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

Employing high-performance liquid chromatography-electrospray mass spectrom etry, we describe a new assay for monitoring 3-hydroxy-3-methylglutaryl-coe nzyme A (HMG-CoA) reductase activity. Incubations were carried out with HMG -CoA reductase (rat liver), HMG-CoA and NADPH, and terminated by the additi on of HCl. The reaction product, mevalonolactone, and internal standard, we re extracted with ethyl acetate, dissolved in methanol, and analyzed by LC- MS. Using an isocratic mobile phase of 10% acetonitrile and 0.1% formic aci d (flow-rate, 0.2 ml/min), the protonated molecules of mevalonolactone at m /z 131 and internal standard, beta,beta -dimethyl-gamma-(hydroxymethyl)-gam ma -butyrolactone, at m/z 145, were detected using selected ion monitoring. The limit of detection was approximately 6.5 pg, and the limit of quantita tion 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 per iod of 3 days and found to be stable. Using this assay, lovastatin and meva statin inhibited HMG-CoA reductase activity with IC50 values 0.24+/-0.02 an d 2.16+/-0.31 muM, respectively. These methods offer some advantages over t hose reported previously which employ radiolabeled substrate and products, and should be useful in searching for compounds that could lower serum chol esterol or alter cell growth and differentiation. (C) 2001 Elsevier Science B.V. All rights reserved.