Development and Validation of LC-MS/MS Method for the Estimation of Lovastatin and its Metabolite (β-Hydroxy Acid) in Pharmaceutical Dosage Form

Abstract

The objective of this study was to validate a simple, specific, accurate and precise Liquid - Liquid extraction high performance liquid chromatographic method with Tandem Mass Spectrometry-Thermo Scientific TSQ Quantum Ultra method for the determination of Lovastatin and Lovastatin acid in human plasma using Atorvastatin as Internal Standard (IS). The precision and accuracy data have to fulfill the requirements for quantification of the analytes in biological matrices to generate data for bioequivalence, bioavailability investigations. A Luna C18, 5µmcolumn having 4.6 x 150 mm internal diameter in isocratic elution mode with flow rate1.0 mL/min of mobile phase containing and Methanol, 5mM ammonium formate in 0.1% formic acid (80:20v/v) were used. The chromatographic separation was achieved by using elution solution consisting of Methanol and 5mM ammonium formate in 0.1% formic acid (80:20%v/v), diluent solution of methanol and water (50:50%,v/v) were monitored on a triple quadrupole massspectrometer, operating in the multiple reaction monitoring (MRM) mode. The method was validated for both Lovastatin and Lovastatin acid over the concentration range of 0.05- 5.00 ng/mL using 300 µL plasma samples. Limit of quantification was found 0.05 ng/mL. The retention time for Lovastatin acid and Lovastatin min 2.84 and 3.45 respectively and Internal Standard were 2.06 min and overall chromatography run time was 5.50 minutes. The mean recovery of Lovastatin (71.00%) and Lovastatin acid (81.20) and IS (79.84%) from spiked plasma samples was consistent and reproducible. The method was validated for linearity, accuracy, precision, specificity, limit of quantification and robustness. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines1. The developed assay method was applied to a clinical pharmacokinetic study in human volunteers.