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

Abstract

A selective, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2 (250 mm×4.6 mm, 5 μm) column. Solid phase extraction of (S)-(-)- and (R)-(+)-metoprolol and rac-metoprolol-d6 as an internal standard (IS) was achieved on Lichrosep DVB HL cartridges employing 200 μL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.24 using 15 mM 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 7.0 min. The precursor→product 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 0.500-500 ng/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 200 mg 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: Chiral column; Chromatographic separation; Human plasma; LC–ESI–MS/MS; R-(+)-metoprolol; S-(−)-metoprolol.

Fig. 1

Product ion mass spectra of: (A) S-(−)-metoprolol (m/z 268.3→116.3, scan range 100–300 amu), (B) R-(+)-metoprolol (m/z 268.3→116.3, scan range 100–300 amu), and (C) Rac-metoprolol-d6, internal standard (m/z 274.2→122.2, scan range 100–300 amu) in positive ionization mode.

Fig. 2

Chromatograms of metoprolol enantiomers (m/z 268.3→116.3) obtained on: (A) protein based Chiral-AGP (250 mm×4.6 mm, 5 μm), (S), (B) Chiralcel^® OD (250 mm×4.6 mm, 5 μm) and (C) Lux Amylose-2 (250 mm×4.6 mm, 5 μm). Mobile phase for column (A) 15 mM ammonium acetate, pH 5.0 adjusted with acetic acid-0.1% diethyl amine in acetonitrile (70:30, v/v), for (B) and (C) 15 mM ammonium acetate, pH 5.0 adjusted with acetic acid-0.1% diethyl amine in acetonitrile (50:50, v/v).

Fig. 3

MRM ion-chromatograms of: (A) double blank plasma (without IS), (B) blank plasma with rac-metoprolol-d6 (IS), (C) S-(−)-metoprolol and R-(+)-metoprolol at LLOQ level (m/z 268.3→116.3) and IS (m/z 274.2→122.2), and (D) real subject sample at 5.0 h after administration of 200 mg extended release metoprolol tablet.

Fig. 4

Post-column analyte infusion MRM LC–MS/MS chromatograms for: (A) S-(−)-metoprolol and R-(+)-metoprolol (m/z 268.3→116.3), and (B) Rac-metoprolol-d6 (m/z 274.2→122.2).

Fig. 5

MRM ion-chromatograms for carry-over test of: (A) double blank plasma (without analyte and IS), (B) S-(−)-metoprolol and R-(+)-metoprolol at ULOQ level and IS, (C) double blank plasma (without analyte and IS) and (D) S-(−)-metoprolol and R-(+)-metoprolol at LLOQ level and IS.

Fig. 6

Mean plasma concentration-time profile of: (A) S-(−)-metoprolol and (B) R-(+)-metoprolol after oral administration of test (200 mg metoprolol succinate extended release tablet from an Indian Pharmaceutical Company, India) and reference (Selo-zok^®, 200 mg metoprolol succinate extended release tablet from AstraZeneca, Denmark) formulations to 14 healthy Indian subjects under fasting conditions.

Fig. 7

Graphical representation of results for 68 incurred samples of S-(−)-metoprolol and R-(+)- metoprolol.