Method Development and Validation of Topotecan Hydrochloride in K2 EDTA Human Plasma by Using HPLC Coupled with Tandem Mass Spectrometry

Abstract

A rapid, sensitive and specific method based on high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) detection with electro spray ionization in positive ionization mode has been developed for the determination of topotecan hydrochloride in K₂ EDTA Human plasma. Sample preparation involved protein precipitation extraction with 0.1 % acetic acid in cold acetonitrile. chromatographic separation was performed on Agilent Eclipse XDB column (C₁₈ 150 × 4.6 mm 5μ), with a mobile phase consisting of 0.1 % acetic acid in acetonitrile and 0.5 % acetic acid in water at a flow rate of 0.7 mL/min. The analytes were then detected by monitoring the transitions m/z 422.2-219.4, 377.0 for topotecan and m/z 587.6- 124.2 for irinotecan using API-4000 LC-MS/MS system (AB Sciex). Calibration curve were linear within the range of 0.5 to 50.00 ng/mL. The lower limit of quantitation was 0.5 ng/mL. The method has been fully validated in K₂EDTA Human plasma. This method can be successfully applied to the pharmacokinetic study sample analysis.