquots.

11.3.3.4 Add 500 mg NA4EDTA.2H2O (Section 7.4.2) to each of the acid

fraction samples and QC aliquots. Cap the bottles and mix by
shaking. Allow the sample and QC aliquots to equilibrate for 1 to 2
hours, with occasional shaking. Proceed to Section 12 for sample
extraction.

11.3.4 Base fraction – typically 500 mL to 1 L

11.3.4.1 Adjust the pH of the second of the two sample bottles to pH 10.0 ±
0.5 with NH4OH while swirling or stirring the water. Re-adjust the
pH as necessary to achieve pH 10.0 ± 0.5.

11.3.4.2 Spike the base fraction (Group 4) native compounds (Section 7.7.2
and Table 10) into the reagent water aliquot that will serve as the
base fraction OPR. Adjust the pH of the OPR aliquot and the blank
aliquot in the same manner as the base fraction of the field sample
(11.3.4.1).

11.3.4.3 Spike the base fraction (Group 4) labeled compounds (Section 7.7.3
and Table 10) into the base fractions of the samples and QC aliquots.

11.3.4.4 Cap the bottles and mix by shaking. Allow the sample and aliquots
to equilibrate for 1 to 2 hours, with occasional shaking. Proceed to
Section 12 for sample extraction.

11.4 Preparation of solid samples and samples from filtered particles and corresponding QC
samples.

Filtered solids from aqueous samples are treated as solid matrices, regardless of whether
they are pourable liquids or solid materials. Two separate aliquots are required to
analyze all of the target analytes in this procedure. If the particle size estimated in
Section 11.2 exceeds 1 mm, use one of the six size-reduction procedures in Section 11.5
first. Following addition of buffer solutions, one aliquot is adjusted to pH 2 ± 0.5 and the
other aliquot is adjusted to pH 11 ± 0.5. Following pH adjustment, each aliquot is
extracted separately per Section 12.

11.4.1 Homogenize the sample in its original container, by shaking samples that are
pourable liquids, or by stirring solids in their original container with a clean
spatula, glass stirring rod, or other suitable implement.

26
December 2007 Method 1694

11.4.2 Using the percent solids data collected in Section 11.1, collect two aliquots of the
well-mixed sample sufficient to provide 1.0 g of dry solids, but do not exceed a
maximum of 5 g wet weight. For biosolids, do not exceed 0.25 g of wet solids.
Place the two sample aliquots in separate clean 50-mL disposable centrifuge
tubes. Designate one of the samples as the acid fraction, the other the base
fraction.

11.4.3 For each sample batch (Section 4.3) to be extracted during the same 12-hour
shift, transfer two 1-g aliquots of peat moss (Section 7.6.3) to clean sample
bottles or flasks. These two peat moss aliquots will be used for the method blank
and the OPR sample for the acid fraction. Transfer two 1-g aliquots of clean
sand (Section 7.6.2) to clean sample bottles or flasks. These two clean sand
aliquots will be used for the method blank and the OPR sample for the base
fraction. (If both acid and base fractions are not required, prepare only the
reference matrix aliquots appropriate for the fraction of interest.)

11.4.4 Acid fraction

11.4.4.1 Add 15 mL of pH 2 phosphate buffer (Section 7.4.1) to the sample,

blank, and OPR. Vortex each for 5 min. Check and adjust the pH to
2.0 ± 0.5 with buffer, vortexing the mixture after each addition.
Maintain the pH above 1.95 to preclude deuterium-hydrogen exchange
on the deuterium-labeled compounds.

11.4.4.2 Spike the acid fraction (Group 1, 2, and 3) native compounds (Section
7.7.2 and Table 10) into the peat moss aliquot that will serve as the
acid fraction OPR. Acidify the OPR aliquot and the blank aliquot in
the same manner as the acid fraction of the field sample (11.4.4.1).

11.4.4.3 Spike the acid fraction (Group 1, 2, and 3) labeled compounds

(Section 7.7.3 and Table 10) into the acid fractions of the samples and
QC aliquots.

11.4.4.4 Vortex the samples and QC aliquots. Proceed to Section 12.3 for
extraction of the solids acid fraction.

11.4.5 Base fraction

11.4.5.1 Add 15 mL of reagent water to the sample, blank, and OPR. Vortex
each for 5 min. Adjust the pH of the sample, blank, and OPR aliquots
to 10.0 ± 0.5 by adding NH4OH solution dropwise. Vortex for 5 min.
Check and adjust the pH to 10.0 ± 0.5 with NH4OH solution, vortexing
the mixture after each addition.

11.4.5.2 Spike the base fraction (Group 4) native compounds (Section 7.7.2 and
Table 10) into one of the QC aliquots. This aliquot will serve as the
OPR. The other will serve as the blank.

27
December 2007 Method 1694

11.4.5.3 Spike the base fraction (Group 4) labeled compounds (Section 7.7.3
and Table 10) into the samples and QC aliquots.

11.4.5.4 Vortex the samples and QC aliquots. Proceed to Section 12.4 for
extraction of the solids base fraction.

11.5 Sample grinding, homogenization, or blending

Samples with particle sizes greater than 1 mm (as determined in Section 11.2) are
subjected to grinding, homogenization, or blending. The method of reducing particle size
to less than 1 mm is matrix-dependent. In general, hard particles can be reduced by
grinding with a mortar and pestle. Softer particles can be reduced by grinding in a Wiley
mill or meat grinder, by homogenization, or in a blender.

11.5.1 Each size-reducing preparation procedure on each matrix must be verified by

running the tests in Section 9.2 before the procedure is employed routinely.

11.5.2 The grinding, homogenization, or blending procedures must be carried out in a

glove box or fume hood to prevent particles from contaminating the work
environment.

11.5.3 Grinding – Amorphous and other solids can be ground in a Wiley mill or heavy
duty meat grinder. In some cases, reducing the temperature of the sample to
freezing or to dry ice or liquid nitrogen temperatures can aid in the grinding
process. Grind the sample aliquots in a clean grinder. Do not allow the sample
temperature to exceed 50 ΕC. Also grind the blank and OPR reference matrix
aliquots using a clean grinder.

11.5.4 Homogenization or blending – Particles that are not ground effectively, or

particles greater than 1 mm in size after grinding, can often be reduced in size by
high speed homogenization or blending. Homogenize and/or blend the particles
or filter for the sample, blank, and OPR aliquots.

11.5.5 After size reduction, return to Section 11.4 for preparation of the sample and QC
aliquots.

12.0 Extraction and Concentration

This method employs solid-phase extraction (SPE) procedures to extract the target analytes from aqueous
samples. Solid samples are extracted using ultrasonic extraction with acetonitrile. The extracts from
solid samples contain significant amount of coextracted interferences which can be removed through the
use of the same SPE procedure employed for the aqueous samples.

12.1 Extraction of aqueous samples absent visible particles, and cleanup of extracts from filtered
solids, solids and biosolids samples.

Extraction of both the acid and base fractions of aqueous samples involve many of the same

28
December 2007 Method 1694

steps, beginning with the conditioning of the SPE cartridges.

12.1.1 Assemble the SPE extraction apparatus and attach the SPE HLB cartridges
(Section 6.5.7).

12.1.2 Condition an SPE HLB cartridge by eluting it with 20 mL of methanol, and 6 mL

of reagent water. Discard these eluants. When extracting the base fraction of a
sample, the conditioning steps stop here. Do not let the cartridge go dry at any
point during the conditioning process.

12.1.3 When extracting the acid fraction of a sample, complete the cartridge conditioning
step by eluting the cartridge with 6 mL reagent water at pH 2.0 ± 0.5. Discard this
eluant.

12.1.4 Using the SPE cartridge appropriate for the sample fraction (acid or base), load the
sample prepared as described in Sections 11.3.3.4 or 11.3.4.4 onto the cartridge at a
flow rate of 5-10 mL/min. Extraction of a 1-L aqueous sample will take 100-200
minutes, thus use of a multi-position extraction manifold is desirable.

12.1.5 Once the entire sample has passed through the cartridge, wash the acid fraction
cartridge with 10 mL of reagent water to remove the EDTA. Do not wash the
cartridge for the base fraction.

12.1.6 Dry the cartridges for either fraction under vacuum for approximately 5 min.

12.2 Cartridge elution

12.2.1 Acid fraction

12.2.1.1 Elute the analytes with 12 mL methanol. Initiate the elution by vacuum
and complete the elution by gravity. Collect the eluant in a clean
centrifuge tube.

12.2.1.2 If triclocarban and triclosan are analytes of interest, elute these two
analytes with 6 mL of acetone:methanol (1:1). Combine with the
methanol eluant.

12.2.1.3 Proceed with concentration of the extract (Section 12.5).

12.2.2 Base fraction

12.2.2.1 Elute the analytes with 6 mL methanol followed by 9 mL of 2% formic

acid solution (Section 7.4.3.1). Initiate the elution by vacuum and
complete the elution by gravity. Collect the eluant in a clean centrifuge
tube.

12.2.2.2 Proceed with concentration of the extract (Section 12.5).

12.3 Acid extraction of solid samples

29
December 2007 Method 1694

12.3.1 Add 20 mL acetonitrile to the solid sample and the QC aliquots, sonicate for 30
min, and centrifuge for approximately 5 min at approximately 3000 rpm.

12.3.2 Decant the extracts (supernatants) of the sample and the QC aliquots into separate,
clean 250-mL round-bottom flasks.

12.3.3 Add 15 mL of phosphate buffer (Section 7.4.1) to the sample and the QC aliquots.
Adjust to pH 2.0 ± 0.5 with HCl. Vortex to resuspend the solids. Check and
adjust the pH to 2.0 ± 0.5 with buffer, vortexing the mixture after the addition.

12.3.4 Perform a second extraction by repeating Sections 12.3.1 and 12.3.2, adding the
extracts to their respective flasks.

12.3.5 For the third extraction, add 15 mL of acetonitrile only to each of the tubes.
Sonicate and centrifuge the tubes, and decant the supernatants into their respective
round-bottom flasks.

12.3.6 If particles are visible in the extract, filter through a 110-mm or larger GF/A filter.
Using squeeze bottles, rinse the filter three times with reagent water, followed by
three rinses with acetonitrile.

12.3.7 Proceed with concentration of the acid extract (Section 12.6) followed by SPE in
12.1 and 12.2.

12.4 Base extraction of solid samples

12.4.1 Add 20 mL acetonitrile to the solid sample and QC aliquots, sonicate for 30 min,
and centrifuge for approximately 5 min at approximately 3000 rpm.

12.4.2 Decant the extracts (supernatants) of the sample and QC aliquots into separate,
clean 250-mL round-bottom flasks.

12.4.3 Add 15 mL of reagent water to the sample and QC aliquots. Add NH4OH
dropwise to the sample and QC aliquots to pH 10.0 ± 0.5. Vortex to resuspend the
solids. Check and adjust the pH to 10.0 ± 0.5 with NH4OH, vortexing the
mixture after the addition.

12.4.4 Perform a second extraction by repeating Sections 12.4.1 and 12.4.2, adding the
extracts to their respective flasks.

12.4.5 For the third extraction, add 15 mL of acetonitrile only to the centrifuge tubes.
Sonicate and centrifuge the tubes, and decant the supernatants into the round-
bottom flasks.

12.4.6 If particles are visible in the extract, filter through a 110-mm or larger GF/A filter.
Using squeeze bottles, rinse the filter three times with reagent water, followed by
three rinses with acetonitrile.

30
December 2007 Method 1694

12.4.7 Proceed with concentration of the base extract (Section 12.6) followed by SPE in
Section 12.1 and 12.2.

12.5 Concentration of aqueous sample extracts

Extracts from the acid and base fractions of aqueous samples are concentrated separately to
near dryness and the solvent exchanged to methanol, as described below. This same
procedure is used to concentration the extracts of solid samples after they have been
subjected to the SPE HLB cleanup procedure in Sections 12.1 - 12.2.

12.5.1 Concentrate the extract to near dryness under a gentle stream of nitrogen in a water
bath held at 50 ± 5 °C.

12.5.2 Add 3 mL of methanol to the concentrated acid and base extracts, including the
blank and OPR aliquots.

12.5.3 Spike the acid extracts with the labeled injection acid internal standards and the
base extracts with the labeled injection base internal standard (Table 10).

