超高效液相色谱-串联质谱法定量分析尿液中色氨酸及其代谢产物

doi:10.3724/SP.J.1123.2020.06022

摘要/Abstract

摘要：

基于超高效液相色谱-串联质谱(UPLC-MS/MS)建立定量分析色氨酸(Trp)及代谢产物3-OH-犬尿氨酸(3-OH-Kyn)、3-OH-邻氨基苯甲酸(3-OH-AA)、黄尿酸(XA)、犬尿氨酸(Kyn)、5-羟基吲哚乙酸(5-HIAA)、犬尿喹啉酸(KA)和5-羟色胺(5-HT)的方法,应用该方法分析其在尿样中的含量,探讨排泄规律。将尿样稀释、离心后,加入丹磺酰氯(DNS-Cl)衍生,经Thermo C₁₈色谱柱(50 mm×3 mm, 2.7 μm)分离和0.1%甲酸和甲醇梯度洗脱后,采用电喷雾电离(ESI)源,在正离子扫描和多反应监测(MRM)模式下检测。以咖啡酸(CA)为内标,定量分析。结果显示,8种目标化合物的线性关系良好,相关系数(R ²)≥0.9740,检测灵敏(LOD为0.005~0.5 ng/mL),回收率高(93.24%~107.65%)。采用本方法检测分析了健康志愿者70个尿液样本,在尿样中检测到Trp原型及其7种代谢产物。结果表明,体内的Trp是通过原型和代谢两种方式排泄:Trp原型的含量为5.22~20.88 μg/mL;尿液中经代谢后排泄的Trp量是原型的124%~268%,即体内的Trp主要经代谢后排出体外。方法主要研究了Trp-5-HT和Trp-Kyn两条途径的代谢产物含量,Trp经Kyn降解生成的3-OH-AA和3-OH-Kyn含量较多,即Trp-Kyn是体内Trp的主要代谢途径。方法通过UPLC-MS/MS实现了尿液中Trp及其代谢产物含量的检测,能为临床检查提供技术和理论支持。

关键词: 超高效液相色谱-串联质谱, 柱前衍生, 色氨酸-犬尿氨酸代谢途径, 尿液

Abstract:

Tryptophan (Trp), also known as α-amino β-indolepropionic acid, is an essential amino acid, which is involved in various physiological processes. Studies have shown that tumors, infectious diseases, and neurological diseases are accompanied by Trp-related metabolic disorders. Understanding the excretion of Trp and its metabolites in normal individuals is of great significance for treating Trp-related diseases and monitoring the health. A rapid quantitative method was developed based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Further, this method was applied to the simultaneous determination of Trp and its metabolites, including kynurenine (Kyn), kynurenic acid (KA), 3-hydroxykynurenine (3-OH-Kyn), 3-hydroxyanthranilic acid (3-OH-AA), xanthurenic acid (XA), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA). The excretion and amount of target compounds in random urine samples collected from healthy participants were studied using this method. Urine samples were collected from healthy male volunteers (between 20-22 years old) without any diet and exercise restrictions. Urine samples were collected between 11∶00-13∶00 daily for 10 d. Thereafter, the urine samples were diluted, centrifuged, and subjected to pre-column derivatization with dansyl chloride (DNS-Cl). Caffeic acid (CA) was used as the internal control. Later, the derivatives were detected using triple quadrupole mass spectrometry with electron pray ionization (ESI) in positive and multi reaction monitoring (MRM) modes. The samples were separated using a Thermo C₁₈ column (50 mm×3 mm, 2.7 μm) with 0.1% aqueous formic acid aqueous solution and methanol as mobile phases at a flow rate of 0.2 mL/min. The three most abundant ions for each derivative were selected for downstream analysis, and the internal control was used for quantification. The polarity and molecular weight of the compounds were found to be altered effectively after DNS-Cl derivatization treatment. The dansyl group effectively altered the polarities of the derivatives, such that their retention behaviors in the reverse elution system were similar and they were well separated. The interference due to impurities was effectively eliminated using the MRM mode. The results showed significant linear correlation, since the correlation coefficients were greater than 0.9740. The recoveries were between 93.24%-107.65%, and the LODs were 0.005-0.5 ng/mL for the eight compounds. Trp prototype and the seven target metabolites, including 3-OH-Kyn, 3-OH-AA, XA, Kyn, KA, 5-HIAA, and 5-HT generated through Trp-5-HT and Trp-Kyn pathways were detected in the urine samples. These results indicated that Trp was excreted in a prototypic form or after being metabolized. The level of the target compounds in random urine samples of individuals were 0.99-3.72 (3-OH-Kyn), 2.51-21.11 (3-OH-AA), 0.25-1.12 (XA), 0.15-1.53 (Kyn), 0.24-2.58 (KA), 0-0.31 (5-HT), and 2.2-17.94 (5-HIAA) μg/mL. For the same individual, in the state of physical health, the fluctuations of Trp and its metabolites in urine were large. Due to these large fluctuations in the absolute content, the difference between individuals was not significant. The data generated using 70 urine samples revealed that the amount of excreted Trp being metabolized was 124%-268% of prototype, which further indicated that the excretion after metabolism was the major underlying mechanism. Upon comparing the levels of metabolites in the Trp-5-HT and Trp-Kyn pathways, the results indicated that the levels of 3-OH-AA and 3-OH-Kyn generated upon Trp degradation through the Kyn pathway was higher than those of the other products. Trp was degraded via Kyn pathway to produce 3-OH-AA, which was the main metabolite of Trp found to be present in the body. This manuscript detected the levels of Trp and its metabolites, as well as summarized the characteristics of excretion using random urine samples, which could provide valuable information for clinical practice.

