A Validated LC-MS/MS Method for Simultaneous Quantification of Simvastatin and Simvastatin Acid in Beagle Plasma: Application to an Absolute Bioavailability Study.

A highly sensitive LC-MS/MS method for simultaneous detection of both simvastatin (SV) and simvastatin acid (SVA) in beagle plasma was developed and successfully applied to an absolute bioavailability study. Lovastatin (LV) was used as internal standard (IS). The analysis was performed using an electrospray ionization (ESI) and selective reaction monitoring (SRM) in positive mode at m/z 441.0 → 325.0 for SV, 459.0 → 343.0 for SVA and 427.0 → 325.0 for IS, respectively. The assay procedure involved a simple liquid-liquid extraction (LLE) of SV, SVA and LV from beagle plasma into methyl tert-butyl ether. Separation of SV, SVA and IS was achieved on a shim-pack VP-ODS column (150 × 2.0 mm, 5 μm) with a binary gradient solvent system of 0.1% formic acid in water and methanol (15:85, v/v) as the mobile phase. The method was validated over the range of 0.25-500 ng/mL for SV (r2 ≥ 0.9923) and 0.24-481.23 ng/mL for SVA (r2 ≥ 0.9987). The results of method validation for accuracy, precision, extraction recovery, matrix effect and stability were within the acceptance criteria. The value of absolute bioavailability of SV and SVA in beagles was 2.97% and 25.40%, respectively. It is the first study developed for the measurement of absolute bioavailability of simvastatin and simvastatin acid in beagles.