12.5.4 Bring the acid and base extracts to a final volume of 4.0 ± 0.1 mL with 0.1%
formic acid solution (Section 7.4.3.2). Vortex to mix.

12.5.5 If visible particles are present in the extract, or if the extract is cloudy, filter
through a 0.2-μm filter (Section 6.6.5.

12.5.6 Transfer 1 mL of each extract to an LC/MS/MS autosampler vial for analysis.

Store the remaining 3 mL of extract as backup in a refrigerator. (Other proportions
of the extract may be used as long as sensitivity is not compromised).

12.5.7 Proceed to Section 14 for analysis.

12.6 Concentration of the solid sample extracts

Extracts from the acid and base fractions of solid samples are concentrated separately prior
to cleanup and the extracts are reconstituted into aqueous solutions that are processed
through the aqueous sample SPE HLB extraction procedures (Sections 12.1 - 12.2) as a
cleanup step.

12.6.1 Concentrate the extracts from the acid and base fractions of the solid samples and
QC aliquots separately, to a final volume of 20 - 30 mL by rotary evaporation at 50
°C. Do not allow the extracts to go dry.

12.6.2 Immediately after concentration, add 200 mL of reagent water and 500 mg of
NA4EDTA.2H2O to the acid fraction extract. Swirl to mix.

12.6.3 Immediately after concentration, add 200 mL of reagent water to the base fraction
extract. Check that the pH is 10.0 ± 0.5. If necessary, adjust dropwise with

31
December 2007 Method 1694

NH4OH solution. Swirl to mix.

12.6.4 Proceed to Section 13 for cleanup of the extracts of all solid samples and associated
QC aliquots.

13.0 Extract Cleanup

As noted in Section 12.6, the extracts from all solid samples are subjected to cleanup using the same SPE
procedure used to extract aqueous samples. In essence, the solvent extract is reconstituted with reagent
water and the pH adjusted to that appropriate for the analytes of interest. The reconstituted sample is
processed through the same SPE procedure and the final extract is concentrated and prepared for
instrumental analysis. Because the volume of the reconstituted solid sample extract is about 200 mL, the
SPE cleanup will take significantly less time than the extraction of a 1-L water sample. Therefore, it is not
recommended that aqueous sample extractions and cleanup of solid sample extracts be performed
simultaneously on the same extraction manifold.

13.1 The acid fraction extract of each solid sample in Section 12.6.2 is processed through the
SPE procedure, beginning at Section 12.1.1 and proceeding through Section 12.2.1.3.
Process the associated QC aliquots (blank and OPR) through the cleanup procedure as well.

13.2 The base fraction extract of each solid sample in Section 12.6.3 is processed through the
SPE procedure, beginning at Section 12.1.1 and proceeding through Section 12.1.6, and
12.2.2.1 through 12.2.2.2, but omitting Sections 12.1.3 and 12.1.5. Process the associated
QC aliquots (blank and OPR) through the cleanup procedure as well.

13.3 After completing the SPE cleanup, concentrate the acid and base extracts of solid samples
and QC aliquots separately per Section 12.5 and proceed to Section 14 for analysis.

14.0 LC/MS/MS Analysis

14.1 Establish the same operating conditions established and optimized in Section 10.1 - 10.3
for the calibration appropriate to the fraction and Group to be analyzed. Analysis is
performed using positive electrospray ionization (ESI+) for the acid fraction Group 1 and 2
analytes and the base fraction Group 4 analytes. Analysis is performed by ESI– for the acid
fraction Group 3 analytes. Retention times (RTs), parent-daughter transitions,
quantitation references, method detection limits, and minimum levels of quantitation for
Groups 1, 2, 3, and 4 are given in Tables 3, 5, 7, and 9, respectively.

14.2 Inject the volume of the concentrated extract specific to the Group into the LC/MS/MS
instrument. The volume injected must be identical to the volume chosen in Section
10.2.1 and used for calibration in Section 10.3.1.

14.2.1 Start the gradient according to the program appropriate for the Group (see Table
2, 4, 6, or 8 for recommended conditions). Start data collection prior to elution
of the first analyte.

32
December 2007 Method 1694

14.2.2 Monitor the daughter m/z's for each analyte throughout its retention time
window. Where known, monitor m/z's associated with interferents expected to
be present.

14.2.3 Stop data collection after elution of the last analyte in each Group. Return the
gradient to the initial mixture for analysis of the next sample extract or standard.

15.0 System and Laboratory Performance

15.1 At the beginning of each 12-hour shift during which analyses are performed, LC/MS/MS
system performance and calibration are verified for all native and labeled compounds.
For these tests, analysis of the CS-3 calibration verification (VER) standard (Section
7.7.5 and Table 11) must be used to verify all performance criteria. Adjustment and/or
recalibration (Section 10) must be performed until all performance criteria are met. Only
after all performance criteria are met may samples, blanks, IPRs, and OPRs be analyzed.

15.2 Calibration verification

15.2.1 Inject the VER (CS-3) calibration standard (Table 10) for the Group being
analyzed using the procedure in Section 14.

15.2.2 The LC peak representing each native and labeled compound in the VER
standard must be present with a S/N of at least 10; otherwise, the LC/MS/MS
system must be adjusted and the verification test repeated.

15.2.3 Compute the concentration of the native compounds that have labeled analogs by
isotope dilution and the concentration of the native compounds that do not have
labeled analogs and of the labeled compounds by the internal standard technique.
These concentrations are computed based on the calibration data in Section 10.

15.2.4 For each compound, compare the concentration with the calibration verification
limit in Table 12. If all compounds meet the acceptance criteria, calibration has
been verified and analysis of standards and sample extracts may proceed. If,
however, any compound fails its respective limit, the measurement system is not
performing properly. In this event, prepare a fresh calibration standard or correct
the problem and repeat the verification (Section 15.2) tests, or recalibrate
(Section 10).

15.3 Retention time

15.3.1 The retention times of the native and labeled compounds in the verification test
(Section 15.2) must be within ± 15 seconds of the respective retention times in the
most recent calibration verification standard.

15.3.2 If the retention time of any compound is not within the limits specified, the LC is
not performing properly. In this event, adjust the LC operating conditions and

33
December 2007 Method 1694

repeat the verification test (Section 15.3) or recalibrate (Section 10), or replace the
LC column and either verify calibration or recalibrate.

15.4 Ongoing precision and recovery

15.4.1 Analyze the extracts of both the acid and base fractions of the ongoing precision
and recovery (OPR) aliquots prior to analysis of samples from the same batch.

15.4.2 Compute the percent recovery of each native compound with a labeled analog by
isotope dilution (Section 10.4). Compute the percent recovery of each native
compound without a labeled analog and of each labeled compound by the
internal standard method (Section 10.5).

15.4.3 For the native and labeled compounds, compare the recovery to the OPR limits
given in Table 12. If all compounds meet the acceptance criteria, system
performance is acceptable and analysis of blanks and samples may proceed. If,
however, any individual concentration falls outside of the range given, the
extraction/concentration processes are not being performed properly for that
compound. In this event, correct the problem, re-prepare, extract, and clean up
the sample batch and repeat the ongoing precision and recovery test (Section
15.4).

15.4.4 If desired, add results that pass the specifications in Section 15.4.3 to initial and
previous ongoing data for each compound in each matrix. Update QC charts to
form a graphic representation of continued laboratory performance. Develop a
statement of laboratory accuracy for each compound in each matrix type by
calculating the average percent recovery (R) and the standard deviation of
percent recovery (SR). Express the accuracy as a recovery interval from R ! 2SR
to R + 2SR. For example, if R = 95% and SR = 5%, the accuracy is 85 to 105%.

15.5 Blank − Analyze the method blank extracted with each sample batch immediately
following analysis of the OPR aliquot to demonstrate that there is no contamination or
carryover from the OPR analysis. If native compounds will be carried from the OPR into
the method blank, analyze one or more aliquots of solvent between the OPR and the
method blank. Results of analysis of the method blank must meet the specifications in
Section 9.5.2 before sample analysis may begin.

16.0 Qualitative Determination

A native or labeled compound is identified in a standard, blank, or sample when the criteria in
Sections 16.1 through 16.2 are met.

16.1 The signal-to-noise ratio (S/N) at the LC peak maximum for each native compound at its
daughter m/z must be greater than or equal to 2.5 for each compound detected in a
sample extract, and greater than or equal to 10 in CALs and VER samples for parent to
daughter transition except S/N of 3 in CS-1.

34
December 2007 Method 1694

16.2 The retention time of the peak for a native compound must be within ± 15 seconds of its
RT in the most recent CS-3 standard (Table 11).

16.3 Because of compound RT overlap and the potential for interfering substances, it is
possible that all of the identification criteria (Sections 16.1 - 16.2) may not be met. If
identification is ambiguous, an experienced spectrometrist (Section 1.5) must determine
the presence or absence of the compound.

16.4 If the criteria for identification in Sections 16.1 - 16.2 are not met, the compound has not
been identified and the result for that compound may not be reported or used for
permitting or regulatory compliance purposes. If interferences preclude identification, a
new aliquot of sample must be analyzed. Refer to Section 18 for guidance.

17.0 Quantitative Determination

17.1 Isotope dilution quantitation

17.1.1 By adding a known amount of a labeled compound to every sample prior to

extraction, correction for recovery of the native analog of that compound can be
made because the native compound and its labeled analog exhibit similar effects
upon extraction, concentration, and chromatography. Relative responses (RRs)
are used in conjunction with the calibration data in Section 10.4 to determine the
concentration in the final extract, as long as labeled compound spiking levels are
constant.

17.1.2 Compute the concentration of each compound in the extract using the RR from
the calibration data (Section 10.4) and following equation:

An C l
C ex (ng / mL) =
Al RR
Where:
Cex = Concentration of the compound in the extract , and the other terms are as
defined in Section 10.4.3

17.2 Internal standard quantitation and labeled compound recovery

17.2.1 Compute the concentration of each native compound that does not have labeled
analog and each labeled compound using the RF from the calibration data
(Section 10.5) and the following equation:
A C
C ex (ng / mL) = s is
Ais RF
Where:
Cex = Concentration of the compound in the extract, and the other terms are as
defined in Section 10.5.1

35
December 2007 Method 1694

17.2.2 Using the concentration in the extract determined above, compute the percent
recovery of the labeled compounds using the following equation:

Concentration found (ng / mL)

Re cov ery (%) = x 100
Concentration spiked (ng / mL)

17.3 The concentration of a native compound in the solid phase of the sample is computed
using the concentration of the compound in the extract and the weight of the solids, as
follows:
C ex Vex
Concentration in solid sample (ng / kg) =
Ws
Where:
Cex = Concentration of the compound in the extract.
Vex = Extract volume in mL.
Ws = Sample weight (dry weight) in kg.

If desired, divide the concentration by 1000 to convert ng/kg to µg/kg.

17.4 The concentration of a native compound in the aqueous phase of the sample is computed
using the concentration of the compound in the extract and the volume of water extracted,
as follows:

Concentration in aqueous phase (ng/L) = 1000 x (Cex x Vex)

Vs

Where:
Cex = Concentration of the compound in the extract.
Vex = Extract volume in mL.
Vs = Sample volume in liters.

17.5 If the SICP area at the daughter quantitation m/z for any compound exceeds the
calibration range of the system, dilute the sample extract by the factor necessary to bring
the concentration within the calibration range, adjust the concentration of the labeled
injection internal standard to the original concentration in the extract, and analyze an
aliquot of this diluted extract. If the compound cannot be measured reliably by isotope
dilution, dilute and analyze an aqueous sample or analyze a smaller portion of a solid, or
other sample. Adjust the compound concentration, detection limit, and minimum level of
quantitation to account for the dilution.

17.6 Reporting of results

17.6.1 Reporting units and levels

17.6.1.1 Aqueous samples – Report results in ng/L (parts-per-trillion).

17.6.1.2 Samples containing solids (aqueous samples containing visible

particles, solids, soils, sediments, filter cake, compost) – Report results
in µg/kg (parts-per-billion) based on the dry weight of the sample.

36
December 2007 Method 1694

Report the percent solids so that the result may be converted to aqueous
units.

17.6.2 Reporting level

17.6.2.1 Report the result for each compound in each sample, blank, or standard
(VER, IPR, OPR) at or above the minimum level of quantitation (ML;
Table 3, 5, 7, or 9) to 3 significant figures. Report the result below the
ML in each sample as <ML (where ML is the concentration at the ML)
or as required by the regulatory authority or permit.

