Liquid chromatography–tandem mass spectrometry assay for the simultaneous quantification of simvastatin, lovastatin, atorvastatin, and their major metabolites in human plasma

• A novel and robust LC–MS/MS assay was developed for multiple statins and metabolites.
• Simple protein precipitation provided high recovery for all analytes.
• The linear quantifiable ranges were validated for 0.1–100 nM and 1–1000 nM.
• The accuracy and precision of the assay met FDA standards.
• The assay was successfully applied to human plasma samples from a clinical study.

Millions of individuals are treated with a variety of statins that are metabolized to a variety of active metabolites. A single assay capable of simultaneously quantifying commonly used statins and their major metabolites has not been previously reported. Herein we describe the development and validation of a novel and robust liquid chromatography–tandem mass spectrometry assay for simultaneously quantifying simvastatin, lovastatin, atorvastatin, and their metabolites, simvastatin acid, lovastatin acid, para-hydroxy atorvastatin, and ortho-hydroxy atorvastatin in human plasma. Plasma samples were processed with a simple protein precipitation technique using acetonitrile, followed by chromatographic separation using an Agilent Zorbax Extend C18 column. A 12.0 min linear gradient elution was used at a flow rate of 400 μL/min with a mobile phase of water and methanol, both modified with 2 mM ammonium formate and 0.2% formic acid. The analytes and internal standard, hesperetin, were detected using the selected reaction monitoring mode on a TSQ Quantum Discovery mass spectrometer with positive electrospray ionization. The assay exhibited a linear range of 1–1000 nM for simvastatin acid and lovastatin acid, and a linear range of 0.1–100 nM for the other analytes in human plasma. The accuracy and the within- and between-day precisions of the assay were within acceptable ranges, and the method was successfully utilized to quantify the statins and their metabolites in human plasma samples collected from an ongoing pharmacokinetic study.