高效液相色谱-质谱联用定量检测人血清5个溶血磷脂酰胆碱的浓度

目的：利用代谢组学代谢靶标分析技术，建立一种定量检测人血清中溶血磷脂酰胆碱（LPC）类物质的方法。方法：以高效液相色谱与串联质谱联用（HPLC-MS/MS）分析平台，利血平为内标物，构建定量检测LPC 14：0、LPC 15：0、LPC 16：0、LPC 17：0和LPC₁₈：0系列物质的检测方法。绘制每种目标LPC对应的标准曲线，分析其相关系数r值，并进一步评估检测平台的灵敏度检测下限（LOD）和线性范围。通过加样回收试验验证检测平台的准确度（加样回收率）和精密度（RSD），并进一步分析该平台在检测人血清标本的适用性。在此基础上，利用该HPLC-MS/MS平台分析了LPC 14：0~LPC₁₈：0 5个LPCs在乙肝相关性肝细胞肝癌（HBV-HCC）患者血清中的浓度。结果：本研究建立了基于HPLC-MS/MS的LPC检测平台，该平台检测稳定性和准确性较高。LPC 14：0~LPC₁₈：0 5个物质的检测下限分别是0.027、0.120、0.320、0.059、0.072μ g·mL^-1；标准曲线方程分别为Y=0.725X-0.002 64、Y=0.212X-0.025 6、Y=1.22X+1.84、Y=0.861X-0.015 5和Y=0.813X+0.558，相关系数分别为0.997 6、0.999 2、0.992 7、0.998 0和0.994 3，测量结果准确度和精密度均控制在85%~115%范围内。利用HPLCMS/MS平台测定的5个LPC类物质，对不同巴塞罗那期HBV-HCC患者具备一定的鉴别能力。结论：本研究利用HPLC-MS/MS分析平台，成功建立了一个稳定、灵敏而可靠的定量检测人血清中LPC 14：0~LPC₁₈：0 5个目标LPC类物质的检测方法，适合临床推广。

Objective: To establish a quantitative detection platform for the identification of lysophosphatidylcholine(LPC) substances in human serum by using targeted metabolomics techniques.Methods: The analytical platform was developed based on a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS),which using reserpine as the internal standard to quantitatively detect LPC substances,including LPC 14:0,LPC 15:0,LPC 16:0,LPC 17:0 and LPC₁₈:0. The standard curve of each LPC was plotted and the linear correlation coefficient was analyzed. The limits of detection (LODs) and the linearity were evaluated. The accuracy(spiked recovery rate) and precision(RSD) of the detection platform were verified by spiked recovery experiments. The applicability of HPLC-MS/MS platform in human serum was analyzed,as well. In addition,the concentrations of five types of LPCs (LPC 14:0-LPC₁₈:0) in patients with hepatitis B-related hepatocellular carcinoma (HBV-HCC) were validated by the current HPLC-MS/MS platform. Results: A LPC detection platform based on HPLC-MS/MS was established,which had high stability and accuracy. The LODs of the five substances LPC 14:0 to LPC₁₈:0 were 0.027, 0.120, 0.320, 0.059 and 0.072 μg·mL^-1, respectively. The standard curves were:Y=0.725X-0.002 64,Y=0.212X-0.025 6,Y=1.22X+1.84,Y=0.861X-0.015 5 and Y=0.813X+0.558, respectively. The correlation coefficients were 0.997 6, 0.999 2, 0.992 7, 0.998 0 and 0.994 3, respectively. In the meantime, the accuracy and precision of the measurement were controlled within the range of 85%-115%. The five types of LPCs determined by HPLC-MS/MS platform could be applied to differentiate HBV-HCC patients with different stages of Barcelona. Conclusions: In the study,a stable,sensitive and reliable quantitative detection platform was successfully established based on HPLC-MS/MS. The platform can quantitatively detect five types of target LPCs(LPC 14:0 to LPC₁₈:0) in human serum, which is suitable for promotion in the clinical study.

