Purpose: Statins are recommended to alleviate high blood cholesterol specially for their effect in decreasing the risk of atherosclerotic cardiovascular disease. Simultaneous analysis of several statins and their active metabolites will facilitate the conduction of clinical pharmacokinetics studies that involve multiple statins in patients prior and post-bariatric surgery. Since triglyceride levels are elevated in most obese patients, matrix effect should be validated in hyperlipidemic plasma (plasma with high triglyceride levels of ≥ 300 mg/dl) to ensure the reliability of the method when used to analyze samples from the obese subjects. The purpose of this study was to develop and validate an LC-MS/MS method for the simultaneous quantifications of simvastatin (SMV), simvastatin acid (SMV-A), atorvastatin (ATV), 2-hydroxy atorvastatin (2-OH-ATV), 4-hydroxy atorvastatin (4-OH-ATV), and rosuvastatin (RSV) in human plasma. The method reliability was ensured by performing matrix effect in hyperlipidemic plasma.
Methods: The analytes were extracted from plasma samples (200 µl) with 1 ml of ethyl acetate. The mixture was vortexed and centrifuged, and the upper layer was collected and dried to be reconstituted with water/acetonitrile (70:30 v/v) upon analysis. The analytes were separated on ACQUITY UPLC BEH C18 column (2.1 × 100 mm I.D., 1.7 µm) using 10 mM ammonium formate and 0.04% formic acid in water as mobile phase A and acetonitrile as mobile phase B with a gradient elution at a flow rate of 0.4 ml/min. LC-MS/MS analysis was performed using an API 5500-Qtrap triple quadrupole mass spectrometer with the transitions of m/z 436.3 → 285.2 for SMV, m/z 437.2 → 303.2 for SMV-A, m/z 559.2 → 440.3 for ATV, m/z 575.4 → 440.3 for 2-OH-ATV and 4-OH-ATV, m/z 482.3 → 258.1 for RSV, and m/z 412.3 → 224.2 for fluvastatin (Internal Standard, IS). The validation was performed according to FDA guidelines 2018 for accuracy, precision, extraction recovery, matrix effect, and stability. Matrix effect was performed in plasma samples with high triglyceride levels (ranged 371-467 mg/dl).
Results: The retention times of SMV (3.8 min), SMV-A (3.6 min), ATV (3.2 min), 2-OH-ATV (3.1 min), 4-OH-ATV (2.8 min), RSV (2.9 min) and the IS (3.2 min) as shown in Figure 1, with no significant interference in the chromatogram of blank plasma. The linearity of the method was determined at the range of 0.25 (LLOQ) -100 ng/ml with a mean correlation coefficient (r2) ≥ 0.98 for all the analytes. Low, medium and, high quality control samples at concentrations of 0.5, 5, and 80 ng/ml, respectively, were used to validate the method. Intra-day accuracy (95-109%) and precision (3-13%) were within the acceptable 15% deviation allowed by FDA. Inter-day accuracy and precision results for all analytes were within the ranges of 96-106% and 5-10%. Extraction recovery for all analytes was within the ranges of 88-100%. Matrix effect for plasma from lean subjects was within the ranges of 93-110%. When matrix effect was performed for plasma samples with high triglyceride levels, the range was 92-103%, not significantly different from that of the plasma from lean subjects. Stability was within 91-100% for freeze and thaw (3 cycles), bench-top (4 hr at room temperature), long-term (1 month at -80ºC), processed samples (24 hr at 20ºC) and stock solution (1 month at -20ºC).
Conclusion: A robust, sensitive, accurate, and specific LC-MS/MS method, for the simultaneous quantifications of simvastatin, simvastatin acid, atorvastatin, 2-hydroxy atorvastatin, 4-hydroxy atorvastatin, and rosuvastatin, was developed and validated. No significant matrix effect was observed even with high triglyceride levels in the plasma. The method can be used in clinical studies that involve multiple statins in subjects’ medications.
Lily Cheung– Associate Professor, Texas Southern University, Houston, Texas
Yang Wang– Postdoctoral Fellow, University of Houston, Houston, Texas
Vadim Sherman– Houston Methodist Surgical Associates, Houston Methodist Research Institute, Houston, Texas
Diana Chow– Professor of Pharmaceutics and Director, Institute of Drug Education and Research, University of Houston, Houston, Texas