高效液相色谱-串联质谱法测定人血浆中色氨酸及其代谢物浓度
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摘要
目的建立一种基于高效液相色谱-串联质谱(LC-MS/MS)技术同时快速测定人血浆中色氨酸及其代谢产物(犬尿氨酸、犬尿喹啉酸和吲哚丙酸)含量的方法。方法采用甲醇/乙腈(v/v,50/50)沉淀蛋白,以色氨酸-d_5为内标,色谱柱:Ultimate XB-C_(18) HPLC柱(2.1 mm×100 mm,3μm),流动相:水-甲醇(45/55,水相含5 mmol/L醋酸铵和0.2%甲酸),流速:0.3 ml/min,柱温:40℃,分析时间共3 min。质谱采用API4000 QTrap三重四极杆质谱仪的正离子多反应监测模式监测。考察该方法的专属性、线性及定量范围、精密度与准确度、回收率与基质效应以及稳定性。结果色氨酸、犬尿氨酸、犬尿喹啉酸及吲哚丙酸的线性范围分别在100～2×10~4、25～5 000、5～1 000、10～2 000 ng/ml;平均回收率均>90%;日内和日间精密度(RSD)和准确度(RE)均小于15%;稳定性良好。该方法成功测定了90例冠心病患者血浆中的色氨酸、犬尿氨酸、犬尿喹啉酸及吲哚丙酸的浓度。结论该方法简便、快捷、灵敏,可用于人体血浆中色氨酸及其代谢产物浓度的测定。
Objective To develop a rapid and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for quantitative determination of tryptophan and its metabolites(kynurenine,kynurenic acid and indole-3-propionic acid) in human plasma.Methods Plasma samples was prepared by protein precipitation with methanol and acetonitrile(v/v,50/50).The tryptophan-d_5 was used as internal standard for tryptophan and its metabolites.Separations were performed on an Ultimate XB-C_(18) HPLC column(2.1 mm×100 mm,3 μm).The mobile phase was composed of water(5 mmol/L ammonium acetate and 0.2% formic acid) and methanol(45/55).The flow rate was 0.3 ml/min.Column temperature was set at 40 ℃.Tryptophan and its metabolites were eluted within 3 min.Quantitative determination was performed by electrospray ionization,operating in positive ion multiple reaction monitoring(MRM) mode.The specificity,standard curve,precision and accuracy,recovery rate,and the matrix effect as well as the stability were investigated.Results The linear ranges of tryptophan,kynurenine,kynurenic acid and indole-3-propionic acid were 100～2×10~4,25～5 000,5～1 000,and 10～2 000 ng/ml,respectively.The average recovery was above 90%.RSD of the intra-and inter-day precision and accuracy of each analyte were below 15% and the stability was good.This method was successfully applied in quantitative determination of tryptophan and its metabolites in human plasma of 90 patients with coronary artery disease.Conclusion A simple,sensitive and accurate LC-MS/MS method is developed and validated of the simultaneous determination of tryptophan and its metabolites in human plasma samples.
Objective To develop a rapid and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for quantitative determination of tryptophan and its metabolites(kynurenine,kynurenic acid and indole-3-propionic acid) in human plasma.Methods Plasma samples was prepared by protein precipitation with methanol and acetonitrile(v/v,50/50).The tryptophan-d_5 was used as internal standard for tryptophan and its metabolites.Separations were performed on an Ultimate XB-C_(18) HPLC column(2.1 mm×100 mm,3 μm).The mobile phase was composed of water(5 mmol/L ammonium acetate and 0.2% formic acid) and methanol(45/55).The flow rate was 0.3 ml/min.Column temperature was set at 40 ℃.Tryptophan and its metabolites were eluted within 3 min.Quantitative determination was performed by electrospray ionization,operating in positive ion multiple reaction monitoring(MRM) mode.The specificity,standard curve,precision and accuracy,recovery rate,and the matrix effect as well as the stability were investigated.Results The linear ranges of tryptophan,kynurenine,kynurenic acid and indole-3-propionic acid were 100～2×10~4,25～5 000,5～1 000,and 10～2 000 ng/ml,respectively.The average recovery was above 90%.RSD of the intra-and inter-day precision and accuracy of each analyte were below 15% and the stability was good.This method was successfully applied in quantitative determination of tryptophan and its metabolites in human plasma of 90 patients with coronary artery disease.Conclusion A simple,sensitive and accurate LC-MS/MS method is developed and validated of the simultaneous determination of tryptophan and its metabolites in human plasma samples.
