高效液相色谱-串联质谱法检测婴幼儿血清中4种双酚类环境激素残留

doi:10.3724/SP.J.1123.2020.03024

摘要/Abstract

摘要：

目前,双酚类化合物是重要的工业原料,常用来制造塑料(奶)瓶、幼儿用吸口杯、食品和饮料(奶粉)罐内侧涂层,其具有类似雌激素的作用,摄入低剂量的双酚类物质便会引起机体尤其婴幼儿体内激素水平的调节。建立了一种同时测定婴幼儿血清中双酚A(BPA)、双酚B(BPB)、双酚F(BPF)、双酚S (BPS)4种双酚类环境激素的高效液相色谱-串联质谱法(HPLC-MS/MS)。以甲基叔丁基醚(MTBE)为提取溶剂,采用液液萃取的方法进行样品处理,同时对影响4种双酚类环境激素提取效率的提取溶剂、提取时间、提取溶剂体积等影响因素进行了优化。100 μL血清样本在40 ℃下经400 μL MTBE提取15 min后,采用Waters ACQUITY UPLC BEH C18柱(50 mm×2.1 mm, 1.7 μm)进行分离,以超纯水和含0.5 mmol/L乙酸铵的甲醇溶液为流动相进行梯度洗脱,流速为0.2 mL/min,采用电喷雾电离、负离子模式扫描,在多反应监测(MRM)模式下测定。BPA、BPB、BPS在0.25~100 μg/L范围内线性关系良好,BPF在1~00 μg/L范围内线性关系良好,相关系数为0.9929~0.9959;方法的检出限分别为0.05、0.05、0.05、0.5 μg/L;在3个添加水平(5、20、100 μg/L)下,4种目标化合物的回收率为84.56%~104.43%,相对标准偏差小于10%。应用该方法对150例婴幼儿血清样品进行检测,结果表明:BPA、BPF、BPS在男童和女童血清中的检出率分别为90.67%和89.33%、6.67%和1.33%、5.33%和16.00%, BPB未被检出。该方法操作简单,回收率好,精密度高,适用于婴幼儿血清中4种双酚类环境激素的同时测定。

关键词: 高效液相色谱-串联质谱, 液液萃取, 环境激素, 婴幼儿血清

Abstract:

Bisphenols are important industrial raw materials that are widely used to produce plastic bottles (feeding bottles), infant cups, and food and beverage (milk powder) cans. Because of the estrogen-like effect of bisphenols, even low-dose intake of these compounds by trace migration affects normal hormone levels in the human body. Therefore, it is imperative to develop a rapid, accurate, and highly sensitive method for the determination of bisphenols in serum. In this study, methyl tert-butyl ether (MTBE) was used as the extraction solvent, and the liquid-liquid extraction pretreatment method was used for sample processing. A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was established for the simultaneous determination of bisphenol A (BPA), bisphenol B (BPB), bisphenol F (BPF), and bisphenol S (BPS) at trace levels in infant serum. The important parameters affecting the extraction efficiency, such as the extraction solvent, extraction time, and extraction solvent volume for the four bisphenol environmental hormones were optimized. Serum samples were extracted by MTBE at 40 ℃ for 15 min. The target compounds were separated on a Waters ACQUITY UPLC BEH C18 column (50 mm×2.1 mm, 1.7 μm) using ultrapure water and methanol solution containing 0.5 mmol/L ammonium acetate as the mobile phases, with gradient elution at a flow rate of 0.2 mL/min. Finally, the analytes were analyzed by HPLC-MS/MS in the negative ion mode. BPA, BPB, and BPS showed good linearity in the range of 0.25-100 μg/L, while BPF showed good linearity in the range of 1-100 μg/L. The correlation coefficients were 0.9929-0.9959, and the limits of detection for BPA, BPB, BPS, and BPF were 0.05, 0.05, 0.05, 0.5 μg/L, respectively. At the three spiked levels (5, 20, 100 μg/L), the average recoveries of BPA, BPB, BPS, and BPF ranged from 84.56% to 104.43%, and the relative standard deviations were less than 10%. The proposed method was successfully applied to the determination of bisphenol contents in 150 infant serum samples. BPA was detected in most serum samples, and the detection rates in the serum of boys and girls were 90.67% and 89.33%, respectively. The detection rates of BPF in the serum of boys and girls were 6.67% and 1.33%, respectively; the corresponding values for BPS were 5.33% and 16.00%. BPB was not detected. The proposed method has the advantages of simple operation, good recovery, and high precision, and it is suitable for the simultaneous determination of the four bisphenol environmental hormones in infant serum.

