Validated Rp-Lc-MS/MS Method for the Simultaneous Determination of Allopurinol and Its Major Metabolite, Oxypurinol, in Human Plasma

Abstract

A rapid and highly sensitive LC-MS=MS method was developed and validated for the determination of allopurinol (AP) and its major metabolite, oxypurinol (OP) in human plasma using lamivudine as an internal standard (IS). The analytes were extracted from human plasma by protein precipitation using acetonitrile. The chromatographic separation was employed on ''waters symmetry shield RP 8 , 150 mm  3.9 mm, 5 lm'' columns using a mixture of 0.01% formic acid in water and acetonitrile in the ratio of 95:05 (v=v) as the mobile phase. Mass spectrometer in the selected reaction-monitoring mode with negative electro spray was used for the detection and quantification of the analyte. Recovery study was performed showing the accuracy at upper and lower limits of quantification. The stability study was also carried out. The described method was found to be linear over a range of 0.01-10 lg mL À1 with a lower limit of quantification of 0.01 lg mL À1 for both AP and OP. The coefficient of variation for the assay precision was found <6.94, and the accuracy was found >96.03. The extraction recoveries for AP and OP were found to be in between 70 and 80%, the accuracy was found to be in between 95 and 106% in human plasma. The dilution integrity test, hemolysis and anticoagulant effect, and matrix effect studies were reported.

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

“…Few methods have reported use of liquid chromatography with mass spectrometric detection for the determination of AP and/or OP in human plasma [13] , [14] , [15] and urine [12] , [14] . In these methods OP has been detected in the negative ESI mode while AP was monitored either in the positive [14] or negative [12] , [13] ionization mode.…”

“…In these methods OP has been detected in the negative ESI mode while AP was monitored either in the positive [14] or negative [12] , [13] ionization mode. While another method reported measurement in the negative mode for both the analytes [15] . As AP and OP are weakly acidic in nature with an ionization constant of 9.4 and 7.7, respectively [2] , ionization was first tried in the negative mode; however, the response was too less for the deprotonated precursor ions of AP ( m/z 135.1, 1.5e5 cps) as compared to OP ( m/z 150.9, 4.2e7 cps).…”

“…However, some of these methods involve lengthy extraction protocols [10] , have chromatographic run time more than 10 min [8] , are less sensitive [8] , [9] and employ large sample volume for processing [3] . Few methods report use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) technique for the determination of OP as a single analyte in human urine [12] and for the simultaneous estimation of AP and OP in human plasma [13] , [14] , [15] and urine [14] . The method described by Ayalasomayajula et al [13] involved a combination of protein precipitation (PPT) and liquid-liquid extraction (LLE) for the extraction of the analytes; however, there was limited information on the method development as the aim was to study the effect of AP on the pharmacokinetics of aliskiren.…”

“…Though the method reported by Liu et al [14] is sensitive but employs two different mobile phases for eluting AP and OP separately and also different electrospray ionization (ESI) modes for mass spectrometric detection. Another LC–MS/MS method developed by Kasawara et al [15] required large plasma volume for processing (500 µL) and both the analytes were detected in the negative ionization mode.…”

Capillary Electrophoresis Hyphenated with Mass Spectrometry for Determination of Inflammatory Bowel Disease Drugs in Clinical Urine Samples

“…LC–mass spectrometry (MS) (LC-MS) was applied for the determination of 6-TG and 6-methylmercaptopurine (6-MMP) in whole blood samples [ 4 ], and 6-MP and its metabolite 6-TG in human plasma [ 5 , 6 ]. ALP and its active metabolite oxypurinol (OXP) have been simultaneously determined via LC-MS/MS in human plasma [ 7 , 8 , 9 ] and in human urine [ 9 , 10 ]. MSL and its metabolite N -acetylmesalazine have been analyzed via LC-MS/MS in human plasma [ 11 , 12 ].…”