Highly sensitive liquid chromatography–tandem mass spectrometry assay for the determination of azathioprine in presence of mercaptopurine and its application to a human pharmacokinetic study

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Abstract
A highly sensitive liquid chromatography–tandem mass spectrometry method was developed to estimate azathioprine in presence of its active metabolite mercaptopurine in human plasma. The analyte and isotope‐labeled internal standards were extracted from 300 μL human plasma by a solid‐phase extraction technique. The method was revealed good linearity over a concentration range of 0.151 to 49.500 ng/mL for azathioprine, containing 50.000 ng/mL of mercaptopurine in all the quality control samples. The detection was established using multiple reaction monitoring method. The method validation was carried out as per the present guidelines on bioanalytical method validation of European medicines agency and United States Food and Drug Administration. The current method was successfully applied to study the pharmacokinetics of azathioprine 50 mg tablets on healthy human volunteers. The results of pharmacokinetic parameters have demonstrated that, estimation of azathioprine in presence of mercaptopurine has no impact on azathioprine results.

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