Simultaneous quantitation of pioglitazone and its metabolites in human serum by liquid chromatography and solid phase...

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of pioglitazone (U-72107) and its potential metabolites (M-1 to M-6) in human serum was developed. The method involved a solid phase extraction (SPE) of pioglitazone, its metabolites, and the internal standard (U-92573) from serum using C18 SPE columns with an elution solvent of 0.5 ml of acetonitrile-water (35:65, v/v). Separation of the eight analytes was achieved within 20 min using a reversed-phase Zorbax RX-C8 analytical column (250 mm x 4.6 mm i.d., 5 microns particle size) with a mobile phase of acetonitrile-water (40:60, v/v) containing 3 ml acetic acid per liter mobile phase (apparent pH 5.5). An ultraviolet detector operated at 269 nm was used with a linear response observed from 0.02 to 2 micrograms ml-1 for these analytes except for M-4 which was best fitted with a polynomial regression. Limit of quantitation was found to be 0.02 microgram ml-1 for pioglitazone, M-3, M-5, and M-6; 0.04 microgram ml-1 for M-2 and M-4; and 0.5 microgram ml-1 for M-1 when using a 0.5 ml serum sample for extraction. Obtained from the method validation, intra- and inter-assay precision was < or = 9% and accuracy ranged from -8.2 to 13.4% for all analytes. The applicability of this method has been demonstrated by successfully analyzing clinical serum samples. The strategies in the HPLC characterization and in the SPE procedure development for this method are discussed as well.