Determination of 16 organophosphate esters in human blood by high performance liquid chromatography-tandem mass spectrometry combined with liquid-liquid extraction and solid phase extraction

doi:10.3724/SP.J.1123.2020.07033

Abstract

Abstract:

Measurement of organophosphate esters (OPEs) in human body fluids is important for understanding human internal exposure to OPEs and for assessing related health risks. Most of the current studies have focused on the determination of OPE metabolites in human urine, as OPEs are readily metabolized into their diester or hydroxylated forms in the human body. However, given the existence of one metabolite across multiple OPEs or multiple metabolites of one OPE, as well as the low metabolic rates of several OPEs in in vitro studies, the reliability of urinary OPE metabolites as biomarkers for specific OPEs is needs to be treated with caution.Human blood is a matrix that is in contact with all body organs and tissues, and the blood levels of compounds may better represent the doses that reach target tissues. Currently, only a few studies have investigated the occurrence of OPEs in human blood by different analytical methods, and the variety of OPEs considered is limited. In this study, a method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of 16 OPEs in human blood, and the extraction efficiency of the solid phase extraction (SPE) column for OPEs was verified. To human blood samples, 10 ng of an internal standard was added, followed by mixing and aging for 30 min. The samples were extracted three times with acetonitrile using a shaker, and then purified on ENVI-18 cartridges with acetonitrile containing 25% dichloromethane as the eluent. Finally, the OPEs were analyzed by high performance liquid chromatography-tandem mass spectrometry. After optimization of the analytical column and mobile phases, the analytes were separated on a BEH C18 column (100 mm×2.1 mm, 1.7 μm) by gradient elution using methanol and 5 mmol/L ammonium acetate in water as the mobile phase. Then, the analytes were ionized in electrospray ionization positive (ESI⁺) mode and detected in the multiple reaction monitoring (MRM) mode. The mass spectral parameters, including the precursor ion, product ion, declustering potential, entrance potential, and collision cell exit potential, were optimized. The results were quantified by the internal standard method. The limits of detection (LOD, S/N=3) of the OPEs were in the range of 0.0038-0.882 ng/mL. The calibration curves for the 16 OPEs showed good linear relationships in the range of 0.1-50 ng/mL, and the correlation coefficients were >0.995. The extraction efficiency of the ENVI-18 column for the 16 OPEs was validated, and the average recoveries of the target compounds were 54.6%-104%. The average recoveries (n=3) of 15 OPEs, except trimethyl phosphate (TMP), in whole blood at three spiked levels were in the range of 53.1%-126%, and the relative standard deviations (RSDs) were in the range of 0.15%-12.6%. The average recoveries of six internal standards were in the range of 66.8%-91.6% except for TMP-d9 (39.1%), with RSDs of 3.52%-6.85%. The average matrix effects of the OPEs in whole blood were 56.4%-103.0%. Significant matrix effects were found for resorcinol bis(diphenyl phosphate) (RDP) (75.8%±1.4%), trimethylphenyl phosphate (TMPP) (68.4%±1.0%), 2-ethylhexyl di-phenyl phosphate (EHDPP) (56.4%±12.4%), and bisphenol-A bis(diphenyl phosphate) (BABP) (58.5%±0.4%). However, these effects could be corrected by similar signal suppressions of the corresponding internal standard (TPHP-d15, 77.4%±7.5%). This method is simple, highly sensitive, and suitable for the determination of OPEs in human blood. Fifteen human whole blood samples were collected to quantify the 16 OPEs using the developed method. The total concentrations of the OPEs ranged from 1.50 to 7.99 ng/mL. The detection frequencies of eight OPEs were higher than 50%. Tri-iso-butyl phosphate (TiBP), tri(2-chloroethyl) phosphate (TCEP), and tri(1-chloro-2-propyl) phosphate (TCIPP) were the dominant OPEs, with median concentrations of 0.813, 0.764, and 0.690 ng/mL, respectively. These results indicated widespread human exposure to OPEs, which should be of concern.

Key words: solid phase extraction (SPE), liquid-liquid extraction (LLE), high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), organophosphate ester, human blood

CLC Number:

O658

HOU Minmin, SHI Yali, CAI Yaqi. Determination of 16 organophosphate esters in human blood by high performance liquid chromatography-tandem mass spectrometry combined with liquid-liquid extraction and solid phase extraction[J]. Chinese Journal of Chromatography, 2021, 39(1): 69-76.

