A novel LC-ESI-MS/MS assay method for the determination of anagrelide in human plasma by using a solid phase extraction technique and its application to a pharmacokinetic study

Abstract

A simple, rapid and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay method has been developed and validated for the determination of anagrelide in human plasma samples using nevirapine as internal standard (IS). The analyte and the IS were extracted from 100 μL of human plasma using solid-phase extraction with no drying, evaporation and reconstitution steps. The chromatographic separation was achieved on a C₁₈ column by using a mixture of methanol and 0.1% formic acid in 5 mM ammonium acetate (80 : 20, v/v) as the mobile phase at a flow rate of 1.0 mL min⁻¹. The linearity of the method was established in the concentration range 0.05–10.0 ng mL⁻¹ with r² ≥ 0.99. Method validation was performed as per FDA and EMEA guidelines and the results met the acceptance criteria. Data acquisition was done by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer in the positive ionization mode. The intra-day and inter-day precision (%CV) and accuracy results in five validation batches across five concentration levels were well within the acceptance limits. The validated method was successfully applied to a pharmacokinetic study in healthy South Indian male subjects under fasting conditions with 0.5 mg anagrelide capsules.