Quantitative Determination Of Atorvastatin And Its Main Metabolites In Human Plasma And Bioequivalence Evaluation

Background Atorvastatin is a potent inhibitor of3-hydroxyl-3-methyglutaryl coenzyme A, it can significantly decrease the synthesisand storage of cholesterol, and is mainly used for the treatment ofhypercholesterolemia. The major metabolites of atorvastatin isortho-hydroxyl atorvastatin （o-OAT） and para-hydroxyl atorvastatin（p-OAT） in vivo, about70%of the pharmacological activity is accountedfor by the two metabolites.Objective A sensitive and specific liquid chromatographic–tandemmass spectrometric （LC–MS/MS） method was to be developed forsimultaneous determination of atorvastatin, its ortho-hydroxyl metabolite（o-OAT） and para-hydroxyl metabolite （p-OAT） in human plasma. Themethod should be applied to the clinical pharmacokinetic andbioequivalence study of atorvastatin. Method In an ice bath condition, the plasma samples （500μL）were spiked with250μL of sodiumformat （pH3;0.1M）, usingd₅-atorvastatin and rosuvastatin as internal standards. The samples wereextracted with diethyl ether-dichloromethane （3:2, v/v） and thenseparated on a Agilent Zorbax SB C₁₈column （150mm×4.6mm I.D.,5μm） using methanol–5mmol·L-1ammonium acetate–formic acid（20:80:0.1, v/v/v） as mobile phase. Detection was performed by tandemmass spectrometry using an electrosprasy source （ESI） in the positive ionmode, operating in the multiple reaction monitoring （MRM） of thetransitions of m/z559440, m/z564445and m/z482258foratorvastatin, d₅-atorvastatin and rosuvastatain, m/z575（440,466） foro-OAT and p-OAT, respectively. The method was successfully applied toa pharmacokinetics and bioequivalence study of atorvastatin in healthyvolunteers.24healthy fasted Chinese males were randomly assigned toreceive20mg of either the test or reference formulation orally, the bloodsamples were collected over a72-hour interval.Results The linear calibration curves for atorvastatin o-OAT andp-OAT were obtained in the concentration range of0.050–15.0ng·mL^-1,0.050–5.00ng·mL^-1and0.050–2.50ng·mL^-1in plasma, respectively.Intra-and inter-day relative standard deviation （RSD） for atorvastatin,o-OAT and p-OAT over the entire concentrations across validation runswere all less than9.1%, and relative error （RE） ranged from-3.6%to5.1%. The90%confidence interval of the test/reference geometric meanratios of C_maxand AUC_0-tfor either AT or o-OAT were all within thebioequivalence criteria range (80%–125%for AUC, and75%–133%forC_max), demonstrating that the two formulations were bioequivalent. TheC_maxfor atorvastatin, o-OAT and p-OAT was10.6,7.64and0.397ng mL^-1, respectively. The AUC_0-tfor atorvastatin, o-OAT and p-OATwas51.7,92.3and13.8ng h mL^-1, respectively. Conclusion This method has been used in the simultaneousdetermination of atorvastatin, o-OAT and p-OAT in human plasma withstable labeled compounds as internal standards. It was suitable for thepharmacokinetic and bioequivalence study of atorvastatin. Thepharmacokinetic results demonstrated that the AUC of atrovastatin inChinese volunteers after oral administration of20mg atrovastatin wassimilar to the result in westerners after oral administration of40mgatrovastatin.