自制混合型小柱净化-高效液相色谱-串联质谱法同时测定尿液中有机磷酸酯代谢物和8-羟基-2'-脱氧鸟苷

doi:10.3724/SP.J.1123.2019.10021

色谱 ›› 2020, Vol. 38 ›› Issue (6): 647-654.DOI: 10.3724/SP.J.1123.2019.10021

自制混合型小柱净化-高效液相色谱-串联质谱法同时测定尿液中有机磷酸酯代谢物和8-羟基-2'-脱氧鸟苷

李栋¹^,², 张芹², 张圣虎², 徐诚², 陈建秋¹, 刘艳华¹, 宋宁慧²^,^*(), 郭瑞昕¹^,^*()

¹ 中国药科大学工学院, 江苏南京 211198
² 生态环境部南京环境科学研究所, 江苏南京 210042

收稿日期:2019-10-22 出版日期:2020-06-08 发布日期:2020-12-10
通讯作者: 宋宁慧,郭瑞昕
作者简介:郭瑞昕.E-mail:snh@nies.org
宋宁慧.E-mail:ruixinguocpu@163.com;
基金资助:
国家自然科学基金(21876207);国家自然科学基金(21507165);环境医学工程教育部重点实验室开放课题项目(2017EME001)

Simultaneous determination of seven metabolites of organophosphate esters and 8-hydroxy-2'-deoxyguanosine in urine by high-performance liquid chromatography-tandem mass spectrometry with homemade mixed column purification

LI Dong¹^,², ZHANG Qin², ZHANG Shenghu², XU Cheng², CHEN Jianqiu¹, LIU Yanhua¹, SONG Ninghui²^,^*(), GUO Ruixin¹^,^*()

¹ School of Engineering, China Pharmaceutical University, Nanjing 211198, China
² Nanjing Institute of Environmental Sciences, Ministry of Ecology and Environment, Nanjing 210042, China

Received:2019-10-22 Online:2020-06-08 Published:2020-12-10
Contact: SONG Ninghui,GUO Ruixin
Supported by:
National Natural Science Foundation of China(21876207);National Natural Science Foundation of China(21507165);the Open Project of Key Laboratory of Environmental Medicine Engineering Ministry of Education(2017EME001)

摘要/Abstract

摘要：

建立了基于自制混合型小柱的样品净化-高效液相色谱-串联质谱同时测定7种有机磷酸酯（OPEs）主要代谢产物及生物标志物8-羟基-2'-脱氧鸟苷（8-OHdG）的分析方法。样品经乙腈提取后用自制小柱富集净化，以乙腈-0.2%（v/v）氨水溶液作为流动相进行梯度洗脱，在多反应监测模式下进行定性和定量分析。结果显示，8种目标物在0.1~200 μg/L范围内呈良好的线性关系，7种OPEs代谢物的回收率为52.36%~114.56%，8-OHdG回收率为88.63%~97.72%。将该方法应用于人体尿液实际样品中，7种OPEs代谢物和8-OHdG的检出范围分别为6.24~46.07 μg/L和5.90~16.71 μg/L，8-OHdG与7种OPEs代谢物总含量之间存在显著相关性。该方法操作简单、灵敏度高、准确性好、重现性强，可为更全面地评价人体内OPEs暴露水平及机体损伤提供可靠的技术支持。

关键词: 高效液相色谱-串联质谱法, 有机磷酸酯, 代谢物, 8-羟基-2'-脱氧鸟苷, 尿液

Abstract:

A method based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with a small homemade mixed column was developed for the simultaneous determination of seven metabolites of organophosphate esters (OPEs) and 8-hydroxy-2'-deoxyguanosine (8-OHdG), an DNA oxidative damage marker, in urine. The urine samples were extracted by acetonitrile and enriched by the self-made column. All the samples were separated by gradient elution with acetonitrile-0.2% (v/v) ammonia solution, and then analyzed by an electrospray ionization source in negative ion multiple reaction monitoring mode. The results showed that the eight targets had good linearities in the range of 0.1-200 μg/L. The recoveries of the seven metabolites of OPEs and 8-OHdG in urine samples were in the ranges of 52.36% to 114.56% and 88.63% to 97.72%, respectively. The established method was successfully applied for the determination of the abovementioned eight target compounds in real urine samples, with the mass concentrations of the seven metabolites of OPEs and 8-OHdG being 6.24-46.07 μg/L and 5.90-16.71 μg/L, respectively. In addition, significant correlations were found between 8-OHdG and the total content of the seven metabolites of OPEs. The established method is simple, sensitive, accurate, and reproducible, and it provides reliable technical support for a more comprehensive evaluation of the level of human exposure to OPEs and the resulting health hazards.

