Development and Validation of a Liquid Chromatographic method for the Analysis of Azithromycin

Citation:
Okaru AO. Development and Validation of a Liquid Chromatographic method for the Analysis of Azithromycin.; 2013.

Abstract:

Azithromycin is a semi-synthetic macrolide antibiotic that is active against a number of Grampositive
and Gram-negative bacteria. Azithromycin is listed in the World Health Organization
and the Ministry of Health, Kenya essential drugs list. Therefore, the quality of azithromycin
products in the market is important because of its role in management of atypical infections.
Liquid chromatography is the method of choice for the analysis of azithromycin in bulk samples
and its formulations. However, the current published liquid chromatographic methods employ
extreme conditions of pH and temperature and also expensive columns for the analysis of
azithromycin.
In this study, a simple, isocratic, rapid, sensitive and robust reverse phase HPLC method was
developed for the analysis of azithromycin in tablets, suspensions and raw materials. The effect
of chromatographic factors including use of inorganic mobile phase buffer, pH, ion-pairing
agent, organic modifier and temperature were investigated during development. Waters XTerra®
C18 column having the dimensions of 250×4.6 mm and 5 μm particle size with mobile phase
containing a mixture of acetonitrile-0.1M KH2PO4 pH 6.5-0.1M tetrabutylammonium hydroxidewater
(25:15:1:59 % v/v/v/v) was employed to achieve separation of azithromycin and its related
substances. The pH of the buffer and ion pairing agent was brought to 6.5 using an equimolar
solution of 0.1M K2HPO4. The mobile phase was delivered at a flow rate of 1 ml/min and the
components were monitored at 215 nm while the column temperature was maintained at 43 °C.
The retention time of azithromycin was found to be about 8 min.
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The proposed method was validated using ICH guidelines for the parameters of linearity,
recovery, precision, sensitivity and robustness. The method was found to be linear in the range
50-150 percent with the coefficient of determination (r2) being 0.997. The LOD and LOQ were
found to be 20 μg and 78 μg respectively. The method was found to exhibit good accuracy with
the percent recovery of azithromycin being 100.7 %. The coefficient of variation for the
repeatability and intermediate precision were within the limits prescribed by ICH.
The robustness ranges for the critical factors of pH, temperature and acetonitrile were
investigated with the aid of Statgraphics Centurion XVI. The robustness range for pH was 6-7
pH units, 22-28% v/v for acetonitrile and 41-45 °C for temperature. At 95% confidence interval,
p=0.05, acetonitrile had the biggest impact on the chromatographic parameters of the critical
peak pair of azithromycin and erythromycin A imino ether.
The degradation of azithromycin in 0.0005% v/v H2O2 was found to be second order (r2=0.994)
with a half life of 13 min. In 0.1M H3PO4 and 0.05M H3PO4, 90% of azithromycin decayed
within 10 min. In 0.025M H3PO4, the degradation was found to be first order with r2 being
0.9907 with a half life of 57.8 h.
The developed method was successfully applied in the analysis of azithromycin raw material and
twenty two commercial products obtained from retail pharmacies within the Central Business
District of the City of Nairobi. Thirteen of these products were tablets while nine were
suspensions of azithromycin. Five of the nine suspensions sampled were premixed suspensions
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while the rest were dry powders for reconstitution. Three tablet brands did not meet the United
States Pharmacopoeia (2012) specifications for assay (90.0-110.0% label claim) while three of
the premixed azithromycin suspensions did not meet the USP (2012) specifications. Similarly,
one of the four dry powders for reconstitution suspensions failed to comply with the assay
specifications.
The developed method is simple, robust, specific, accurate and stability indicating. It can
therefore be used by quality control laboratories in the routine analysis of azithromycin bulk
samples and formulations as well as in stability studies. The method can also be applied in post
market surveillance to monitor the quality of products in circulation and detect counterfeits.

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