17.6.2.2 Blanks – Report the result for each compound below the ML but above
the MDL to 2 significant figures. Report results below the MDL as
<MDL (where MDL is the concentration at the MDL) or as required by
the regulatory authority or permit. In addition to reporting results for
the samples and blank(s) separately, the concentration of each
compound in a method blank or field blank associated with the sample
may be subtracted from the results for that sample, or must be
subtracted if requested or required by a regulatory authority or in a
permit.

17.6.2.3 Results for a compound in a sample that has been diluted are reported at
the least dilute level at which the area at the quantitation m/z is within
the calibration range (Section 17.5).

17.6.2.4 For a compound having a labeled analog, report results at the least
dilute level at which the area at the quantitation m/z is within the
calibration range (Section 17.5) and the labeled compound recovery is
within the normal range for the method (Section 9.3 and Table 12).

17.6.2.5 Results from tests performed with an analytical system that is not in
control must not be reported or otherwise used for permitting or
regulatory compliance purposes, but do not relieve a discharger or
permittee of reporting timely results.

18.0 Analysis of Complex Samples

18.1 Some samples may contain high levels (>1 μg/L; >1 mg/kg) of the compounds of
interest, interfering compounds, and/or polymeric materials. The concentration of
analytes and/or interferences in some extracts may overload the LC column and/or mass
spectrometer.

18.2 Analyze a smaller aliquot of the sample (Section 17.5) when the interferences preclude
analysis of the full sample volume or amount. If a smaller aliquot of a solid, biosolid, or
mixed-phase sample is analyzed, attempt to assure that the smaller aliquot is
representative.

37
December 2007 Method 1694

18.3 Perform integration of peak areas and calculate concentrations manually when
interferences preclude computerized calculations.

18.4 Signal suppression – Coextracted interferences in the sample may suppress signals for the
compounds of interest. To detect signal suppression, the labeled injection internal
standard(s) must be monitored in the analysis. If the signal for the labeled injection
internal standard is suppressed by more than 30%, as compared to the average signal for
the labeled injection internal standard in the 5-point calibration, the sample must be
further cleaned up and reanalyzed. If the sample cannot be cleaned up further, the sample
or extract must be diluted, and a diluted sample or extract must be analyzed (Section
17.5).

18.5 Recovery of labeled compounds – For most samples, recoveries of the labeled
compounds will be similar to those from reagent water or from the alternate matrix
(Section 7.6 and Table 12).

18.5.1 If the recovery of any of the labeled compounds is outside of the normal range
(Table 12), a diluted sample must be analyzed (Section 17.5).

18.5.2 If the recovery of any of the labeled compounds in the diluted sample is outside
of normal range, the calibration verification standard (Section 7.7.5 and Table
11) must be analyzed and calibration verified (Section 15.2).

18.5.3 If the calibration cannot be verified, a new calibration must be performed and the
original sample extract reanalyzed.

18.5.4 If calibration is verified and the diluted sample does not meet the limits for
labeled compound recovery, this method does not apply to the sample being
analyzed and the result may not be reported or used for permitting or regulatory
compliance purposes. In this case, alternate extraction and cleanup procedures in
this method or an alternate LC column must be employed to resolve the
interference. If all cleanup procedures in this method and an alternate LC
column have been employed and labeled compound recovery remains outside of
the normal range, extraction and/or cleanup procedures that are beyond this scope
of this method will be required to analyze the sample.

19.0 Pollution Prevention

19.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity
or toxicity of waste at the point of generation. Many opportunities for pollution
prevention exist in laboratory operation. EPA has established a preferred hierarchy of
environmental management techniques that places pollution prevention as the
management option of first choice. Whenever feasible, laboratory personnel should use
pollution prevention techniques to address waste generation. When wastes cannot be
reduced at the source, the Agency recommends recycling as the next best option.

19.2 The compounds in this method are used in extremely small amounts and pose little threat
to the environment when managed properly. Standards should be prepared in volumes

38
December 2007 Method 1694

consistent with laboratory use to minimize the disposal of excess volumes of expired
standards.

19.3 For information about pollution prevention that may be applied to laboratories and
research institutions, consult Less is Better: Laboratory Chemical Management for Waste
Reduction, available from the American Chemical Society's Department of Governmental
Relations and Science Policy, 1155 16th Street NW, Washington DC 20036, 202/872­
4477.

20.0 Waste Management

20.1 The laboratory is responsible for complying with all Federal, State, and local regulations
governing waste management, particularly the hazardous waste identification rules and
land disposal restrictions, and to protect the air, water, and land by minimizing and
controlling all releases from fume hoods and bench operations. Compliance is also
required with any sewage discharge permits and regulations. An overview of
requirements can be found in Environmental Management Guide for Small Laboratories
(EPA 233-B-98-001).

20.2 Samples at pH <2, or pH >12 are hazardous and must be neutralized before being poured
down a drain, or must be handled as hazardous waste.

20.3 The compounds in this method decompose above 500 ΕC. Low-level waste such as
absorbent paper, tissues, animal remains, and plastic gloves may be burned in an
appropriate incinerator. Gross quantities (milligrams) should be packaged securely and
disposed of through commercial or governmental channels that are capable of handling
toxic wastes.

20.4 For further information on waste management, consult The Waste Management Manual
for Laboratory Personnel and Less is Better-Laboratory Chemical Management for
Waste Reduction, available from the American Chemical Society's Department of
Government Relations and Science Policy, 1155 16th Street N.W., Washington, D.C.
20036.

21.0 Method Performance

Method 1694 was validated and preliminary data were collected in a single laboratory (Reference
2). Performance data are given in Table 14.

22.0 References
1 EPA Methods 610, 1668A, and 8321A

2 "Analytical Procedure for the Analysis of Pharmaceutical Compounds in Solid and

Aqueous Samples by LC-MS/MS," Axys Analytical Services proprietary.

39
December 2007 Method 1694

3 Previous work on pharmaceuticals and personal-care products

3a Castiglioni at al., A Multiresidue Analytical Method Using Solid-Phase Extraction and

High-Pressure Liquid Chromatography Tandem Mass Spectrometry to Measure
Pharmaceuticals of Different Therapeutic Classes in Urban Wastewaters. J.
Chromatogr.A 1092 (2005), 206-215.

3b Dana W. Kolpin, Edward T. Furlong etc., Pharmaceuticals, Hormones, and Other

Organic Wastewater Contaminants in US Streams, 1999-2000: A National
Reconnaissance, Environ.Sci.Technol. 2002, 36,1202-1211.

3c Michele E. Lindsey, Michael Meyer, and E.M.Thurman, Analysis of Trace Levels of

Sulfonamide and Tetracycline Antimicrobials in Groundwater and Surface Water
Using Solid-Phase Extraction and Liquid Chromatography/Mass Spectrometry, Anal.
Chem. 2001, 73, 4640-4646.

3d Roman Hirsch, Tomas A. Ternes, etc., Determination of Antibiotics in Different Water

Compartments via LC/ESI-MS/MS, Journal of Chromatography A, 815(1998) 213­
223.

3e Fiese, E.F., and Steffen, S.H., Comparison of the acid stability of azithromycin and
erythromycin A, J. Antimicrobial Chemotherapy, 25 Suppl. A(1990) 39-47.

4 "Working with Carcinogens," Department of Health, Education, & Welfare, Public

Health Service, Centers for Disease Control, NIOSH, Publication 77-206, August 1977,
NTIS PB-277256.

5 "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910.

6 "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety,

1979.

7 "Standard methods for the Examination of Water and Wastewater," 18th edition and later
revisions, American Public Health Association, 1015 15th St, N.W., Washington, DC
20005, 1-35: Section 1090 (Safety), 1992.

8 Provost, L.P., and Elder, R.S., "Interpretation of Percent Recovery Data," American
Laboratory, 15: 56-83, 1983.

9 "Standard Practice for Sampling Water," ASTM Annual Book of Standards, ASTM, 1916
Race Street, Philadelphia, PA 19103-1187, 1980.

10 A) Paul E. Stackelberg, Jacob Gibs, Edward T. Furlong, Michael T. Meyer, Steven D.

Zaugg, R. Lee Lippincott. Science of the Total Environment 377 (2007) 255–272. B)
Zhengqi Ye and Howard S. Weinberg and Michael T. Meyer. Anal. Chem., 79 (3), 1135 ­
1144, 2007.

11 "Handbook of Analytical Quality Control in Water and Wastewater Laboratories,"

40
December 2007 Method 1694

USEPA EMSL, Cincinnati, OH 45268, EPA-600/4-79-019, March 1979.

41
December 2007 Method 1694

23.0 Tables and Flowchart

Table 1. Names and CAS Registry numbers for pharmaceuticals and personal-care products (PPCPs)
determined by isotope dilution and internal standard HPLC/MS/MS

Compound CAS Registry Labeled analog CAS Registry

Acetaminophen 103-90-2 13C2­ 15N-Acetaminophen

Albuterol 18559-94-9 Albuterol-d3
Ampicillin 69-53-4
Anhydrochlortetracycline (ACTC) 4497-08-9
Anhydrotetracycline (ATC) 4496-85-9
Azithromycin 83905-01-5
Caffeine 58-08-2 13C3-Caffeine
Carbadox 6804-07-5
Carbamazepine 298-46-4
Cefotaxime 63527-52-6
Chlortetracycline (CTC) 57-62-5
Cimetidine 51481-61-9
Ciprofloxacin 85721-33-1 13C3­ 15N-Ciprofloxacin
Clarithromycin 81103-11-9
Clinafloxacin 105956-97-6
Cloxacillin 61-72-3
Codeine 76-57-3
Cotinine 486-56-6 Cotinine-d3
Dehydronifedipine 67035-22-7
Demeclocycline 127-33-3
Digoxigenin 1672-46-4
Digoxin 20830-75-5
Diltiazem 42399-41-7
1,7-Dimethylxanthine 611-59-6
Diphenhydramine 58-73-1
Doxycycline 564-25-0
Enrofloxacin 93106-60-6
4-Epianhydrochlortetracycline 158018-53-2
(EACTC)
4-Epianhydrotetracycline (EATC) 4465-65-0
4-Epichlortetracycline (ECTC) 14297-93-9
4-Epioxytetracycline (EOTC) 14206-58-7
4-Epitetracycline (ETC) 23313-80-6
Erythromycin 114-07-8
Erythromycin anhydrate 59319-72-1 13C2-Erythromycin anhydrate
Flumequine 42835-25-6
Fluoxetine 54910-89-3 Fluoxetine-d5
Gemfibrozil 25812-30-0 Gemfibrozil-d6

42
December 2007 Method 1694

Compound CAS Registry Labeled analog CAS Registry

Ibuprofen 15687-27-1 13C3-Ibuprofen
Isochlortetracycline (ICTC) 514-53-4
Lincomycin 154-21-2
Lomefloxacin 98079-51-7
Metformin 657-24-9 Metformin-d6
Miconazole 22916-47-8
Minocycline 10118-91-8
Naproxen 22204-53-1 13C-Naproxen-d3
Norfloxacin 70458-96-7
Norgestimate 35189-28-7
Ofloxacin 82419-36-1
Ormetoprim 6981-18-6
Oxacillin 66-79-5
Oxolinic acid 14698-29-4
Oxytetracycline (OTC) 79-57-2
Penicillin V 87-08-1
Penicillin G 61-33-6
Ranitidine 66357-35-5
Roxithromycin 80214-83-1
Sarafloxacin 98105-99-8
Sulfachloropyridazine 80-32-0
Sulfadiazine 68-35-9
Sulfadimethoxine 122-11-2
Sulfamerazine 127-79-7
Sulfamethazine 57-68-1 13C6-Sulfamethazine
Sulfamethizole 144-82-1
Sulfamethoxazole 723-46-6 13C6-Sulfamethoxazole
Sulfanilamide 63-74-1
Sulfathiazole 72-14-0
Tetracycline (TC) 60-54-8
Thiabendazole 148-79-8 Thiabendazole-d6
Triclocarban 101-20-2 13C6-Triclocarban
Triclosan 3380-34-5 13C12-Triclosan
Trimethoprim 738-70-5 13C3-Trimethoprim
Tylosin 1401-69-0
Virginiamycin 11006-76-1
Warfarin 81-81-2 Warfarin-d5
Other standards
Unlabeled compound spiked into sample and used for recovery correction
Meclocycline
Labeled injection internal standard spiked into sample extract prior to injection into LC/MS/MS
13
C3-Atrazine
13
C6-2,4,5-Trichlorophenoxyacetic acid
(13C6-TCPAA)

