QuEChERS-液相色谱-串联质谱法快速测定肝细胞癌血液中3种小分子代谢物

目的：通过建立QuEChERS（快速、简易、廉价、有效、稳定、安全）前处理方法，结合液相色谱-三重四极杆质谱联用技术，实现肝细胞癌血液中3种小分子代谢物的同时检测。方法：血清样品经乙腈提取，无水硫酸镁盐析及N-丙基乙二胺（PSA）吸附剂净化后，取上清液采用Phenomenex Luna Omega 3 μm PS C₁₈ 100&#197;（150 mm&#215;2.1 mm）色谱柱，以甲醇-0.1%乙酸水溶液为流动相，梯度洗脱。电喷雾离子源正离子扫描，多反应监测模式（MRM）进行定量分析。结果：十四酰胺的检测下限为0.6 ng&#183;g^-1，定量下限为2 ng&#183;g^-1；油酸酰胺的检测下限为0.3 ng&#183;g^-1，定量下限为1 ng&#183;g^-1。3种小分子代谢物质量浓度在10~100 ng&#183;mL^-1范围内呈线性，其中目标物线性相关系数（R²）均大于0.991 7，基质效应（ME）在0.79~1.55之间；3个水平加样回收率在77.5%~105.1%之间，日内精密度（n=5）在6.9%~11.8%之间，日间精密度在7.4%~11.1%之间；利用该方法扩大样本量检测，3种小分子代谢物在健康组血清中的浓度（2.591、0.221、7.485 mg&#183;kg^-1）和肝癌组血清中的浓度（9.599、0.266、5.063 mg&#183;kg^-1）差异显著（P<0.01）。结论：该方法简单快捷，准确可靠，适合于大批量肝细胞癌血液样品中3种小分子代谢物的快速检测。

Objective:To establish an LC-MS method for the simultaneous determination of 3 small-molecule metabolites in the hepatocellular carcinoma blood by establishing quEChERS (quick, easy, cheap, effective, rugged, safe) coupled with liquid chromatography-mass spectrometer.Methods:Serum samples were extracted with acetonitrile and anhydrous sodium sulfate were used for salting-out process.After purified by PSA, the supernatant was applied on a Phenomenex Luna Omega 3 μm PS C₁₈ 100&#197; (150 mm&#215;2.1 mm) column with methanol-0.1% acetic acid solution as mobile phase and subjected to gradient elution.For quantitation, electrospray ionization (ESI) source was applied and the mass spectrometer was operated in multiple reaction monitoring (MRM) modes. Results:The limits of detection (LOD) and the limits of quantification (LOQ) for n-tetradecanamide were 0.6 ng&#183;g^-1 and 2 ng&#183;g^-1 respectively.LOD and LOQ for oleamide were 0.3 ng&#183;g^-1 and 1 ng&#183;g^-1 respectively.There was a good linear relationship in the range of 10-100 ng&#183;mL^-1 of the 3 small-molecule metabolites.The target linear correlation coefficient (R²) was higher than 0.991 7.The matrix effect (ME) was between 0.79 and 1.55.The method recovery was between 77.5% and 105.1% in three spike levels.The intraday precision (n=5) was between 6.9% and 11.8% and daytime precision was between 7.4% and 11.1%.Using this method to expand the sample size detection, the concentration of three small-molecule metabolites in healthy group (2.591, 0.221, 7.485 mg&#183;kg^-1) and cancer group (9.599, 0.266, 5.063 mg&#183;kg^-1) were significantly different (P<0.01).Conclusion:This method is simple, accurate and reliable, and is suitable for the rapid detection of three small-molecule metabolites in large quantities of the hepatocellular carcinoma blood samples.

