Solid phase extraction and high performance liquid chromatography for the determination of azithromycin in human plasma.

ABSTRACT

An effective, selective and sensitive method for the determination of azithromycin in plasma using high performance liquid chromatography-electrochemical detection (HPLC-ECD) extracted by solid phase extraction was developed and validated. Clarithromycin was used as an internal standard. Azithromycin was extracted from 1 ml of plasma using an Oasis HLB solid-phase extraction cartridge and the elution was evaporated to dryness, dissolved in 100 microl mobile phase and 40 microl auto-injected into an HPLC system with a 200 microl loop. The mobile phase was acetonitrile, methanol, phosphate buffer, 0.05M, pH 6.0 (202060, v/v/v) with a flow rate of 1.0 ml/minute. The lower limit of quantitation (LLOQ) was 10 ng/ml without interfering peaks. The calibration curve was linear (r2 = 0.9998) over a concentration range 10 to 400 ng/ml. The accuracy and precision were acceptable. The mean recoveries at 30, 100 and 200 ng/ml were 85.3 +/- 5.5%, 80.1 +/- 6.8% and 82.9 +/- 2.5%, respectively. Azithromycin was stable in plasma for at least 6 hours at room temperature and 6 months storage at -80 degrees C. The post-preparation stability of spiked samples was more than 24 hours after preparation. The cartridge can be used two times providing a deviation of less than 4%. This method has adequate sensitivity, specificity, accuracy and precision to measure azithromycin in human plasma and is free from interference from the plasma matrix. The validated method was to quantify azithromycin plasma concentrations in five healthy Thai volunteers after the administration of 500 mg azithromycin capsules.