Key words: high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), organophosphate ester (OPEs), metabolites, 8-hydroxy-2'-deoxyguanosine (8-OHdG), urine

李栋, 张芹, 张圣虎, 徐诚, 陈建秋, 刘艳华, 宋宁慧, 郭瑞昕. 自制混合型小柱净化-高效液相色谱-串联质谱法同时测定尿液中有机磷酸酯代谢物和8-羟基-2'-脱氧鸟苷[J]. 色谱, 2020, 38(6): 647-654.

LI Dong, ZHANG Qin, ZHANG Shenghu, XU Cheng, CHEN Jianqiu, LIU Yanhua, SONG Ninghui, GUO Ruixin. Simultaneous determination of seven metabolites of organophosphate esters and 8-hydroxy-2'-deoxyguanosine in urine by high-performance liquid chromatography-tandem mass spectrometry with homemade mixed column purification[J]. Chinese Journal of Chromatography, 2020, 38(6): 647-654.

图/表 13

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Compound	Retention time/min	Parent ion (m/z)	Daughter ion (m/z)	Collision energy/V	Declustering potential/V	Entrance potential/V	Collision cell exit potential/V
* Quantitative ion.
DEP	1.13	153.1	78.9^*	-24	-42	-10	-7
			124.7	-14	-42	-10	-7
DnBP	5.14	210	78.8^*	-7	-57	-34	-7
			153.8	-7	-57	-20	-7
DPhP	5.66	248.9	92.6^*	-43	-83	-7	-7
			155.2	-29	-83	-7	-7
BCEP	5.46	222.8	62.8^*	-51	-86	-7	-7
			81.0	-34	-86	-7	-7
BCPP	4.71	250.9	36.7^*	-24	-25	-7	-7
			35.0	-24	-25	-7	-7
BBOEP	6.00	299.1	79.0^*	-58	-98	-7	-7
			198.9	-23	-98	-7	-7
BDCPP	5.86	318.2	36.8^*	-37	-30	-7	-7
			34.9	-37	-30	-7	-7
8-OHdG	1.31	282.6	192.8	-70	-27	-10	-10

Compound	Retention time/min	Parent ion (m/z)	Daughter ion (m/z)	Collision energy/V	Declustering potential/V	Entrance potential/V	Collision cell exit potential/V
* Quantitative ion.
DEP	1.13	153.1	78.9^*	-24	-42	-10	-7
			124.7	-14	-42	-10	-7
DnBP	5.14	210	78.8^*	-7	-57	-34	-7
			153.8	-7	-57	-20	-7
DPhP	5.66	248.9	92.6^*	-43	-83	-7	-7
			155.2	-29	-83	-7	-7
BCEP	5.46	222.8	62.8^*	-51	-86	-7	-7
			81.0	-34	-86	-7	-7
BCPP	4.71	250.9	36.7^*	-24	-25	-7	-7
			35.0	-24	-25	-7	-7
BBOEP	6.00	299.1	79.0^*	-58	-98	-7	-7
			198.9	-23	-98	-7	-7
BDCPP	5.86	318.2	36.8^*	-37	-30	-7	-7
			34.9	-37	-30	-7	-7
8-OHdG	1.31	282.6	192.8	-70	-27	-10	-10

Compound	Regression equation	R²	Intra-day RSD/% (n=3)	Inter-day RSD/% (n=3)	LOD/ (ng/L)	LOQ/ (ng/L)
y: peak area; x: mass concentration, μg/L.
DEP	y=1.75×10⁴x+6.89×10⁴	0.9978	7.6	6.9	4.16	12.5
DnBP	y=6.24×10³x-2.29×10⁴	0.9985	3.9	5.6	0.03	0.1
DPhP	y=4.25×10⁴x-9.52×10⁴	0.9991	4.3	7.0	5.00	15.0
BCEP	y=2.04×10⁵x-4.29×10⁵	0.9981	6.2	4.5	0.03	0.10
BCPP	y=9.56×10²x-2.88×10³	0.998	9.6	4.4	18.33	55.0
BBOEP	y=7.18×10³x-2.05×10⁴	0.9985	9.0	4.0	6.66	20.0
BDCPP	y=8.17×10³x-1.08×10⁴	0.9995	8.3	6.0	13.86	41.6
8-OHdG	y=2.60×10²x-1.60×10²	0.9997	8.1	8.8	13.03	40.0

Compound	Regression equation	R²	Intra-day RSD/% (n=3)	Inter-day RSD/% (n=3)	LOD/ (ng/L)	LOQ/ (ng/L)
y: peak area; x: mass concentration, μg/L.
DEP	y=1.75×10⁴x+6.89×10⁴	0.9978	7.6	6.9	4.16	12.5
DnBP	y=6.24×10³x-2.29×10⁴	0.9985	3.9	5.6	0.03	0.1
DPhP	y=4.25×10⁴x-9.52×10⁴	0.9991	4.3	7.0	5.00	15.0
BCEP	y=2.04×10⁵x-4.29×10⁵	0.9981	6.2	4.5	0.03	0.10
BCPP	y=9.56×10²x-2.88×10³	0.998	9.6	4.4	18.33	55.0
BBOEP	y=7.18×10³x-2.05×10⁴	0.9985	9.0	4.0	6.66	20.0
BDCPP	y=8.17×10³x-1.08×10⁴	0.9995	8.3	6.0	13.86	41.6
8-OHdG	y=2.60×10²x-1.60×10²	0.9997	8.1	8.8	13.03	40.0

