| Objective In order to accurately detect glycerophospholipids in human plasma,LC-MS/MS technology was adopted and the optimization of sample pretreatment was carried out.Finally,a glycerophospholipids detection method with good repeatability,high recovery and relatively low cost was established.The glycerophospholipids including lysophosphatidylcholine(LPC),phosphatidylcholine(PC)and phosphatidylethanolamine(PE)were detected in this study.Method(1)Optimization of mass spectrometry conditions: the mass spectrometry conditions of each analyte were optimized by mass spectrometry direct injection technique.(2)Optimization of chromatographic conditions: The chromatographic conditions such as mobile phase type and elution gradient were optimized by LC-MS/MS technology.(3)Sample pretreatment optimization: the standard samples and plasma samples were used to optimize the pretreatment process to obtain the best pretreatment scheme by LC-MS/MS technology.The sample pretreatment process included the selection of deproteinized solvent,the selection of extraction solvent,the selection of extraction times,the selection of reconstitution solvent and its proportion,and the selection of the p H of the reconstitution solvent.(4)Method verification: recovery,matrix effect,linear range,limit of detection,limit of quantification,and intra-day and inter-day precision of the method were investigated.Results(1)The MRM conditions of various phospholipids were obtained by mass spectrometry direct injection and LC-MS/MS technology,including precursor / product ion pairs and their CE,DP,CEP,EP,CXP,etc.(2)From the point of view of the deproteinization effect,acetonitrile was selected as the final deproteinization solvent.By comparing the extraction effect of different kinds of solvents,the extraction effects of dichloromethane/methanol(2:1,V/V)and chloroform/methanol(2:1,V/V)were comprehensively considered,and chloroform/methanol(2:1,V/V)was selected as the optimal extraction solvent,and the extraction twice was selected as the final extraction times by comparing the signal response intensity of each substance extracted once and twice.(3)According to the corresponding signal intensity,peak shape and stability in different acid and base environments,methanol-isopropanol(9:1,V/V,0.1% FA)was the most suitable solvent for redissolution.(4)Linear range was at 5-1000 ng/m L for LPC,PC and PE.Through the investigation of the recovery of the method,the recovery of the three compounds in the range of low,medium and high concentrations was 53.78%-55.04%,80.87%-93.90%,92.60%-103.0%,and CV was 0.38%-7.74%.The coefficient of variation of intra-day precision was 2.26%-7.12%,the variation of inter-day precision was less than 9.82%,and the coefficient of variation was 0.61%-9.82%.ConclusionsThe method for detection of glycerophospholipids(LPC?PC and PE)based on LC-MS/MS technique was established and evaluated in this study.Experimental results showed that the method was simple,without complicated pretreatment methods,with high sensitivity and convenient for the detection of LPC,PC and PE in plasma.The experiment is suitable for detection in medical laboratory.In addition,the abnormalities of LPC,PC and PE phospholipids are related to metabolic diseases such as diabetes,hypertension and atherosclerosis.The method established in this study can provide technical means for the study of some metabolic diseases. |
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