Key words: ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), pre-column derivation, tryptophan-kynurenine pathway, urine

中图分类号:

O658

李慧, 崔兰冲, 章国磊, 张萌萌, 焦丽丽, 吴巍. 超高效液相色谱-串联质谱法定量分析尿液中色氨酸及其代谢产物[J]. 色谱, 2021, 39(5): 518-525.

LI Hui, CUI Lanchong, ZHANG Guolei, ZHANG Mengmeng, JIAO Lili, WU Wei. Quantitative analysis of tryptophan and its metabolites in urine by ultra performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2021, 39(5): 518-525.

图/表 6

参考文献

[1]	Zhang J W, Zhang X. J Alloy Compd, 2020,842(25):155934
[2]	Friedman M. Int J Tryptophan Res, 2018,11:1
[3]	Qin X P, An Y T. Tianjin Journal of Traditional Chinese Medicine, 2019,36(10):1031
[3]	秦袖平, 安雅婷. 天津中医药, 2019,36(10):1031
[4]	Li J X, Zhang X M, Xu Q S. Amino Acids and Biological Resources, 2005,27(3):58
[4]	李剑欣, 张绪梅, 徐琪寿. 氨基酸和生物资源, 2005,27(3):58
[5]	Platten M Nollen E A A Rohrig U F, et al. Nat Rev Drug Discov, 2019,18(5):379
[6]	Li S S, Yang X Z, Chang Y, et al. Chinese Journal of Pharmacology and Toxicology, 2019,33(9):710
[6]	李素素, 杨雪枝, 常艳, 等. 中国药理学与毒理学杂志, 2019,33(9):710
[7]	Zhang J, Tang A G. Practical Preventive Medicine, 2012,19(4):633
[7]	张将, 唐爱国. 实用预防医学, 2012,19(4):633
[8]	Karu N McKercher C Nichols D S, et al. BMC Nephrol, 2016,17(1):171
[9]	Yang Y L, Cui P P, Tan T, et al. Journal of Analytical Science, 2016,32(5):634
[9]	杨永丽, 崔盼盼, 谭婷, 等. 分析科学学报, 2016,32(5):634
[10]	Sidorova A A, Kartsova L A. J Anal Chem, 2011,66(3):322
[11]	Martin G E, Sherman A D. Neurochem Res, 1980,5(3):223
[12]	Gregory O, Marieke H, Julien R, et al. Diabetes Metab J, 2017,1(4):1
[13]	Cheng Y, Li Y, Benkowitz P, et al. Sci Rep, 2020,10(1):12675
[14]	Heilman P L, Wang E W, Lewis M M, et al. Mov Disord, 2020. DOI: 10.1002/mds.28202
[15]	Wu Z M, Zhang L, Li N. Chongqing Medicine, 2014,43(25):3369
[15]	吴智明, 张林, 李楠. 重庆医学, 2014,43(25):3369
[16]	Yu M Q, Wei B, Li Q G, et al. Journal of Nanjing Sports Institute, 2019,2(4):41
[16]	俞孟辰, 魏冰, 李奇庚, 等. 南京体育学院学报, 2019,2(4):41
[17]	Han Q, Robinson H, Li J Y. Biochem J, 2012,446(2):253
[18]	Martin G E, Sherman A D. Neurochem Res, 1980,5(3):223
[19]	Tabara Y, Kohara K, Okada Y, et al. Clin Nutr, 2020,39(6):1857
[20]	de Jong W H A, Wilkens M H L I, de Vries E G E, et al. Anal Bioanal Chem, 2010,396(7):2609
[21]	Jong W H A D, Smit R, Bakker S J L, et al. J Chromatogr B, 2009,877(7):603

