Simultaneous analysis of the total plasma concentration of atorvastatin and its five metabolites and the unbound plasma concentration of atorvastatin: Application in a clinical pharmacokinetic study of single oral dose

Atorvastatin (ATV) and its two active metabolites, o-hydroxy atorvastatin acid (o-OH-ATV) and p-hydroxy atorvastatin acid (p-OH-ATV) are responsible for its HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme-A) reductase inhibitory activity, while its corresponding inactive lactone forms (LAC) are related to the manifestation of myopathy. The present study reports the development and validation of a method for the simultaneous analysis of ATV and its five metabolites (o-OH-ATV, p-OH-ATV, ATV-LAC, o-OH-ATV-LAC, p-OH-ATV-LAC) as total plasma concentration and ATV as unbound plasma concentration using UPLC-MS/MS. The method was applied in a pharmacokinetic study following administration of a single oral 20, 40 or 80 mg ATV dose in healthy volunteers (n = 15). ATV and its five metabolites were separated on a C18 column using as mobile phase a mixture of 0.2% formic acid and acetonitrile (55:45, v/v) at a flow of 0.4 mL/min. The method showed linearity from 25 pg/mL to 200 ng/mL plasma as total concentration and from 6.25 pg to 25 ng/mL plasma ultrafiltrate as ATV unbound concentration. The coefficients of variation and the relative standard errors of the accuracy and precision analyses were <15%. The method allowed quantification of plasma concentrations of ATV and its five metabolites up to 36 h after 20, 40 or 80 mg ATV administration. The pharmacokinetic parameters dose normalized to 20 mg are presented as follow (n = 15, mean): unbound fraction 9.38%, maximum plasma concentration 9.52 ng/mL, time to reach maximum plasma concentration 0.98 h, apparent total clearance 742.90 L/h, apparent distribution volume 9005 L, and AUC metabolite/ATV ratios 0.06 for p-OH-ATV, 0.94 for o-OH-ATV, 1.43 for ATV-LAC, 0.25 for p-OH-ATV-LAC and 1.75 for o-OH-ATV-LAC. In conclusion, the methods for simultaneous analysis of ATV and its five metabolites as total plasma concentration and ATV as the unbound plasma concentration showed sensitivity, linearity, precision and accuracy compatible with application in pharmacokinetic studies of single oral dose of 20, 40 or 80 mg ATV.