Figures/Tables 6

References

[1]	Wei G L, Li D Q, Zhuo M N, et al. Environ Pollut, 2015,196:29
[2]	Wang Y, Hou M, Zhang Q, et al. Environ Sci Technol, 2017,51(19):10991
[3]	Stapleton H M, Sharma S, Getzinger G, et al. Environ Sci Technol, 2012,46(24):13432
[4]	Zhang T, Bai X Y, Lu S Y, et al. Environ Int, 2018,121(Pt 2):1363
[5]	Suhring R, Diamond M L, Scheringer M, et al. Environ Sci Technol, 2016,50(14):7409
[6]	Wang Y, Wu X, Zhang Q, et al. Chemosphere, 2018,211:335
[7]	Xing L, Zhang Q, Sun X, et al. Sci Total Environ, 2018,636:632
[8]	He M J, Lu J F, Wei S Q. Environ Pollut, 2019,244:388
[9]	Wang Y, Yao Y, Li W, et al. Sci Total Environ, 2019,671:528
[10]	Liao C, Kim U J, Kannan K. Sci Total Environ, 2020,704:135328
[11]	Cristale J, Hurtado A, Gomez-Canela C, et al. Environ Res, 2016,149:66
[12]	Cristale J, Aragao Bele T G, Lacorte S, et al. Environ Pollut, 2018,237:695
[13]	Bekele T G, Zhao H, Wang Q. J Hazard Mater, 2020,401:123410
[14]	van der Veen I, de Boer J. Chemosphere, 2012,88(10):1119
[15]	Behl M, Hsieh J H, Shafer T J, et al. Neurotoxicol Teratol, 2015,52(Pt B):181
[16]	Meeker J D, Stapleton H M. Environ Health Perspect, 2010,118(3):318
[17]	Zhang Q, Ji C, Yin X, et al. Environ Pollut, 2016,210:27
[18]	Zhang Q, Lu M, Dong X, et al. Environ Sci Technol, 2014,48(12):6995
[19]	Araki A, Saito I, Kanazawa A, et al. Indoor Air, 2014,24(1):3
[20]	He C, Toms L L, Thai P, et al. Environ Int, 2018,111:124
[21]	Wang Y, Li W, Martinez-Moral M P, et al. Environ Int, 2019,122:213
[22]	Zhao F, Wan Y, Zhao H, et al. Environ Sci Technol, 2016,50(16):8896
[23]	Ya M, Yu N, Zhang Y, et al. Environ Int, 2019,131:105056
[24]	Hou M, Shi Y, Jin Q, et al. Environ Int, 2020,139:105698
[25]	Wang X, Liu Q, Zhong W, et al. Environ Int, 2020,134:105321
[26]	Kucharska A, Cequier E, Thomsen C, et al. Environ Int, 2015,83:50
[27]	Liu L Y, He K, Hites R A, et al. Environ Sci Technol, 2016,50(6):3065
[28]	He M J, Lu J F, Ma J Y, et al. Environ Pollut, 2018,237:143
[29]	Kim J W, Isobe T, Muto M, et al. Chemosphere, 2014,116:91
[30]	Ma J, Zhu H, Kannan K. Environ Sci Technol Lett, 2019,6(9):525
[31]	Van den Eede N, Maho W, Erratico C, et al. Toxicol Lett, 2013,223(1):9
[32]	Su G, Letcher R J, Crump D, et al. Environ Sci Tech Let, 2015,2(4):100
[33]	Van den Eede N, Erratico C, Exarchou V, et al. Toxicol Appl Pharmacol, 2015,284(2):246
[34]	Greaves A K, Su G, Letcher R J. Toxicol Appl Pharmacol, 2016,308:59
[35]	Li D, Zhang Q, Zhang S H, et al. Chinese Journal of Chromatography, 2020,38(6):647
[35]	李栋, 张芹, 张圣虎, 等. 色谱, 2020,38(6):647
[36]	Ballesteros-Gomez A, Erratico C A, Eede N V, et al. Toxicol Lett, 2015,232(1):203
[37]	Ballesteros-Gomez A, Van den Eede N, Covaci A. Environ Sci Technol, 2015,49(6):3897
[38]	Phillips A L, Hammel S C, Hoffman K, et al. Environ Int, 2018,116:176
[39]	He C, Wang X, Tang S, et al. Environ Sci Technol, 2018,52(21):12765
[40]	Tan H, Yang L, Yu Y, et al. Environ Sci Technol, 2019,53(9):4784
[41]	Henriquez-Hernandez L A, Carreton E, Camacho M, et al. Front Vet Sci, 2017,4:79
[42]	Li P, Jin J, Wang Y, et al. Chemosphere, 2017,171:654
[43]	Gao D, Yang J, Bekele T G, et al. Environ Sci Pollut Res Int, 2020,27(3):2721
[44]	Gao L H. [PhD Dissertation]. Beijing: University of Science and Technology Beijing, 2016
[44]	高立红. [博士学位论文]. 北京: 北京科技大学, 2016