Compound	t_R/min	M_r	Number of dansyl group	Precursor ion (m/z)	Product ion (m/z)	Collision energy/eV	RF lens
3-OH-Kyn	3.86	224.08	2	691.14	171.14^*	37.54	98.31
					456.15	27.48
					349.37	14.98
3-OH-AA	4.25	153.04	1	387.09	171.15^*	17.59	85.73
					216.19	15.36
					341.50	15.21
XA	4.78	205.04	1	439.06	170.25^*	19.81	154.31
					268.16	9.89
					393.14	12.37
Kyn	5.30	208.08	1	442.12	170.15^*	22.21	80.25
					251.14	11.32
					271.04	9.22
Trp	5.38	203.80	1	438.18	170.89^*	23.33	122.21
					200.02	17.63
					283.56	16.51
5-HIAA	6.22	191.06	1	425.11	171.15^*	22.39	240.83
					205.89	19.33
					380.35	12.54
KA	6.51	189.04	1	423.51	170.44^*	18.72	120.39
					252.10	11.65
					378.22	12.25
5-HT	7.75	179.06	2	643.34	171.13^*	37.95	88.75
					380.10	15.66
					393.02	28.77
CA (IS)	7.78	182.06	2	647.09	171.11^*	27.39	145.28
					234.57	20.72
					413.07	18.70

Compound	t_R/min	M_r	Number of dansyl group	Precursor ion (m/z)	Product ion (m/z)	Collision energy/eV	RF lens
3-OH-Kyn	3.86	224.08	2	691.14	171.14^*	37.54	98.31
					456.15	27.48
					349.37	14.98
3-OH-AA	4.25	153.04	1	387.09	171.15^*	17.59	85.73
					216.19	15.36
					341.50	15.21
XA	4.78	205.04	1	439.06	170.25^*	19.81	154.31
					268.16	9.89
					393.14	12.37
Kyn	5.30	208.08	1	442.12	170.15^*	22.21	80.25
					251.14	11.32
					271.04	9.22
Trp	5.38	203.80	1	438.18	170.89^*	23.33	122.21
					200.02	17.63
					283.56	16.51
5-HIAA	6.22	191.06	1	425.11	171.15^*	22.39	240.83
					205.89	19.33
					380.35	12.54
KA	6.51	189.04	1	423.51	170.44^*	18.72	120.39
					252.10	11.65
					378.22	12.25
5-HT	7.75	179.06	2	643.34	171.13^*	37.95	88.75
					380.10	15.66
					393.02	28.77
CA (IS)	7.78	182.06	2	647.09	171.11^*	27.39	145.28
					234.57	20.72
					413.07	18.70

