Chlorambucil EUROPEAN PHARMACOPOEIA 6.

added at the beginning of the titration, the volume of 0.5 M Reference solution (b). Dilute 25 ml of reference solution (a)
sulphuric acid used in the 1st titration and two-fifteenths of
to 100 ml with acetone R.
the volume of 0.1 M silver nitrate used in the 2nd titration.Apply separately to the plate 5 µl of each solution. Develop
1 ml of 1 M sodium hydroxide is equivalent to 0.1654 g over a path of 10 cm using a mixture of 20 volumes of
of C2H3Cl3O2. methyl ethyl ketone R, 20 volumes of heptane R, 25 volumes
of methanol R and 40 volumes of toluene R. Examine in
STORAGE ultraviolet light at 254 nm. Any spot in the chromatogram
In an airtight container. obtained with the test solution, apart from the principal
spot, is not more intense than the spot in the chromatogram
01/2008:0137 obtained with reference solution (a) (2.0 per cent) and
corrected 6.0 at most 1 such spot is more intense than the spot in the
chromatogram obtained with reference solution (b) (0.5 per
cent).
CHLORAMBUCIL
Water (2.5.12). Not more than 0.5 per cent, determined on
1.000 g by the semi-micro determination of water.
Chlorambucilum
Sulphated ash (2.4.14). Not more than 0.1 per cent,
determined on 1.0 g.
ASSAY
Dissolve 0.200 g in 10 ml of acetone R and add 10 ml of
water R. Titrate with 0.1 M sodium hydroxide, using 0.1 ml
of phenolphthalein solution R as indicator.
1 ml of 0.1 M sodium hydroxide is equivalent to 30.42 mg
C14H19Cl2NO2 Mr 304.2 of C14H19Cl2NO2.
[305-03-3] STORAGE
DEFINITION Store protected from light.
Chlorambucil contains not less than 98.5 per cent
and not more than the equivalent of 101.0 per cent of 01/2008:0071
4-4-[di(2-chloroethyl)amino]phenylbutyric acid, calculated corrected 6.0
with reference to the anhydrous substance.
CHARACTERS
CHLORAMPHENICOL
A white or almost white, crystalline powder, practically Chloramphenicolum
insoluble in water, freely soluble in acetone and in alcohol.
IDENTIFICATION
First identification : A, B.
Second identification : A, C, D.
A. Melting point (2.2.14) : 64 °C to 67 °C.
B. Examine by infrared absorption spectrophotometry C11H12Cl2N2O5 Mr 323.1
(2.2.24), comparing with the spectrum obtained with [56-75-7]
chlorambucil CRS.
C. To 0.4 g add 10 ml of dilute hydrochloric acid R, mix DEFINITION
and allow to stand for 30 min, shaking from time to time. Chloramphenicol is 2,2-dichloro-N-[(1R,2R)-2-hydroxy-1-
Filter and wash the precipitate with 2 quantities, each of (hydroxymethyl)-2-(4-nitrophenyl)ethyl]acetamide, produced
10 ml, of water R. To 10 ml of the combined filtrate and by the growth of certain strains of Streptomyces venezuelae
washings add 0.5 ml of potassium tetraiodomercurate in a suitable medium. It is normally prepared by synthesis.
solution R. A pale-brown precipitate is formed. To another It contains not less than 98.0 per cent and not more than
10 ml of the combined filtrate and washings add 0.2 ml of the equivalent of 102.0 per cent of C11H12Cl2N2O5, calculated
potassium permanganate solution R. The colour of the with reference to the dried substance.
latter is discharged immediately.
D. Dissolve 50 mg in 5 ml of acetone R and dilute to 10 ml CHARACTERS
with water R. Add 0.05 ml of dilute nitric acid R and A white, greyish-white or yellowish-white, fine, crystalline
0.2 ml of silver nitrate solution R2. No opalescence is powder or fine crystals, needles or elongated plates, slightly
produced immediately. Heat the solution on a water-bath ; soluble in water, freely soluble in alcohol and in propylene
an opalescence develops. glycol.
A solution in ethanol is dextrorotatory and a solution in
TESTS ethyl acetate is laevorotatory.
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel GF254 R as the coating substance. IDENTIFICATION
Carry out all operations as rapidly as possible protected First identification : A, B.
from light. Prepare the solutions immediately before use. Second identification : A, C, D, E.
Test solution. Dissolve 0.2 g of the substance to be examined A. Melting point (2.2.14) : 149 °C to 153 °C.
in acetone R and dilute to 10 ml with the same solvent. B. Examine by infrared absorption spectrophotometry
Reference solution (a). Dilute 1 ml of the test solution to (2.2.24), comparing with the spectrum obtained with
50 ml with acetone R. chloramphenicol CRS.

