HILIC-MS/MS法测定人尿中内源性糖类化合物浓度

目的：利用亲水色谱-质谱联用技术对人尿中15个内源性糖类化合物进行快速测定。方法：分别对色谱条件、质谱条件和样品前处理方法进行优化，最终以含内标的甲醇-乙腈（50∶50，v/v）为沉淀剂，按体积比5∶1加入到尿液中除杂，以含10 mmol·L^-1醋酸铵的水-乙腈（95∶5，v/v）和含10 mmol·L^-1醋酸铵的乙腈-水（95∶5，v/v）为流动相，梯度洗脱，样品经ACQUITY UPLC BEH Amide（2.1 mm×100 mm，1.7μm）色谱柱分离，在多反应监测（MRM）模式下，采用电喷雾离子化源、负离子扫描模式进行定量分析。结果：人尿中糖类物质线性关系良好，大部分糖类物质r>0.99，最低检测限在0.075~30 nmol·L^-1之间；日内、日间精密度范围分别为2.5%~7.6%和3.3%~7.3%，均小于15%；回收率范围为85.5%~115%；大部分化合物的基质效应在80%~120%之内，样品上清液4℃稳定性好，72 h内浓度变化范围为93.1%~106%，最终测得20例健康人尿液中各糖的含量分别为（平均值±标准误差）：楝二糖（2 668.1±134.4）nmol·L^-1，蔗糖（5 070.6±277.6）nmol·L^-1，麦芽糖（15 214.5±442.5）nmol·L^-1，麦芽三糖（849.7±42.6）nmol·L^-1，葡萄糖（173 965.0±4 918.9）nmol·L^-1，果糖（22 172.5±1 463.6）nmol·L^-1，棉子糖（292.1±17.6）nmol·L^-1，肌苷（2 083.5±93.4）nmol·L^-1，腺苷（2 645.5±78.2）nmol·L^-1，甘露糖醇（83 925.0±3 938.6）nmol·L^-1，赤藓糖醇（375 805.0±12 094.6）nmol·L^-1，阿拉伯糖醇（192 921.1±5 689.2）nmol·L^-1，麦芽四糖、木糖和核糖均低于定量限（分别低于60、600和3 000 nmol·L^-1）。结论：该方法能快速测定人尿液中15个内源性糖类化合物含量，对代谢组学后期的靶标验证、代谢机理研究等具有重要意义。

Objective: To quickly determine 15 endogenous carbohydrates in human urine by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.Methods: Chromatography conditions, mass spectrometry conditions and sample preparation method were all optimized.The methanol-acetonitrile(50:50, v/v)mixture was used to effectively remove the protein and impurities in urine, with the ratio of 5 times volume of organic solvents to 1 volume of urine sample.The chromatographic separation for endogenous carbohydrates in urine was accomplished by using an ACQUITY UPLC BEH Amide column(2.1 mm×100 mm,1.7 μm).Mobile phase A was composed of 10 mmol·L^-1 ammonium acetate in water-acetonitrile(95:5,v/v),and mobile phase B consisted of 10 mmol·L^-1 ammonium acetate in acetonitrile-water(95:5,v/v),and the gradient elution mode was applied to chromatographic separation.The compounds of interest were detected by MS/MS in negative ion multiple reaction monitoring(MRM)mode.Results: Method validation including linearity, precision, matrix effects, recovery and stability was performed.The calibration curves showed a good linearity(correlation coefficient of r> 0.99)with the LOD in the range of 0.075-30 nmol·L^-1 for most of compounds.The intra-and inter-day precision (RSD)were befow 15%,ranging from 2.5%-7.6% and 3.3%-7.3%, respectively.The recovery was in the range of 85.5%-115%.The matrix effect of most chemicals was in the range of 80%-120%.The stability data in the range of 93.1%-106% suggesting that post-preparative samples stored at 4℃ for 72 h was stable under routine laboratory analysis.Finally,concentrations of fifteen carbohydrates in human urine(mean±SE):melibiose(2 668.1±134.4)nmol·^-1; sucrose(5 070.6±277.6)nmol·L^-1; maltose(15 214.5±442.5)nmol·L^-1; maltotriose (849.7±42.6)nmol·L^-1; glucose(173 965.0±4 918.9)nmol·L^-1; fructose(22 172.5±1 463.6) nmol·L^-1; raffinose(292.1±17.6)nmol·L^-1; inosine(2 083.5±93.4)nmol·L^-1; adenosine(2 645.5±78.2)nmol·L^-1; mannitol(83 925.0±3 938.6)nmol·L^-1; erythritol(375 805.0±12 094.6)nmol·L^-1; arabitol(192 921.1±5 689.2)nmol·L^-1;maltotetraose, xylose and ribose were below the limit of quantitation (60,600 and 3000 nmol·L^-1,respectively).Conclusion: LC-MS/MS method for determination of 15 endogenous carbohydrates in human urine was developed and validated.The method was applied to the analysis of normal human urine, and could be used in the validation of biomarker and metabolic mechanism research in the future.

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