ABSTRACT
A simple, rapid, specific, sensitive and liquid chromatography coupled with tandem mass spectrophotometric method was developed and validated for the estimation of azathioprine and its metabolite 6-mercaptopurine in human plasma by using lamivudine and 6-mercaptopurine D3 as the internal standard. Azathioprine and 6-mercaptopurine were extracted from human plasma by solid-phase extraction (SPE)-Evaporation method, using Oasis MCX cartridge for cleaning procedure. The stationary phase was chromatographed on a ZORBAX SB CN, (75X50 mm, 5 μ) column where as mobile phase constitutes of acetonitrile: 2mM ammonium acetate (70:30 v/v) at a flow rate of 0.800 ml/min. The detection was performed with an Applied Biosystems Sciex API 4000 mass spectrome-ter by multiple reaction monitoring (MRM). The method validation proofs were carried out as per the USFDA guidelines as described, showing a linearity system (r2 > 0.99) over a range of 2.455 ng/mL to 106.568 ng/mL for azathioprine and 1.165 ng/mL to 101.143 ng/mL concentrations for 6-mercaptopurine and a recovery shows 99.36% and 100.44% for azathioprine and 6-mercaptopurine respectively. The results show that this proposed approach is effective and can be applied to the extraction and analysis of other pharmaceutical compounds.
ABSTRACT
A simple, rapid, specific, sensitive and liquid chromatography coupled with tandem mass spectrophotometric method was developed and validated for the estimation of azathioprine and its metabolite 6-mercaptopurine in human plasma by using lamivudine and 6-mercaptopurine D3 as the internal standard. Azathioprine and 6-mercaptopurine were extracted from human plasma by solid-phase extraction (SPE)-Evaporation method, using Oasis MCX cartridge for cleaning procedure. The stationary phase was chromatographed on a ZORBAX SB CN, (75X50 mm, 5 μ) column where as mobile phase constitutes of acetonitrile: 2mM ammonium acetate (70:30 v/v) at a flow rate of 0.800 ml/min. The detection was performed with an Applied Biosystems Sciex API 4000 mass spectrome-ter by multiple reaction monitoring (MRM). The method validation proofs were carried out as per the USFDA guidelines as described, showing a linearity system (r2 > 0.99) over a range of 2.455 ng/mL to 106.568 ng/mL for azathioprine and 1.165 ng/mL to 101.143 ng/mL concentrations for 6-mercaptopurine and a recovery shows 99.36% and 100.44% for azathioprine and 6-mercaptopurine respectively. The results show that this proposed approach is effective and can be applied to the extraction and analysis of other pharmaceutical compounds.