Synopsis of Thesis
Title: Simultaneous profiling of polar lipids by supercritical fluid chromatography/tandem mass spectrometry with derivatization for lipidomics
(誘導体化法適用超臨界流体クロマトグラフィー/質量分析による極性脂質の一斉プロファイリング)
Name of Applicant: Lee Jae Won
Chapter 1: General introduction
Lipidomics is a systems-level analysis of lipid species, their abundance, biological activities, and subcellular localization. Many studies have reported the aberrant metabolism of polar lipids in human diseases. To characterize the altered polar lipids: phospholipids (PLs), lysophospholipids (LPLs), and sphingolipids (SLs), an effective analytical method is needed. In particular, comprehensive analysis of polar lipids is hindered by the peak tailing and low detection sensitivity of acidic PLs: phosphatidylserine (PS) and phosphatidic acid (PA). Peak tailing must be advanced because it can decrease the detection sensitivity of compounds. To solve these issues, adjusted mobile phase or EDTA-prewashed column were used previously. However, the modification of chromatographic conditions was insufficient due to the low repeatability to treat and the needs to use the non-metal system.
In this study, the derivatization was applied for the modification of characteristics in polar lipids to advance the peak shape and sensitivity. The main factor of peak tailing was found as the metal-affinity of polar head groups. To block several functional groups in polar lipids, various derivatization can be used. In the selection of derivatizations, acylation and esterification were excluded due to their needs of additional extraction step and the removal of water. It can cause time-consuming and handy-error. Finally, at first, TMS silylation was used due to its simplicity and wide applicability to various functional groups. Upon silylation, the analysis of several polar lipids improved, but it was insufficient to derivatize phosphate group that has high metal-affinity to cause peak tailing. Secondly, TMSD methylation was used to derivatize phosphate for the advanced analysis of PLs, LPLs, and SLs. SFC/MS/MS was effective to analyze derivatized polar lipids with the increased hydrophobicity.
Chapter 2: Development of a polar lipid profiling by SFC/MS/MS with silylation
Of various derivatizations, TMS silylation which is highly reactive, simple and widely applicable to various functional groups containing reactive hydrogen (e.g. –OH, -COOH, -NH2) was used to advance the polar lipid profiling by SFC/MS/MS. By silylation, the metal-affinity of acidic PLs was suppressed by blocking the functional groups in polar head groups. In addition, the silylation increased the hydrophobicity of polar lipids so that their separation was also advanced in SFC using non-polar CO2. Especially, hydroxyl groups were silylated mainly, hence, the analysis of phosphatidylinositol (PI) was improved well. However, this method was insufficient to derivatize phosphate group that has high metal-affinity and to improve PS analysis. Thus, more effective method should be selected for polar lipid profiling.
Chapter 3: Development of a polar lipid profiling by SFC/MS/MS with methylation
Methylation is an effective method to derivatize the phosphate group for polar lipid profiling. Diazomethane is a conventional reagent for the methylation, but this reagent needs care due to its explosive and carcinogenic. Thus, in this study, trimethylsilyldiazomethane (TMSD) which is relatively safe, stable, and easy to treat, was used. Finally, 6 PLs, 6 LPLs, and 4 SLs were methylated by the optimized conditions, and the comprehensive profiling of polar lipids was achieved with sharp peaks. Advanced peak shapes provided the increased detection sensitivity. Detail and reliable quantification of various polar lipid species in mouse liver was obtained successfully.
Chapter 4: Conclusions
In this study, two derivatizations (silylation & methylation) were used for the advanced polar lipid profiling by SFC/MS/MS. Of two methods, methylation was better than silylation for the comprehensive profiling of polar lipids. However, in the analysis of PI, silylation was better than methylation. Finally, according to the purpose, silylation and methylation are expected to be useful for the polar lipid profiling. Simultaneous analysis is helpful for the detail characterization of various polar lipids. For the practical uses, high-throughput analysis is also effective when the samples are too many. In lipidomics, this method will be useful for the detail phenotype of polar lipids in the study of biomarker and drug development.
List of publications:
1. Lee J. W., Yamamoto T., Uchikata T., Matsubara A., Fukusaki E., Bamba T., Development of a polar lipid profiling method by supercritical fluid chromatography/mass spectrometry, Journal of Separation Science, 34, 3553-3560 (2011).
2. Lee J. W., Nishiumi S., Yoshida M., Fukusaki E., Bamba T., Simultaneous profiling of polar lipids by supercritical fluid chromatography/tandem mass spectrometry with methylation, Journal of Chromatography A, 1279, 98-107 (2013).