Simultaneous analysis of allopurinol and oxypurinol using a validated liquid chromatography-tandem mass spectrometry method in human plasma

Abstract

The present study describes a simple, reliable and reproducible liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for the simultaneous determination of allopurinol and its active metabolite, oxypurinol in human plasma for a pharmacokinetic/bioequivalence study. After protein precipitation (PPT) of 100 µL plasma sample with 1.0% formic acid in acetonitrile, the recovery of the analytes and allopurinol-d2 as an internal standard ranged from 85.36% to 91.20%. The analytes were separated on Hypersil Gold (150 mm×4.6 mm, 5 µm) column using 0.1% formic acid-acetonitrile (98:2, v/v) as the mobile phase. Quantification was done using electrospray ionization in the positive mode. The calibration concentration range was established from 60.0 to 6000 ng/mL for allopurinol and 80.0-8000 ng/mL for oxypurinol. Matrix effect in human plasma, expressed as IS-normalized matrix factors ranged from 1.003 to 1.030 for both the analytes. The developed method was found suitable for a clinical study with 300 mg allopurinol tablet formulation in healthy subjects.

Keywords: Allopurinol; Bioequivalence study; Liquid chromatography–tandem mass spectrometry; Oxypurinol.

Fig. 1

Product ion mass spectra of (A) allopurinol (m/z 137.0 → 109.9, scan range 50–150 Da), (B) oxypurinol (m/z 153.1 → 136.0, scan range 100–200 Da) and (C) allopurinol-d2, IS (m/z 139.0 → 111.9, scan range 100–200 Da) in the positive ionization mode.

Fig. 2

MRM ion-chromatograms of (A) double blank plasma, without allopurinol-d2, IS, (B) blank plasma spiked with IS, (C) allopurinol, allopurinol-d2 and oxypurinol at lower limit of quantification and (D) a real subject sample at C_max after oral administration of 300 mg allopurinol tablet formulation.

Fig. 3

Post-column analyte infusion chromatograms of (A) allopurinol, (B) oxypurinol and (C) allopurinol-d2 at upper limit of quantification while injecting extracted blank plasma.

Fig. 4

Mean plasma concentration-time profile of (A) allopurinol and (B) oxypurinol after oral administration of 300 mg allopurinol tablet formulation (test) and reference formulation to 44 healthy Indian subjects. The plasma-time profile of allopurinol is presented up to 24 h as the concentration was below the limit of quantitation beyond this time. None of the subjects had a measurable concentration after this time point.