title: Development and optimization of ultra-high performance supercritical fluid chromatography mass spectrometry method for high-throughput determination of tocopherols and tocotrienols in human serum.
abstract: The goal of this study was to develop an effective supercritical fluid chromatography method using single quadrupole MS for analysis of all isomeric forms of vitamin E. Finally, two fast and effective methods, the high resolution one and the high speed one, for the determination of 8 vitamin E isomers in human serum were developed. Rapid high-throughput liquid-liquid extraction was selected as a sample preparation step. Sample pretreatment of 100 μL human serum was consisted of protein precipitation with 200 μL ethanol and liquid-liquid extraction by 400 μL hexane/dichloromethane (80/20, v/v). The separation was performed on BEH 2-EP (3.0 × 100 mm, 1.7 μm) stationary phase, using isocratic elution with carbon dioxide and 10 mM ammonium formate in methanol in the ratio 98:2 for high resolution method with run time 4.5 min and in the ratio 95:5 for high speed method, where the run time was 2.5 min. The method development included optimization of key parameters: the choice of the suitable stationary phase and the composition of mobile phase, where an influence of various modifiers, their ratio and additives were tested, and optimization of fine tunning parameters including BPR pressure, flow-rate and column temperature. Quantification of all isomeric forms was performed using SIM (single ion monitoring) experiments in ESI positive ion mode. Both high speed and high resolution chromatographic methods were validated in terms of precision, accuracy, range, linearity, LOD, LOQ and matrix effects using the same LLE procedure. The high resolution method provided more sensitive results (LOD: 0.017-0.083 μg mL(-1)) and better linearity (r(2) > 0.9930) than the high speed one (LOD: 0.083-0.25 μg mL(-1), r(2) > 0.9877) at the cost of double time of analysis.
authors: Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05, Hradec Králové, Czech Republic.
authors: Veronika
authors: V
authors: Pilařová
authors: Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05, Hradec Králové, Czech Republic.
authors: Tomáš
authors: T
authors: Gottvald
authors: Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05, Hradec Králové, Czech Republic.
authors: Pavel
authors: P
authors: Svoboda
authors: Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany CAS & Faculty of Science of Palacký University, Šlechtitelů 27, 783 71, Olomouc, Czech Republic.
authors: Ondřej
authors: O
authors: Novák
authors: Research Institute of Brewing and Malting, Malting Institute Brno, Mostecká 7, 614 00, Brno, Czech Republic.
authors: Karolína
authors: K
authors: Benešová
authors: Research Institute of Brewing and Malting, Malting Institute Brno, Mostecká 7, 614 00, Brno, Czech Republic.
authors: Sylvie
authors: S
authors: Běláková
authors: Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05, Hradec Králové, Czech Republic. Electronic address: nol@email.cz.
authors: Lucie
authors: L
authors: Nováková
mesh_terms: D025924:Chromatography, Supercritical Fluid; D006801:Humans; D059625:Liquid-Liquid Extraction; D013058:Mass Spectrometry; D008968:Molecular Conformation; D024505:Tocopherols / Q000097:blood* / Q000737:chemistry; D024508:Tocotrienols / Q000097:blood* / Q000737:chemistry
chemical_list: D024508:Tocotrienols; D024505:Tocopherols
keywords: Human serum; Liquid liquid extraction; Mass spectrometry; Tocopherols; Tocotrienols; Ultra-high performance supercritical fluid chromatography