43
December 2007 Method 1694

Table 2. Group 1 – Acidic extraction, positive electrospray ionization (ESI+) instrument conditions

Instrument Waters 2690 HPLC or Waters 2795 HPLC, Micromass Quattro Ultima MS/MS
LC Column Waters Xtera C18, 10.0 cm, 2.1 mm i.d., 3.5 µm particle size
Ionization Positive Ion Electrospray
Acquisition MRM mode, unit resolution
Injection Volume 15 μL

LC Gradient Program LC Flow Rate General LC Conditions

Time (mL/min) Column Temp 40 °C
1
(min) Flow Mixture Gradient
95% Solvent A 0.15 – 0.30
0.0 0.150 1 Flow Rate
5% Solvent B mL/min
95% Solvent A
4.0 0.250 6 Max Pressure 345 Bar
5% Solvent B
12% Solvent A Autosampler tray
22.5 0.300 6 4ºC
88% Solvent B temperature
23.0 100% Solvent B 0.300 6 MS Conditions
26.0 100% Solvent B 0.300 6 Source Temp 140°C
95% Solvent A
26.5 0.150 6 Desolvation Temp 350°C
5% Solvent B
95% Solvent A Cone / Desolvation 80 L/hr /
33.0 0.150 6
5% Solvent B Gas Rate 400 L/hr
1
Solvent A = 0.3% Formic Acid and 0.1% Ammonium Formate in HPLC water
Solvent B = 1:1 Acetonitrile:Methanol

44
December 2007 Method 1694

Table 3. Group 1 acidic extraction, positive electrospray ionization (ESI+) compound retention times (RTs), parent-daughter transitions,
quantitation references, method detection limits, and minimum levels of quantitation.

Parent- Detection limits and minimum levels

daughter Water (ng/L) Other (μg/kg) Extract (ng/ΦL)
Analyte RT (min) m/zs Quantitation reference MDL ML MDL ML MDL ML
Group 1 Analytes Extracted Under Acidic Conditions and Analyzed Using Positive Electrospray Ionization (+) ESI
Native compounds
13
Sulfanilamide 2.5 190.0 - 155.8 C6-Sulfamethazine 8.9 50 48 200 2.2 12.5
Cotinine 2.8 177.0 - 98.0 Cotinine-d3 3.4 5 1.1 5 0.9 1.25
13
Acetaminophen 4.6 152.2 - 110.0 C2­ 15N-Acetaminophen 27 200 35 200 6.7 50
13
Sulfadiazine 6.0 251.2 - 156.1 C6-Sulfamethazine 0.4 5 2.7 10 0.1 1.25
13
1,7-Dimethylxanthine 6.9 181.2 - 124.0 C3-Caffeine 120 500 270 1000 30 125
13
Sulfathiazole 7.7 256.3 - 156.0 C6-Sulfamethoxazole 0.5 5 1.9 50 0.1 1.25
13
Codeine 8.3 300.0 - 152.0 C3-Trimethoprim 1.5 10 3.4 10 0.4 2.5
13
Sulfamerazine 8.7 265.0 - 156.0 C6-Sulfamethazine 0.3 2 1.4 5 0.1 0.5
13
Lincomycin 9.3 407.5 - 126.0 C3-Trimethoprim 0.8 10 4.7 10 0.2 2.5
13
Caffeine 9.3 195.0 - 138.0 C3 Caffeine 15 50 5.4 50 3.6 12.5
13
Sulfamethizole 10.0 271.0 - 156.0 C6-Sulfamethoxazole 0.4 2 0.88 5 0.1 0.5
13
Trimethoprim 10.0 291.0 - 230.0 C3-Trimethoprim 1.1 5 3.3 10 0.3 1.25
Thiabendazole 10.0 202.1 - 175.1 Thiabendazole-d6 0.7 5 2.1 10 0.2 1.25
13
Sulfamethazine 10.1 279.0 - 156.0 C6-Sulfamethazine 0.6 2 0.83 5 0.2 0.5
13
Cefotaxime 10.2 456.4 - 396.1 C3-Trimethoprim 10 20 18 50 2.5 5
13
Carbadox 10.5 263.2 - 231.2 C3-Trimethoprim 2.3 5 2.1 10 0.6 1.25
13
Ormetoprim 10.5 275.3 - 259.1 C3-Trimethoprim 0.3 2 0.50 2 0.1 0.5
13
Norfloxacin 10.7 320.0 - 302.0 C3 15N-Ciprofloxacin 28 50 15 50 7.0 12.5
13
Sulfachloropyridazine 10.8 285.0 - 156.0 C6-Sulfamethazine 1.2 5 1.9 5 0.3 1.25
13
Ofloxacin 10.8 362.2 - 318.0 C3 15N-Ciprofloxacin 1.8 5 3.4 10 0.4 1.25
13
Ciprofloxacin 10.9 332.2 - 314.2 C3 15N-Ciprofloxacin 5.1 20 8.1 20 1.3 5
13
Sulfamethoxazole 11.2 254.0 - 156.0 C6-Sulfamethoxazole 0.4 2 1.2 5 0.1 0.5
13
Lomefloxacin 11.2 352.2 - 308.1 C3 15N-Ciprofloxacin 4.9 10 4.4 10 1.2 2.5
13
Enrofloxacin 11.5 360.0 - 316.0 C3 15N-Ciprofloxacin 5.2 10 3.1 10 1.3 2.5
13
Sarafloxacin 11.9 386.0 - 299.0 C3 15N-Ciprofloxacin 170 200 -- 200 42 12.5
13
Clinafloxacin 12.1 366.3 - 348.1 C3 15N-Ciprofloxacin 6.9 20 14 50 1.7 5

45
December 2007 Method 1694

Parent- Detection limits and minimum levels

daughter Water (ng/L) Other (ng/g) Extract (ng/ΦL)
Analyte RT (min) m/zs Quantitation reference MDL ML MDL ML MDL ML
13
Digoxigenin 12.6 391.2 - 355.2 C3-Trimethoprim 5.7 20 9.4 20 1.4 5
13
Oxolinic acid 13.1 261.8 - 243.8 C3-Trimethoprim 0.6 2 0.62 2 0.2 0.5
13
Sulfadimethoxine 13.2 311.0 - 156.0 C6-Sulfamethoxazole 0.1 1 0.55 2 0.03 0.25
13
Diphenhydramine 14.5 256.8 - 168.1 C3-Trimethoprim 0.4 2 0.66 2 0.1 0.5
13
Penicillin G 14.6 367.5 - 160.2 C3-Trimethoprim 2.4 10 13 50 0.6 2.5
13
Azithromycin 14.8 749.9 - 591.6 C3-Trimethoprim 1.3 5 1.6 5 0.3 1.25
13
Flumeqine 15.2 262.0 - 173.7 C3-Trimethoprim 2.7 5 1.4 5 0.7 1.25
13
Ampicillin 15.3 350.3 - 160.2 C3-Trimethoprim -- 5 -- 5 -- 1.25
13
Diltiazem 15.3 415.5 - 178.0 C3-Trimethoprim 0.6 2 0.30 2 0.2 0.25
13
Carbamazepine 15.3 237.4 - 194.2 C3-Trimethoprim 1.4 5 1.6 5 0.4 1.25
13
Penicillin V 15.4 383.4 - 160.2 C3-Trimethoprim 4.4 20 19 50 1.1 5
13
Erythromycin 15.9 734.4 - 158.0 C2-Erythromycin -- 1 -- 2 -- 0.25
13
Tylosin 16.3 916.0 - 772.0 C2-Erythromycin anhydrate 13 50 8.1 50 3.2 5
13
Oxacillin 16.4 434.3 - 160.1 C3-Trimethoprim 3.3 10 9.4 20 0.8 2.5
13
Dehydronifedipine 16.5 345.5 - 284.1 C3-Trimethoprim 0.6 2 0.41 2 0.2 0.5
13
Digoxin 16.6 803.1 - 283.0 C3-Trimethoprim -- 50 -- 100 -- 12.5
Fluoxetine 16.9 310.3 - 148.0 Fluoxetine-d5 3.7 10 2.8 10 0.9 1.25
13
Cloxcillin 16.9 469.1 - 160.1 C3-Trimethoprim 4.3 10 9.2 20 0.1 2.5
13
Virginiamycin 17.3 508.0 - 355.0 C3-Trimethoprim 3.6 10 3.4 10 0.9 2.5
13
Clarithromycin 17.5 748.9 - 158.2 C2-Erythromycin anhydrate 1.0 5 1.2 5 0.3 1.25
13
Erythromycin anhydrate 17.7 716.4 - 158.0 C2-Erythromycin anhydrate 0.4 2 0.46 2 0.1 0.25
13
Roxithromycin 17.8 837.0 - 679.0 C2-Erythromycin anhydrate 0.2 1 0.22 1 0.05 0.25
13
Miconazole 20.1 417.0 - 161.0 C3-Trimethoprim 1.3 5 0.90 5 0.3 1.25
13
Norgestimate 21.7 370.5 - 124.0 C3-Trimethoprim 2.5 10 1.4 10 0.6 2.5
Labeled compounds spiked into each sample
13
Cotinine-d3 2.8 180.0 - 79.9 C3 Atrazine
13
C2­ 15N-Acetaminophen 4.5 155.2 - 111.0 13
C3 Atrazine
13 13
C3 Caffeine 9.3 198.0 - 140.0 C3 Atrazine
13
Thiabendazole-d6 9.8 208.1 - 180.1 C3 Atrazine
13 13
C3-Trimethoprim 10.0 294.0 - 233.0 C3 Atrazine
13 13
C6 Sulfamethazine 10.1 285.1 - 162.0 C3 Atrazine
13
C3 15N-Ciprofloxacin 10.9 336.1 - 318.0 13
C3 Atrazine
13 13
C6-Sulfamethoxazole 11.2 260.0 - 162.0 C3 Atrazine
13 13
C2-Erythromycin 15.9 736.4 - 160.0 C3 Atrazine

46
December 2007 Method 1694

Parent- Detection limits and minimum levels

daughter Water (ng/L) Other (Φg/g) Extract (ng/ΦL)
Analyte RT (min) m/zs Quantitation reference MDL ML MDL ML MDL ML
13
Fluoxetine-d5 16.8 315.3 - 153.0 C3 Atrazine
13 13
C2-Erythromycin anhydrate 17.7 718.4 - 160.0 C3 Atrazine
Injection internal standard
13 219.5 - 176.9
C3 Atrazine 15.9 External standard
(134.0)

47
December 2007 Method 1694

Table 4. Group 2 – Acidic extraction positive electrospray ionization (ESI+) instrument conditions

Instrument Waters 2690 HPLC or Waters 2795 HPLC, Micromass Quattro Ultima MS/MS
LC Column Waters Xtera C18, 10.0 cm, 2.1 mm i.d., 3.5 µm particle size
Ionization Positive Ion Electrospray
Acquisition MRM mode, unit resolution
Injection Volume 5 µL

LC Gradient Program LC Flow General LC Conditions

Time Rate Column Temp 40 °C
1
(min) Flow Mixture (mL/min) Gradient
10% Solvent A 0.20 – 0.23
0.0 0.20 1 Flow Rate
90% Solvent B mL/min
10% Solvent A
1.0 0.20 6 Max Pressure 345 Bar
90% Solvent B
40% Solvent A Autosampler tray
18.0 0.23 6 4ºC
60% Solvent B temperature
90% Solvent A
20.0 0.23 6 MS Conditions
10% Solvent B
90% Solvent A
24.0 0.23 6 Source Temp 120°C
10% Solvent B
10% Solvent A
24.3 0.20 6 Desolvation Temp 400°C
90% Solvent B
10% Solvent A Cone / Desolvation 70 L/hr /
28 0.20 6
90% Solvent B Gas Rate 450 L/hr
1
Solvent A = 1:1 acetonitrile:methanol, with 5 mM Oxalic Acid
Solvent B = HPLC H2O, with 5 mM Oxalic Acid

48
December 2007 Method 1694

Table 5. Group 2 acidic extraction positive electrospray ionization (ESI+) compound retention times (RTs), parent-daughter transitions,
quantitation references, method detection limits, and minimum levels of quantitation.

