Liquid chromatography-tandem mass spectrometric assay for pravastatin and two isomeric metabolites in mouse plasma and tissue homogenates

A bioanalytical assay for pravastatin and two isomeric metabolites, 3′α-isopravastatin and 6′-epipravastatin, was developed and validated. Mouse plasma and tissue homogenates from liver, kidney, brain and heart were pre-treated using protein precipitation with acetonitrile containing deuterated internal standards of the analytes. The extract was diluted with water and injected into the chromatographic system. This system consisted of a polar embedded octadecyl silica column using isocratic elution with formic acid in a water–acetonitrile mixture. The eluate was transferred to an electrospray interface using negative ionization and the analytes were detected and quantified with the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was successfully validated in a 3.4–7100 ng/ml concentration range for pravastatin, 1.3–2200 ng/ml for 3′α-isopravastatin and 0.5–215 ng/ml for 6′-epipravastatin using only plasma for calibration. For plasma samples, subjected to full validation, within and between day precisions were 1–7% (9–18% at the LLQ level) and accuracies were between 91% and 103%. For tissue homogenates, subjected to partial validation, within and between day precisions were 2–12% (6–19% at the LLQ level) and accuracies were between 87% and 113% (81 and 113% at the LLQ level). Drug and metabolites were shown to be chemically stable under most relevant analytical conditions. Finally, the assay was successfully applied for a pilot study in mice. After intravenous administration of the drug, all isomeric compounds were found in plasma; however, in liver and kidney homogenate only the parent drug showed levels exceeding the LLQ.