Liquid chromatography-tandem mass spectrometric assay for pravastatin and two isomeric metabolites in mouse plasma and tissue homogenates.
ABSTRACT A bioanalytical assay for pravastatin and two isomeric metabolites, 3'α-isopravastatin and 6'-epipravastatin, was developed and validated. Mouse plasma and tissue homogenates from liver, kidney, brain and heart were pre-treated using protein precipitation with acetonitrile containing deuterated internal standards of the analytes. The extract was diluted with water and injected into the chromatographic system. This system consisted of a polar embedded octadecyl silica column using isocratic elution with formic acid in a water-acetonitrile mixture. The eluate was transferred to an electrospray interface using negative ionization and the analytes were detected and quantified with the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was successfully validated in a 3.4-7100ng/ml concentration range for pravastatin, 1.3-2200ng/ml for 3'α-isopravastatin and 0.5-215ng/ml for 6'-epipravastatin using only plasma for calibration. For plasma samples, subjected to full validation, within and between day precisions were 1-7% (9-18% at the LLQ level) and accuracies were between 91% and 103%. For tissue homogenates, subjected to partial validation, within and between day precisions were 2-12% (6-19% at the LLQ level) and accuracies were between 87% and 113% (81 and 113% at the LLQ level). Drug and metabolites were shown to be chemically stable under most relevant analytical conditions. Finally, the assay was successfully applied for a pilot study in mice. After intravenous administration of the drug, all isomeric compounds were found in plasma; however, in liver and kidney homogenate only the parent drug showed levels exceeding the LLQ.
- [Show abstract] [Hide abstract]
ABSTRACT: A simple and reproducible method for the determination of pravastatin and pravastatin lactone in rat plasma and urine by means of ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) using deuterium labeled internal standards for quantification is reported. Separation of analytes was performed on BEH C(18) analytical column (50 mm × 2.1mm, 1.7 μm), using gradient elution by mobile phase consisting of acetonitrile and 1mM ammonium acetate at pH 4.0. Run time was 2 min. Quantification of analytes was performed using the SRM (selected reaction monitoring) experiment in ESI negative ion mode for pravastatin and in ESI positive ion mode for pravastatin lactone. Sample treatment consisted of a protein precipitation by ACN and microextraction by packed sorbent (MEPS) for rat plasma. Simple MEPS procedure was sufficient for rat urine. MEPS was implemented using the C8 sorbent inserted into a microvolume syringe, eVol hand-held automated analytical syringe and a small volume of sample (50 μl). The analytes were eluted by 100 μl of the mixture of acetonitrile: 0.01 M ammonium acetate pH 4.5 (90:10, v:v). The method was validated and demonstrated good linearity in range 5-500 nmol/l (r(2)>0.9990) for plasma and urine samples. Method recovery was ranged within 97-109% for plasma samples and 92-101% for the urine samples. Intra-day precision expressed as the % of RSD was lower than 8% for the plasma samples and lower than 7% for the urine samples. The method was validated with sensitivity reaching LOD 1.5 nmol/l and LOQ 5 nmol/l in plasma and urine samples. The method was applied for the measurement of pharmacokinetic plots of pravastatin and pravastatin lactone in rat plasma and urine samples.Talanta 02/2012; 90:22-9. · 3.50 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Background & Aims A genome wide association study and multiple pharmacogenetic studies have implicated the hepatic uptake transporter organic anion transporting polypeptide-1B1 (OATP1B1) in the pharmacokinetics and musculoskeletal toxicity of statin drugs. Other OATP uptake transporters can participate in the transport of pravastatin, partially compensating for the loss of OATP1B1 in patients carrying the polymorphism. Nonalcoholic steatohepatitis (NASH) in humans and in a diet-induced rodent model alter the expression of multiple OATP transporters. Methods To determine how genetic alteration in one Oatp transporter can interact with NASH-associated changes in Oatp expression we measured the disposition of intravenously administered pravastatin in Slco1b2 knockout (Slco1b2-/-) and wild-type (WT) mice fed either a control or a methionine and choline deficient (MCD) diet to induce NASH. Results Genetic loss of Oatp1b2, the rodent ortholog of human OATP1B transporters, caused a modest increase in pravastatin plasma concentrations in mice with healthy livers. Although a diet-induced model of NASH decreased the expression of multiple hepatic Oatp transporters, it did not alter the disposition of pravastatin compared to WT control mice. In contrast, the combination of NASH-associated decrease in compensatory Oatp transporters and Oatp1b2 genetic loss caused a synergistic increase in plasma area under the curve (AUC) and tissue concentrations in kidney and muscle. Conclusions Our data show that NASH alters the expression of multiple hepatic uptake transporters which, due to overlapping substrate specificity among the OATP transporters, may combine with the pharmacogenetic loss of OATP1B1 to increase the risk of statin-induced adverse drug reactions.Journal of Hepatology 01/2014; · 9.86 Impact Factor