Compound	Abbreviation	Formula	M_r	CAS No.
Trimethyl phosphate	TMP	C₃H₉O₄P	140.08	512-56-1
Triethyl phosphate	TEP	C₆H₁₅O₄P	182.16	78-40-0
Tripropyl phosphate	TPrP	C₉H₂₁O₄P	224.23	513-08-06
Tri-n-butyl phosphate	TnBP	C₁₂H₂₇O₄P	266.31	126-73-8
Tri-iso-butyl phosphate	TiBP	C₁₂H₂₇O₄P	266.31	126-71-6
Tris(2-ethylhexyl) phosphate	TEHP	C₂₄H₅₁O₄P	434.63	78-42-2
Tri(2-butoxyethyl) phosphate	TBOEP	C₁₈H₃₉O₇P	398.47	78-51-3
Tri(1-chloro-2-propyl) phosphate	TCIPP	C₉H₁₈Cl₃O₄P	327.57	13674-84-5
Tri(2-chloroethyl) phosphate	TCEP	C₆H₁₂Cl₃O₄P	285.49	115-96-8
Tri(1,3-dichloro-2-propyl) phosphate	TDCPP	C₉H₁₅Cl₆O₄P	430.90	13674-87-8
Tri-phenyl phosphate	TPHP	C₁₈H₁₅O₄P	326.28	115-86-6
Trimethylphenyl phosphate	TMPP	C₂₁H₂₁O₄P	368.36	563-04-2
Cresyl diphenyl phosphate	CDPP	C₁₉H₁₇O₄P	340.31	26444-49-5
2-Ethylhexyl di-phenyl phosphate	EHDPP	C₂₀H₂₇O₄P	362.41	1241-94-7
Resorcinol bis(diphenyl phosphate)	RDP	C₃₀H₂₄O₈P₂	574.45	57583-54-7
Bisphenol-A bis(diphenyl phosphate)	BABP	C₃₉H₃₄O₈P₂	692.63	5945-33-5

Compound	Abbreviation	Formula	M_r	CAS No.
Trimethyl phosphate	TMP	C₃H₉O₄P	140.08	512-56-1
Triethyl phosphate	TEP	C₆H₁₅O₄P	182.16	78-40-0
Tripropyl phosphate	TPrP	C₉H₂₁O₄P	224.23	513-08-06
Tri-n-butyl phosphate	TnBP	C₁₂H₂₇O₄P	266.31	126-73-8
Tri-iso-butyl phosphate	TiBP	C₁₂H₂₇O₄P	266.31	126-71-6
Tris(2-ethylhexyl) phosphate	TEHP	C₂₄H₅₁O₄P	434.63	78-42-2
Tri(2-butoxyethyl) phosphate	TBOEP	C₁₈H₃₉O₇P	398.47	78-51-3
Tri(1-chloro-2-propyl) phosphate	TCIPP	C₉H₁₈Cl₃O₄P	327.57	13674-84-5
Tri(2-chloroethyl) phosphate	TCEP	C₆H₁₂Cl₃O₄P	285.49	115-96-8
Tri(1,3-dichloro-2-propyl) phosphate	TDCPP	C₉H₁₅Cl₆O₄P	430.90	13674-87-8
Tri-phenyl phosphate	TPHP	C₁₈H₁₅O₄P	326.28	115-86-6
Trimethylphenyl phosphate	TMPP	C₂₁H₂₁O₄P	368.36	563-04-2
Cresyl diphenyl phosphate	CDPP	C₁₉H₁₇O₄P	340.31	26444-49-5
2-Ethylhexyl di-phenyl phosphate	EHDPP	C₂₀H₂₇O₄P	362.41	1241-94-7
Resorcinol bis(diphenyl phosphate)	RDP	C₃₀H₂₄O₈P₂	574.45	57583-54-7
Bisphenol-A bis(diphenyl phosphate)	BABP	C₃₉H₃₄O₈P₂	692.63	5945-33-5

