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.