[1] FITIAN AI, CABRERA R. Disease monitoring of hepatocellular carcinoma through metabolomics[J]. World J Hepatol,2017,9(1):1
[2] LIU Y, HONG Z, TAN G, et al. NMR and LC/MS-based global metabolomics to identify serum biomarkers differentiating hepatocellular carcinoma from liver cirrhosis[J]. Int J Cancer, 2014, 135(3):658
[3] RESSOM HW,XIAO JF,TULI L,et al. Utilization of metabolomics to identify serum biomarkers for hepatocellular carcinoma in patients with liver cirrhosis[J]. Anal Chim Acta,2012, 743(18):90
[4] ZENG J, HUANG X, ZHOU L, et al. Metabolomics identifies biomarker pattern for early diagnosis of hepatocellular carcinoma:from diethylnitrosamine treated rats to patients[J]. Sci Rep,2015, 10(5):16101
[5] MATSUMOTO T, KOBAYASHI T, KAMATA K. Role of lysophosphatidylcholine(LPC) in atherosclerosis[J]. Curr Med Chem, 2007, 14(30):3209
[6] KRAUTBAUER S, EISINGER K, WIEST R, et al. Systemic saturated lysophosphatidylcholine is associated with hepatic function in patients with liver cirrhosis[J]. Prostaglandins Other Lipid Mediat,2016, 6(124):27
[7] SONOMURA K,KUDOH S,SATO TA, et al. Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry[J]. J Sep Sci, 2015, 38(12):2033
[8] 张虹.加标回收率的测定和结果判断[J].石油与天然气化工, 2000, 29(1):50 ZHANG H. Study on the determination and recovery of spike recovery[J]. Petrol Nat Gas Chem Ind,2000,29(1):50
[9] 朱超,梁琼麟,王义明,等.代谢组学的整合化发展及其新进展[J].分析化学, 2010, 38(7):1060 ZHU C, LIANG QL, WANG YM, et al. Integrative development of metabolomics and its new progress[J]. Chin J Anal Chem, 2010, 38(7):1060
[10] PENG B,LI H,PENG XX. Functional metabolomics:from biomarker discovery to metabolome reprogramming[J]. Protein Cell, 2015, 6(9):628
[11] GAO P, XU G. Mass-spectrometry-based microbial metabolomics:recent developments and applications[J]. Anal Bioanal Chem, 2015, 407(3):669
[12] RAMAUTAR R,SOMSEN GW,de JONG GJ. CE-MS for metabolomics:developments and applications in the period 2012-2014[J]. Electrophoresis,2015,36(1):212
[13] WOLFENDER JL, MARTI G, THOMAS A, et al. Current approaches and challenges for the metabolite profiling of complex natural extracts[J]. J Chromatogr A,2015, 1382(1):136
[14] GODFREY AR, JONES L, DAVIES M, et al. Miltefosine:a novel internal standard approach to lysophospholipid quantitation using LC-MS/MS[J]. Anal Bioanal Chem,2017, 409(11):2791
[15] 王中华,陈艳华,徐婧,等.血清脂质组学研究中多重离子化液相色谱-质谱方法的比较[J].分析化学, 2017, 45(5):674 WANG ZH, CHEN YH, XU J, et al. Comparison of multiple ionization liquid chromatography-mass spectrometry in serum lipid group study[J]. Chin J Anal Chem,2017, 45(5):674
[16] XIA YQ, JEMAL M. Phospholipids in liquid chromatography/mass spectrometry bioanalysis:comparison of three tandem mass spectrometric techniques for monitoring plasma phospholipids, the effect of mobile phase composition on phospholipids elution and the association of phospholipids with matrix effects[J]. Rapid Commun Mass Spectrom,2009, 23(14):2125
[17] QUEHENBERGER O, ARMANDO AM, BROWN AH, et al. Lipidomics reveals a remarkable diversity of lipids in human plasma[J]. J Lipid Res,2010, 51(11):3299
[18] 亢爱春,霍勇,齐丽彤.溶血磷脂酰胆碱在动脉粥样硬化中的作用[J].中国动脉硬化杂志, 2006, 12(14):1083 KANG AC, HUO Y, QI LT. The role of lysophosphatidylcholine in atherosclerosis[J]. Chin J Arterioscler, 2006, 12(14):1083