Enantioselective determination of metoprolol and its metabolites in human urine high-performance liquid chromatography with fluorescence detection (HPLC–FLD) and tandem mass spectrometry (MS/MS)

•Development method for determination of enantiomers of metoprolol and metabolites.•The chromatographic conditions were optimized.•SPE was used for isolation of metoprolol and its metabolites from urine.•The method was used for the determination of enantiomers in human urine.

A sensitive, stereoselective assay using solid phase extraction and high-performance liquid chromatography (HPLC) with fluorescence detection (FLD) was developed and validated for the analysis of enantiomers of metoprolol and its metabolites (α-hydroxymetoprolol, O-desmethylmetoprolol). Chiral separation was achieved using a CHIRALCEL OD-RH column, packed with cellulose tris-(3,5-dimethylphenyl-carbamate) stationary phase, employing a mobile phase composed by a mixture of 0.2% diethylamine in water and acetonitrile in gradient elution mode. Linear calibration curves were obtained over the range of 0.025–2.0 μg/mL (R2 > 0.994) in urine for both enantiomers of metoprolol and its metabolites with quantitation limit of 0.025 μg/mL. Intra and inter-day precision and accuracy were below 15% for both metoprolol and metabolites enantiomers. The recovery of enantiomer of metoprolol and its metabolite was greater than 68.0%, utilizing a SPE procedure. The method was tested with urine quality control samples and human urine fractions after administration of 50 mg rac-metoprolol.