Determination of polycyclic aromatic hydrocarbon metabolites in urine by liquid-liquid extraction-high resolution gas chromatography-high resolution dual-focus magnetic mass spectrometry

doi:10.3724/SP.J.1123.2019.10031

Abstract

Abstract:

A sensitive and accurate method was developed to quantify eight polycyclic aromatic hydrocarbon (PAH) metabolites in human urine by liquid-liquid extraction-high resolution gas chromatography-high resolution dual-focus magnetic mass spectrometry (LLC-HRGC-HRMS). About 2 mL urine was mixed with a deuterium- or ¹³C-labeled isotopic internal standard, and the conjugated targets were enzymatically digested in the presence of ascorbic acid. The free compounds were extracted with toluene-pentane (1:4, v/v) and condensed to near dryness. Then, the target compounds were redissolved in toluene. After derivatization, they were separated and quantified by HRGC-HRMS. The linear ranges of the 1-hydroxynaphthalene, 1-hydroxyphenanthrene, and 2-hydroxyphenanthrene were 0.14-41.6 μg/L, 0.05-8.33 μg/L, and 0.04-8.33 μg/L, respectively, and those for the other five PAH metabolites were 0.02-8.33 μg/L. The correlation coefficients were greater than 0.99. The limits of detection were in the range of 0.006-0.042 μg/L, and the recoveries were 81.4%-127.0%. The intra-day and inter-day relative standard deviations (RSDs) were less than 6.9% and 10.9%, respectively. This method was utilized for the determination of 330 human urine samples. The results showed that 3-hydroxychrysene and 6-hydroxychrysene were not detected, and the detection rate of the other six PAH metabolites was 100%. This method is sensitive, accurate and stable, and it is suitable for the determination of the eight PAH metabolites in human urine.

Key words: high resolution gas chromatography-high resolution dual-focus magnetic mass spectrometry (HRGC-HRMS), liquid-liquid extraction (LLE), biological monitoring, hydroxyl polycyclic aromatic metabolites, urine

FU Hui, LU Yifu, HU Xiaojian, ZHANG Xu, YANG Yanwei, ZHU Ying. Determination of polycyclic aromatic hydrocarbon metabolites in urine by liquid-liquid extraction-high resolution gas chromatography-high resolution dual-focus magnetic mass spectrometry[J]. Chinese Journal of Chromatography, 2020, 38(6): 715-721.

Figures/Tables 6

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Compound	Abbreviation	Retention time/min	Monitoring ion [M⁺] (m/z)	IS
1-Hydroxynaphthalene	1-OHN	9.40	216.0970	1-OHN-d₇
2-Hydroxyfluorene	2-OHF	16.34	254.1127	2-OHF-d₉
3-Hydroxyfluorene	3-OHF	15.94	254.1127	3-OHF-d₉
1-Hydroxyphenanthrene	1-OHP	20.30	266.1127	1-OHP-d₉
2-Hydroxyphenanthrene	2-OHP	21.32	266.1127	2-OHP-d₉
1-Hydroxypyrene	1-OHPyr	25.73	290.1127	1-OHPyr-d₉
3-Hydroxychrysene	3-OHC	27.52	316.1283	3-OHC-d₁₁
6-Hydroxychrysene	6-OHC	27.03	316.1283	6-OHC-¹³C₆
1-Hydroxynaphthalene-d₇	1-OHN-d₇	9.35	223.1410
2-Hydroxyfluorene-d₉	2-OHF-d₉	16.24	263.1692
3-Hydroxyfluorene-d₉	3-OHF-d₉	15.86	263.1692
1-Hydroxyphenanthrene-d₉	1-OHP-d₉	20.17	275.1692
2-Hydroxyphenanthrene-d₉	2-OHP-d₉	21.19	275.1692
1-Hydroxypyrene-d₉	1-OHPyr-d₉	25.70	299.1692
3-Hydroxychrysene-d₁₁	3-OHC-d₁₁	27.48	327.1975
6-Hydroxychrysene-¹³C₆	6-OHC-¹³C₆	27.03	322.1485

