Quantitative determination of azithromycin in human plasma by liquid chromatography-mass spectrometry and its application in a bioequivalence study.

Abstract A sensitive, rapid liquid chromatographic–electrospray ionization mass spectrometric method for determination of azithromycin in human plasma was developed and validated. Azithromycin in plasma (0.2 mL) was extracted with methyl tert -butyl ether–hexane (50:50, v/v), organic phase was transferred to another clear 1.5 mL Eppendorf tube and evaporated to dryness at 40 °C and dissolved in mobile phase, samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150 mm × 2.1 mm i.d., 5 μm), together with a mobile phase containing of 20 mM ammonium acetate (pH 5.2)–acetonitrile–methanol (50:40:10, v/v/v) and was isocratically eluted at a flow rate of 0.2 mL/min. Azithromycin and its internal standard, clarithromycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 2 to 1000 ng/mL with r  = 0.9977. The limit of quantification for azithromycin in plasma was 2 ng/mL with good accuracy and precision. The higher mean extraction recovery, say 81.2% and 75.5% for azithromycin and internal standard (IS), respectively, was obtained in this work. The intra-day and inter-day precision ranged from 4.8% to 8.6% and 6.4% to 10.7% (R.S.D.), respectively. The established method has been successfully applied to bioequivalence study of 2 azithromycin formulations for 24 healthy volunteers.