Simultaneous determination of colchicine and febuxostat in rat plasma: Application in a rat pharmacokinetic study.

A selective, sensitive and rapid LC-MS/MS method has been developed and validated as per FDA regulatory guideline for the simultaneous quantitation of colchicine and febuxostat in rat plasma. From the rat plasma, colchicine and febuxostat were extracted using 10% tert-butyl methyl ether in ethyl acetate using colchicine-d6 as an internal standard (IS). The chromatographic separation of colchicine, febuxostat and the IS was achieved using an isocratic a mobile phase [comprising of 5 mM ammonium formate with and 0.025% formic acid in acetonitrile (20:80, v/v)] the separation of colchicine, febuxostat and the IS was achieved in isocratic mode on an Eclipse XDB-C18 column. The injection volume and flow-rate were 5.0 μL and 0.9 mL/min, respectively. Colchicine and febuxostat were detected by positive electro-spray ionization in multiple reaction monitoring using transition pair (Q1→Q3) of m/z 400.10→358.10, 317.05→ 261.00, respectively. The assay was linear in the range of 0.25-254 and 2.60-622 ng/mL for colchicine and febuxostat, respectively. The inter- and intra-day precision values ranged between 0.58-13.0% and 1.03-4.88% for colchicine and febuxostat, respectively. No matrix and carryover effects were observed during the validation. Both analytes were stable on bench-top, in auto-sampler and storage (freeze-thaw cycles and long-term storage at -80°C) conditions. A pharmacokinetic study in rats was done to show the applicability of the validated method.