[1] HANAHAN D,WEINBERG R. The hallmarks of cancer[J]. Cell, 2000, 100(1):57
[2] FERLAY J, SOERJOMATARAM I, ERVIK M, et al. GLOBOCAN 2012 v1. 0, Cancer Incidence and Mortality Worldwide:IARC Cancer Base No. 11[EB/OL]. Lyon:International Agency for Research on Cancer,2013.(2014-01-20)[2015-03-15]. http://globocan.iarc.fr
[3] BOYLE P,LEVIN B. World Cancer Report 2008[M]. Lyon:IARC Press, 2008:55
[4] 沈鹏,季国忠.肝细胞癌血清标志物的研究进展[J].医学综述, 2013, 19(2):278 SHEN P,JI GZ. Reserch progress in serum markers of hepatocellular carcinoma[J]. Med Recapit,2013, 19(2):278
[5] WANT EJ, O'MAILLE G, SMITH CA, et al. Solvent-dependent metabolite distribution, clustering, and protein extraction for serum profiling with mass spectrometry[J]. Anal Chem, 2006, 78(3):743
[6] NICHOLSON JK, LINDON JC, HOLMES E. Metabonomics:understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data[J]. Xenobiotica, 1999, 29(11):1181
[7] ARITA Y, KIHARA S, OUCHI N, et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity[J]. Biochem Biophys Res Commun,1999, 257(1):79
[8] KADOWAKI T, YAMAUCHI T. Adiponectin and adiponectin receptors[J]. Endocr Rev,2005, 26(3):439
[9] MIYOSHI Y, FUNAHASHI T, KIHARA S, et al. Association of serum adiponectin levels with breast cancer risk[J]. Clin Cancer Res, 2003,9(15):5699
[10] YIN P, WAND D, ZHAO X, et al. Metabonomic study of intestinal fistulas based on ultra performance liquid chromatography coupled with Q-TOF mass spectrometry(UPLC/Q-TOF MS)[J]. J Proteome Res, 2006, 5(9):2135
[11] YU K,SHENG GP, SHENG JF, et al. A metabonomic investigation on the biochemical perturbation in liver failure patients caused by hepatitis bavirus[J]. J Prot Res, 2007,6(7):2413
[12] LI H, FAN SF, WANG Y, et al. Rapid detection of small molecule metabolites in serum of hepatocellular carcinoma patients using ultrafast liquid chromatography-ion trap-time of flight tandem mass spectrometry[J]. Anal Sci Int J Japan Soc Anal Chem, 2017, 33(5):573
[13] 杨阳,王志鹏,高守红,等.代谢组学在结直肠癌肿瘤标志物发现中的研究进展[J].药物分析杂志,2018, 38(6):915 YANG Y, WANG ZP, GAO SH, et al. Research advances of discovery of tumor markers in colorectal cancer based on metabolomics[J]. Chin J Pharm Anal, 2018, 38(6):915
[14] XUE R, LIN Z,DENG C, et al. A serum metabolomic investigation followed by gas chromatography mass spectrometry[J]. Rapid Commun Mass Spectrom,2008, 22(19):3061
[15] CHEN T, XIE G, WANG X, et al. Serum and urine metabolite profiling reveals potential biomarkers of human hepatocellular carcinoma[J]. Mol Cell Proteomics, 2015,10(7):M110. 004945
[16] GAO H, HUANG H, XU W, et al. Fecal metabolome profiling of liver cirrhosis and hepatocellular carcinoma patients by ultra performance liquid chromatography-mass spectrometry[J]. Anal Chim, 2011, 691(1):68
[17] PATTERSON AD, MAURHOFER O, BEYOGL D, et al. Aberrant lipid metabolism in hepatocellular carcinome revealed by plasma metabolomics and lipid profiling[J]. Cancer Res, 2011, 71(21):6590
