Determination of allopurinol and oxypurinol in human plasma and urine by liquid chromatography-tandem mass spectrometry

Allopurinol is used widely for the treatment of gout, but its pharmacokinetics is complex and some patients show hypersensitivity, necessitating careful monitoring and improved detection methods. In this study, a sensitive and reliable liquid chromatography-tandem mass spectrometry method was developed to determine the concentrations of allopurinol and its active metabolite oxypurinol in human plasma and urine using 2,6-dichloropurine as the internal standard (IS). Analytes and the IS were extracted from 0.5 ml aliquots of plasma or urine using ethyl acetate and separated on an Agilent Eclipse Plus C18 column using methanol and ammonium formate–formic acid buffer containing 5 mM ammonium formate and 0.1% formic acid (95:5, v/v) as the mobile phase (A) for allopurinol or methanol plus 5 mM ammonium formate aqueous solution (95:5, v/v) as the mobile phase (B) for oxypurinol. Allopurinol was detected in positive ion mode and the analysis time was about 7 min. The calibration curve was linear from 0.05 to 5 μg/mL allopurinol in plasma and 0.5–30 μg/mL in urine. The lower limit of quantification (LLOQ) was 0.05 μg/mL in plasma and 0.5 μg/mL in urine. The intra- and inter-day precision and relative errors of quality control (QC) samples were ≤11.1% for plasma and ≤ 8.7% for urine. Oxypurinol was detected in negative mode with an analysis time of about 4 min. The calibration curve was linear from 0.05 to 5 μg/mL in plasma (LLOQ, 0.05 μg/mL) and from 1 to 50 μg/mL in urine (LLOQ, 1 μg/mL). The intra- and inter-day precision and relative errors were ≤7.0% for plasma and ≤9.6% for urine. This method was then successfully applied to investigate the pharmacokinetics of allopurinol and oxypurinol in humans.