Key words: high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), liquid-liquid extraction, environmental hormones, infant serum

中图分类号:

O658

刘芳, 孟桃于, 陈练, 吴雅君, 熊顺, 丁利. 高效液相色谱-串联质谱法检测婴幼儿血清中4种双酚类环境激素残留[J]. 色谱, 2020, 38(12): 1381-1387.

LIU Fang, MENG Taoyu, CHEN Lian, WU Yajun, XIONG Shun, DING Li. Determination of four bisphenol environmental hormone residues in infant serum by liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2020, 38(12): 1381-1387.

图/表 8

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Analyte	Parent ion (m/z)	Product ion (m/z)	Declustering potential/ V	Collision energy/ V
Bisphenol A (BPA)	227.0	212.0^*	-85	-26
		133.0	-85	-40
Bisphenol B (BPB)	241.0	210.9^*	-85	-35
		146.9	-85	-35
Bisphenol S (BPS)	248.9	108.0^*	-95	-45
		155.9	-95	-30
Bisphenol F (BPF)	198.9	93.1^*	-80	-35
		105.1	-80	-35

Analyte	Parent ion (m/z)	Product ion (m/z)	Declustering potential/ V	Collision energy/ V
Bisphenol A (BPA)	227.0	212.0^*	-85	-26
		133.0	-85	-40
Bisphenol B (BPB)	241.0	210.9^*	-85	-35
		146.9	-85	-35
Bisphenol S (BPS)	248.9	108.0^*	-95	-45
		155.9	-95	-30
Bisphenol F (BPF)	198.9	93.1^*	-80	-35
		105.1	-80	-35

Compound	Added/ (μg/L)	Intra-day assay		Inter-day assay
Compound	Added/ (μg/L)	Recovery/%	RSD/%	Recovery/%	RSD/%
BPA	5	99.74	5.91	91.95	8.49
	20	102.99	6.75	102.34	8.73
	100	101.75	4.45	99.03	5.34
BPB	5	86.32	5.24	93.06	7.41
	20	84.91	4.24	93.48	8.38
	100	88.13	6.58	92.43	5.63
BPF	5	101.83	6.71	104.43	7.80
	20	93.80	5.72	101.20	8.36
	100	93.58	7.53	100.01	6.44
BPS	5	84.56	4.18	88.38	6.14
	20	89.71	8.33	92.43	7.87
	100	91.82	5.99	91.47	5.10

Compound	Added/ (μg/L)	Intra-day assay		Inter-day assay
Compound	Added/ (μg/L)	Recovery/%	RSD/%	Recovery/%	RSD/%
BPA	5	99.74	5.91	91.95	8.49
	20	102.99	6.75	102.34	8.73
	100	101.75	4.45	99.03	5.34
BPB	5	86.32	5.24	93.06	7.41
	20	84.91	4.24	93.48	8.38
	100	88.13	6.58	92.43	5.63
BPF	5	101.83	6.71	104.43	7.80
	20	93.80	5.72	101.20	8.36
	100	93.58	7.53	100.01	6.44
BPS	5	84.56	4.18	88.38	6.14
	20	89.71	8.33	92.43	7.87
	100	91.82	5.99	91.47	5.10

Compound	Male						Female
Compound	Detection rate/%	Age/y		Number of positive samples	Content/ (μg/L)		Detection rate/%	Age/y		Number of positive samples	Content/ (μg/L)
BPA	90.67	0-	0.5	14	1.15	-30.50	89.33	0-	0.5	14	0.28-46.03
		0.5-	1	12	0.35	-53.70		0.5-	1	11	0.45-41.17
		1-	2	12	0.25	-32.70		1-	2	12	0.29-24.77
		3-	4	15	0.29	-33.88		3-	4	15	0.30-28.31
		≥	5	15	0.41	-28.57		≥	5	15	0.61-36.50
BPB	-	-		-		-	-	-		-	-
BPF	6.67	0-	0.5	-		-	1.33	0-	0.5	-	-
		0.5-	1	1	1.17			0.5-	1	-	-
		1-	2	-		-		1-	2	-	-
		3-	4	2	1.12	, 2.67		3-	4	-	-
		≥	5	2	1.03	, 1.16		≥	5	1	1.52
BPS	5.33	0-	0.5	1	0.32		16.00	0-	0.5	2	0.28, 0.73
		0.5-	1	-		-		0.5-	1	2	0.31, 0.45
		1-	2	-		-		1-	2	-	-
		3-	4	-		-		3-	4	-	-
		≥	5	3	0.40	-0.97		≥	5	8	0.25-1.71