多功能离子液体分散液液微萃取结合高效液相色谱法检测人尿中5种邻苯二甲酸酯代谢物

doi:10.3724/SP.J.1123.2019.09026

摘要/Abstract

摘要：

将多功能离子液体与分散液液微萃取（DLLME）技术相结合，建立了测定尿液中5种邻苯二甲酸酯类（PAEs）物质代谢产物的高灵敏度新方法。对影响DLLME效率的各单因素进行了优化，包括萃取剂的种类及体积、分散剂的种类及体积、萃取温度、超声时间、冷却时间、离心时间和盐效应等条件，经过严格的优化，最佳的萃取条件分别为：萃取剂[C₈MIM]PF₆]35 μL，分散剂[BSO₃HMIm]OTf]30 μL和[C₄MIM]BF₆]120 μL，萃取温度为35℃，超声时间5 min，冷却时间5 min，离心时间5 min，盐析剂NH₄PF₆ 0.1 g。在最佳的萃取条件下，5种PAEs代谢物在0.5~1000 μg/L范围内具有良好的线性关系，决定系数（R²）均大于0.9955，方法检出限为0.16~0.19 μg/L，尿液中添加低中高水平（5、20、100 μg/L）的PAEs代谢物，其回收率为92.9%~105.0%，日内精密度及日间精密度的相对标准偏差（RSD）均小于5.96%，方法学验证各指标及稳定性均符合分析要求。对所采集的10份糖尿病患者的尿液进行检测，并对该人群PAEs代谢物的暴露水平进行评价。结果表明，各PAEs代谢物均有检出，其中邻苯二甲酸单（2-乙基己基）酯（MEHP）的检出率为100%。总之，该方法萃取过程中未添加有毒的有机试剂，均使用多功能离子液体作为萃取剂、分散剂和盐析剂，萃取过程绿色环保，简单高效；方法的灵敏度较高，稳定性较好，适用于人体尿液中痕量PAEs代谢物的检测。

关键词: 分散液液微萃取, 高效液相色谱, 邻苯二甲酸酯类代谢产物, 尿液, 多功能型离子液体

Abstract:

A novel sensitive method for the determination of the five primary metabolites of phthalate esters (PAEs) in urine was developed by combining dispersive liquid-liquid microextraction (DLLME) using ionic liquids with high performance liquid chromatography (HPLC). The factors affecting the efficiency of DLLME were optimized. The types and proportions of extraction solvent and dispersants, as well the ultrasonic extraction time, cooling time, and centrifugal time, were determined. The optimal conditions were as follows:extraction solvent[C₈MIM]PF₆] 35 μL; dispersants[BSO₃HMIm]OTf] 30 μL, [C₄MIM]BF₆] 120 μL; NH₄PF₆ 0.1 g, extraction at 35℃, ultrasonic dispersion for 5 min, cooling in ice water for 5 min, centrifugation at 4000 r/min for 5 min. After optimization, the five primary metabolites of PAEs were determined. The method showed a good linear relationship within the concentration range of 0.5-1000 μg/L. The determination coefficients (R²) were greater than 0.9955. The detection limit was in the range of 0.16-0.19 μg/L. Under the optimized conditions, the extraction recoveries for the PAEs were 92.9%-105.0%, and the relative standard deviations (RSDs) of the intra- and inter-day precisions were < 5.96%. Urine samples collected from 10 diabetic patients were tested, and the exposure level of the population to the PAE metabolites was evaluated. All the PAE metabolites were detected in these samples, and the detection rate of 2-ethylhexyl hydrogen phthalate (MEHP) was 100%. In conclusion, no toxic organic reagents were added during the extraction process in this method, and multifunctional ionic liquids were used as the extraction agent, dispersant, and salting-out agent. In other words, the extraction process was demonstrated to be green, simple, and efficient. The developed method has high sensitivity and stability, and it is suitable for the determination of trace PAE metabolites in human urine.

Key words: dispersive liquid-liquid microextraction (DLLME), high performance liquid chromatography (HPLC), phthalate metabolites, urine, multiple functional ionic liquids

孙倩, 戴浩强, 陈佩佩, 佘慧, 武佳. 多功能离子液体分散液液微萃取结合高效液相色谱法检测人尿中5种邻苯二甲酸酯代谢物[J]. 色谱, 2020, 38(8): 929-936.

SUN Qian, DAI Haoqiang, CHEN Peipei, SHE Hui, WU Jia. Combination of dispersive liquid-liquid microextraction using multifunctional ionic liquids with high performance chromatography for determination of phthalate ester metabolites in human urine sample[J]. Chinese Journal of Chromatography, 2020, 38(8): 929-936.

图/表 10

图1 萃取剂(a)种类和(b)体积对PAEs代谢物回收率的影响(n=6)

Fig. 1 Effect of (a) the type and (b) the volume of extraction solvent on recoveries of PAE metabolites (n=6) MEP: monoethyl phthalate; MBP: mono-n-butyl phthalate; MIBP: mono-iso-butyl phthalate; MBZP: monobenzyl phthalate; MEHP: mono(2-ethylhexyl) phthalate.Conditions: spiked urine sample volume 5.0 mL; dispersants [BSO3HMIm][OTf] 30 μL, [C4MIM][BF6] 120 μL; NH4PF6 0.1 g; extraction at 35 ℃; ultrasonic dispersion for 5 min; cooling in ice water for 5 min; and centrifugation at 4000 r/min for 5 min. For Fig. 1a, extraction solvent volume 35 μL.

