Rapid and Stable Liquid Chromatographic Tandem Mass Spectrometric Method for Simultaneous Estimation of Pioglitazone, Keto Pioglitazone and Hydroxy Pioglitazone in Human Plasma: Application to Bioequivalence Study

Abstract

High performance liquid chromatographic tandem mass spectrometric method for the determination of pioglitazone, keto pioglitazone (M-III) and hydroxy pioglitazone (M-IV) in human plasma has been developed and validated using pioglitazone-D4, keto pioglitazone-D4 and hydroxy pioglitazone-D5 as internal standards. Solid phase extraction was carried out for sample preparation to extract analyte and internal standard from human plasma. The extracted sample was injected through autosampler in HPLC connected with Hypersil Gold, 100 mm × 4.6 mm, 5 μm using mobile phase consisting of methanol:solution A:solution B - 80:10:10 v/v/v). Pioglitazone, keto pioglitazone (M-III) and hydroxy pioglitazone (M-IV) were chromatographically separated and detected using the MS detector. The best-fit lines using weighting factor (1/concentration2) linear least square regression analysis were obtained by peak area ratio of pioglitazone, keto pioglitazone (M-III) and hydroxy pioglitazone (M-IV) with their internal standards pioglitazone-D4, keto pioglitazone-D4 and hydroxy pioglitazone-D5, respectively. This report provides the results of various validation parameters including stability studies and extended precision and accuracy which is required for long study samples batch analysis. This analytical method is valid for the determination of pioglitazone in the range of 18.9 ng/mL to 2994.4 ng/mL), keto pioglitazone (M-III) (3.23 ng/mL to 512.60 ng/mL), hydroxy pioglitazone (M-IV) 10.1 ng/mL to 1603.8 ng/mL) and using pioglitazone-D4, keto pioglitazone-D4, hydroxy pioglitazone-D5, respectively as internal standards in human plasma using a Hypersil Gold, 100 mm × 4.6 mm, 5 μm column.