Rapid profiling and quantification of phospholipid molecular species in human plasma based on chemical derivatization...

In this study, we developed a novel strategy using solid-phase extraction (SPE) coupled with shotgun mass spectrometry (MS) based on trimethylsilyldiazomethane (TMSCHN₂) stable-isotope derivatization for rapid profiling and accurate quantification of phospholipids (PLs) in human plasma. HybridSPE-Phospholipid (HybridSPE-PL, zirconia coated silica stationary phase) was used for sample pretreatment via the Lewis acid-base interaction between zirconia and phosphate moiety of PLs. This step allows rapid enrichment and recovery of PLs from human plasma. Afterward, PLs were derivatized with TMSCHN₂, which leads to methylation of hydroxyl and amino groups in PLs and allows highly sensitive PL analysis by shotgun MS in positive ionization mode (limit of detection decreased up to 116.67 fold compared to underived PLs). We developed an accuracy quantification method for determination of PL molecular species in biological samples. Two or more PL standards were selected for each PL class and derivatized with TMSCHN₂ without stable-isotope coding. They were then used as the internal standards. PLs in biological samples were isotopic derivatized via acid-catalyzed H/D exchange and methanolysis of TMSCHN₂. For accurate quantification, a calibration curve for each class of PLs was typically constructed by using the internal standards to normalize the non-uniformity response caused by the differential fragmentation kinetics resulting from the distinct chemical constitution of individual PL species in the biological samples. This newly developed method was used to comprehensively analyze PL molecular species in human plasma samples. It is a promising methodology for rapid profiling and accurate quantification of complex lipid molecules in biological samples.