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

doi:10.3724/SP.J.1123.2019.10021

Abstract

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

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.

Figures/Tables 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