Optimization, validation and application of an assay for the activity of HMG-CoA reductase in vitro by LC-MS/MS
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 vali...
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| Main Authors: | , , , , , , , |
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| Format: | Book |
| Published: |
Elsevier,
2015-12-01T00:00:00Z.
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| Online Access: | Connect to this object online. |
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| Summary: | 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. |
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| Item Description: | 2095-1779 10.1016/j.jpha.2015.06.002 |