Liquid chromatography-tandem mass spectrometric assay for pravastatin and two isomeric metabolites in mouse plasma and tissue homogenates
Universiteit Utrecht, Faculty of Science, Department of Pharmaceutical Sciences, Section of Biomedical Analysis, Division of Drug Toxicology, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (Impact Factor: 2.73). 10/2010; 878(28):2751-9. DOI: 10.1016/j.jchromb.2010.08.015
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.
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ABSTRACT: An Ultra Flow liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for estimation of pravastatin in human plasma. Pravastatin and omperazole (internal standard) were extracted from human plasma using a solid phase extraction procedure with Strata X cartridges. Samples were chromatographed on Hypurity Advance C18, 50 × 4.6 mm, 5μm column using a mobile phase consisting of (80:20, v/v), acetonitrile and 2 mm ammonium formate. Pravastatin and the internal standard were ionised using the electro spray interface operating in negative ion mode. The characteristic ion dissociation transitions m/z 423.1→321.2 and m/z 344→193.8 was monitored for pravastatin and internal standard respectively. The limit of quantitation was 5.078 ng/mL using 250 μl of plasma. Inter and intra batch precision expressed by relative standard deviation was less than 9%. The assay was robust, sensitive, and highly specific and there was no interference from human plasmaobserved. With a total run-time of 2 minutes, the method was suitable for supporting clinical studies and applied to the analysis of samples from a bioequivalence study.
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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.
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ABSTRACT: A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for the JAK2 inhibitor CYT387 was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing cediranib as internal standard. The extract was directly injected into the chromatographic system after dilution with water. This system consisted of a sub-2 μm particle, trifunctional bonded octadecyl silica column with a gradient using 0.005% (v/v) of formic acid in a mixture of water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 0.25-1000 ng/ml calibration range. Within day precisions were 3.0-13.5%, between day precisions 5.7% and 14.5%. Accuracies were between 96% and 113% for the whole calibration range. The drug was stable under all relevant analytical conditions. Finally, the assay was successfully used to assess drug levels in mice.
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