Simultaneous LC-MS/MS analysis of simvastatin, atorvastatin, rosuvastatin and their active metabolites for plasma samples of obese patients underwent gastric bypass surgery

Abstract

Statins, HMG-CoA reductase inhibitors, are considered the first line treatment of hyperlipidemia to reduce the risk of atherosclerotic cardiovascular diseases. The prevalence of hyperlipidemia and the risk of atherosclerotic cardiovascular diseases are higher in obese patients. Published methods for the quantification of statins and their active metabolites did not test for matrix effect of or validate the method in hyperlipidemic plasma. A sensitive, specific, accurate, and reliable LC-MS/MS method for the simultaneous quantification of simvastatin (SMV), active metabolite of simvastatin acid (SMV-A), atorvastatin (ATV), active metabolites of 2-hydroxy atorvastatin (2-OH-ATV), 4-hydroxy atorvastatin (4-OH-ATV), and rosuvastatin (RSV) was developed and validated in plasma with low (52-103 mg/dl, <300 mg/dl) and high (352-403 mg/dl, >300 mg/dl) levels of triglyceride. The column used in this method was ACQUITY UPLC BEH C18 column (2.1 × 100 mm I.D., 1.7 μm). A gradient elution of mobile phase A (10 mM ammonium formate and 0.04% formic acid in water) and mobile phase B (acetonitrile) was used with a flow rate of 0.4 ml/min and run time of 5 min. 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) were determined by Selected Reaction Monitoring (SRM) method to detect transitions ions in the positive ion mode. The assay has a linear range of 0.25 (LLOQ) -100 ng/ml for all six analytes. Accuracy (87-114%), precision (3-13%), matrix effect (92-110%), and extraction recovery (88-100%) of the assay were within the 15% acceptable limit of FDA Guidelines in variations for plasma with both low and high triglyceride levels. The method was used successfully for the quantification of SMV, ATV, RSV, and their active metabolites in human plasma samples collected for an ongoing clinical pharmacokinetic and pharmacodynamic study on patients prior to and post gastric bypass surgery (GBS).

Keywords: Atorvastatin and metabolites; Hyperlipidemic plasma; LC–MS/MS; Obese subjects from GBS; Rosuvastatin; Simvastatin and metabolite.

Figure 1.

Representative SRM positive product ion mass spectra for (a) SMV, (b) SMVA, (c) ATV, (d) 2-OH-ATV and 4-OH-ATV, (e) RSV, and (f) IS. The transitions were 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 IS

Figure 2.

Representative chromatograms of (a) blank plasma, (b) IS, and (c) the six analytes at LLOQ of 0.25 ng/ml in plasma with low (left panel) and high (right panel) triglyceride levels. The retention times for SMV, SMV-A, ATV, 2-OH-ATV, 4-OH-ATV, RSV, and IS are 3.8, 3.6, 3.2, 3.1, 2.8, 2.9, and 3.3 min, respectively.

Figure 3.

Plasma concentration profiles from obese patients for (a) simvastatin and simvastatin acid, (b) atorvastatin, 2-hydroxy atorvastatin, and 4-hydroxy atorvastatin, and (c) rosuvastatin pre- and post-surgery at baseline, 3 M, 6 M and 12 M post GBS. Molar concentrations were normalized by dose/ body weight [(nM)/(mg/Kg). *Pt: patient ID number; 3 M, 6 M and 12 M: 3, 6, and 12 months follow-up visit after GBS