[18] PATIERSON AD, LI H, EICHLER GS, et al. UPLC-ESI-TOFMSbased metabolomics and gene expression dynamics inspector selforganizing metabolomic maps as tools for understanding the cellular response to ionizing radiation[J]. Anal Chem,2008, 80(3):665
[19] GAO H, LU Q, LIU X, et al. Application of H-1 NMR-based metabonomics in the study of metabolic profiling of human hepatocellular carcinoma and liver cirrhosis[J]. Cancer Sci, 2010, 100(4):782
[20] SHARIFF MIF, LADEP NG, COXIJ, et al. Characterization of urinary biomarkers of hepatocellular carcinoma using megnetic resonance spectroscopy in a Nigerian population[J]. J Prot Res, 2010, 9(2):1096
[21] SHARIFF MIF, GOMAA AI, COX IJ, et al. Urinary metabolic biomarkers of hepatocellular carcinoma in an Egyption population:a validation study[J]. J Prot Res, 2011,10(4):1828
[22] 徐玲燕,杜娟,吴伟,等. LC-MS/MS在临床内源性类固醇激素分析中的方法研究和应用进展[J].药物分析杂志, 2018, 38(7):1106 XU LY, DU J, WU W, et al. Research and application progress of LC-MS/MS in clincals endogenous steroid analysis[J]. Chin J Pharm Anal, 2018, 38(7):1106
[23] 谢增瑞. QuEChERS提取法提取血液样品中常见毒物的应用[J].中国法医学杂志, 2016, 31(z1):156 XIE ZR. The application of QuEChERS to extract common poisons in blood samples[J]. Chin J Forensic Med, 2016, 31(z1):156
[24] 冶廷祥.血液检材中常见安眠镇静类药物的QuEChERS处理与GC-MS分析[D].兰州:兰州大学, 2016 YE TX. QuEChERS Treatment and GC-MS Analysis of Common Hypnotic Sedative Drugs in Blood Samples[D]. Lanzhou:Lanzhou University, 2016
[25] 于天晓,韩彦洁,张瑶,等.基于Fe₃O₄磁性纳米粒子的QuEChERS 预处理方法联合GC-MS对血液中多种苯二氮类药物检测[J].分析试验室,2018,37(4):450 YU TX, HAN YJ, ZHANG Y, et al. Fe₃O₄ magnetic nanoparticles based QuEChERS pretreatment method combined with GC-MS technology for simultaneous detection of multiple benzodiazepine drugs in blood[J]. Chin J Anal Lab,2018, 37(4):450
[26] 成玉,刘玉敏,黄凤杰,等.基于超高效液相色谱-四极杆飞行时间高分辨质谱的高通量血清代谢组学方法[J].高等学校化学学报, 2013, 34(1):77 CHENG Y,LIU YM, HUANG FJ, et al. Robust and high throughput UPLC-QTOFMS method for the global metabolic profiling study of human serum[J]. Chem J Chin Univ, 2013, 34(1):77
[27] FRANK JS, STEVEN JL, VICTOR V. Comparison of solid-phase extraction sorbent for cleanup in pesticide residue analysis of fresh fruits and vegetables[J]. J Sep Sci, 2002, 25(14):883
[28] 易江华,段振娟,方国臻,等. QuEChERS方法在食品农兽药残留检测中的应用[J].中国食品学报, 2013, 13(2):153 YI JH, DUAN ZJ, FANG GZ, et al. Study of QuEChERS for the detection of pesticide and veterinary drug residues in food[J]. J Chin Ins Food Sci Technol,2013, 13(2):153
[29] 王连珠,周昱,黄小燕,等.基于QuEChERS提取方法优化的液相色谱-串联质谱法测定蔬菜中51种氨基甲酸酯类农药残留[J].色谱, 2013,31(12):1167 WANG LZ, ZHOU Y, HUANG XY, et al. Determination of 51 carbamate pesticide residues in vegetables by liquid chromatographytandem mass spectrometry based on optimization of QuEChERS sample preparation method[J]. Chin J Chromatogr,2013,31(12):1167
[30] 罗辉泰,黄晓兰,吴惠勤,等. QuEChERS/液相色谱-串联质谱法同时测定鱼肉中30种激素类及氯霉素类药物残留[J].分析测试学报, 2011,30(12):1329 LUO HT, HUANG XL, WU HQ, et al. Simultaneous determination of 30 hormones and chloramphenicols residues in fish using QuEChERS sample preparation and liquid chromatography-tandem mass spectrometry[J]. J Instrum Anal, 2011, 30(12):1329