Parent- Detection limits and minimum levels

RT daughter Water (ng/L) Other (ng/g) Extract (ng/μL)
Analyte (min) m/zs Quantitation reference MDL ML MDL ML MDL ML
Group 2 Analytes Extracted Under Acidic Conditions and Analyzed Using Positive Electrospray Ionization (+) ESI.
Native compounds
Minocycline 5.1 458.0 - 441.0 Thiabendazole-d6 51 200 -- 200 13 50
Epitetracycline 8.1 445.2 - 410.2 Thiabendazole-d6 3.6 20 8.6 20 0.9 5
Epioxytetracycline (EOTC) 8.6 461.2 - 426.2 Thiabendazole-d6 4.1 20 18 50 1.0 5
Oxytetracycline (OTC) 9.4 461.2 - 426.2 Thiabendazole-d6 2.1 20 2.2 20 0.5 5
Tetracycline (TC) 9.9 445.2 - 410.2 Thiabendazole-d6 1.9 20 2.8 20 0.5 5
Demeclocycline 11.7 465.0 - 430.0 Thiabendazole-d6 6.6 50 7.9 50 1.7 12.5
Isochlortetracycline (ICTC) 1 11.9 479.0 - 462.2 Thiabendazole-d6 1.7 20 3.5 20 0.4 5
Epichlortetracycline (ECTC) 1 12.0 479.0 - 444.0 Thiabendazole-d6 7.7 50 26 100 1.9 12.5
Chlortetracycline (CTC) 14.1 479.0 - 444.0 Thiabendazole-d6 1.2 20 2.3 20 0.3 5
Doxycycline 16.7 445.2 - 428.2 Thiabendazole-d6 2.8 20 2.3 20 0.7 5
Epianhydrotetracycline (EATC) 17.0 426.8 - 409.8 Thiabendazole-d6 7.7 50 14 50 1.9 12.5
Anhydrotetracycline (ATC) 18.8 426.8 - 409.8 Thiabendazole-d6 4.6 50 7.1 50 1.2 12.5
Epianhydrochlortetracycline (EACTC) 20.7 461.2 - 444.0 Thiabendazole-d6 28 200 23 200 7.0 50
Anhdrochlortetracycline (ACTC) 22.1 461.2 - 444.0 Thiabendazole-d6 5.2 50 11 50 1.3 12.5
Labeled compound spiked into each sample
13
Thiabendazole-d6 7.0 208.1 - 180.1 C3 Atrazine
Injection internal standard
13 219.5 - 176.9
C3 Atrazine 10.5 External standard
(134.0)
1. Isochlortetracycline (ICTC) is reported as the sum ICTC + ECTC due to a common transition ion.

49
December 2007 Method 1694

Table 6. Group 3 – Acidic extraction negative electrospray ionization (ESI-) instrument conditions

Instrument Waters 2690 HPLC or Waters 2795 HPLC, Micromass Quattro Ultima MS/MS
LC Column Waters Xtera C18MS, 10.0 cm, 2.1 mm i.d., 3.5 µm particle size
Ionization Negative Ion Electrospray
Acquisition MRM mode, unit resolution
Injection Volume 15 μL

LC Gradient Program LC Flow General LC Conditions

Time Rate
1
(min) Flow Mixture (mL/min) Gradient Column Temp 40°C
60% Solvent A,
0.0 0.2 1 Flow Rate 0.200 mL/min
40% Solvent B
60% Solvent A,
0.5 0.2 6 Max Pressure 345 Bar
40% Solvent B
Autosampler tray
7.0 100% Solvent B 0.2 6 4ºC
temperature
12.5 100%Solvent B 0.2 6 MS Conditions
60% Solvent A,
12.7 0.2 6 Source Temp 100°C
40% Solvent B
60% Solvent A,
16.0 0.2 1 Desolvation Temp 350°C
40% Solvent B
Cone / Desolvation 50L/hr /
Gas Rate 300 L/hr

1. Solvent A = 0.1% Ammonium Acetate and 0.1% Acetic Acid in HPLC water
Solvent B = 1:1 MethanolAcetonitrile

50
December 2007 Method 1694

Table 7. Group 3 acidic extraction negative electrospray ionization (ESI-) compound retention times (RTs), parent-daughter transitions,
quantitation references, method detection limits, and minimum levels of quantitation

Detection limits and minimum levels

RT Parent- daughter Water (ng/L) Other (Φg/g) Extract (ng/ΦL)
Analyte (min) m/zs Quantitation reference MDL ML MDL ML MDL ML
Group 3 Analytes Extracted Under Acidic Conditions and Analyzed Using Negative Electrospray Ionization (-) ESI.
Native compounds
13
Naproxen 6.7 228.9 - 168.6 C-Naproxen-d3 3.9 10 6.1 20 1.0 2.5
Warfarin 7.1 307.0 - 117.0 Warfarin-d5 0.9 5 1.6 5 0.2 1.25
13
Ibuprofen 8.4 205.1 - 161.1 C3-Ibuprofen 6.0 50 11 50 1.5 12.5
Gemfibrozil 9.5 249.0 - 121.0 Gemfibrozil-d6 0.8 5 1.2 5 0.2 1.25
13
Triclocarban 9.6 312.9 - 159.7 C6-Triclocarban 2.1 10 2.7 10 0.5 2.5
13
Triclosan 9.7 286.8 - 35.0 C12-Triclosan 92 200 56 200 23 50
Labeled compounds spiked into samples
13 13
C-Naproxen-d3 6.6 232.9 - 168.6 C6-TCPAA
13
Warfarin-d5 7.0 312.0 - 161.0 C6-TCPAA
13 13
C3-Ibuprofen 8.5 208.2 - 163.1 C6-TCPAA
13
Gemfibrozil-d6 9.5 255.0 - 121.0 C6-TCPAA
13 13
C6-Triclocarban 9.6 318.9 - 159.7 C6-TCPAA
13 13
C12-Triclosan 9.7 298.8 - 35.0 C6-TCPAA
Injection Internal Standard
13
C6-TCPAA 4.9 258.8 - 200.7 External standard

51
December 2007 Method 1694

Table 8. Group 4 – Basic extraction positive electrospray ionization (ESI+) instrument conditions

Instrument Waters 2690 HPLC or Waters 2795 HPLC, Micromass Quattro Ultima MS/MS
LC Column Waters Atlantis HILIC, 10 cm, 2.1 mm i.d., 3.0 µm particle size
Ionization Positive Ion Electrospray
Acquisition MRM mode, unit resolution
Purge Solvent 100% CH3CN (changed from H2O)
Injection Volume 2.0 μL

LC Gradient Program LC Flow General LC Conditions

Time Rate
1
(min) Flow Mixture (mL/min) Gradient Column Temp 40 °C
2% Solvent A
0.0 0.25 1 Flow Rate 0.25 mL/min
98% Solvent B
30% Solvent A
5.0 0.25 6 Max Pressure 345 Bar
70% Solvent B
30% Solvent A Autosampler tray
12.0 0.25 6 4ºC
70% Solvent B temperature
2% Solvent A
12.5 0.25 6 MS Conditions
98% Solvent B
2% Solvent A
16.0 0.25 6 Source Temp 120°C
98% Solvent B
Desolvation Temp
350°C
Cone / Desolvation 70L/hr /
Gas Rate 400 L/hr

1. Solvent A = 0.1% Acetic Acid/Ammonium Acetate Buffer

Solvent B = Acetonitrile

52
December 2007 Method 1694

Table 9. Group 4 basic extraction positive electrospray ionization (ESI+) compound retention times (RTs), parent-daughter transitions,
quantitation references, method detection limits, and minimum levels of quantitation

Parent- Detection limits and minimum levels

RT daughter Water (ng/L) Other (ng/g) Extract (ng/ΦL)
Quantitation reference
Analyte (min) m/zs MDL ML MDL ML MDL ML
Group 4 Analytes Extracted Under Basic Conditions and Analyzed Using Positive Electrospray Ionization (+) ESI
Native compounds
Cimetidine 6.9 253.1 - 159.0 Albuterol-d3 0.6 2 0.78 2 0.2 0.5
Albuterol 9.4 240.0 - 148.0 Albuterol-d3 0.9 2 0.39 2 0.2 0.5
Ranitidine 10.3 315.0 - 175.9 Albuterol-d3 0.7 2 1.1 2 0.2 0.5
Metformin 11.0 131.1 - 60.1 Metformin-d6 23 100 38 100 5.8 25
Labeled compounds spiked into samples
Albuterol-d3 9.4 243.0 - 151.0 Cotinine-d3
Metformin-d6 11.0 285.1 - 162.0 Cotinine-d3
Injection internal standard
Cotinine-d3 5.9 180.0 - 79.9 External standard
219.5 - 176.9
13C3-Atrazine 2.0 External Standard
(134.0)

53
December 2007 Method 1694

Table 10. Nominal concentrations of native compounds, labelled compounds, and instrument internal
standard solutions 1

Typical amount spiked

Spiking solution
Compound Name into sample
concentration (μg/mL)
(ng)
Native compound spike solutions for acid (Typical spiking volume
extracted analytes (Groups 1 and 3) into sample: 30 µL)
Acetaminophen 100 3000
Azithromycin 2.5 75
Caffeine 25 750
Carbodox 2.5 75
Carbamazapine 2.5 75
Cefotaxime 10 300
Clarithromycin 2.5 75
Cloxacillin 5 150
Codeine 5 150
Cotinine 2.5 75
Dehydronifedipine (Oxidized Nifedipine) 1 30
Diphenhydramine 1 30
Diltiazem 0.5 15
Digoxin 25 750
Digoxigenin 10 300
Erythromycin 0.5 15
Flumequine 2.5 75
Fluoxetine 2.5 75
Lincomycin 5 150
Miconazole 2.5 75
Norgestimate 5 150
Ormetoprim 1 30
Oxacillin 5 150
Oxolinic acid 1 30
Penicillin G 5 150
Penicillin V 5 150
Roxithromycin 0.5 15
Sulfachloropyridazine 2.5 75
Sulfadiazine 2.5 75
Sulfadimethoxine 0.5 15
Sulfamerazine 1 30
Sulfamethazine 1 30
Sulfamethizole 1 30
Sulfamethoxazole 1 30
Sulfanilamide 25 750

54
December 2007 Method 1694

Typical amount spiked

Spiking solution
Compound Name into sample
concentration (μg/mL)
(ng)
Sulfathiazole 2.5 75
Thiabendazole 2.5 75
Trimethoprim 2.5 75
Tylosin 10 300
Virginiamycin 5 150
1,7-Dimethylxanthine 250 7500
Ampicillin 2.5 75
Ciprofloxacin 8.75 263
Clinafloxacin 10 300
Enrofloxacin 5 150
Lomefloxacin 5 150
Norfloxacin 25 750
Ofloxacin 2.5 75
Sarafloxacin 22.8 684
Gemfibrozil 2.5 75
Ibuprofen 25 750
Naproxen 5 150
Triclocarban 5 150
Triclosan 100 3000
Warfarin 2.5 75
Native compound spike solutions for (Typical spiking volume
tetracyclines (Group 2) into sample: 200 µL)
Tetracycline (TC) 0.5 100
Oxytetracycline (OTC) 0.5 100
Doxycycline 0.5 100
Chlortetracycline (CTC) 0.5 100
Anhydrochlortetracycline (ACTC) 1.25 250
Anhydrotetracycline (ATC) 1.25 250
4-Epianhydrochlortetracycline (EACTC) 5 1000
4-Epianhydrotetracycline (EATC) 1.25 250
4-Epichlortetracycline (ECTC) 1.25 250
4-Epioxytetracycline (EOTC) 0.5 100
4-Epitetracycline (ETC) 0.5 100
Isochlortetracycline (ICTC) 0.5 100
Demeclocycline 1.25 250
Minocycline 5 1000
Native compound spike solutions for base (Typical spiking volume
extracted analytes (Group 4) into sample: 15 µL)
Albuterol 1 15
Cimetidine 2 30