Compound	Linear equation	R²	Linear range/ (μg/mL)		LOD/ (ng/mL)	Spiked content/ (μg/mL)	Recovery/%	RSDs (n=6)/%
Compound	Linear equation	R²	Linear range/ (μg/mL)		LOD/ (ng/mL)	Spiked content/ (μg/mL)	Recovery/%	Intra-day	Inter-day
3-OH-Kyn	Y=0.0082X-0.046	0.9764	0.1-	5.0	0.01	0.1	105.41	1.31	0.61
						2.5	106.22	2.54	1.23
						5	98.47	3.11	3.26
3-OH-AA	Y=0.0009X-0.0310	0.9873	0.5-	25	0.1	0.5	93.24	0.78	0.78
						10	106.24	3.11	3.11
						20	102.61	2.21	2.21
XA	Y=0.0645X -0.0683	0.9740	0.1-	5.0	0.05	0.1	102.92	0.51	0.51
						2.5	104.23	2.14	2.14
						5	96.97	1.16	1.16
Kyn	Y=2.9064X-0.1972	0.9941	0.5-	25	0.1	1	95.96	1.23	1.23
						12	105.44	2.11	2.11
						25	103.12	1.11	1.11
Trp	Y=0.5550X+0.1972	0.9954	1-	50	0.5	2	107.65	2.14	3.59
						20	105.94	3.12	3.12
						40	102.21	1.45	1.45
5-HIAA	Y=4.5184X+0.8491	0.9797	0.2-	10	0.01	0.2	96.77	0.85	0.85
						4	104.33	2.33	2.33
						8	107.25	2.54	2.54
KA	Y=0.0677X-0.0153	0.9908	0.1-	5.0	0.05	0.1	96.77	0.75	0.75
						2	98.99	2.69	2.69
						4	102.21	1.78	1.78
5-HT	Y=0.0105X+0.0457	0.9998	0.01-	0.5	0.005	0.02	97.47	0.99	0.99
						0.25	104.43	1.23	1.23
						0.5	103.32	1.25	1.25

Compound	Linear equation	R²	Linear range/ (μg/mL)		LOD/ (ng/mL)	Spiked content/ (μg/mL)	Recovery/%	RSDs (n=6)/%
Compound	Linear equation	R²	Linear range/ (μg/mL)		LOD/ (ng/mL)	Spiked content/ (μg/mL)	Recovery/%	Intra-day	Inter-day
3-OH-Kyn	Y=0.0082X-0.046	0.9764	0.1-	5.0	0.01	0.1	105.41	1.31	0.61
						2.5	106.22	2.54	1.23
						5	98.47	3.11	3.26
3-OH-AA	Y=0.0009X-0.0310	0.9873	0.5-	25	0.1	0.5	93.24	0.78	0.78
						10	106.24	3.11	3.11
						20	102.61	2.21	2.21
XA	Y=0.0645X -0.0683	0.9740	0.1-	5.0	0.05	0.1	102.92	0.51	0.51
						2.5	104.23	2.14	2.14
						5	96.97	1.16	1.16
Kyn	Y=2.9064X-0.1972	0.9941	0.5-	25	0.1	1	95.96	1.23	1.23
						12	105.44	2.11	2.11
						25	103.12	1.11	1.11
Trp	Y=0.5550X+0.1972	0.9954	1-	50	0.5	2	107.65	2.14	3.59
						20	105.94	3.12	3.12
						40	102.21	1.45	1.45
5-HIAA	Y=4.5184X+0.8491	0.9797	0.2-	10	0.01	0.2	96.77	0.85	0.85
						4	104.33	2.33	2.33
						8	107.25	2.54	2.54
KA	Y=0.0677X-0.0153	0.9908	0.1-	5.0	0.05	0.1	96.77	0.75	0.75
						2	98.99	2.69	2.69
						4	102.21	1.78	1.78
5-HT	Y=0.0105X+0.0457	0.9998	0.01-	0.5	0.005	0.02	97.47	0.99	0.99
						0.25	104.43	1.23	1.23
						0.5	103.32	1.25	1.25