A Simple and Sensitive HPLC-UV Method for Quantitation of Lovastatin in Human Plasma: Application to a Bioequivalence Study

Abstract

Lovastatin (structure shown in Fig. 1), a highly effective cholesterol-lowering agent, belongs to a class of the most powerful lipid lowering agents generally called the statins and is a competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase.1) Furthermore, it has been reported that lovastatin has a good effect in reducing lethality in coronary heart disease.2) After oral administration of lovastatin, the plasma levels of the drug has been reported to be very low, which is mainly attributed to the documented extensive hepatic first-pass effect of the drug. 3,4)Although a series of sensitive and accurate methods have been described for determination of lovastatin in biological fluids, but all these methods require the use of sophisticated and relatively non-popular instrumentations such as gas chromatography/mass spectrometry [5][6][7] or, more recently, highperformance liquid chromatography/mass spectrometry, 8,9) the availability of a simple, popular and cost-effective method for this purpose remains a limiting factor against pharmacokinetic studies on this drug. The purpose of this investigation was to develop a rapid, popular, and sensitive analytical method, based on HPLC with UV detection of lovastatin in human plasma in order to, primarily, be used throughout a bioequivalence study. Preparation of Calibration Standards and Quality Control Samples In order to prepare stock standard solution of lovastatin, 100 mg of lovastatin was dissolved in 100 ml methanol. The stock solution was then further diluted with methanol to obtain the different working solutions ranging 40, 100, 200, 400, 1000, 2000, 4000 ng/ml, from which the spiked plasma samples were prepared by appropriate dilution. Quality control (QC) samples were prepared at low (10.0 ng/ml), medium (25.0 ng/ml) and high (50.0 ng/ml) concentrations in the same way as the plasma samples for calibration. MATERIALS AND METHODS ChemicalsSamples Preparation A liquid-liquid extraction procedure was used in this study for isolation of lovastatin from the plasma samples. For this purpose, 20 ml of atorvastatin (internal standard; IS) methanolic solution with concentration of 100 mg/ml was added to 2 ml plasma and after vortexmixing for 30 s, 4 ml of diethyl ether was added while the mixture was vortex-mixed for another 1 min. The organic and aqueous layers were then separated by centrifugation at 5000ϫg for 10 min, the organic supernatant was transferred to another tube and evaporated at 40°C to dryness. Finally, the residue was reconstituted in 200 ml mobile phase and following a brief orbital shaking, 50 ml was injected onto the HPLC system. Chromatographic Condition The analytical column was Symmetry C 18 (Waters, U.S.A.) (particle size 5 mm; 250ϫ4.6 mm) with the corresponding guard column An available, simple, sensitive, and rapid method has been developed for determination of the 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor, lovastatin in human plasma. The analytical procedure involves a one-step liquid-liquid extr...

A Review of Current Trends and Advances in Analytical Methods for Determination of Statins: Chromatography and Capillary Electrophoresis

“…In most of the methods protein precipitation reagent is used as a dilution solvent for internal standard in order to reduce the number of reagent additions [71][72][73][74]. Still, Apostolou and coworkers [75] suggested addition of protein precipitation reagent after the internal standard in order to ensure a more satisfying binding of internal standard molecules with plasma proteins, simulating the binding of proteins with analytes in real human plasma.…”

“…Hence, Hamidi and co-workers [73] tested a wide spectrum of organic solvents from various physicochemical categories with different volume fractions as well as combinations for extraction of lovastatin from human plasma. The best extraction efficacy was obtained using diethyl ether as extraction solvent.…”

“…0.01% formic acid/10 mM ammonium formate (pH 4.1) was finally chosen as appropriate buffer. Uncommon pH was used for quantification of lovastatin in human plasma [73]. Mobile phase consisted of acetonitrile and 0.05 M phosphate buffer with pH 7, adjusted with phosphoric acid.…”

“…Their typical plasma concentrations are in ng/mL levels. However, several sensitive LC/DAD methods for determination of pravastatin [31], atorvastatin [71], lovastatin [73], rosuvastatin [81], and atorvastatin with rosuvastatin [90] have been developed with limit of quantification (LOQ) in range of 1 -10 ng/mL. Less sensitive LC/DAD method for quantification of lovastatin in human plasma was developed [91].…”

“…In some cases one of the statins has been used as internal standard [73,75,77,84], while other works utilized internal standards of various structure, including hydrochlorothiazide [79], estrone [80], naproxen [90], gemfibrozil [91], pioglitazone [95] etc. The best internal standards for precise and accurate quantification in MS or tandem MS are stable-isotopelabeled standards.…”