Compound	Abbreviation	Retention time/min	Monitoring ion [M⁺] (m/z)	IS
1-Hydroxynaphthalene	1-OHN	9.40	216.0970	1-OHN-d₇
2-Hydroxyfluorene	2-OHF	16.34	254.1127	2-OHF-d₉
3-Hydroxyfluorene	3-OHF	15.94	254.1127	3-OHF-d₉
1-Hydroxyphenanthrene	1-OHP	20.30	266.1127	1-OHP-d₉
2-Hydroxyphenanthrene	2-OHP	21.32	266.1127	2-OHP-d₉
1-Hydroxypyrene	1-OHPyr	25.73	290.1127	1-OHPyr-d₉
3-Hydroxychrysene	3-OHC	27.52	316.1283	3-OHC-d₁₁
6-Hydroxychrysene	6-OHC	27.03	316.1283	6-OHC-¹³C₆
1-Hydroxynaphthalene-d₇	1-OHN-d₇	9.35	223.1410
2-Hydroxyfluorene-d₉	2-OHF-d₉	16.24	263.1692
3-Hydroxyfluorene-d₉	3-OHF-d₉	15.86	263.1692
1-Hydroxyphenanthrene-d₉	1-OHP-d₉	20.17	275.1692
2-Hydroxyphenanthrene-d₉	2-OHP-d₉	21.19	275.1692
1-Hydroxypyrene-d₉	1-OHPyr-d₉	25.70	299.1692
3-Hydroxychrysene-d₁₁	3-OHC-d₁₁	27.48	327.1975
6-Hydroxychrysene-¹³C₆	6-OHC-¹³C₆	27.03	322.1485

Compound	Linear range/(μg/L)	Regression equation	r	LOD/(μg/L)	LOQ/(μg/L)
y: peak area ratio of quantitative ion of the analyte to IS; x: mass concentration ratio of the analyte to IS.
1-OHN	0.14-41.6	y=0.9321x+0.3733	0.994	0.042	0.139
2-OHF	0.02-8.33	y=0.9665x+0.1833	0.992	0.006	0.020
3-OHF	0.02-8.33	y=0.9796x-0.0449	0.997	0.006	0.020
1-OHP	0.05-8.33	y=0.9605x+0.0383	0.993	0.015	0.050
2-OHP	0.04-8.33	y=0.9154x-0.0010	0.992	0.012	0.040
1-OHPyr	0.02-8.33	y=0.9762x+0.0291	0.997	0.006	0.020
3-OHC	0.02-8.33	y=0.9580x-0.0698	0.997	0.006	0.020
6-OHC	0.02-8.33	y=0.9955x+0.0732	0.998	0.006	0.020

Compound	Linear range/(μg/L)	Regression equation	r	LOD/(μg/L)	LOQ/(μg/L)
y: peak area ratio of quantitative ion of the analyte to IS; x: mass concentration ratio of the analyte to IS.
1-OHN	0.14-41.6	y=0.9321x+0.3733	0.994	0.042	0.139
2-OHF	0.02-8.33	y=0.9665x+0.1833	0.992	0.006	0.020
3-OHF	0.02-8.33	y=0.9796x-0.0449	0.997	0.006	0.020
1-OHP	0.05-8.33	y=0.9605x+0.0383	0.993	0.015	0.050
2-OHP	0.04-8.33	y=0.9154x-0.0010	0.992	0.012	0.040
1-OHPyr	0.02-8.33	y=0.9762x+0.0291	0.997	0.006	0.020
3-OHC	0.02-8.33	y=0.9580x-0.0698	0.997	0.006	0.020
6-OHC	0.02-8.33	y=0.9955x+0.0732	0.998	0.006	0.020

Compound	0.125 μg/L		1.25 μg/L		6.25 μg/L		Intra-day RSD/%	Inter-day RSD/%
Compound	Recovery/%	RSD/%	Recovery/%	RSD/%	Recovery/%	RSD/%	Intra-day RSD/%	Inter-day RSD/%
1-OHN	94.3	2.9	82.9	6.1	100.0	8.3	5.8	9.1
2-OHF	127.0	3.1	110.5	1.7	111.4	8.2	4.4	9.7
3-OHF	120.2	2.2	117.8	2.8	117.1	9.5	4.8	6.8
1-OHP	115.3	9.0	98.6	4.1	107.1	7.8	6.9	8.6
2-OHP	121.4	0.2	98.8	5.2	104.8	4.9	3.4	9.7
1-OHPyr	121.9	4.2	84.9	2.1	96.2	1.9	2.7	10.3
3-OHC	95.4	5.6	123.7	7.4	126.1	7.6	6.9	5.1
6-OHC	115.7	6.8	99.2	7.5	81.4	2.2	5.5	10.9