55
December 2007 Method 1694

Typical amount spiked

Spiking solution
Compound Name into sample
concentration (μg/mL)
(ng)
Metformin 100 1500
Ranitidine 2 30
Labeled compound solutions for acid (Typical spiking volume
extracted analytes (Groups 1, 2 and 3) into sample: 100 µL)
Meclocycline 8 800
d10-Carbamazepine-10,11-epoxide 2 200
d3-Cotinine 2 200
d5-Fluoxetine 1 100
d6-Gemfibrozil 1 100
13
C2, 15N-Acetaminophen 4 400
13
C6-Sulfamethoxazole 1 100
13
C, d3-Naproxen 3 300
13
C6-Triclocarban 0.5 50
13
C3-Trimethoprim 1 100
d6-Thiabendazole 1 100
13
C3-Caffeine 3 300
13
C2-Erythromycin 1 100
13
C12-Triclosan 4 400
d5-Warfarin 1 100
13
C6-Sulfamethazine 1 100
13
C3, 15N-Ciprofloxacin 4 400
13
C3-Ibuprofen 4 400
Labeled compound solutions for base (Typical spiking volume
extracted analytes (Group 4) into sample: 100 µL)
d3-Albuterol 1 100
d6-Metformin 4 400
Instrument internal standard solutions for (Typical spiking volume
acid extracted analytes (Groups 1, 2 and 3) into extract: 80 µL)
13
C3-Atrazine 2.5 200
13
C6-2,4,5-Trichlorophenoxyacetic acid 2.5 200
Instrument internal standard solutions for (Typical spiking volume
base extracted analytes (Group 4) into extract: 100 µL)
13
C3-Atrazine 2 200
d3-Cotinine 2 200
1. See Sections 7.8 – 7.9 for solution details

56
December 2007 Method 1694

Tables 11a-c. Concentrations of calibration solutions (ng/mL)

Table 11a Concentrations of calibration standards for Group 1 and Group 3 compounds (ng/mL) (Acid
extraction, positive and negative ESI). CS=calibration standard.
Compound CS-1 CS-2 CS-3 (VER) CS-4 CS-5
Acetaminophen 50 150 750 2500 10000
Azithromycin 1.25 3.75 18.7 62.5 250
Caffeine 12.5 37.5 187. 625 2500
Carbadox 1.25 3.75 18.7 62.5 250
Carbamazapine 1.25 3.75 18.7 62.5 250
Cefotaxime 5 15 75 250 1000
Clarithromycin 1.25 3.75 18.7 62.5 250
Cloxacillin 2.5 7.5 37.5 125 500
Codeine 2.5 7.5 37.5 125 500
Cotinine 1.25 3.75 18.7 62.5 250
Dehydronifedipine (Oxidized Nifedipine) 0.5 1.5 7.5 25 100
Diphenhydramine 0.5 1.5 7.5 25 100
Diltiazem 0.25 0.75 3.75 12.5 50
Digoxin 12.5 37.5 187 625 2500
Digoxigenin 5 15 75 250 1000
Erythromycin 0.25 0.75 3.75 12.5 50
Erythromycin anhydrate 0.25 0.75 3.75 12.5 50
Flumequine 1.25 3.75 18.7 62.5 250
Fluoxetine 1.25 3.75 18.7 62.5 250
Lincomycin 2.5 7.5 37.5 125 500
Miconazole 1.25 3.75 18.7 62.5 250
Norgestimate 2.5 7.5 37.5 125 500
Ormetoprim 0.5 1.5 7.5 25 100
Oxacillin 2.5 7.5 37.5 125 500
Oxolinic acid 0.5 1.5 7.5 25 100
Penicillin G 2.5 7.5 37.5 125 500
Penicillin V 5 15 75 250 1000
Roxithromycin 0.25 0.75 3.75 12.5 50
Sulfachloropyridazine 1.25 3.75 18.7 62.5 250
Sulfadiazine 1.25 3.75 18.7 62.5 250
Sulfadimethoxine 0.25 0.75 3.75 12.5 50
Sulfamerazine 0.5 1.5 7.5 25 100
Sulfamethazine 0.5 1.5 7.5 25 100
Sulfamethizole 0.5 1.5 7.5 25 100
Sulfamethoxazole 0.5 1.5 7.5 25 100
Sulfanilamide 12.5 37.5 187.5 625 2500
57
December 2007 Method 1694

Compound CS-1 CS-2 CS-3 (VER) CS-4 CS-5

Sulfathiazole 1.25 3.75 18.7 62.5 250
Thiabendazole 1.25 3.75 18.7 62.5 250
Trimethoprim 1.25 3.75 18.7 62.5 250
Tylosin 5 15 75 250 1000
Virginiamycin 2.5 7.5 37.5 125 500
1,7-Dimethylxanthine 125 375 1870 6250 25000
Ampicillin 1.25 3.75 18.7 62.5 250
Ciprofloxacin 4.4 13.1 65.6 218. 875
Clinafloxacin 5 15 75 250 1000
Enrofloxacin 2.5 7.5 37.5 125 500
Lomefloxacin 2.5 7.5 37.5 125 500
Norfloxacin 12.5 37.5 187 625 2500
Ofloxacin 1.25 3.75 18.7 62.5 250
Sarafloxacin 11.4 34.2 171 570 2280
Gemfibrozil 1.25 3.75 18.7 62.5 250
Ibuprofen 12.5 37.5 187 625 2500
Naproxen 2.5 7.5 37.5 125 500
Triclocarban 2.5 7.5 37.5 125 500
Triclosan 50 150 750 2500 10000
Warfarin 1.25 3.75 18.7 62.5 250
Labeled compounds
d3-Cotinine 50 50 50 50 50
d5-Fluoxetine 25 25 25 25 25
d6-Gemfibrozil 25 25 25 25 25
13 15
C2, N-Acetaminophen 100 100 100 100 100
13
C6-Sulfamethoxazole 25 25 25 25 25
13
C-d3-Naproxen 75 75 75 75 75
13
C6-Triclocarban 12.5 12.5 12.5 12.5 12.5
13
C3-Trimethoprim 25 25 25 25 25
d6-Thiabendazole 25 25 25 25 25
13
C3-Caffeine 75 75 75 75 75
13
C2-Erythromycin 25 25 25 25 25
13
C12-Triclosan 90 90 90 90 90
d5-Warfarin 25 25 25 25 25
13
C6-Sulfamethazine 25 25 25 25 25
13 15
C3, N-Ciprofloxacin 100 100 100 100 100
13
C3-Ibuprofen 100 100 100 100 100

58
December 2007 Method 1694

Compound CS-1 CS-2 CS-3 (VER) CS-4 CS-5

Instrument internal standards
13
C3-Atrazine 50 50 50 50 50
13
C6-2,4,5-Trichlorophenoxyacetic acid 50 50 50 50 50

Table 11b Concentrations of calibration standards for Group 2 compounds (ng/mL) (Acid extraction,
positive ESI). CS=calibration standard.
Compound name CS-1 CS-2 CS-3 (VER) CS-4 CS-5
Tetracycline (TC) 5 12.5 25 50 150
Oxytetracycline (OTC) 5 12.5 25 50 150
Doxycycline 5 12.5 25 50 150
Chlortetracycline (CTC) 5 12.5 25 50 150
Anhydrochlortetracycline (ACTC) 12.5 31.25 62.5 125 375
Anhydrotetracycline (ATC) 12.5 31.25 62.5 125 375
4-Epianhydrochlortetracycline (EACTC) 50 125 250 500 1500
4-Epianhydrotetracycline (EATC) 12.5 31.2 62.5 125 375
4-Epichlortetracycline (ECTC) 12.5 31.2 62.5 125 375
4-Epioxytetracycline (EOTC) 5 12.5 25 50 150
4-Epitetracycline (ETC) 5 12.5 25 50 150
Isochlortetracycline (ICTC) 5 12.5 25 50 150
Demeclocycline 12.5 31.2 62.5 125 375
Minocycline 50 125 250 500 1500
Labeled compounds
d3-Cotinine 50 50 50 50 50
d5-Fluoxetine 25 25 25 25 25
d6-Gemfibrozil 25 25 25 25 25
13 15
C2, N-Acetaminophen 100 100 100 100 100
13
C6-Sulfamethoxazole 25 25 25 25 25
13
C, d3-Naproxen 75 75 75 75 75
13
C6-Triclocarban 12.5 12.5 12.5 12.5 12.5
13
C3-Trimethoprim 25 25 25 25 25
1
d6-Thiabendazole 25 25 25 25 25
13
C3-Caffeine 75 75 75 75 75
13
C2-Erythromycin 25 25 25 25 25
13
C12-Triclosan 90 90 90 90 90
d5-Warfarin 25 25 25 25 25
13
C6-Sulfamethazine 25 25 25 25 25
13 15
C3, N-Ciprofloxacin 100 100 100 100 100
13
C3-Ibuprofen 100 100 100 100 100
Instrument internal standards

59
December 2007 Method 1694

Compound name CS-1 CS-2 CS-3 (VER) CS-4 CS-5

13
C3-Atrazine1 50 50 50 50 50
13
C6-2,4,5-Trichlorophenoxyacetic acid 50 50 50 50 50
1. Note: The Group 2, acid extracted positive ESI (tetracyclines) contains the same labeled compounds as
for Group 1 and 3, acid extracted positive and negative ESI, yet the only labeled compounds used in
determination of the Group 2 are Thiabendazole-d6 and 13C3-Atrazine. This minimizes the work
required to prepare solutions. Some of those surrogates are used to quantify the Group 1 and 2 and some
Group 3 in separate runs of the same extract. This is not a requirement.

Table 11c Concentrations of calibration standards for Group 4 (ng/mL) compounds (Base extraction,
positive ESI). CS=calibration standard.
Compound name CS-1 CS-2 CS-3 (VER) CS-4 CS-5
Albuterol 0.25 0.75 3.75 12.5 50
Cimetidine 0.5 1.5 7.5 25 100
Metformin 25 75 375 1250 5000
Ranitidine 0.5 1.5 7.5 25 100
Labeled compounds
d3-Albuterol 25 25 25 25 25
d6-Metformin 100 100 100 100 100
Instrument internal standards
13
C3-Atrazine 50 50 50 50 50
d3-Cotinine 50 50 50 50 50

60
December 2007 Method 1694

Table 12. QC acceptance criteria for PPCPs in VER, IPR, OPR, and samples.

IPR
VER RSD X OPR Labeled compound
Compound (%) (%) (%) (%) recovery in samples (%)
Acetaminophen 70 - 130 30 55 - 108 50 - 120
Albuterol 70 - 130 30 55 - 120 50 - 133
Ampicillin 70 - 130 70 6 - 180 5 - 200
Anhydrochlortetracycline (ACTC) 70 - 130 30 55 - 121 50 - 135
Anhydrotetracycline (ATC) 70 - 130 30 8 - 127 7 - 141
Azithromycin 70 - 130 30 36 - 108 33 - 120
Caffeine 70 - 130 30 55 - 111 50 - 124
Carbadox 70 - 130 30 36 - 130 33 - 144
Carbamazepine 70 - 130 30 23 - 123 21 - 137
Cefotaxime 70 - 130 36 9 - 168 8 - 186
Chlortetracycline (CTC) 70 - 130 31 49 - 155 45 - 172
Cimetidine 70 - 130 47 6 - 108 5 - 120
Ciprofloxacin 70 - 130 30 55 - 108 50 - 120
Clarithromycin 70 - 130 30 8 - 139 8 - 154
Clinafloxacin 70 - 130 37 6 - 180 5 - 200
Cloxacillin 70 - 130 30 6 - 180 5 - 200
Codiene 70 - 130 30 37 - 116 34 - 129
Cotinine 70 - 130 30 55 - 112 50 - 124
Dehydronifedipine 70 - 130 30 47 - 108 42 - 120
Demeclocycline 70 - 130 30 6 - 180 5 - 200
Digoxigenin 70 - 130 30 8 - 165 8 - 183
Digoxin 70 - 130 45 6 - 133 5 - 148
Diltiazem 70 - 130 48 13 - 108 11 - 120
1,7-Dimethylxanthine 70 - 130 30 55 - 124 50 - 138
Diphenhydramine 70 - 130 30 53 - 108 48 - 120
Doxycycline 70 - 130 30 24 - 149 22 - 166
Enrofloxacin 70 - 130 30 55 - 113 50 - 125
4-Epianhydrochlortetracycline (EACTC) 70 - 130 30 20 - 108 18 - 120
4-Epianhydrotetracycline (EATC) 70 - 130 30 6 - 180 5 - 200
4-Epichlortetracycline (ECTC) 70 - 130 30 55 - 135 50 - 150
4-Epioxytetracycline (EOTC) 70 - 130 30 55 - 127 50 - 142
4-Epitetracycline (ETC) 70 - 130 30 55 - 156 50 - 173
Erythromycin hydrate 70 - 130 30 55 - 142 50 - 158
Flumequine 70 - 130 30 39 - 180 36 - 200
Fluoxetine 70 - 130 30 54 - 112 49 - 125
Gemfibrozil 70 - 130 30 55 - 108 50 - 120
Ibuprofen 70 - 130 30 55 - 108 50 - 120
Isochlortetracycline (ICTC) 70 - 130 30 6 - 180 5 - 200
Lincomycin 70 - 130 60 6 - 108 5 - 120
Lomefloxacin 70 - 130 33 19 - 180 17 - 200
Metformin 70 - 130 30 55 - 134 50 - 149
Miconazole 70 - 130 30 29 - 108 27 - 120
Minocycline 70 - 130 30 6 - 159 5 - 176

