Simultaneous quantification of prodrug oseltamivir and its metabolite oseltamivir carboxylate in human plasma by LC–MS/MS to support a bioequivalence study

AbstractA simple, precise and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the simultaneous determination of oseltamivir and oseltamivir carboxylate, a neuraminidase inhibitor, using their deuterated analogs as internal standards (ISs). The method involved solid phase extraction of the analytes and ISs from 200μL human plasma with no reconstitution and drying steps. The chromatographic separation was achieved on a Symmetry C18 (100mm×4.6mm, 5μm) column using 10mM ammonium formate and acetonitrile (30:70, v/v) as the mobile phase in a run time of 2.0min. Quantitation of analytes and ISs were done by multiple reaction monitoring on a triple quadrupole mass spectrometer in the positive ionization mode. The linearity of the method was established in the concentration range of 0.5–200ng/mL and 2.0–800ng/mL for oseltamivir and oseltamivir carboxylate respectively. The mean extraction recovery for oseltamivir (94.4%) and oseltamivir carboxylate (92.7%) from spiked plasma samples was consistent and reproducible. The application of this method was demonstrated by a bioequivalence study in 42 healthy Indian subjects with 75mg oseltamivir phosphate capsules. The assay reproducibility was established by reanalysis of 151 incurred subject samples

Development of a sensitive and rapid method for quantitation of (S)-(â)- and (R)-(+)-metoprolol in human plasma by chiral LCâESIâMS/MS

A selective, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LCâESIâMS/MS) method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2 (250mmÃ4.6mm, 5μm) column. Solid phase extraction of (S)-(â)- and (R)-(+)-metoprolol and rac-metoprolol-d6 as an internal standard (IS) was achieved on Lichrosep DVB HL cartridges employing 200μL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.24 using 15mM ammonium acetate in water, pH 5.0 and 0.1% (v/v) diethyl amine in acetonitrile (50:50, v/v) as the mobile phase within 7.0min. The precursorâproduct ion transitions for the enantiomers and IS were monitored in the multiple reaction monitoring and positive ionization mode. The method was validated over the concentration range of 0.500â500ng/mL for both the enantiomers. Matrix effect was assessed by post-column analyte infusion experiment and the mean extraction recovery was greater than 94.0% for both the enantiomers at all quality control levels. The stability of analytes was evaluated in plasma and whole blood under different storage conditions. The method was successfully applied to a clinical study in 14 healthy volunteers after oral administration of 200mg metoprolol tablet under fasting conditions. The assay reproducibility is shown by reanalysis of 68 incurred samples. The suitability of the developed method was assessed in comparison with different chromatographic methods developed for stereoselective analysis of metoprolol in biological matrices. Keywords: S-(â)-metoprolol, R-(+)-metoprolol, Chiral column, Chromatographic separation, LCâESIâMS/MS, Human plasm

Validation of an LC-MS/MS method for the quantitative determination of Mavoglurant (AFQ056) in human plasma

A simple, sensitive and selective liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) method was validated for the identification and quantification of Mavoglurant (AFQ056) in human plasma. The chromatographic separation was performed using a Cosmosil 5 C18 (150 mm × 4.6 mm, 5 µm) column at 40 ± 0.5 oC with a mobile phase consisted of acetic acid in water (0.1% v/v): methanol (10:90, v/v) with a flow rate of 1.0 mL/min followed by quantification with tandem mass spectrometer, operated with electrospray ionization (ESI) in positive ion mode and applying multiple reaction monitoring (MRM). The validated method described in this paper presents high absolute recovery with precision and accuracy meeting the acceptance criteria. The method was precise and accurate for 2- and 10-fold dilution of samples. The method was validated using sodium heparin as specific anticoagulant and cross-validated using lithium heparin and K3EDTA. The method was specific for mavoglurant within the given criteria for acceptance (apparent peak area for mavoglurant in zero samples ≤ 20% of mean peak area at LLOQ) in human plasma. The method was fully validated for the quantitative determination of mavoglurant in human plasma between the range of 2.00 ng/mL and 2500 ng/mL

Validation of a liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood

A liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) method was developed and validated for the quantification of Sotrastaurin (AEB071) and N-desmethyl-sotrastaurin (AEE800) in human blood. The validation of the analytical procedure was assessed according to the latest Food and Drug Administration “Guidance for Industry, Bioanalytical Method Validation”. Chromatographic separation was performed using an RP C18 column at 40±3.0 oC with a mobile phase consisted of 2mM ammonium acetate in water (pH 4.5): methanol: acetonitrile (25:15:60 v/v) of a flow rate of 1 mL/min followed by quantification with mass spectrometer, operated in electrospray ionization (ESI) positive ion mode and applying multiple reaction monitoring (MRM). The LC–MS/MS method described in this paper presents high absolute recovery (the overall mean recovery of Sotrastaurin and N-desmethly-sotrastaurin was 115.9% and 110.4% respectively), with a sensitivity of 3.00 ng/mL as lower limit of quantitation using a sample volume of 300 µL, low inter-day bias and precision (for Sotrastaurin, 0.4 to -4.4% and 1.8 to 5.2% and for N-desmethyl-sotrastaurin, ranged from 2.3 to -1.6% and 3.9 to 2.7%, respectively), with a short runtime of 3.5 min. The method was validated using K3EDTA as specific anticoagulant and cross-validated using Li-Heparin and Na-Heparin. The method was specific for Sotrastaurin and N-desmethly-sotrastaurin within the given criteria of acceptance (apparent peak area for Sotrastaurin and N-desmethly-sotrastaurin in zero samples ≤ 20% of mean peak area at LLOQ) in human blood. The method was fully validated for the quantitative determination of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood between the range of 3 ng/mL and 1200 ng/mL