Isocyanate derivatization coupled with phospholipid removal microelution-solid phase extraction for the simultaneous quantification of (S)-metoprolol and (S)-α-hydroxymetoprolol in human plasma with LC–MS/MS

•Novel sensitive method developed and validated for quantifying (S)-metoprolol and one of its metabolite in human plasma.•Phospholipid removal microelution-solid phase extraction minimizes matrix effects and allows good recovery using minimal sample volumes.•Excellent chiral separation using isocyanate derivatization.•Successful application on a clinical cohort of patients taking metoprolol. A sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS) assay was developed and validated for the quantification of (S)-metoprolol (MET) and its main metabolite, (S)-α-hydroxymetoprolol (OH-MET). Human plasma samples (50 μL) were spiked with both analytes and their deuterated internal standards (IS) (S)-MET-(d7) and α-OH-MET-(d5). Phospholipid removal microelution-solid phase extraction (PRM-SPE) was performed using a 4-step protocol with Oasis PRiME MCX μElution 96-well cartridges. The eluates were reconstituted in 100 μL of acetonitrile with 50 μg/mL (S)-α-methylbenzyl isocyanate (MBIC) for chiral derivatization. After 60 min at room temperature, the reaction was quenched using 100 μL of water 2 % formic acid. Chromatographic separation of the derivatized analytes was performed on a Kinetex phenyl-hexyl core-shell stationary phase with an elution gradient. Mobile phases were composed of a mixture of water and methanol, with ammonium formate and formic acid as buffers. Total runtime was 15 min. Analyte detection was performed by an AB/SCIEX 4000 QTRAP mass spectrometer with multiple reaction monitoring. Chromatograms showed MBIC successfully reacted with racemic MET, α-OH-MET, and their respective IS. Detection by positive electrospray ionization did not reveal derivatized by-products. Quantification ranges were validated for (S)-MET and (S)-α-OH-MET between 0.5–500 and 1.25−500 ng/mL, respectively, with correlation coefficients (r2) >0.9906. The PRM-SPE assay showed low matrix effects (86.9–104.0 %) and reproducible recoveries (69.4–78.7 %) at low, medium, and high quality control (QC) levels. Precision and accuracy were all comprised between 85–115 % for all three QCs, and between 80–120 % for the lower limit of quantification, for intra- and inter-day values (n = 6, 3 consecutive days). Non-derivatized analytes were stable at room temperature, after 3 freeze-thaw cycles, and stored for 30 days at −80 °C (n = 4). Reinjection reproducibility of a previously validated batch was achieved after 8 days under auto-sampler conditions, indicating the stability