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

Determination of ergocalciferol in human plasma after Diels-Alder derivatization by LCâMS/MS and its application to a bioequivalence study

An accurate, sensitive and selective method is developed for determination of ergocalciferol (vitamin D2) in human plasma using LCâMS/MS. After liquid-liquid extraction with n-hexane, ergocalciferol was derivatized by reacting with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), a strong dienophile based on Diels-Alder reaction. Ergocalciferol and its deuterated internal standard, ergocalciferol-d6, were analyzed on X Select CSH C18 (100 mmÃ4.6 mm, 2.5 µm) column using acetonitrile and 0.1% (v/v) formic acid in water containing 0.14% methylamine within 6.0 min under gradient elution mode. Tandem mass spectrometry in positive ionization mode was used to quantify ergocalciferol by multiple reaction monitoring (MRM). Entire data processing was done using Watson LIMS⢠software which provided excellent data integrity and high throughput with improved operational efficiency. The major advantage of this method includes higher sensitivity (0.10 ng/mL), superior extraction efficiency (â¥83%) and small sample volume (100 µL) for processing. The method was linear in the concentration range of 0.10â100 ng/mL for ergocalciferol. The intra-batch and inter-batch accuracy and precision (% CV) values varied from 97.3% to 109.0% and 1.01% to 5.16%, respectively. The method was successfully applied to support a bioequivalence study of 1.25 mg ergocalciferol capsules in 12 healthy subjects. Keywords: Ergocalciferol, Diels-Alder reaction, 4-phenyl-1,2,4-triazoline-3,5-dione, LCâMS/MS, Human plasm