Bierhanzl Václav Matěj, Čabala Radomír, Ston Martin, Kubinec Róbert, Szabó Alexandra Hengerics, Podolec Peter
Faculty of Science, Department of Analytical Chemistry, Charles University in Prague, Prague, Czech Republic.
J Sep Sci. 2015 Jan;38(1):67-72. doi: 10.1002/jssc.201400657. Epub 2014 Dec 4.
A new, rapid, sensitive, robust, and reliable method has been developed for the qualitative analysis of phosphoserine, phosphoethanolamine, phosphoglycerol, and phosphate using gas chromatography with mass spectrometry and two-step trimethylsilylation. The method employs hexamethyldisilazane for silylation of the phosphate and hydroxyl groups in the first phase and bis(trimethylsilyl)trifluoroacetamide for silylation of the less-reactive amino groups in the second phase. This order is of key importance for the method because of the different reactivities of the two reagents and the mechanism of derivatization of the active groups of the analytes. Trimethylsilylated derivatives of the analytes were identified on the basis of their retention times and mass spectra. The probable structures of the major fragments were identified in the spectra of the trimethylsilylated derivatives and characteristic m/z fragments were selected for each analyte. Fragments with m/z 73 and 299 occurred in the spectra of all the analytes. The characteristic retention data were employed to calculate the retention indices of the individual silylated phosphorylated substances in the hydrocarbon range C12-C19 for the DB-5ms column. The method was employed to measure the polar fraction of the hydrolysate of the cytoplasmic membrane of Bacillus subtilis. The detection limits vary between 5 μg/mL (trimethylsilylated phosphate) and 72 μg/mL (trimethylsilylated phosphoethanolamine).
已开发出一种新的、快速、灵敏、稳健且可靠的方法，用于使用气相色谱-质谱联用和两步三甲基硅烷化法定性分析磷酸丝氨酸、磷酸乙醇胺、磷酸甘油和磷酸盐。该方法在第一阶段使用六甲基二硅氮烷对磷酸盐和羟基进行硅烷化，在第二阶段使用双（三甲基硅基）三氟乙酰胺对反应活性较低的氨基进行硅烷化。由于两种试剂的反应活性不同以及分析物活性基团的衍生化机制，这种顺序对该方法至关重要。根据分析物的保留时间和质谱图鉴定其三甲硅烷基化衍生物。在三甲硅烷基化衍生物的质谱图中鉴定出主要碎片的可能结构，并为每种分析物选择特征性的m/z碎片。m/z 73和299的碎片出现在所有分析物的质谱图中。利用特征保留数据计算了DB-5ms柱上碳氢化合物范围C12-C19内各个三甲硅烷基化磷酸化物质的保留指数。该方法用于测量枯草芽孢杆菌细胞质膜水解产物的极性部分。检测限在5 μg/mL（三甲硅烷基化磷酸盐）至72 μg/mL（三甲硅烷基化磷酸乙醇胺）之间变化。