Analyte	Precursor ion (m/z)	Product ion (m/z)	Declustering potential (DP)/V	Entrance potential (EP)/V	Collision cell exit potential (CXP)/V
TMP	141.1	109.1^*	60	22	10
		79.0	60	29	6
TEP	183.0	99.0^*	54	24	7
		81.0	60	50	8
TPrP	225.4	99.0^*	60	22	7
		141	60	24	10
TnBP	267.4	99.0^*	60	20	10
		155	60	12	10
TiBP	267.4	99.0^*	60	20	10
		155	60	12	10
TEHP	435.3	99.0^*	140	22	9
		113.1	120	16	8
TBOEP	399.3	299.3^*	95	19	10
		199.0	95	21	10
TCIPP	327.0	99.0^*	70	30	10
	329.1	99.0	70	28	10
TCEP	285.0	63.0^*	80	42	10
		99.2	75	30	10
TDCPP	431.1	98.9^*	85	35	9
		208.9	84	20	8
TPHP	327.1	152.0^*	130	42	11
		77.1	130	65	7
TMPP	369.2	166.1^*	147	37	11
		90.9	147	61	8
CDPP	341.1	152.1^*	135	40	10
		165.1	135	40	10
EHDPP	363.2	76.9^*	70	71	7
		251.0	72	12	9
RDP	575.2	419.2^*	190	46	15
		481.1	183	46	15
BABP	693.2	367.0^*	200	45	15
		693.3	200	12	15
TMP-d9	150.1	83.1	90	31	7
TEP-d15	198.1	101.9	65	27	8
TPrP-d21	246.4	102	120	25	9
TCEP-d12	299.1	102	75	30	6
TnBP-d27	294.4	101.9	140	25	10
TPHP-d15	342.3	160	135	47	10
TCIPP-d18	345.1	101.9	75	30	8

Analyte	Precursor ion (m/z)	Product ion (m/z)	Declustering potential (DP)/V	Entrance potential (EP)/V	Collision cell exit potential (CXP)/V
TMP	141.1	109.1^*	60	22	10
		79.0	60	29	6
TEP	183.0	99.0^*	54	24	7
		81.0	60	50	8
TPrP	225.4	99.0^*	60	22	7
		141	60	24	10
TnBP	267.4	99.0^*	60	20	10
		155	60	12	10
TiBP	267.4	99.0^*	60	20	10
		155	60	12	10
TEHP	435.3	99.0^*	140	22	9
		113.1	120	16	8
TBOEP	399.3	299.3^*	95	19	10
		199.0	95	21	10
TCIPP	327.0	99.0^*	70	30	10
	329.1	99.0	70	28	10
TCEP	285.0	63.0^*	80	42	10
		99.2	75	30	10
TDCPP	431.1	98.9^*	85	35	9
		208.9	84	20	8
TPHP	327.1	152.0^*	130	42	11
		77.1	130	65	7
TMPP	369.2	166.1^*	147	37	11
		90.9	147	61	8
CDPP	341.1	152.1^*	135	40	10
		165.1	135	40	10
EHDPP	363.2	76.9^*	70	71	7
		251.0	72	12	9
RDP	575.2	419.2^*	190	46	15
		481.1	183	46	15
BABP	693.2	367.0^*	200	45	15
		693.3	200	12	15
TMP-d9	150.1	83.1	90	31	7
TEP-d15	198.1	101.9	65	27	8
TPrP-d21	246.4	102	120	25	9
TCEP-d12	299.1	102	75	30	6
TnBP-d27	294.4	101.9	140	25	10
TPHP-d15	342.3	160	135	47	10
TCIPP-d18	345.1	101.9	75	30	8

Analyte	Recoveries/%				RSD/%
Analyte	1	2	3	Mean	RSD/%
TMP	52.7	59.3	51.6	54.6	4.2
TEP	63.0	68.8	67.3	66.3	3.0
TPrP	79.5	79.5	84.3	81.1	2.7
TnBP	76.2	77.2	80.5	78.0	2.3
TiBP	77.6	80.3	78.5	78.8	1.4
TEHP	73.3	78.8	74.9	75.7	2.8
TBOEP	81.4	89.3	94.9	88.5	6.8
TCEP	80.8	88.1	73.7	80.9	7.2
TCIPP	87.9	91.5	87.9	89.1	2.1
TDCPP	66.3	74.8	70.8	70.6	4.2
TPHP	82.6	83.0	81.4	82.3	0.8
EHDPP	69.7	90.5	81.8	80.7	10
TMPP	76.8	83.0	82.6	80.8	3.5
CDPP	101	106	104	104	2.6
RDP	85.1	92.0	92.3	89.8	4.1
BABP	79.1	82.3	89.2	83.5	5.1