Abstract
A rapid and highly sensitive LC-MS/MS method was developed and validated for the determination of allopurinol (AP) and its major metabolite, oxypurinol (OP) in human plasma using lamivudine as an internal standard (IS). The analytes were extracted from human plasma by protein precipitation using acetonitrile. The chromatographic separation was employed on "waters symmetry shield RP
8
, 150 mm × 3.9 mm, 5 μm" columns using a mixture of 0.01% formic acid in water and acetonitrile in the ratio of 95:05 (v/v) as the mobile phase. Mass spectrometer in the selected reaction-monitoring mode with negative electro spray was used for the detection and quantification of the analyte. Recovery study was performed showing the accuracy at upper and lower limits of quantification. The stability study was also carried out. The described method was found to be linear over a range of 0.01-10 μg mL
−1
with a lower limit of quantification of 0.01 μg mL
−1
for both AP and OP. The coefficient of variation for the assay precision was found <6.94, and the accuracy was found >96.03. The extraction recoveries for AP and OP were found to be in between 70 and 80%, the accuracy was found to be in between 95 and 106% in human plasma. The dilution integrity test, hemolysis and anticoagulant effect, and matrix effect studies were reported.