Method Validation of a Solid Phase Extraction, High Performance Liquid Chromatography and Electrochemical Detection Method for Determination of Azithromycin in Human Plasma

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Sukanya Supattanapong
Julraht Konsil
Bung-orn Sripanidkulchai
Jeffery Roy Johns


An effective and sensitive method for the determination of azithromycin in plasma with high performance liquid chromatography (HPLC) and electrochemical detection extracted by solid phase extraction has been developed and validated. Clarithromycin was used as an internal standard. Azithromycin was extracted from 1 ml of plasma by using an Oasis® HLB (Hydrophilic-Lipophilic Balance) solid-phase extraction cartridge. After eluting with 1 ml of methanol, the sample solution was evaporated to dryness, dissolved in 100 μl phosphate buffer-methanol-acetonitrile (60:20:20, v/v/v) and 40 μl injected into the HPLC system. The lower limit of quantitation (LLOQ) was 10 ng/ml without interfering peaks. The calibration curve was linear (r² = 0.9998) over the concentration range from 10 to 400 ng/ml. The accuracy and precision were in acceptable range according to criteria of guideline for Industry: Bioanalytical Method Validation of the US FDA (2001). 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 h at room temperature and 6 months stored at -80°C. The post-preparative stability of spiked-samples was stable more than 24 h after preparation. The cartridge can be used two times providing deviation less than 4%. The method can be used for determination of azithromycin in plasma in pharmacokinetic study, bioavailability and bioequivalence.


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