EUROPEAN PHARMACOPOEIA 11.

0 Azithromycin

01/2018:1649 Reference solution (b). Dissolve the contents of a vial of

corrected 9.6 azithromycin for system suitability CRS (containing impurities
F, H and J) in 1.0 mL of the solvent mixture and sonicate for
5 min.
Reference solution (c). Dissolve 8.0 mg of azithromycin for
peak identification CRS (containing impurities A, B, C, E, F, G,
AZITHROMYCIN I, J, L, M, N, O and P) in 1.0 mL of the solvent mixture.
Column :
Azithromycinum – size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl amorphous
organosilica polymer for chromatography R (5 μm) ;
– temperature : 60 °C.
Mobile phase :
– mobile phase A : 1.80 g/L solution of anhydrous disodium
hydrogen phosphate R adjusted to pH 8.9 with dilute
phosphoric acid R or with dilute sodium hydroxide
solution R ;
– mobile phase B : methanol R2, acetonitrile R1 (25:75 V/V);
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 25 50 → 45 50 → 55
C38H72N2O12,xH2O Mr 749 (anhydrous substance)
with x = 1 or 2 25 - 30 45 → 40 55 → 60
Azithromycin monohydrate : [121470-24-4] 30 - 80 40 → 25 60 → 75
Azithromycin dihydrate : [117772-70-0]
80 - 81 25 → 50 75 → 50
DEFINITION 81 - 93 50 50
(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-
C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl- Flow rate : 1.0 mL/min.
3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6- Detection : spectrophotometer at 210 nm.
trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-1- Injection : 50 μL.
oxa-6-azacyclopentadecan-15-one. The degree of hydration is
1 or 2. Identification of impurities : use the chromatogram supplied
with azithromycin for peak identification CRS and the
Semi-synthetic product derived from a fermentation product. chromatogram obtained with reference solution (c) to identify
Content : 96.0 per cent to 102.0 per cent (anhydrous substance).the peaks due to impurities A, B, C, E, F, G, I, J, L, M, N, O
and P ; use the chromatogram supplied with azithromycin for
CHARACTERS system suitability CRS and the chromatogram obtained with
Appearance : white or almost white powder. reference solution (b) to identify the peak due to impurity H.
Solubility : practically insoluble in water, freely soluble in Relative retention with reference to azithromycin
anhydrous ethanol and in methylene chloride. (retention time = 45-50 min) : impurity L = about 0.29 ;
impurity M = about 0.37 ; impurity E = about 0.43 ;
IDENTIFICATION
impurity F = about 0.51 ; impurity D = about 0.54 ;
Infrared absorption spectrophotometry (2.2.24). impurity J = about 0.54 ; impurity Q = about 0.54 ;
Comparison : azithromycin CRS. impurity I = about 0.61 ; impurity C = about 0.73 ;
If the spectra obtained in the solid state show differences, impurity N = about 0.76 ; impurity H = about 0.79 ;
prepare further spectra using 90 g/L solutions in methylene impurity A = about 0.83 ; impurity P = about 0.92 ;
chloride R. impurity O = about 1.23 ; impurity G = about 1.26 ;
impurity B = about 1.31.
TESTS System suitability : reference solution (b) :
Solution S. Dissolve 0.500 g in anhydrous ethanol R and dilute – peak-to-valley ratio : minimum 1.4, where H = height
p
to 50.0 mL with the same solvent. above the baseline of the peak due to impurity J and
Appearance of solution. Solution S is clear (2.2.1) and Hv = height above the baseline of the lowest point of the
colourless (2.2.2, Method II). curve separating this peak from the peak due to impurity F.
pH (2.2.3) : 9.0 to 11.0. Limits :
Dissolve 0.100 g in 25.0 mL of methanol R and dilute to – correction factors : for the calculation of content,
50.0 mL with carbon dioxide-free water R. multiply the peak areas of the following impurities by
the corresponding correction factor : impurity F = 0.3 ;
Specific optical rotation (2.2.7): − 49 to − 45 (anhydrous impurity G = 0.2 ; impurity H = 0.1 ; impurity L = 2.3 ;
substance), determined on solution S. impurity M = 0.6 ; impurity N = 0.7 ;
Related substances. Liquid chromatography (2.2.29). – impurity B : not more than twice the area of the principal
Solvent mixture. Prepare a 1.73 g/L solution of ammonium peak in the chromatogram obtained with reference
dihydrogen phosphate R adjusted to pH 10.0 with ammonia R. solution (a) (2.0 per cent);
Transfer 350 mL of this solution to a suitable container. Add – impurities A, C, E, F, H, I, L, M, N, O, P : for each impurity,
300 mL of acetonitrile R and 350 mL of methanol R. Mix well. not more than 0.5 times the area of the principal peak in
Test solution. Dissolve 0.200 g of the substance to be examined the chromatogram obtained with reference solution (a)
in the solvent mixture and dilute to 25.0 mL with the solvent (0.5 per cent);
mixture. – sum of impurities D, J and Q : not more than 0.5 times the
Reference solution (a). Dilute 1.0 mL of the test solution to area of the principal peak in the chromatogram obtained
100.0 mL with the solvent mixture. with reference solution (a) (0.5 per cent) ;

