Development and Validation of a Rapid HPLC Method for the Direct Determination of Colchicine in Pharmaceuticals and Biological Fluids

Abstract

A reversed‐phase high performance liquid chromatographic (RP‐HPLC) method is developed and validated for the determination of the colchicine, an anti‐gout alkaloid. The analytical column, an Inertsil ODS‐2, 5 µm, 250×4 mm, was operated at ambient temperature. Isocratic elution with A: 48% CH3COONH4 0.05 M and B: 52% CH3OH, was used at a flow rate of 1.0 mL/min. Inlet pressure was 270 kg/cm2. UV detection was performed at 245 nm. The Limit of Detection was 0.5 ng, per 20 µL injection volume, while linearity held up to 2.5 ng/µL. Codeine was used as Internal Standard at a concentration level of 10 ng/µL. The statistical evaluation of the method was performed in terms of intra‐day (n=8) and inter‐day calibration (n=8) and was found to be satisfactory, with high accuracy and precision results. The method was successfully applied to commercial pharmaceuticals and biological fluids. Blood serum samples, after deproteinization with acetonitrile, yielded high recovery rates from 97.2 to 104.6%, while direct analysis of urine after a 10‐fold dilution provided recovery rates ranging from 89.5 to 104.0%.