Determination of pravastatin and pravastatin lactone in rat plasma and urine using UHPLC–MS/MS and microextraction by packed sorbent

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 C18 analytical column (50mm×2.1mm, 1.7μm), using gradient elution by mobile phase consisting of acetonitrile and 1mM ammonium acetate at pH 4.0. Run time was 2min. 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 (50μl). The analytes were eluted by 100μl of the mixture of acetonitrile: 0.01M ammonium acetate pH 4.5 (90:10, v:v). The method was validated and demonstrated good linearity in range 5–500nmol/l (r2>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 1.5nmol/l and LOQ 5nmol/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.