General Notices (1) apply to all monographs and other texts 2025
Azithromycin EUROPEAN PHARMACOPOEIA 11.0

– impurity G : not more than 0.2 times the area of the

principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent) ;
– any other impurity : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent) ;
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(3.0 per cent) ;
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (a) A. 6-demethylazithromycin,
(0.1 per cent) ; disregard the peaks eluting before impurity L
and after impurity B.
Water (2.5.12) : 1.8 per cent to 6.5 per cent, determined on
0.200 g.
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 g.
ASSAY
Liquid chromatography (2.2.29).
Solution A. Mix 60 volumes of acetonitrile R and 40 volumes
of a 6.7 g/L solution of dipotassium hydrogen phosphate R
adjusted to pH 8.0 with phosphoric acid R.
Test solution. Dissolve 53.0 mg of the substance to be
examined in 2 mL of acetonitrile R and dilute to 100.0 mL B. 3-deoxyazithromycin (azithromycin B),
with solution A.
Reference solution (a). Dissolve 53.0 mg of azithromycin CRS
in 2 mL of acetonitrile R and dilute to 100.0 mL with
solution A.
Reference solution (b). Dissolve 5 mg of the substance to be
examined and 5 mg of azithromycin impurity A CRS in 0.5 mL
of acetonitrile R and dilute to 10 mL with solution A.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl vinyl polymer for
chromatography R (5 μm) ;
– temperature : 40 °C.
C. 3″-O-demethylazithromycin (azithromycin C),
Mobile phase : mix 60 volumes of acetonitrile R1 and
40 volumes of a 6.7 g/L solution of dipotassium hydrogen
phosphate R adjusted to pH 11.0 with a 560 g/L solution of
potassium hydroxide R.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 210 nm.
Injection : 10 μL.
Run time : 1.5 times the retention time of azithromycin.
Retention time : azithromycin = about 10 min.
System suitability : reference solution (b) :
– resolution : minimum 3.0 between the peaks due to
impurity A and azithromycin.
Calculate the percentage content of C38H72N2O12 taking into D. 14-demethyl-14-(hydroxymethyl)azithromycin
account the assigned content of azithromycin CRS. (azithromycin F),

STORAGE
In an airtight container.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H, I, J, L, M, N, O, P, Q.
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): K. E. 3′-(N,N-didemethyl)azithromycin (aminoazithromycin),

2026 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 11.0 Azithromycin

F. 3′-N-demethyl-3′-N-formylazithromycin,
K. C14,1″-epoxyazithromycin (azithromycin E),

G. 3′-N-demethyl-3′-N-[(4-methylphenyl)sulfonyl]- L. azithromycin 3′-N-oxide,

azithromycin,

M. 3′-(N,N-didemethyl)-3′-N-formylazithromycin,
H. 3′-N-[[4-(acetylamino)phenyl]sulfonyl]-3′-N-
demethylazithromycin,

N. 3′-de(dimethylamino)-3′-oxoazithromycin,

I. 3′-N-demethylazithromycin,

O. 2-desethyl-2-propylazithromycin,

J. 13-O-decladinosylazithromycin, P. unknown structure,

General Notices (1) apply to all monographs and other texts 2027
Azithromycin EUROPEAN PHARMACOPOEIA 11.0

Q. 3′-N-[[4-(acetylamino)phenyl]sulfonyl]-3′-(N,N-
didemethyl)azithromycin.

2028 See the information section on general monographs (cover pages)