EUROPEAN PHARMACOPOEIA 10.
0 Chloramphenicol
IMPURITIES
Specified impurities : B, E, G.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use): A, C, D, F.
A. 4-[4-[(2-chloroethyl)(2-hydroxyethyl)amino]phenyl]buta-
noic acid,
B. 4-[4-[(2-chloroethyl)amino]phenyl]butanoic acid,
C. 4-[4-[[2-[[bis(2-chloroethoxy)phosphoryl]oxy]ethyl](2-
chloroethyl)amino]phenyl]butanoic acid,
D. 2-chloroethyl 4-[4-[bis(2-chloroethyl)amino]phenyl]-
butanoate,
E. 4-[4-[[2-[[4-[4-[bis(2-chloroethyl)amino]phenyl]-
butanoyl]oxy]ethyl](2-chloroethyl)amino]phenyl]butanoic
acid,
F. 4-[4-[[2-[[4-[4-[[2-[[4-[4-[bis(2-chloroethyl)amino]-
phenyl]butanoyl]oxy]ethyl](2-chloroethyl)amino]-
phenyl]butanoyl]oxy]ethyl](2-chloroethyl)amino]-
phenyl]butanoic acid,
General Notices (1) apply to all monographs and other texts
G. 4-[2-[bis(2-chloroethyl)amino]phenyl]butanoic acid or
4-[3-[bis(2-chloroethyl)amino]phenyl]butanoic acid (meta
or ortho chlorambucil).
04/2019:0071
corrected 10.0
CHLORAMPHENICOL
Chloramphenicolum
C11H12Cl2N2O5 Mr 323.1
[56-75-7]
DEFINITION
2,2-Dichloro-N-[(1R,2R)-1,3-dihydroxy-1-(4-
nitrophenyl)propan-2-yl]acetamide.
Content : 97.5 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance : white, greyish-white or yellowish-white, fine,
crystalline powder or fine crystals, needles or elongated plates.
Solubility : slightly soluble in water, freely soluble in ethanol
(96 per cent) and in propylene glycol.
A solution in anhydrous ethanol is dextrorotatory and a
solution in ethyl acetate is laevorotatory.
IDENTIFICATION
First identification : A.
Second identification : B.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : chloramphenicol CRS.
B. Melting point (2.2.14).
Determination A : determine the melting point of the
substance to be examined.
Results A : 149 °C to 153 °C.
Determination B : mix equal parts of the substance to be
examined and chloramphenicol CRS and determine the
melting point of the mixture.
Results B : the absolute difference between the melting point
of the mixture and the value obtained in determination A
is not greater than 2 °C.
TESTS
Acidity or alkalinity. To 0.1 g add 20 mL of carbon
dioxide-free water R, shake and add 0.1 mL of bromothymol
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) : + 18.5 to + 20.5.
Dissolve 1.50 g in anhydrous ethanol R and dilute to 25.0 mL
with the same solvent.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
2167
Chloramphenicol palmitate EUROPEAN PHARMACOPOEIA 10.0

Solution A. Dissolve 2.0 g of sodium heptanesulfonate R in Chlorides (2.4.4) : maximum 100 ppm.
900 mL of water for chromatography R. Add 6.8 g of potassium To 1.00 g add 10 mL of nitric acid R and 20 mL of water R
dihydrogen phosphate R and 5 mL of triethylamine R. Adjust and shake for 5 min. Filter through a filter paper previously
to pH 2.5 with phosphoric acid R and dilute to 1000 mL with washed by filtering 5 mL portions of water R until 5 mL of
water for chromatography R. filtrate no longer becomes opalescent on addition of 0.1 mL of
Test solution (a). Dissolve 20.0 mg of the substance to be nitric acid R and 0.1 mL of silver nitrate solution R1. 15 mL of
examined in 10 mL of methanol R and dilute to 200.0 mL with the filtrate complies with the test.
mobile phase A. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Test solution (b). Dissolve 25.0 mg of the substance to be on 1.000 g by drying in an oven at 105 °C.
examined in 5 mL of methanol R and dilute to 50.0 mL with Sulfated ash (2.4.14): maximum 0.1 per cent, determined on
mobile phase A. 2.0 g.
Reference solution (a). Dissolve 20.0 mg of chlorampheni-
col CRS in 10 mL of methanol R and dilute to 200.0 mL with ASSAY
mobile phase A. Liquid chromatography (2.2.29) as described in the test for
Reference solution (b). Dilute 1.0 mL of test solution (b) to related substances with the following modifications.
100.0 mL with mobile phase A. Dilute 1.0 mL of this solution Mobile phase : mobile phase A.
to 10.0 mL with mobile phase A. Injection : test solution (a) and reference solution (a).
Reference solution (c). Dissolve 12.5 mg of 4-nitrobenzal- Run time : 1.5 times the retention time of chloramphenicol.
dehyde R (impurity B) in 2 mL of methanol R and dilute to
50 mL with mobile phase A. Calculate the percentage content of C11H12Cl2N2O5 taking into
account the assigned content of chloramphenicol CRS.
Reference solution (d). Dissolve 5 mg of chloramphenicol for
peak identification CRS (containing impurity A) in 1 mL of STORAGE
methanol R, add 1 mL of reference solution (c) and dilute to Protected from light. If the substance is sterile, the container
10 mL with mobile phase A. is also sterile, airtight and tamper-evident.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ; IMPURITIES
Specified impurities : A.
– stationary phase : base-deactivated end-capped octadecylsilyl
silica gel for chromatography R (5 μm) ; Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
– temperature : 25 °C.
the tests in the monograph. They are limited by the general
Mobile phase : acceptance criterion for other/unspecified impurities and/or
– mobile phase A : methanol R, solution A (32:68 V/V); by the general monograph Substances for pharmaceutical
– mobile phase B : methanol R ; use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Time Mobile phase A Mobile phase B Control of impurities in substances for pharmaceutical use) : B.
(min) (per cent V/V) (per cent V/V)
0 - 13 100 0
13 - 25 100 → 60 0 → 40
25 - 33 60 40

Flow rate : 1.0 mL/min. A. (1R,2R)-2-amino-1-(4-nitrophenyl)propane-1,3-diol,

Detection : spectrophotometer at 277 nm.
Injection : 10 μL of test solution (b) and reference solutions (b)
and (d).
Identification of impurities : use the chromatogram supplied
with chloramphenicol for peak identification CRS and the B. 4-nitrobenzaldehyde.
chromatogram obtained with reference solution (d) to identify
the peaks due to impurities A and B.
Relative retention with reference to chloramphenicol 01/2008:0473
(retention time = about 14 min) : impurity A = about 0.7 ; corrected 10.0
impurity B = about 0.9.
System suitability : reference solution (d) :
– resolution : minimum 2.0 between the peaks due to
impurity B and chloramphenicol.
Calculation of percentage contents : CHLORAMPHENICOL PALMITATE
– correction factor : multiply the peak area of impurity A by
0.7 ; Chloramphenicoli palmitas
– for each impurity, use the concentration of chloramphenicol
in reference solution (b).
Limits :
– impurity A : maximum 0.2 per cent ;
– unspecified impurities : for each impurity, maximum
0.10 per cent ;
– total : maximum 0.5 per cent ; C27H42Cl2N2O6 Mr 561.6
– reporting threshold : 0.05 per cent. [530-43-8]

2168 See the information section on general monographs (cover pages)