Enantiomeric separation of mirtazapine and its metabolites by nano-liquid chromatography with UV-absorption and mass spectrometric detection.
Mirtazapine (MIR) and two of its main metabolites, namely, 8-hydroxymirtazapine and N-desmethylmirtazapine, were separated in totheir enantiomers by nanoLC in a laboratory-made fused-silica capillary column (75 microm ID) packed with a vancomycin-modified silica stationary phase. The simultaneous separation of the three couples of the studied enantiomers was achieved in less than 33 min, using an experimentally optimized mobile phase delivered in the isocratic mode. Optimization of the mobile-phase composition was achieved by testing the influence of the buffer pH and concentration, the water concentration, the organic modifier type and concentration, and on the retention and resolution of the analytes. The optimum mobile-phase composition contained 500 mM ammonium acetate pH 4.5/water/MeOH/MeCN, 1:14:40:45 v/v/v/v. Using a UV detector at 205 nm, the method was validated studying several experimental parameters such as LOD and LOQ, intraday and interday repeatability, and linearity. Good results were achieved: LOD and LOQ were in the range 5-15 and 10-40 microg/mL, respectively (the highest value was obtained for the DEMIR enantiomers); correlation coefficients, 0.9993-0.9999; the intraday and interday precision was acceptable (RSD < 2%) using an internal standard. The method was tested for the separation of the studied enantiomers in an extracted (solid-phase) serum sample spiked with standard racemic mixture of MIR and its two metabolites. Finally, the nanoLC system was connected to a mass spectrometer through a nanoelectrospray interface and the MS, MS2, and MS3 spectra were acquired showing the potential of the system used for characterization and identification of the separated analytes.
Fanali
Salvatore
S
Istituto di Metodologie Chimiche, Consiglio Nazionale delle Ricerche, Monterotondo Scalo, Roma, Italy. fanali@imc.cnr.it
Aturki
Zeineb
Z
Kasicka
Václav
V
Raggi
Maria Augusta
MA
D'Orazio
Giovanni
G
D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000379:methods; D008803:Mianserin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification; D000078785:Mirtazapine; D008958:Models, Molecular; D000644:Quaternary Ammonium Compounds; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation / Q000379:methods; D013237:Stereoisomerism
D007202:Indicators and Reagents; D000644:Quaternary Ammonium Compounds; D008803:Mianserin; D000078785:Mirtazapine