基于RP-UPLC-MS技术的人血清样品脂质组学分析
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摘要
目的建立基于RP-UPLC-MS技术的人血清样本脂质组学分析方法。方法以人血清为样本,利用甲醇-氯仿(体积比为2∶1,含质量分数为0.01%的BHT)混合溶剂进行脂质提取,优化液相色谱条件及质谱检测参数并对人血清中甘油三酯、甘油磷脂酰胆碱、溶血性甘油磷脂酰胆碱等代表性脂质类成分进行方法学考察。结果建立了以Acquity BEH C_(18)(5 cm×1.0 mm,1.7μm)为分析柱,柱温50℃,以乙腈-异丙醇(体积比为5∶2,含质量分数为1%的甲酸铵)为流动相A,超纯水(含质量分数为1%的甲酸铵)为流动相B,梯度方式洗脱,采用正负离子检测模式电喷雾电离(±ESI)方式进行质谱检测的RP-UPLC-MS人血清样本脂质组学分析方法,且方法学考察结果表明该方法精密度、稳定性及重复性均良好。结论该方法适用于人血清样本的脂质组学研究,为临床疾病脂类生物标志物的寻找奠定了基础。
Objective To develop ananalysis method based on RP-UPLC-MS for human serum lipidomics.Methods The samples ere pretreated by lipid extraction ith methanol-chloroform( volume ratio of 2 ∶ 1,containing 0. 01%(w) BHT). The method as developed and validated. Typical lipid molecules such as triglyceride( TG),glycerol phosphatidyl choline( GPC) and Lyso-glycerol phosphatidyl choline( lyso-GPC)had been detected. Results The chromatographic separation as performed on an Acquity BEH C_(18)column( 5 cm × 1. 0 mm,1. 7 μm,50 ℃)ith mixture solution( B) as mobile phasehich consists of 0. 1%(w)ammonium formateultra pure ater solution( A) and acetonitrile-isopropyl alcohol( V ∶ V = 5 ∶ 2) in a linear gradient elution manner. The MS detection as carried out by the integrated ionization approach using electrospray ionization( ESI) in both positive and negative ion modes. The methodw as proved to possess good precision,reliable stability and high reproducibility. Conclusions The method could be applied in the lipidomics study for finding the biomarkers of lipid biomarkers in clinical diseases.
Objective To develop ananalysis method based on RP-UPLC-MS for human serum lipidomics.Methods The samples ere pretreated by lipid extraction ith methanol-chloroform( volume ratio of 2 ∶ 1,containing 0. 01%(w) BHT). The method as developed and validated. Typical lipid molecules such as triglyceride( TG),glycerol phosphatidyl choline( GPC) and Lyso-glycerol phosphatidyl choline( lyso-GPC)had been detected. Results The chromatographic separation as performed on an Acquity BEH C_(18)column( 5 cm × 1. 0 mm,1. 7 μm,50 ℃)ith mixture solution( B) as mobile phasehich consists of 0. 1%(w)ammonium formateultra pure ater solution( A) and acetonitrile-isopropyl alcohol( V ∶ V = 5 ∶ 2) in a linear gradient elution manner. The MS detection as carried out by the integrated ionization approach using electrospray ionization( ESI) in both positive and negative ion modes. The methodw as proved to possess good precision,reliable stability and high reproducibility. Conclusions The method could be applied in the lipidomics study for finding the biomarkers of lipid biomarkers in clinical diseases.
引文
[1]HAN X,GROSS R W.Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry:a bridge to lipidomics[J].Lipid Reasearch,2003,44(6):1071-1079.
[2]朱超,胡坪,梁琼麟,等.脂组学及其研究进展[J].分析化学,2009,9(39):1390-1396.
[3]KHALIL M B,HOU W,ZHOU H,et al.Lipidomics era:accomplishments and challenges[J].Mass spectrum Reviews,2010,29(6):877-929.
[4]YANG S,LUSH,YUAN Y J,et al.Lipidomic analysis reveals differential defense responses of Taxus cuspidate sells to two elicitors,methyljasmonate and cerium(Ce4+)[J].Biochimica Et Biophysica Acta,2008,1781(3):123-134.
[5]BLIGH E G,DYERWJ.A rapid method of tatal lipid extraction and purification[J].Can J Biochem Physiol,1959,37(8):911-917.
[6]PANG L Q,LIANG Q L,WANG Y M,et al.Simultaneous determination and quantification og seven major phospholipid classes in human blood using normal-phase liquid chromatography coupled with electrospray mass spectrometry and the application in diabetes nephropathy[J].J Chromatogr B,2008,869(1/2):118-125.
[7]WATSON A D.Thematic review series:systems biology approsches to metabolic and cardiovascular disorders[J].J Lioid Res,2006,47(10):2101-2111.
[8]SCHMID P,HUNTER E.Extraction and purification of lipids.II.Why is chloroform-methanol such a good lipid solvent?[J].Physiol Chem Phy,1973,5:151-155.
[2]朱超,胡坪,梁琼麟,等.脂组学及其研究进展[J].分析化学,2009,9(39):1390-1396.
[3]KHALIL M B,HOU W,ZHOU H,et al.Lipidomics era:accomplishments and challenges[J].Mass spectrum Reviews,2010,29(6):877-929.
[4]YANG S,LUSH,YUAN Y J,et al.Lipidomic analysis reveals differential defense responses of Taxus cuspidate sells to two elicitors,methyljasmonate and cerium(Ce4+)[J].Biochimica Et Biophysica Acta,2008,1781(3):123-134.
[5]BLIGH E G,DYERWJ.A rapid method of tatal lipid extraction and purification[J].Can J Biochem Physiol,1959,37(8):911-917.
[6]PANG L Q,LIANG Q L,WANG Y M,et al.Simultaneous determination and quantification og seven major phospholipid classes in human blood using normal-phase liquid chromatography coupled with electrospray mass spectrometry and the application in diabetes nephropathy[J].J Chromatogr B,2008,869(1/2):118-125.
[7]WATSON A D.Thematic review series:systems biology approsches to metabolic and cardiovascular disorders[J].J Lioid Res,2006,47(10):2101-2111.
[8]SCHMID P,HUNTER E.Extraction and purification of lipids.II.Why is chloroform-methanol such a good lipid solvent?[J].Physiol Chem Phy,1973,5:151-155.