A selective, sensitive and rapid LC-MS/MS method has been developed and validated as per US Food and Drug Administration regulatory guidelines for the simultaneous quantitation of colchicine and febuxostat in rat plasma. Colchicine and febuxostat were extracted from the rat plasma 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 a mobile phase comprising 5 mm ammonium formate and 0.025% formic acid in acetonitrile (20:80, v/v) 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 electrospray ionization in multiple reaction monitoring mode using transition pairs (Q1 → Q3) of m/z 400.10 → 358.10 and 317.05 → 261.00, respectively. The assay was linear in the ranges of 0.25-254 and 2.60-622 ng/ml for colchicine and febuxostat, respectively. The inter- and intra-day precision values were 0.58-13.0 and 1.03-4.88% for colchicine and febuxostat, respectively. No matrix or carryover effects were observed during the validation. Both analytes were stable on the bench-top, in the autosampler and in storage (freeze-thaw cycles and long-term storage at -80°C). A pharmacokinetic study in rats was performed to show the applicability of the validated method.
Keywords: LC-MS/MS; colchicine; febuxostat; method validation; pharmacokinetics; rat plasma.
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