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

• Published in: Journal of pharmaceutical analysis 2015-12, Vol.5 (6), p.383-388
• Main Authors: Wang, Jing, Sun, Ji-Ye, Sha, Chun-Jie, Shao, Yu-Feng, Liu, Yan-Hong, Li, You-Xin, Duan, Zhen-Wen, Liu, Wan-Hui
• Format: Article
• Language: English
• Subjects: HMG-CoA reductase; HMG-CoA还原酶; LC-MS/MS; Mevalonolactone; Original; Quality control; Xuezhikang; 优化; 体外活性测定; 应用; 洛伐他汀; 液相色谱-串联质谱; 验证
• ISSN: 2095-1779; 2214-0883
• DOI: 10.1016/j.jpha.2015.06.002

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 lag of HMGR, 20 nM of NADPH with 50 rain of reaction time. The method was validated by several intraand 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-DT, respectively. The accuracy and precision of the method were evaluated over the concentration range of 0.005- 1.000 lag/mL for MVAL and 0.010-0.500 lag/mL for lovastatin acid in three validation batch runs. The lower limit of quantitation was found to be 0.005 lag/mL for MVAL and 0.010 lag/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 anDlied to the oualitv control study of Xuezhikanu caosule for the first time.