61
December 2007 Method 1694

IPR
Naproxen 70 - 130 30 55 - 108 50 - 120
Norfloxacin 70 - 130 30 55 - 121 50 - 135
Norgestimate 70 - 130 30 39 - 108 36 - 120
Ofloxacin 70 - 130 30 55 - 180 50 - 200
Ormetoprim 70 - 130 30 55 - 108 50 - 120
Oxacillin 70 - 130 30 6 - 180 5 - 200
Oxolinic acid 70 - 130 30 46 - 112 42 - 124
Oxytetracycline (OTC) 70 - 130 30 55 - 165 50 - 183
Penicillin V 70 - 130 30 6 - 180 5 - 200
Penicillin G 70 - 130 30 6 - 180 5 - 200
Ranitidine 70 - 130 41 26 - 144 24 - 160
Roxithromycin 70 - 130 30 42 - 108 38 - 120
Sarafloxacin 70 - 130 32 18 - 180 17 - 200
Sulfachloropyridazine 70 - 130 30 55 - 180 50 - 200
Sulfadiazine 70 - 130 30 6 - 180 5 - 200
Sulfadimethoxine 70 - 130 30 55 - 108 50 - 120
Sulfamerazine 70 - 130 30 55 - 133 50 - 148
Sulfamethazine 70 - 130 30 55 - 128 50 - 142
Sulfamethizole 70 - 130 30 55 - 108 50 - 120
Sulfamethoxazole 70 - 130 30 55 - 108 50 - 120
Sulfanilamide 70 - 130 71 6 - 170 5 - 189
Sulfathiazole 70 - 130 30 45 - 108 41 - 120
Tetracycline (TC) 70 - 130 30 55 - 139 50 - 155
Thiabendazole 70 - 130 30 55 - 108 50 - 120
Triclocarban 70 - 130 30 55 - 108 50 - 120
Triclosan 70 - 130 30 55 - 108 50 - 120
Trimethoprim 70 - 130 30 55 - 114 50 - 126
Tylosin 70 - 130 30 17 - 134 16 - 149
Virginiamycin 70 - 130 33 6 - 170 5 - 189
Warfarin 70 - 130 30 55 - 108 50 - 120

13
C2­ 15N-Acetaminophen 70 - 130 30 6 - 180 5 - 200 19 – 200
Albuterol-d3 70 - 130 30 38 - 109 35 - 121 39 – 141
13
C3-Caffeine 70 - 130 46 6 - 180 5 - 200 31 – 200
13
C3­ 15N-Ciprofloxacin 70 - 130 34 6 - 180 5 - 200 37 – 181
Cotinine-d3 70 - 130 84 6 - 108 5 - 120 5 – 145
13
C2-Erythromycin hydrate 70 - 130 30 55 - 108 50 - 120 23 – 120
Fluoxetine-d5 70 - 130 30 55 - 113 50 - 126 40 – 148
Gemfibrozil-d6 70 - 130 30 42 - 110 38 - 122 21 – 123
13
C3-Ibuprofen 70 - 130 30 31 - 109 28 - 122 29 – 127
Metformin-d6 70 - 130 30 6 - 127 5 - 141 5 – 200
13
C-Naproxen-d3 70 - 130 30 37 - 118 34 - 131 14 – 132
13
C6-Sulfamethazine 70 - 130 30 6 - 141 5 - 157 12 – 120
13
C6-Sulfamethoxazole 70 - 130 30 55 - 131 50 - 146 40 – 129
Thiabendazole-d6 (A Pos) 70 - 130 30 55 - 132 50 - 146 32 – 140
Thiabendazole-d6 (TCY) 70 - 130 30 55 - 108 50 - 120 30 – 132
13
C6-Triclocarban 70 - 130 30 6 - 155 5 - 172 5 – 147

62
December 2007 Method 1694

IPR
13
C12-Triclosan 70 - 130 30 6 - 151 5 - 168 5 – 153
13
C3-Trimethoprim 70 - 130 30 55 - 162 50 - 180 50 – 172
Warfarin-d5 70 - 130 30 55 - 159 50 - 177 50 – 200

63
December 2007 Method 1694

Table 13. Suggested sample quantities to be extracted for various matrices 1

Percent Quantity
Sample matrix2 Example Phase
solids extracted
Single-phase
Aqueous Drinking water
Groundwater No visible
Treated wastewater particles Aqueous 1000 mL
Solid Dry soil
Filter cake
Compost >20 Solid 1g

Multi-phase
Liquid/Solid
Aqueous/solid2 Wet soil 1 - 30
Untreated effluent 1-5 1g
Aqueous and
Municipal sludge 1 - 30 solid 0.25 g

1. The quantity of sample to be extracted is adjusted to provide 1 g of solids (dry weight). One liter of aqueous
samples containing 0.1% solids will contain 1 gram of solids. For aqueous samples containing greater than
0.1% solids, a lesser volume is used so that 1 gram of solids (dry weight) will be prepared.
2. 1 g of solids (0.25 g for biosolids), or 5 g wet weight if solids content is <20%.

64
 December 2007 Method 1694

Table 14. Performance Data from single laboratory validation.

Solid-Based on 5 samples Reagent Water-Based on 5 samples Biosolids-Based on 6 samples

Solids Water Biosolids

Solids Solids Relative Water Water Relative Biosolids Biosolids Relative
Average Standard Standard Average Standard Standard Average Standard Standard
Analyte Recovery Deviation Deviation Recovery Deviation Deviation Recovery Deviation Deviation
Group 3 acidic extraction ESI-
Warfarin 90.76 5.96 6.57 86.52 3.59 4.15 119.64 13.67 11.42
Ibuprofen 103.16 3.63 3.52 97.43 3.70 3.80 93.82 7.96 8.48
Gemfibrozil 97.94 2.84 2.90 98.11 2.45 2.50 78.35 21.19 27.05
Naproxen 96.57 6.23 6.45 95.41 5.03 5.27 99.94 10.10 10.10
Triclocarban 100.60 2.36 2.34 106.09 4.81 4.53 265.00 190.08 71.73
Triclosan 97.52 6.78 6.95 93.14 3.35 3.60 359.73 500.34 139.09
d5-Warfarin 113.44 8.21 7.23 143.13 8.22 5.74 145.03 23.97 16.52
13C3-Ibuprofen 59.25 2.34 3.95 90.20 8.04 8.91 74.30 21.35 28.74
d6-Gemfibrozil 65.37 2.85 4.36 94.94 3.16 3.33 65.80 28.35 43.09
13C-d3-Naproxen 65.52 5.45 8.31 98.95 3.99 4.03 55.18 20.45 37.06
13C6-Triclocarban 20.36 1.38 6.78 100.55 3.10 3.09 54.18 36.10 66.62
13C12-Triclosan 42.75 3.75 8.77 108.28 5.80 5.36 71.62 34.48 48.15
Group 1 acidic extraction ESI+
Acetaminophen 104.50 3.53 3.38 100.85 1.17 1.16 94.25 4.97 5.27
Azithromycin 66.08 11.99 18.15 60.95 6.80 11.15 86.53 25.39 29.34
Caffeine 96.41 10.08 10.45 99.14 6.38 6.44 86.08 5.95 6.91
Carbadox 107.42 3.78 3.52 69.50 7.48 10.76 52.91 13.50 25.52
Carbamazepine 98.84 6.85 6.93 59.14 4.92 8.32 91.50 19.27 21.06
Cefotaxime 122.83 4.32 3.52 71.75 23.92 33.33 173.69 15.86 9.13
Ciprofloxacin 95.76 3.54 3.70 99.66 2.20 2.21 73.93 69.08 93.44
Clarithromycin 54.67 4.07 7.44 106.90 2.64 2.47 69.53 12.17 17.50
Clinafloxacin 172.32 31.99 18.57 76.12 5.13 6.74 171.73 24.34 14.17
Cloxacillin 261.54 15.78 6.03 60.77 3.90 6.42 166.24 11.42 6.87
Codeine 97.65 1.79 1.83 64.66 5.36 8.29 141.78 11.87 8.38
Cotinine 96.04 3.79 3.94 102.27 9.75 9.53 92.34 4.56 4.94
Dehydronifedipine 84.14 6.80 8.09 66.50 7.17 10.78 126.82 13.52 10.66
Diphenhydramine 66.76 2.94 4.40 68.99 8.42 12.20 103.43 20.40 19.73
Diltiazem 66.96 3.47 5.19 55.23 20.89 37.83 160.04 114.83 71.75
Digoxin 92.33 13.28 14.39 52.22 18.64 35.69 22.60 18.11 80.13
Digoxigenin 126.01 1.96 1.55 64.36 8.55 13.28 79.57 11.63 14.61
Enrofloxacin 97.44 11.24 11.54 96.37 7.08 7.34 108.72 6.71 6.17
Erythromycin-H2O 136.67 3.12 2.28 113.95 3.09 2.71 100.45 7.46 7.42
Flumequine 151.31 7.43 4.91 91.15 4.08 4.48 92.35 14.79 16.02
Fluoxetine 88.09 16.86 19.14 85.89 4.27 4.97 100.48 19.64 19.55
Lincomycin 55.95 15.38 27.49 17.70 1.70 9.58 198.99 13.38 6.72
Lomefloxacin 179.94 32.28 17.94 106.07 6.19 5.83 79.59 9.63 12.10
Miconazole 51.31 4.73 9.23 73.81 6.50 8.81 55.79 17.71 31.74
Norfloxacin 101.34 6.89 6.80 114.79 5.07 4.41 63.02 7.17 11.37
Norgestimate 58.06 3.52 6.06 48.26 3.61 7.48 49.20 7.61 15.47

