Analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors using liquid chromatography-electrospray mass spectrometry

Eun Jung Park, Dongho Lee, Young Geun Shin, Daniel D. Lantvit, Richard B. Van Breemen, A. Douglas Kinghorn, John M. Pezzuto

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Employing high-performance liquid chromatography-electrospray mass spectrometry, we describe a new assay for monitoring 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. Incubations were carried out with HMG-CoA reductase (rat liver), HMG-CoA and NADPH, and terminated by the addition of HCl. The reaction product, mevalonolactone, and internal standard, were extracted with ethyl acetate, dissolved in methanol, and analyzed by LC-MS. Using an isocratic mobile phase of 10% acetonitrile and 0.1% formic acid (flow-rate, 0.2 ml/min), the protonated molecules of mevalonolactone at m/z 131 and internal standard, β,β-dimethyl-γ-(hydroxymethyl)-γ-butyrolactone, at m/z 145, were detected using selected ion monitoring. The limit of detection was approximately 6.5 pg, and the limit of quantitation was approximately 16.3 pg. Extraction recovery was >90%. The relative standard deviations for intra- and inter-day assays were approximately 4.1±2.7 and 9.4±3.4%, respectively. Mevalonolactone was examined over a period of 3 days and found to be stable. Using this assay, lovastatin and mevastatin inhibited HMG-CoA reductase activity with IC50 values 0.24±0.02 and 2.16±0.31 μM, respectively. These methods offer some advantages over those reported previously which employ radiolabeled substrate and products, and should be useful in searching for compounds that could lower serum cholesterol or alter cell growth and differentiation.

Original languageEnglish
Pages (from-to)327-332
Number of pages6
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume754
Issue number2
DOIs
Publication statusPublished - 2001 Apr 25

Keywords

  • 3-Hydroxy-3-methylglutaryl-coenzyme A reductase

ASJC Scopus subject areas

  • Chemistry(all)

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