Optimization, validation and application of an assay for the activity of HMG-CoA reductase in vitro by LC-MS/MS

Abstract

A stable HMG-CoA reductase (HMGR) reaction in vitro was developed by a sensitive, selective and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The optimized enzyme reaction condition contained 1.5 μg of HMGR, 20 nM of NADPH with 50 min of reaction time. The method was validated by several intra- and inter-day assays. The production transitions of m/z 147.0/59.1 and m/z 154.0/59.1 were used to detect and quantify mevalonolactone (MVAL) and MVAL-D₇, respectively. The accuracy and precision of the method were evaluated over the concentration range of 0.005-1.000 μg/mL for MVAL and 0.010-0.500 μg/mL for lovastatin acid in three validation batch runs. The lower limit of quantitation was found to be 0.005 μg/mL for MVAL and 0.010 μg/mL for lovastatin acid. Intra-day and inter-day precision ranged from 0.95% to 2.39% and 2.26% to 3.38% for MVAL, 1.46% to 3.89% and 0.57% to 5.10% for lovastatin acid, respectively. The results showed that the active ingredients in Xuezhikang capsules were 12.2 and 14.5 mg/g, respectively. This assay method could be successfully applied to the quality control study of Xuezhikang capsule for the first time.

Keywords: HMG-CoA reductase; LC–MS/MS; Mevalonolactone; Quality control; Xuezhikang.

Fig. 1

Relation of HMG CoA reductase activity (substrate transformation) and the amount of HMG-CoA reductase (A), NADPH (B), pre-incubation time (C) and reaction time (D).

Fig. 2

Representative MRM chromatograms of MVAL in enzyme reaction matrix. (A) blank matrix; (B) blank matrix spiked with 0.010 μg/mL of MVAL (left) and MVAL-D₇ (right); (C) real matrix sample generated by enzyme reaction (left) and MVAL-D₇ (right).