Rapid, Selective, and Rugged Method Development and Validation of Atorvastatin and its Active Metabolites, o-Hydroxy Atorvastatin and p-Hydroxy Atorvastatin, in Stabilized Plasma Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Abstract

A simple, sensitive, selective, and rugged liquid chromatography coupled with mass spectrometry (LC-MS/MS) method for the quantification of atorvastatin and its active metabolites, p-hydroxy atorvastatin and o-hydroxy atorvastatin, in human plasma was developed and validated. Isocratic elution of atorvastatin and its metabolites were achieved in 4.8 min using Luna 5 μ, C18, 100 mm × 4.60 mm column having a mobile phase of acetonitrile: 0.20% formic acid (65:35% v/v). The flow rate was 0.60 mL/min at a column temperature of 40 ± 5°C. Electron spray ionization technique in negative mode was selected to improve the selectivity and sensitivity required for this application. The retention times of atorvastatin, p-hydroxy atorvastatin and o-hydroxy atorvastatin were 3.73, 2.34, and 3.33 min, respectively. The method was validated for linearity, precision, accuracy, specificity, sensitivity, matrix effect, dilution integrity, ruggedness, reinjection reproducibility, and stability. Calibration curves during the course of validation were found to be linear for atorvastatin, p-hydroxy atorvastatin, and o-hydroxy atorvastatin in the ranges of 204.554–202,809.991, 197.836–196,149.459, and 202.316–200,590.719 pg/mL with correlation coefficient ≥0.9969, 0.9958, and 0.9970, respectively, and by using a 1/x² weighted least square regression analysis of standard plots associated with ten-point calibration standards. The precision and mean accuracy were within the acceptable limits.

Keywords:

• atorvastatin
• LC-MS/MS
• o-hydroxy atorvastatin
• p-hydroxy atorvastatin
• validation