Validated HPLC method for determination of PAT-5A, an insulin sensitizing agent, in rat plasma

A high performance liquid chromatographic method for the determination of PAT-5A (a potent insulin sensitizer) using DRF-2095 (a thiazolidinedione) as internal standard (I.S.) is described. A 1:1 v/v ethylacetate and dichloromethane solvent mixture was used for extraction of PAT-5A from plasma. A Kromasil KR100-5C18-250A, 5 microm, 4.6 x 250 mm SS column was used for the analysis. Mobile phase consisting of sodium dihydrogen phosphate (pH 4.0, 0.05 M) and methanol mixture (25:75, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector set at 345 nm. Ratio of peak area of analyte to I.S. was used for quantification of plasma samples. Using this method the absolute recovery of PAT-5A from rat plasma was > 90% and the limit of quantification was 0.05 microg/ml. The intra-day relative standard deviation (RSD) ranged from 2.19 to 4.98% at 1.0 microg/ml, 1.05 to 3.68% at 10.0 microg/ml and 3.14 to 5.08% at 50 microg/ml. The inter-day RSD were 1.6, 2.24 and 1.54% at 1, 10 and 50 microg/ml, respectively. The method was applied to measure the plasma concentrations of PAT-5A in pharmacokinetic and bioavailability studies in male Wistar rats.