引文
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[18] Tong Q,Song J,Yang G,et al.Simultaneous determination of tryptophan,kynurenine,kynurenic acid,xanthurenic acid and 5-hydroxytryptamine in human plasma by LC-MS/MS and its application to acute myocardial infarction monitoring[J].Biomed Chromatogr,2018,32(4).
[2] Dodd D,Spitzer MH,Van Treuren W,et al.A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites[J].Nature,2017,551(7682):648-652.
[3] Song P,Ramprasath T,Wang H,et al.Abnormal kynurenine pathway of tryptophan catabolism in cardiovascular diseases[J].Cell Mol Life Sci,2017,74(16):2899-2916.
[4] Zuo H,Ueland PM,Ulvik A,et al.Plasma biomarkers of inflammation,the kynurenine pathway,and risks of all-cause,cancer,and cardiovascular disease mortality[J].AM J Epidemiol,2016,183(4):249-258.
[5] Yu E,Ruiz-Canela M,Guasch-Ferré M,et al.Increases in plasma tryptophan are inversely associated with incident cardiovascular disease in the prevención con dieta mediterránea (PREDIMED) study[J].J Nutr,2017,147(3):314-322.
[6] Sulo G,Vollset SE,■,et al.Neopterin and kynurenine-tryptophan ratio as predictors of coronary events in older adults,the Hordaland Health Study[J].Int J Cardiol,2013,168(2):1435-1440.
[7] Polyzos KA,Ketelhuth DF.The role of the kynurenine pathway of tryptophan metabolism in cardiovascular disease.An emerging field[J].Hamostaseologie,2015,35(2):128-136.
[8] de Mello VD,Paananen J,Lindstr?m J,et al.Indolepropionic acid and novel lipid metabolites are associated with a lower risk of type 2 diabetes in the Finnish Diabetes Prevention Study[J].Sci Rep,2017,7:46337.
[9] Cason CA,Dolan KT,Sharma G,et al.Plasma microbiome-modulated indole- and phenyl-derived metabolites associate with advanced atherosclerosis and postoperative outcomes[J].J Vasc Surg,2018,68(5):1552-1562.
[10] Wikoff WR,Anfora AT,Liu J,et al.Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites[J].Proc Natl Acad Sci U S A,2009,106(10):3698-3703.
[11] Zhu W,Stevens AP,Dettmer K,et al.Quantitative profiling of tryptophan metabolites in serum,urine,and cell culture supernatants by liquid chromatography-tandem mass spectrometry[J].Anal Bioanal Chem,2011,401(10):3249-3261.
[12] Hényková E,Vránová HP,Amakorová P,et al.Stable isotope dilution ultra-high performance liquid chromatography-tandem mass spectrometry quantitative profiling of tryptophan-related neuroactive substances in human serum and cerebrospinal fluid[J].J Chromatogr A,2016,1437:145-157.
[13] Ilona Sadok AGMM.Chromatographic analysis of tryptophan metabolites[J].J Sep Sci,2017,40(15):3020-3045.
[14] Fuertig R,Ceci A,Camus SM,et al.LC-MS/MS-based quantification of kynurenine metabolites,tryptophan,monoamines and neopterin in plasma,cerebrospinal fluid and brain[J].Bioanalysis,2016,8(18):1903-1917.
[15] Thakare R,Chhonker YS,Gautam N,et al.Quantitative analysis of endogenous compounds[J].J Pharmaceut Biomed,2016,128:426-437.
[16] Agency EM.Guideline on bioanalytical method validation[J].药物评价研究,2012,35(5):396-398.
[17] Midttun ?,Hustad S,Ueland PM.Quantitative profiling of biomarkers related to B-vitamin status,tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry[J].Rapid Commun Mass Spectrom,2009,23(9):1371-1379.
[18] Tong Q,Song J,Yang G,et al.Simultaneous determination of tryptophan,kynurenine,kynurenic acid,xanthurenic acid and 5-hydroxytryptamine in human plasma by LC-MS/MS and its application to acute myocardial infarction monitoring[J].Biomed Chromatogr,2018,32(4).