Compound	5 μg/L		50 μg/L		100 μg/L
Compound	Recovery/ %	RSD/ %	Recovery/ %	RSD/ %	Recovery/ %	RSD/ %
DEP	62.05	7.9	58.74	1.5	52.36	6.5
DnBP	88.23	3.5	78.55	4.2	72.91	1.9
DPhP	114.56	2.3	100.03	7.5	96.13	4.8
BCEP	84.71	6.4	80.61	3.7	83.8	9.4
BCPP	98.94	9.0	89.51	3.6	83.77	7.2
BBOEP	108.25	5.5	86.89	8.6	102.96	2.1
BDCPP	93.37	6.9	83.43	3.2	79.67	8.3
8-OHdG	89.84	3.6	97.72	4.3	88.63	5.8

自制混合型小柱净化-高效液相色谱-串联质谱法同时测定尿液中有机磷酸酯代谢物和8-羟基-2'-脱氧鸟苷

Simultaneous determination of seven metabolites of organophosphate esters and 8-hydroxy-2'-deoxyguanosine in urine by high-performance liquid chromatography-tandem mass spectrometry with homemade mixed column purification

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Metrics

Sample No.	Mass concentrations/(μg/L)
Sample No.	DEP	DnBP	DPhP	BCEP	BCPP	BBOEP	BDCPP	Total	8-OHdG
Total: total mass concentration of the seven metabolites of OPEs; ND: not detected.
1	0.03	3.79	0.54	0.53	2.89	ND	0.30	8.08	6.01
2	ND	5.51	0.58	0.52	1.62	ND	0.44	8.66	6.30
3	1.64	3.84	0.58	0.52	2.88	ND	0.40	9.86	8.23
4	ND	2.74	0.47	0.49	2.58	ND	0.64	6.92	5.93
5	ND	4.29	1.11	0.53	4.59	ND	0.48	11.00	6.27
6	0.02	3.74	0.60	0.52	2.05	ND	0.36	7.30	6.14
7	ND	3.88	0.55	0.53	0.99	ND	0.35	6.30	5.97
8	1.96	3.63	0.65	0.51	7.05	0.56	0.59	14.96	5.97
9	ND	3.88	0.69	0.53	1.15	ND	0.28	6.52	6.10
10	ND	3.91	0.53	0.52	1.65	ND	0.39	7.00	16.71
11	ND	2.28	0.49	0.48	41.84	ND	0.98	46.07	43.04
12	ND	4.36	0.60	0.53	1.34	ND	0.31	7.14	5.90
13	0.50	3.12	0.54	0.51	0.96	ND	0.62	6.24	6.20
14	0.36	3.93	0.73	0.50	1.67	0.71	0.92	8.82	6.17
15	ND	4.28	0.79	0.53	1.94	ND	0.29	7.84	5.93

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Compound	DEP	DnBP	DPhP	BCEP	BCPP	BBOEP	BDCPP	Total	8-OHdG
* Significant at p＜0.05 level; ** significant at p＜0.01 level.
DEP	1	-0.094	-0.039	-0.118	-0.031	0.489	0.133	0.037	-0.135
DnBP		1	0.395	0.704^**	-0.578^*	-0.008	-0.556^*	-0.533^*	-0.548^*
DPhP			1	0.398	-0.187	0.164	-0.11	-0.148	-0.289
BCEP				1	-0.647^**	-0.311	-0.901^**	-0.642^**	-0.608^*
BCPP					1	-0.038	0.629^*	0.995^**	0.942^**
BBOEP						1	0.518^*	0.027	-0.137
BDCPP							1	0.646^**	0.564^*
Total								1	0.926^**
8-OHdG									1

Compound	DEP	DnBP	DPhP	BCEP	BCPP	BBOEP	BDCPP	Total	8-OHdG
* Significant at p＜0.05 level; ** significant at p＜0.01 level.
DEP	1	-0.094	-0.039	-0.118	-0.031	0.489	0.133	0.037	-0.135
DnBP		1	0.395	0.704^**	-0.578^*	-0.008	-0.556^*	-0.533^*	-0.548^*
DPhP			1	0.398	-0.187	0.164	-0.11	-0.148	-0.289
BCEP				1	-0.647^**	-0.311	-0.901^**	-0.642^**	-0.608^*
BCPP					1	-0.038	0.629^*	0.995^**	0.942^**
BBOEP						1	0.518^*	0.027	-0.137
BDCPP							1	0.646^**	0.564^*
Total								1	0.926^**
8-OHdG									1