Liquid Chromatography Mass-spectrometry for Determination of Azithromycin in Plasma and Application for Pharmacokinetic Study

Abstract

A simple, sensitive, selective and reproducible method based on high performance
liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/MS) was
developed for the determination of azithromycin (AZM) in human plasma. The internal
standard (IS: roxithromycin) was separated from AZM on a Hypersil Gold C18 column, with
retention times of 10.71 and 13.67 min, respectively. The mobile phase consisted of a
mixture of 20 mM ammonium acetate buffer (pH 5.2), acetonitrile and methanol (50:40:10,
v/v/v), running through the column at a flow rate of 0.3 mL/min. Sample preparation was
prepared by liquid-liquid extraction with a mixture of 7:3 (v/v) diethylether:
dichloromethane. The precision of the method based on within-day repeatability and
reproducibility (day-to-day variation) was below 5% (% coefficient of variations: % CV).
Good accuracy was observed for both the intra-day or inter-day assays. Limit of
quantification was accepted as 0.5 ng using 200 μL plasma samples. The mean recoveries for
AZM and the IS were greater than 85%. The method was applied successfully to the
investigation of the pharmacokinetics of AZM when given as oral doses of 750 mg twelve
hourly for 3 days in a total of 5 Thai male patients with acute uncomplicated falciparum
malaria.