A validated liquid chromatography tandem mass spectrometry method for simultaneous determination of pioglitazone, hydroxypioglitazone, and ketopioglitazone in human plasma and its application to a clinical study

• LC/MS/MS-method developed for analysis of pioglitazone and both active metabolites.
• Samples process by protein precipitation/phospholipid depletion in 96-well format.
• Method was successfully applied in a clinical trial in fatty liver disease.

A simple and rapid high performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of pioglitazone and its active metabolites hydroxypioglitazone and ketopioglitazone in human plasma. Samples were processed by protein precipitation with acetonitrile and selective phospholipid depletion in a 96-well plate format. The method used deuterated internal standards for each analyte. Chromatographic separation was achieved with gradient elution on a Hypersil GOLD C18 column. The mass spectrometer was operated in electrospray positive ion mode with detection by selected reaction monitoring using the transitions m/z 357.1 > 134.0 for pioglitazone, m/z 373.1 > 150.0 for hydroxypioglitazone, and m/z 371.0 > 148.0 for ketopioglitazone. A linear standard curve was established for the range of 10–1800 ng/mL for all three analytes. Intra-run and inter-run precision and accuracy (relative error) were less than 15%, and the mean extraction recoveries of all analytes were more than 87.8%. The validated method is sensitive and selective and was successfully applied to analyze clinical samples obtained from patients with nonalcoholic fatty liver disease taking pioglitazone.