Abstract
A fixed dose combination of lesinurad and allopurinol has been recently approved by USFDA and EMA for treatment of gout-associated hyperuricemia in patients who have not achieved target serum uric acid levels with allopurinol alone. In this study, an ultra-performance hydrophilic interaction liquid chromatography (UPHILIC) coupled with tandem mass spectrometry method was developed and validated for simultaneous determination of allopurinol, oxypurinol and lesinurad in rat plasma. Liquid liquid extraction using ethyl acetate as extracting agent was used for samples extraction procedure. Acquity UPLC HILIC column (100 mm x 2.1, 1.7μm) was used for separation of allopurinol, oxypurinol, lesinurad and internal standard (5-Florouracil). The mobile phase consisting of acetonitrile, water and formic acid (95:5:0.1, v/v/v), were eluted at 0.3 mL/min flow rate having total chromatographic run time of 3 min per sample. The analytes were detected on Acquity triple quadrupole mass spectrometer equipped with a Z-Spray electrospray ionization (ESI). The ESI source was operated in negative mode and multiple reaction monitoring was used for ion transition for all compounds. The precursor to product ion transition of m/z 134.94 > 64.07 for allopurinol, 150.89 > 41.91 for oxypurinol, 401.90 > 176.79 for lesinurad and 128.85 >41.92 for internal standard were used for identification and quantification. The calibration curves for all analytes were found to be linear with weighing factor of 1/x2 using regression analysis. The developed assay was successfully applied in an oral pharmacokinetic study of allopurinol, oxypurinol and lesinurad in rats.
Highlights
Gout is a form of inflammatory arthritis characterized by the deposition of monosodium urate crystals in the joints due to elevated levels of serum uric acid (SUA), known as hyperuricaemia [1,2]
ALP and OXP were detected by both electrospray ionization (ESI) positive or negative mode, whereas LES was only measured by ESI positive mode
Negative ionization mode was selected for monitoring of all three analytes (ALP, OPX and LES) which helped in curtailing time required for stabilization of high voltages during polarity switching
Summary
Gout is a form of inflammatory arthritis characterized by the deposition of monosodium urate crystals in the joints due to elevated levels of serum uric acid (SUA), known as hyperuricaemia [1,2]. Allopurinol (ALP), a xanthine oxidase inhibitor (XOI), is one of the most commonly prescribed medicine for the treatment of hyperuricaemia and gout [3,4]. It acts by inhibiting the xanthine oxidase (XO) enzyme which catalyzes the formation of xanthine from hypoxanthine and further to uric acid [3,5]. Like ALP, OXP inhibits XO enzyme and has much longer serum half-life (⁓23 h) compared to ALP (⁓1.2 h) and responsible for most of the pharmacological effects of ALP [6,7,8]. The main therapeutic effect produces by OXP, but due to poor absorption of OXP preparation, parent drug (ALP) is still used as main formulation [9]
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