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Ofloxacin 166.98 26.78 16.04 127.81 12.16 9.51 78.44 34.96 44.57
Ormetoprim 64.83 2.86 4.41 66.94 3.44 5.14 78.76 5.96 7.56
Oxacillin 168.38 15.50 9.20 60.02 6.22 10.36 163.73 11.69 7.14
Oxolinic Acid 96.72 2.48 2.56 69.17 6.30 9.10 108.07 12.09 11.19
Penicillin G 214.04 14.66 6.85 58.83 6.77 11.51 99.09 14.24 14.37
Penicillin V 195.93 9.43 4.81 61.80 7.81 12.63 157.80 9.85 6.24
Roxithromycin 61.28 3.61 5.89 85.57 2.23 2.60 83.70 19.61 23.42
Sarafloxacin 146.84 25.89 17.63 87.70 4.25 4.84 108.36 8.57 7.91
Sulfachloropyridazine 158.30 8.72 5.51 115.36 3.88 3.36 90.24 9.86 10.92
Sulfadiazine 158.51 17.49 11.03 80.11 2.33 2.91 107.55 12.80 11.90
Sulfadimethoxine 78.65 3.44 4.37 87.00 2.95 3.39 67.87 9.14 13.46
Sulfamerazine 115.08 13.12 11.40 90.48 1.01 1.12 136.01 9.46 6.96
Sulfamethazine 119.60 5.59 4.67 100.67 6.19 6.15 103.35 11.96 11.57
Sulfamethizole 75.61 8.69 11.49 93.86 4.52 4.82 70.14 3.00 4.28
Sulfamethoxazole 103.21 2.97 2.88 88.17 3.63 4.12 102.56 12.66 12.34
Sulfanilamide 99.94 29.94 29.96 20.71 0.95 4.59 130.84 8.89 6.79
Sulfathiazole 59.06 3.39 5.74 76.73 3.22 4.20 92.10 8.07 8.76
Thiabendazole 106.47 1.44 1.35 99.83 1.92 1.93 81.89 6.03 7.36
Trimethoprim 103.24 3.23 3.13 80.82 5.65 6.99 98.81 5.35 5.42
Tylosin 60.99 9.93 16.28 103.48 9.59 9.27 47.80 14.56 30.46
Virginiamycin 116.39 10.65 9.15 43.62 15.26 34.99 172.33 42.36 24.58
1,7 DimethylXanthine 100.64 16.81 16.70 95.73 7.16 7.48 137.15 38.02 27.72
13C2-15N-Acetaminophen 258.79 19.66 7.60 112.20 4.48 3.99 137.17 28.54 20.80
13C3-Caffeine 203.04 50.76 25.00 115.82 7.57 6.54 119.47 9.50 7.95
d3-Cotinine 28.08 9.20 32.76 3.20 0.35 10.92 68.57 19.19 27.99
13C3-N15-Ciprofloxacin 66.74 18.81 28.19 144.50 16.99 11.75 132.12 13.74 10.40
13C2-Erythromycin-H2O 97.49 7.73 7.93 86.25 2.83 3.28 54.62 13.12 24.03
d5-Fluoxetine 92.68 8.40 9.07 103.69 6.05 5.84 94.67 35.78 37.79
13C6-Sulfamethazine 54.80 7.77 14.17 105.70 11.12 10.52 50.78 7.28 14.35
13C6-Sulfamethoxazole 85.21 9.29 10.90 111.77 9.32 8.34 72.67 10.63 14.63
d6-Thiabendazole 92.75 8.63 9.30 117.76 4.64 3.94 66.75 5.19 7.78
13C3-Trimethoprim 121.40 12.12 9.98 144.35 9.96 6.90 94.08 12.15 12.91
Group 4 basic extraction ESI+
Albuterol 100.43 7.12 7.09 90.04 14.09 15.65 96.58 1.88 1.95
Cimetidine 37.37 10.69 28.60 64.93 12.83 19.75 52.77 14.30 27.09
Metformin 115.61 9.40 8.13 103.72 13.16 12.69 89.06 3.32 3.72
Ranitidine 79.99 13.90 17.37 103.66 22.67 21.87 71.15 7.22 10.15
d3-Albuterol 91.52 8.47 9.25 63.83 2.41 3.78 105.42 19.25 18.26
d6-Metformin 94.38 13.05 13.83 51.66 6.86 13.28 161.13 67.48 41.88
Group 2 acidic extraction ESI+
Chlortetracycline 121.24 6.98 5.75 95.24 22.32 23.44 114.43 45.67 39.91
4-Epichlortetracycline 112.41 8.71 7.75 96.83 15.65 16.16 95.59 32.60 34.11
Anhydrochlortetracycline 92.62 10.43 11.27 102.22 11.51 11.26 50.40 21.73 43.12
4-Epianhydrochlortetracycline 53.57 1.87 3.49 82.28 13.31 16.18 33.88 8.30 24.49
Isochlortetracycline 65.88 5.01 7.61 149.37 16.06 10.75 91.65 25.51 27.83
Demeclocycline 54.53 1.96 3.59 136.58 3.18 2.33 76.03 31.01 40.79
Doxycycline 67.83 2.96 4.36 119.65 1.01 0.85 87.03 34.42 39.55
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 December 2007 Method 1694

Oxytetracycline 112.85 3.12 2.77 148.84 5.76 3.87 74.46 16.46 22.10
4-Epioxytetracycline 119.40 6.94 5.81 122.38 6.25 5.11 83.55 18.09 21.65
Tetracycline 93.41 3.95 4.23 124.79 4.69 3.76 77.98 19.24 24.68
4-Epitetracycline 138.95 3.42 2.46 102.11 4.02 3.94 97.37 37.03 38.03
4-Epianhydrotetracycline 70.11 6.87 9.80 170.82 22.25 13.02 67.87 23.27 34.29
Anhydrotetracycline 50.21 4.13 8.23 98.14 2.50 2.55 86.20 34.27 39.76
d6-Thiabendazole 77.07 4.76 6.18 64.80 3.21 4.95 89.82 15.10 16.81

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December 2007 Method 1694

Aqueous Sample Solid Portion (If required) Solid

Filter, or
Up to 1 g dry equivalent, not exceeding 5 g wet
Centrifuge + filter

15 mL pH 2.0 15 mL water
Phosphate Buffer NH4OH to pH 10.0

Aqueous Portion
Add labeled Add labeled
Up to 1000 mL
compounds, vortex compounds, vortex

Adjust to pH 2.0 Adjust to pH 10.0 20 mL CH3CN, 20 mL CH3CN,

with HCl with NH4OH sonicate 30 min, sonicate 30 min,
centrifuge centrifuge

Add labeled Add labeled 15 mL pH 2.0 Phosphate Buffer 15 mL water NH4OH to pH 10.0
compounds, stir compounds, stir 20 mL CH3CN, vortex 20 mL CH3CN, vortex
sonicate, centrifuge sonicate, centrifuge

500 mg Na4EDTA 20 mL CH3CN, vortex 20 mL CH3CN, vortex

sonicate, centrifuge sonicate, centrifuge

Rotary evaporation Rotary evaporation

Dilute
(200 mL reagent water), Dilute
no pH adjustment (200 mL reagent water)

500 mg Na4EDTA Check/adjust to pH 10.0

SPE HLB 20 cc/1 g SPE HLB 20 cc/1 g

condition condition

Load, Load,
Acid wash 10 mL water,
dry 5 min,
dry 5 min,
elute with 6 mL MeOH, Base
elute with 12 mL MeOH 9 mL 2% FA in MeOH

Elute with 6 mL acetone:MeOH 1:1

N2 blowdown, N2 blowdown,
reconstitute in 3 mL MeOH, reconstitute in 3 mL MeOH,
add injection internal standards, add injection internal standards,
dilute to 4 mL with 0.1% FA buffer, dilute to 4 mL with 0.1% FA buffer,
vortex vortex

(+) ESI (+) ESI (-) ESI (+) ESI

Group 1 Group 2 Group 3 Group 4

Figure 1 Flow chart for determination of pharmaceuticals and personal-care products by LC/MS/MS

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December 2007 Method 1694

24.0 Glossary
These definitions and purposes are specific to this method but have been conformed to
common usage to the extent possible.

24.1 Units of weight and measure and their abbreviations

24.1.1 Symbols

ΕC degrees Celsius
ΦL microliter
Φm micrometer
< less than
> greater than
% percent

24.1.2 Abbreviations (in alphabetical order)

cm centimeter
g gram
h hour
ID inside diameter
in. inch
L liter
M Molecular ion
m mass or meter
mg milligram
min minute
mL milliliter
mm millimeter
m/z mass-to-charge ratio
N normal; gram molecular weight of solute divided by hydrogen
equivalent of solute, per liter of solution
OD outside diameter
pg picogram
ppb part-per-billion
ppm part-per-million
ppq part-per-quadrillion
ppt part-per-trillion
psig pounds-per-square inch gauge
v/v volume per unit volume
w/v weight per unit volume

24.2 Definitions and acronyms (in alphabetical order)

Analyte – A pharmaceutical or personal-care product tested for by this method.

The analytes are listed in Table 1.

Calibration standard (CAL) – A solution prepared from a secondary standard

and/or stock solution and used to calibrate the response of the HPLC/MSMS

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December 2007 Method 1694

instrument.

Calibration verification standard (VER) – The mid-point calibration standard (CS­

4) that is used to verify calibration. See Table 4.

CS-1, CS-2, CS-3, CS-4, CS-5, CS-6 – See Calibration standards and Table 4.

Field blank – An aliquot of reagent water or other reference matrix that is placed in
a sample container in the field, and treated as a sample in all respects, including
exposure to sampling site conditions, storage, preservation, and all analytical
procedures. The purpose of the field blank is to determine if the field or sample
transporting procedures and environments have contaminated the sample.

GPC – Gel permeation chromatograph or gel permeation chromatography

HPLC – High performance liquid chromatograph or high performance liquid

chromatography

Labeled injection internal standard – A labeled spiked into the concentrated extract
immediately prior to injection of an aliquot of the extract into the LC/MS/MS.

Internal standard – a labeled compound used as a reference for quantitation of other

labeled compounds and for quantitation of a native compound other than the
compound of which it is a labeled analog. See Internal standard quantitation.

Internal standard quantitation – A means of determining the concentration of (1) a

naturally occurring (native) compound by reference to a compound other than its
labeled analog and (2) a labeled compound by reference to another labeled
compound.

IPR – Initial precision and recovery; four aliquots of a reference matrix spiked with
the analytes of interest and labeled compounds and analyzed to establish the ability
of the laboratory to generate acceptable precision and recovery. An IPR is
performed prior to the first time this method is used and any time the method or
instrumentation is modified.

Isotope dilution quantitation – A means of determining a naturally occurring

(native) compound by reference to the same compound in which one or more
atoms has been isotopically enriched. In this method, labeled are enriched with
deuterium to produce 2H labeled analogs or carbon-13 to produce 13C-labeled
analogs. The labeled analogs are spiked into each sample to allow identification
and correction of the concentration of the native compounds in the analytical
process.

Labeled compound – A molecule in which one or more of the atoms is isotopically

entriched, thereby increasing the mass of the molecule

Laboratory blank – See method blank

Laboratory control sample (LCS) – See Ongoing precision and recovery standard
(OPR)

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December 2007 Method 1694

Laboratory reagent blank – See method blank

May – This action, activity, or procedural step is neither required nor prohibited.

May not – This action, activity, or procedural step is prohibited.

Method blank – An aliquot of reagent water that is treated exactly as a sample

including exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with samples. The method blank is used to
determine if analytes or interferences are present in the laboratory environment, the
reagents, or the apparatus.

Method detection limit (MDL) – The lowest concentration at which an analyte can
be detected under routine operating conditions (see 40 CFR 136, appendix B).
MDLs are listed in Table 3, 5, 7, and 9.

Minimum level (ML) – The greater of a multiple of the MDL or the lowest
calibration point (see 68 FR 11790, March 12, 2003.) MLs are listed in Tables 3,
5, 7, and 9.

MS – Mass spectrometer or mass spectrometry

Must – This action, activity, or procedural step is required.

Native compound – A molecule in which the atoms all have naturally occuring
isotopic abundances

OPR – Ongoing precision and recovery standard (OPR); a method blank spiked
with known quantities of analytes. Also known as a “laboratory control sample”
(LCS). The OPR is analyzed exactly like a sample. Its purpose is to assure that the
results produced by the laboratory remain within the limits specified in this method
for precision and recovery.

Preparation blank – See method blank

Quality control check sample (QCS) – A sample containing all or a subset of the
analytes at known concentrations. The QCS is obtained from a source external to
the laboratory or is prepared from a source of standards different from the source of
calibration standards. It is used to check laboratory performance with test
materials prepared external to the normal preparation process.

Reagent water – water demonstrated to be free from the analytes of interest and
potentially interfering substances at the method detection limit for the analyte.

Relative standard deviation (RSD) – The standard deviation times 100 divided by
the mean. Also termed "coefficient of variation."

RF – Response factor. See Section 10.5

RR – Relative response. See Section 10.4

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December 2007 Method 1694

RSD – See relative standard deviation

Signal-to-noise ratio (S/N) – The height of the signal as measured from the mean
(average) of the noise to the peak maximum divided by the width of the noise.

Should – Although this action, activity, or procedural step is suggested and not
required, you may be asked to explain why you changed or omitted this action,
activity, or procedural step.

SICP – Selected ion current profile; the line described by the signal at an exact
m/z.

SPE – Solid-phase extraction; an extraction technique in which an analyte is

extracted from an aqueous solution by passage over or through a material capable
of reversibly adsorbing the analyte. Also termed liquid-solid extraction.

Stock solution – A solution containing an analyte that is prepared using a reference

material traceable to EPA, the National Institute of Science and Technology
(NIST), or a source that will attest to the purity and authenticity of the reference
material.

VER – See Calibration verification.

72