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 mu L human serum was consisted of protein precipitation with 200 mL ethanol and liquid-liquid extraction by 400 mu L hexane/dichloromethane (80/20, v/v).
The separation was performed on BEH 2-EP (3.0 x 100 mu m, 1.7 mu m) stationary phase, using isocratic elution with carbon dioxide and 10 mu M 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 mu g mL(-1)) and better linearity (r(2) > 0.9930) than the high speed one (LOD: 0.083-0.25 mg mL(-1), r(2) > 0.9877) at the cost of double time of analysis.