Simultaneous determination of azathioprine and its metabolite 6-mercaptopurine in human plasma using solid phase extraction-evaporation and liquid chromatography–positive electrospray tandem mass spectrometry

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 spectrometer 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.DOI: http://dx.doi.org/10.3329/icpj.v1i11.12059 International Current Pharmaceutical Journal 2012, 1(11): 342-352

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

“…The method offers a high throughput run time of just 2.50 min. The methods reported by authors [6–13] did not apply for pharmacokinetics of azathioprine, the reported methods were for simultaneous quantification of azathioprine and mercaptopurine. They did not evaluate the metabolite impact / back conversion on azathioprine results, which are non‐selective methods compared to the current method.…”

“…To the best of our knowledge, few LC‐MS/MS and HPLC methods were reported for simultaneous estimation of azathioprine and its active metabolites in human blood, blood spots, plasma and urine [6–13]. The reported methods are only suitable for azathioprine's simultaneous estimation and active metabolites in human blood and plasma.…”

Quantification of Thiopurine Nucleotides in Erythrocytes and Clinical Application to Pediatric Acute Lymphoblastic Leukemia

“…Two more articles described application of solid-phase extraction for sample preparation, in which 6MP metabolites in plasma (not in RBC) were measured and a limited number of patients' samples were analyzed without sufficient information for clinical application. 32,33…”