Busulfan

EUROPEAN PHARMACOPOEIA 5.0

STORAGE
In an airtight container, protected from light, at a
temperature of 2 C to 8 C.
If the substance is sterile, store in an airtight, sterile,
tamper-proof container.

A. Examine by infrared absorption spectrophotometry

(2.2.24), comparing with the spectrum obtained with
busulfan CRS.
B. Examine by thin-layer chromatography (2.2.27), using
silica gel G R as the coating substance.
Test solution. Dissolve 20 mg of the substance to be
LABELLING
examined in 2 ml of acetone R.
The label states :
Reference solution. Dissolve 20 mg of busulfan CRS in
the mass of peptide in the container,
2 ml of acetone R.
where applicable, that the substance is free from bacterial
Apply separately to the plate 5 l of each solution.
endotoxins.
Develop over a path of 15 cm using a mixture of
equal volumes of acetone R and toluene R. Dry the
IMPURITIES
plate in a stream of hot air and spray with anisaldehyde
Specified impurities : A, B, C, D, E.
solution R. Heat the plate at 120 C. The principal spot
in the chromatogram obtained with the test solution is
similar in position, colour and size to the principal spot in
the chromatogram obtained with the reference solution.
C. To 0.1 g add 5 ml of 1 M sodium hydroxide. Heat until
a clear solution is obtained. Allow to cool. To 2 ml of
the solution add 0.1 ml of potassium permanganate
solution R. The colour changes from purple through
A. X2 = D-His, X4 = L-Ser, X5 = L-Tyr : [2-D-histidine]buserelin,
violet to blue and finally to green. Filter and add 1 ml
of ammoniacal silver nitrate solution R. A precipitate
B. X2 = L-His, X4 = D-Ser, X5 = L-Tyr : [4-D-serine]buserelin,
is formed.
D. X2 = L-His, X4 = L-Ser, X5 = D-Tyr : [5-D-tyrosine]buserelin,
D. To 0.1 g add 0.1 g of potassium nitrate R and 0.25 g of
sodium hydroxide R, mix and heat to fusion. Allow to
cool and dissolve the residue in 5 ml of water R. Adjust to
pH 1 to 2 using dilute hydrochloric acid R. The solution
gives reaction (a) of sulphates (2.3.1).
C. buserelin-(3-9)-peptide,

E. [1-(5-oxo-D-proline)]buserelin.

BUSULFAN
Busulfanum

TESTS
Appearance of solution. Dissolve 0.25 g in 20 ml of
acetonitrile R, dilute to 25 ml with water R and examine
immediately. The solution is clear (2.2.1) and not more
intensely coloured than reference solution B7 (2.2.2,
Method II).
Acidity. Dissolve 0.20 g with heating in 50 ml of ethanol R.
Add 0.1 ml of methyl red solution R. Not more than 0.05 ml
of 0.1 M sodium hydroxide is required to change the colour
of the indicator.
01/2005:0542 Loss on drying (2.2.32). Not more than 2.0 per cent,
determined on 1.000 g by drying in vacuo at 60 C.
Sulphated ash (2.4.14). Not more than 0.1 per cent,
determined on 1.0 g.

ASSAY
To 0.250 g add 50 ml of water R. Shake. Boil under a reflux
condenser for 30 min and, if necessary, make up to the
initial volume with water R. Allow to cool. Using 0.3 ml of
phenolphthalein solution R as indicator, titrate with 0.1 M
C6H14O6S2
Mr 246.3 sodium hydroxide until a pink colour is obtained.
1 ml of 0.1 M sodium hydroxide is equivalent to 12.32 mg
DEFINITION
of C6H14O6S2.
Busulfan contains not less than 99.0 per cent and not more
STORAGE
than the equivalent of 100.5 per cent of butane-1,4-diyl
di(methanesulphonate), calculated with reference to the
Store in an airtight container, protected from light.
dried substance.
CHARACTERS
A white or almost white, crystalline powder, very slightly
soluble in water, freely soluble in acetone and in acetonitrile,
very slightly soluble in alcohol.
It melts at about 116 C.
IDENTIFICATION
First identification : A.
Second identification : B, C, D.
1138

01/2005:1847

BUTCHERS BROOM
Rusci rhizoma
DEFINITION
Dried, whole or fragmented underground parts of Ruscus
aculeatus L.

See the information section on general monographs (cover pages)

Butchers broom

EUROPEAN PHARMACOPOEIA 5.0

Content : minimum 1.0 per cent of total sapogenins,

expressed as ruscogenins [mixture of neoruscogenin
(C27H40O4 ; Mr 428.6) and ruscogenin (C27H42O4 ; Mr 430.6)]
(dried drug).

Top of the plate

Several zones of various colours
Stigmasterol: a violet zone

A violet zone

_______

_______

CHARACTERS
Macroscopic and microscopic characters described under
identification tests A and B.

