To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of atorvastatin, two activity-related hydroxy statin metabolites and three toxicity-related statin lactones in human plasma, and its application to the study of pharmacokinetics in healthy subjects and the analysis of concentrations in patients.

After acidification, plasma samples were treated by protein precipitation. The LC separation was performed on a Zorbarx SB-C₁₈(50 mm×2.1 mm, 5 μm) column. Methanol-acetonitrile (1∶1) water-methanol-acetonitrile (9∶0.5∶0.5) containing 0.05% formic acid were used as the mobile phases for gradient elution, and the flow rate was 0.35 mL·min^-1. The electric spray ionization source, positive ion mode and multi-reaction monitoring scanning were adopted for MS detection. The m/z of each targeted analyte was 559.3→440.2 for atorvastatin, 575.1→440.3 for 2-hydroxy atorvastatin acid (2-HAT) and 4-hydroxy atorvastatin acid (4-HAT), 540.9→448.2 for atorvastatin lactone (ATL), 557.2→448.2 for 2-hydroxy atorvastatin lactone (2-HATL) and 4-hydroxy atorvastatin lactone (4-HATL), and 422.2→290.0 for the internal standard of pitavastatin. After a full method validation, the developed LC-MS/MS method was used to determine the plasma samples of healthy subjects and patients after taking atorvastatin calcium tablets, and the pharmacokinetic characteristics of atorvastatin and five metabolites were analyzed.

The calibration curves of atorvastatin and its metabolites presented a good linear relationship in the range of 0.1-25 nmol·L^-1. The RSD of intra-and inter-day precision and the RE of accuracy were all less than 15%, and the stability was well tolerated under different conditions. In healthy subjects after oral administration of 20 mg atorvastatin calcium tablets, the respective mean values of C_max for atorvastatin, 2-HAT, 4-HAT, ATL, 2-HATL and 4-HATL were 11.48, 4.71, 0.28, 1.71, 2.52 and 2.31 nmol·L^-1, AUC_0-∞ were 87.31, 58.79, 8.60, 28.75, 45.76, 31.49 nmol·h·L^-1, t_1/2 were 7.96, 7.93, 19.58, 8.76, 8.98 and 21.37 h. After 12 h of administration, the average blood concentrations of atorvastatin, 2-HAT, 4-HAT, ATL, 2-HATL and 4-HATL in the patient were (4.16±1.31) nmol·L^-1, (2.65±1.33) nmol·L^-1, (1.15±1.16) nmol·L^-1, (2.96±1.83) nmol·L^-1, (4.27±2.00) nmol·L^-1 and (3.70±1.74) nmol·L^-1.

The method for the simultaneous quantitative determination of atorvastatin and five metabolites in human plasma established in this study is accurate, rapid, sensitive and stable, and can be used for clinical pharmacokinetics research and plasma drug concentration monitoring. The clinical studies revealed that toxicity related lactone metabolites have a high level of exposure in humans, which requires attention to the possible risk of side effects.