Simultaneous determination of atorvastatin, metformin and glimepiride in human plasma by LC-MS/MS and its application to a human pharmacokinetic study

Abstract

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and fully validated for the simultaneous quantification of atorvastatin, metformin and glimepiride in human plasma. Carbamazepine was used as internal standard (IS). The analytes were extracted from 200 μL aliquots of human plasma via protein precipitation using acetonitrile. The reconstituted samples were chromatographed on a Alltima HP C18 column by using a 60:40 (v/v) mixture of acetonitrile and 10 mM ammonium acetate (pH 3.0) as the mobile phase at a flow rate of 1.1 mL/min. The calibration curves obtained were linear (r² ≥0.99) over the concentration range of 0.50-150.03 ng/mL for atorvastatin, 12.14-1207.50 ng/mL for metformin and 4.98-494.29 ng/mL for glimepiride. The API-4000 LC-MS/MS in multiple reaction monitoring (MRM) mode was used for detection. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. All the analytes were found to be stable in a battery of stability studies. The method is precise and sensitive enough for its intended purpose. A run time of 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The developed assay method was successfully applied to a pharmacokinetic study in human male volunteers.

Keywords: Atorvastatin; Glimepiride; Human plasma; LC–MS/MS; Metformin; Pharmacokinetics.

Fig. 1

Chemical structures of atorvastatin, metformin, glimepiride and carbamazepine (IS).

Fig. 2

Typical MRM chromatograms of atorvastatin (left panel) and IS (right panel) in human blank plasma (A), and human plasma spiked with IS (B), a LLOQ sample along with IS (C).

Fig. 3

Typical MRM chromatograms of metformin (left panel) and IS (right panel) in human blank plasma (A), and human plasma spiked with IS (B), a LLOQ sample along with IS (C).

Fig. 4

Typical MRM chromatograms of glimepiride (left panel) and IS (right panel) in human blank plasma (A), and human plasma spiked with IS (B), a LLOQ sample along with IS (C).

Fig. 5

Mean plasma concentration–time profile of atorvastatin (A), metformin (B), and glimepiride (C) in human plasma following oral dosing of atorvastatin (40 mg), metformin (500 mg ) and glimepiride (2 mg) tablet to healthy volunteers (n=6).