1492 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.0 Chloramphenicol palmitate

C. Examine the chromatograms obtained in the test test for pyrogens. Inject per kilogram of the rabbit’s mass
for related substances. The principal spot in the 2.5 ml of a solution containing per millilitre 2 mg of the
chromatogram obtained with 1 µl of the test solution is substance to be examined.
similar in position and size to the principal spot in the
chromatogram obtained with reference solution (a). ASSAY
Dissolve 0.100 g in water R and dilute to 500.0 ml with the
D. Dissolve about 10 mg in 1 ml of alcohol (50 per
same solvent. Dilute 10.0 ml of this solution to 100.0 ml with
cent V/V) R, add 3 ml of a 10 g/l solution of calcium
water R. Measure the absorbance (2.2.25) at the maximum
chloride R and 50 mg of zinc powder R and heat on a
at 278 nm.
water-bath for 10 min. Filter the hot solution and allow
to cool. Add 0.1 ml of benzoyl chloride R and shake for Calculate the content of C11H12Cl2N2O5 taking the specific
1 min. Add 0.5 ml of ferric chloride solution R1 and absorbance to be 297.
2 ml of chloroform R and shake. The aqueous layer is STORAGE
coloured light violet-red to purple.
Store protected from light. If the substance is sterile, store
E. To 50 mg in a porcelain crucible add 0.5 g of anhydrous in a sterile, airtight, tamper-proof container.
sodium carbonate R. Heat over an open flame for 10 min.
Allow to cool. Take up the residue with 5 ml of dilute
nitric acid R and filter. To 1 ml of the filtrate add 1 ml
of water R. The solution gives reaction (a) of chlorides 01/2008:0473
(2.3.1). corrected 6.0

TESTS CHLORAMPHENICOL PALMITATE

Acidity or alkalinity. To 0.1 g add 20 ml of carbon
dioxide-free water R, shake and add 0.1 ml of bromothymol Chloramphenicoli palmitas
blue solution R1. Not more than 0.1 ml of 0.02 M
hydrochloric acid or 0.02 M sodium hydroxide is required
to change the colour of the indicator.
Specific optical rotation (2.2.7). Dissolve 1.50 g in ethanol R
and dilute to 25.0 ml with the same solvent. The specific
optical rotation is + 18.5 to + 20.5.
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel GF254 R as the coating substance.
C27H42Cl2N2O6 Mr 561.6
Test solution. Dissolve 0.10 g of the substance to be [530-43-8]
examined in acetone R and dilute to 10 ml with the same
solvent. DEFINITION
Reference solution (a). Dissolve 0.10 g of Chloramphenicol palmitate contains not less than 98.0 per
chloramphenicol CRS in acetone R and dilute to cent and not more than the equivalent of 102.0 per
10 ml with the same solvent. cent of (2R,3R)-2-[(dichloroacetyl)amino]-3-hydroxy-3-(4-
nitrophenyl)propyl hexadecanoate, calculated with reference
Reference solution (b). Dilute 0.5 ml of reference solution (a) to the dried substance.
to 100 ml with acetone R.
Semi-synthetic product derived from a fermentation product.
Apply separately to the plate 1 µl and 20 µl of the test
solution, 1 µl of reference solution (a) and 20 µl of reference CHARACTERS
solution (b). Develop over a path of 15 cm using a mixture A white or almost white, fine, unctuous powder, practically
of 1 volume of water R, 10 volumes of methanol R and insoluble in water, freely soluble in acetone, sparingly soluble
90 volumes of chloroform R. Allow the plate to dry in air in ethanol (96 per cent), very slightly soluble in hexane.
and examine in ultraviolet light at 254 nm. Any spot in the It melts at 87 °C to 95 °C.
chromatogram obtained with 20 µl of the test solution, apart
from the principal spot, is not more intense than the spot It shows polymorphism (5.9). The thermodynamically stable
in the chromatogram obtained with reference solution (b) form has low bioavailability following oral administration.
(0.5 per cent). IDENTIFICATION
Chlorides (2.4.4). To 1.00 g add 20 ml of water R and A. Examine by thin-layer chromatography (2.2.27), using
10 ml of nitric acid R and shake for 5 min. Filter through a TLC silanised silica gel plate R.
filter paper previously washed by filtering 5 ml portions of
Test solution. Dissolve 50 mg of the substance to be
water R until 5 ml of filtrate no longer becomes opalescent
examined in a mixture of 1 ml of 1 M sodium hydroxide
on addition of 0.1 ml of nitric acid R and 0.1 ml of silver
and 5 ml of acetone R and allow to stand for 30 min. Add
nitrate solution R1. 15 ml of the filtrate complies with the
1.1 ml of 1 M hydrochloric acid and 3 ml of acetone R.
limit test for chlorides (100 ppm).
Reference solution (a). Dissolve 10 mg of
Loss on drying (2.2.32). Not more than 0.5 per cent, chloramphenicol CRS in acetone R and dilute to
determined on 1.000 g by drying in an oven at 105 °C. 5 ml with the same solvent.
Sulphated ash (2.4.14). Not more than 0.1 per cent, Reference solution (b). Dissolve 10 mg of palmitic acid R
determined on 2.0 g. in acetone R and dilute to 5 ml with the same solvent.
Pyrogens (2.6.8). If intended for use in the manufacture Reference solution (c). Dissolve 10 mg of the substance
of a parenteral dosage form without a further appropriate to be examined in acetone R and dilute to 5 ml with the
procedure for the removal of pyrogens, it complies with the same solvent.

General Notices (1) apply to all monographs and other texts 1493