A specific, accurate, precise and reproducible high-performance liquid chromatographic (HPLC) method was developed and validated for the simultaneous quantitation of five 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase inhibitors, viz. atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin, in pharmaceutical formulations and extended the application to in vitro metabolism studies of these statins. Ternary gradient elution at a flow rate of 1 mL/min was employed on an Intertisl ODS 3V column (4.6 x 250 mm, 5 microm) at ambient temperature. The mobile phase consisted of 0.01 m ammonium acetate (pH 5.0), acetonitrile and methanol. Theophylline was used as an internal standard (IS). The HMG-CoA reductase inhibitors and their metabolites were monitored at a wavelength of 237 nm. Drugs were found to be 89.6-105.6% of their label's claim in the pharmaceutical formulations. For in vitro metabolism studies the reaction mixtures were extracted with simple liquid-liquid extraction using ethyl acetate. Baseline separation of statins and their metabolites along with IS free from endogenous interferences was achieved. Nominal retention times of IS, atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin were 7.5, 17.2, 21.6, 28.5, 33.5 and 35.5 min, respectively. The proposed method is simple, selective and could be applicable for routine analysis of HMG-CoA reductase inhibitors in pharmaceutical preparations as well as in vitro metabolism studies.

The aim of this study was to study the effect of 1-aminobenzotriazole (ABT) on in vitro metabolism, oral, and intravenous (IV) pharmacokinetics of chlorzoxazone (CZX) in rats. Enzyme kinetics of CZX was performed with rat and human liver microsomes and pure isozyme (CYP2E1) with and without ABT. The enzyme kinetics (V(max) and K(m)) of the formation of 6-hydroxychlorzoxazone (OH-CZX) was found to be similar among rat liver microsomes (3486 pmol mg protein(-1) min(-1) and 345 microM), human liver microsomes (3194 pmol mg protein(-1) min(-1) and 335 microM) and pure isozyme (3423 pmol mg protein(-1) min(-1) and 403 microM), but K(I) and K(inact) values for ABT towards the ability to inhibit the formation of OH-CZX from CZX varied between liver microsomes (rat: 32.09 microM and 0.12 min(-1); human: 27.19 microM and 0.14 min(-1)) and pure isozyme (3.18 microM and 0.29 min(-1)). The novel robust analytical method was capable of quantifying CZX, OH-CZX, and ABT simultaneously in a single run, and the method was used for both in vitro and in vivo studies. Pre-treatment of rats with ABT prior to oral and IV administration of CZX significantly decreased the clearance (threefold) and consequently increased the AUC of CZX (approx. three- to fourfold). When rats were pre-treated with ABT, the formation of OH-CZX was completely blocked after oral and IV administration; however, we were able to measure OH-CZX in rats administered with CZX by oral and IV routes without pre-treatment of ABT. The oral bioavailability of CZX was approximately 71% when dosed alone and reached 100% under pre-treatment with ABT. The t(1/2) values of CZX was significantly prolonged for oral dosing compared with IV dosing under pre-treated conditions with ABT, suggesting an involvement of pre-systemic component in the disposition of CZX. The pharmacokinetic parameters of ABT did not change when it was dosed along with CZX (oral and IV), indicating that either CZX or OH-CZX had no effect on disposition of ABT. The plasma concentrations of ABT were above and beyond the required levels to inhibit CYP2E1 enzyme for at least 36 h post-treatment.