A violet zone
Ruscogenins : a yellow zone

A yellow zone (ruscogenins)

Several zones of various colours

_______

IDENTIFICATION
A. The rhizome consists of yellowish, branched, articulated,
somewhat knotty pieces, cylindrical or subconical, about
5 cm to 10 cm long and about 5 mm thick. The surface
is marked with thin annulations about 1 mm to 3 mm
wide, separated from one another ; rounded scars of the
aerial stems are present on the upper surface. On the
lower surface numerous roots, or their scars, occur ; the
roots are about 2 mm in diameter and similar in colour to
the rhizome. The outer layer is easily detached, revealing
a yellowish-white, very hard central cylinder.

_______

Reference solution

Test solution

TESTS
Foreign matter (2.8.2) : maximum 5 per cent.
Loss on drying (2.2.32) : maximum 12.0 per cent, determined
on 1.000 g of the powdered drug (355) by drying in an oven
at 100-105 C for 2 h.
Total ash (2.4.16) : maximum 12.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1) : maximum
5.0 per cent.

B. Reduce to a powder (355). The powder is yellowish.

Examine under a microscope using chloral hydrate
solution R. The powder shows groups of sclereids from
the ground tissue of the rhizome, variously-shaped cells,
ranging from rounded to elongated or rectangular ; the
walls are moderately thickened and distinctly beaded, with
large, rounded to oval pits. Fragments of the endodermis
composed of a single layer of irregularly-thickened cells.
Groups of rounded parenchymatous cells, thickened at
the corners, with small, triangular intercellular spaces ;
thin-walled parenchyma containing raphides of calcium
oxalate. Groups of thick-walled fibres and small vessels,
up to about 50 m in diameter, the walls showing
numerous small, slit-shaped pits.

ASSAY
Liquid chromatography (2.2.29).
Test solution. To 2.000 g of the powdered drug (355), add
60 ml of ethanol R, 15 ml of water R and 0.2 g of potassium
hydroxide R. Extract under reflux on a water-bath for 4 h.
Allow to cool and filter into a 100 ml volumetric flask. Rinse
the extraction flask and the residue in the filter with 3
quantities, each of 10 ml, of ethanol R and add the rinsings
to the volumetric flask. Dilute to 100.0 ml with ethanol R.
Introduce 25.0 ml of the solution into a round-bottomed
flask fitted to a rotary evaporator and evaporate to dryness.
Dissolve the residue in 10 ml of butanol R, add 3 ml of
hydrochloric acid R1 and 8 ml of water R. Heat under reflux
on a water-bath for 1 h. Allow to cool and transfer the liquid
C. Thin-layer chromatography (2.2.27)
into a separating funnel, rinse the round-bottomed flask with
Test solution. Introduce 1.0 g of the powdered drug (355) 2 quantities, each of 10 ml, of butanol R. Add the rinsings
to the separating funnel. Extract with 3 quantities, each of
and 50 ml of dilute hydrochloric acid R into a 100 ml
20 ml, of butanol R saturated with water R. Combine the
flask with a ground-glass neck. Heat on a water-bath
butanolic
extracts and evaporate to dryness using a rotary
under a reflux condenser for 40 min. Allow to cool and
evaporator.
Dissolve the residue in 20 ml of methanol R and
extract the unfiltered mixture with 3 quantities, each of
transfer to a 100 ml volumetric flask. Rinse the extraction
25 ml, of methylene chloride R. Combine the organic
flask with 20 ml, 20 ml and 10 ml of methanol R and add
solutions and dry over anhydrous sodium sulphate R.
the rinsings to the volumetric flask. Dilute to 100 ml with
Filter and evaporate to dryness. Dissolve the residue in
methanol R.
5 ml of methanol R.
Reference solution. Dissolve 5.0 mg of ruscogenins R in
Reference solution. Dissolve 1 mg of ruscogenins R and 100 ml of methanol R.
1 mg of stigmasterol R in methanol R and dilute to 5 ml Column :
with the same solvent.
size : l = 0.25 m, = 4.6 mm,
Plate : TLC silica gel plate R.
stationary phase : octadecylsilyl silica gel for
chromatography R (5 m).
Mobile phase : methanol R, methylene chloride R
Mobile
phase :
(7:93 V/V).
mobile phase A : water R,
Application : 10 l, as bands.
mobile phase B : acetonitrile for chromatography R,
Development : over a path of 15 cm.
Time
Mobile phase A
Mobile phase B
Drying : in air.
Detection : spray with vanillin reagent R, dry the plate in
an oven at 100-105 C for 1 min and examine in daylight.
Results : see below the sequence of the zones present in
the chromatograms obtained with the reference solution
and the test solution. Furthermore, other weak zones
may be present in the chromatogram obtained with the
test solution.
General Notices (1) apply to all monographs and other texts

(min)

(per cent V/V)

(per cent V/V)

0 - 25

40

60

25 - 27

40 0

60 100

27 - 37

100

37 - 39

0 40

100 60

39 - 42

40

60

Flow rate : 1.2 ml/min.