图2 分散剂(a)种类和(b, c)体积对PAEs代谢物回收率的影响(n=6)

Fig. 2 Effect of (a) the type and (b, c) the volume of dispersive solvent on recoveries of PAE metabolites (n=6) Extraction solvent [C8MIM][PF6] 35 μL; other conditions are the same as in Fig. 1.

图3 萃取体系中NH4PF6的加入量对PAEs代谢物回收率的影响(n=6)

Fig. 3 Effects of amount of NH4PF6 on recoveries of PAE metabolites (n=6) Extraction solvent [C8MIM][PF6] 35 μL; other conditions are the same as in Fig. 1.

图4 萃取条件对PAEs代谢物回收率的影响(n=6)

Fig. 4 Effect of extraction conditions on the recoveries of PAE metabolites (n=6) a. extraction temperature; b. sonication time; c. cooling time; d. centrifugation time.Extraction solvent [C8MIM][PF6] 35 μL; other conditions are the same as in Fig. 1.

表1 尿液中5种PAEs代谢物的线性范围、回归方程、决定系数和检出限

Table 1 Linear ranges, regression equations, determination coefficients (R2) and LODs of the five PAE metabolites

Compound	Linear range/(μg/L)	Regression equation	R²	LOD/(μg/L)
y: peak area; x: mass concentration, μg/L; linear range: 0.5-1000 μg/L.
MEP	0.5-1000	y=58.166x-0.5410	0.9985	0.17
MIBP	0.5-1000	y=62.253x-0.8688	0.9963	0.19
MBP	0.5-1000	y=58.292x-0.6217	0.9979	0.16
MBZP	0.5-1000	y=61.934x-0.9530	0.9994	0.18
MEHP	0.5-1000	y=59.979x+1.8258	0.9955	0.19

表2 尿样中5种PAEs代谢物在3个加标水平下的回收率及RSDs(n=6)

Table 2 Recoveries and RSDs of the five PAE metabolites in urine samples at three spiked levels (n=6)

Analyte	Background/(μg/L)	5 μg/L		20 μg/L		100 μg/L		Intra-day RSD	Inter-day RSD
Analyte	Background/(μg/L)	Recovery/%	RSD/%	Recovery/%	RSD/%	Recovery/%	RSD/%	Intra-day RSD	Inter-day RSD
nd: not detected.
MEP	nd	99.6	5.02	104.5	0.57	98.1	1.59	2.98	3.56
MBP	nd	105.0	7.23	101.4	4.42	93.0	1.88	2.16	3.8
MIBP	10.47±0.27	102.4	3.38	99.6	1.80	98.6	1.39	3.68	3.32
MBZP	nd	97.4	5.34	92.9	7.32	98.5	2.05	3.56	1.23
MEHP	20.68±0.58	103.9	2.94	100.5	0.49	99.0	0.16	5.96	3.52

图5 (a) 加标尿液样品和(b)实际尿液样品的色谱图

Fig. 5 Chromatograms of a spiked urine sample and a real urine sample a. a blank urine sample spiked with 20 μg/L of five PAE metabolites; b. a real urine sample.

表3 新方法和已发表方法的比较

Table 3 Comparison of the proposed method with other published methods for determination of PAE metabolites

Extraction method	Instrument	Sample preparation	Linear range/(μg/L)	LOD/(μg/L)(S/N=3)	Reference
SPE: solid-phase extraction; IL-DLLME: ionic liquid-based dispersive liquid-liquid microextraction; IL-CIA-DLLME: ionic liquid cold-induced aggregation dispersive liquid-liquid microextraction.
SPE	GC-MS	enzymatic hydrolysis→SPE→azeotropic distillation→derivatisation	1.0-200	0.8-1.4	[25]
SPE	LC-MS	enzymatic hydrolysis→SPE →nitrogen-blow (drying out)	0.2-200	0.06	[5]
IL-DLLME	HPLC	ionic liquid-based DLLME→ice-water bath→centrifugation	50-600	3.3	[26]
IL-CIA-DLLME	HPLC	sample was heated to 50 ℃→ionic liquid cold-induced aggregation dispersive liquid-liquid microextraction→centrifugation	2.0-100	0.7-1.4	[27]
IL-DLLME	HPLC	enzymatic hydrolysis→DLLME→sonication→cooling in ice→ centrifugation	0.5-1000	0.16-0.19	this method

表4 糖尿病患者尿样中PAEs代谢物的含量

Table 4 Contents of PAE metabolites in diabetes patient urine samples μg/L

Sample No.	MEP	MIBP	MBP	MBZP	MEHP
nd: not detected.
1	nd	18.0	nd	nd	11.8
2	nd	nd	nd	22.1	23.7
3	nd	nd	nd	nd	26.3
4	nd	35.9	nd	nd	24.1
5	nd	38.7	nd	28.2	25.3
6	nd	33.2	49.2	nd	35.2
7	6.05	31.8	nd	nd	45.7
8	nd	nd	nd	nd	34.7
9	nd	51.6	79.6	nd	47.0
10	nd	35.3	nd	29.1	45.8

表5 糖尿病患者尿样中5种物质的检测水平及HMDB数据库中正常人群参考范围

Table 5 Detected PAE metabolite levels by proposed method and normal reference ranges in urine in HMDB database

Analyte	HMDB ID	This method(μmol/mmol creatinine)	Database(μmol/mmol creatinine)
MEP	HMDB0002120	0.00173-0.0107	0.0522-0.0623
MIBP	HMDB0002056	0.00448-0.077	0.00346-0.00415
MBP	HMDB0013247	0.0122-0.119	0.00829-0.0977
MBZP	HMDB0061744	0.00416-0.0328	0.00253-0.00310
MEHP	HMDB0013248	0.00236-0.056	0.000971-0.00123

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