1139

Butyl parahydroxybenzoate

EUROPEAN PHARMACOPOEIA 5.0

Appearance of solution. Solution S is clear (2.2.1) and

not more intensely coloured than reference solution BY6
(2.2.2, Method II).
Acidity. To 2 ml of solution S add 3 ml of alcohol R, 5 ml of
carbon dioxide-free water R and 0.1 ml of bromocresol green
solution R. Not more than 0.1 ml of 0.1 M sodium hydroxide
is required to change the colour of the indicator to blue.
Related substances. Thin-layer chromatography (2.2.27).
Test solution (a). Dissolve 0.10 g of the substance to be
examined in acetone R and dilute to 10 ml with the same
solvent.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with
acetone R.
Reference solution (a). Dilute 0.5 ml of test solution (a) to
100 ml with acetone R.
01/2005:0881
Reference solution (b). Dissolve 10 mg of butyl
parahydroxybenzoate CRS in acetone R and dilute to
BUTYL PARAHYDROXYBENZOATE 10 ml with the same solvent.
Reference solution (c). Dissolve 10 mg of propyl
Butylis parahydroxybenzoas
parahydroxybenzoate R in 1 ml of test solution (a) and
dilute to 10 ml with acetone R.
Plate : suitable octadecylsilyl silica gel with a fluorescent
indicator having an optimal intensity at 254 nm as the
coating substance.
Mobile phase : glacial acetic acid R, water R, methanol R
(1:30:70 V/V/V).
C11H14O3
Mr 194.2 Application : 2 l.
Development : over a path of 15 cm.
DEFINITION
Drying : in air.
Butyl 4-hydroxybenzoate.
Detection : examine in ultraviolet light at 254 nm.
Content : 98.0 per cent to 102.0 per cent.
System suitability : the chromatogram obtained with
reference solution (c) shows 2 clearly separated principal
CHARACTERS
spots.
Appearance : white or almost white, crystalline powder or
Limits :
colourless crystals.
any impurity : any spot in the chromatogram obtained
Solubility : very slightly soluble in water, freely soluble in
with test solution (a), apart from the principal spot, is not
alcohol and in methanol.
more intense than the spot in the chromatogram obtained
with reference solution (a) (0.5 per cent).
IDENTIFICATION
First identification : A, B.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
Second identification : A, C, D.
Detection : spectrophotometer at 203 nm.
Injection : 20 l.
Retention time with reference to neoruscogenin (retention
time = about 16 min) : ruscogenin = about 1.2.
System suitability : reference solution :
resolution : minimum 1.5 between the peaks due to
neoruscogenin and ruscogenin.
Calculate the percentage content of sapogenins expressed
as ruscogenins (neoruscogenin and ruscogenin) in the
test solution by comparing the areas of the peaks in the
chromatograms obtained with the test solution and the
reference solution.

A. Melting point (2.2.14) : 68 C to 71 C.

B. Infrared absorption spectrophotometry (2.2.24).
Comparison : butyl parahydroxybenzoate CRS.
C. Examine the chromatograms obtained in the test for
related substances.
Results : the principal spot in the chromatogram obtained
with test solution (b) is similar in position and size to
the principal spot in the chromatogram obtained with
reference solution (b).
D. To about 10 mg in a test-tube add 1 ml of sodium
carbonate solution R, boil for 30 s and cool (solution A).
To a further 10 mg in a similar test-tube add 1 ml of
sodium carbonate solution R ; the substance partly
dissolves (solution B). Add at the same time to solution A
and solution B 5 ml of aminopyrazolone solution R
and 1 ml of potassium ferricyanide solution R and mix.
Solution B is yellow to orange-brown. Solution A is
orange to red, the colour being clearly more intense than
any similar colour which may be obtained with solution B.
TESTS
Solution S. Dissolve 1.0 g in alcohol R and dilute to
10 ml with the same solvent.
1140

ASSAY
To 1.000 g add 20.0 ml of 1 M sodium hydroxide. Heat at
about 70 C for 1 h. Cool rapidly in an ice bath. Prepare a
blank in the same manner. Carry out the titration on the
solutions at room temperature. Titrate the excess sodium
hydroxide with 0.5 M sulphuric acid, continuing the titration
until the second point of inflexion and determining the
end-point potentiometrically (2.2.20).
1 ml of 1 M sodium hydroxide is equivalent to 194.2 mg of
C11H14O3.
IMPURITIES

A. R = H : 4-hydroxybenzoic acid,
B. R = CH3 : methyl 4-hydroxybenzoate,
C. R = CH2-CH3 : ethyl 4-hydroxybenzoate,
D. R = CH2-CH2-CH3 : propyl 4-hydroxybenzoate.

See the information section on general monographs (cover pages)