牛奶中全氟烷基酸的简便萃取与定量研究

李城华; 汪嫣; 蒋可志

doi:10.16495/j.1006-3757.2023.01.009

牛奶中全氟烷基酸的简便萃取与定量研究

doi: 10.16495/j.1006-3757.2023.01.009

杭州师范大学材料与化学化工学院有机硅化学与材料技术重点实验室，浙江杭州　311121

基金项目: 浙江省分析测试基金项目（LGC20B050011）

详细信息

作者简介:
李城华（1996−），女，硕士研究生，研究方向：色质谱分析，E-mail：lchcbz@163.com

通讯作者:
蒋可志（1980−），男，副教授，研究方向：色质谱分析，E-mail：jiangkezhi@hznu.edu.cn

中图分类号: O657. 63
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- 被引次数: 0
出版历程
- 收稿日期: 2023-01-31
- 录用日期: 2023-03-02
- 修回日期: 2023-03-02
- 刊出日期: 2023-03-31

Simple Extraction and Determination of Perfluoroalkyl Acids in Milk

College of Material, Chemistry and Chemical Engineering, Key Laboratory of Organosilicon Chemistry and Material Technology of Ministry of Education, Hangzhou Normal University, Hangzhou 311121, China

Funds: Analysis and Detection Foundation of Science and Technology Department in Zhejiang Province (LGC20B050011)

摘要

摘要: 采用阴离子交换树脂和硅胶作为混合填料对牛奶中的全氟烷基酸进行固相萃取，并结合液相色谱质谱联用技术，建立了一种简便的牛奶中21种全氟烷基酸同时定量方法. 在牛奶样中加入乙腈析出大量蛋白，萃取液在自制固相萃取小柱采用1.5 mL 80%乙腈水溶液进行洗脱纯化. 对纯化液进行液相色谱质谱联用分析，分析条件如下：以乙腈-5 mmol的醋酸铵水溶液为流动相，在XDB-C₁₈色谱柱上进行梯度洗脱，采用电喷雾负离子模式电离（ESI），质量扫描模式为多反应监测（MRM）模式检测. 方法中全氟烷基酸（PFAAs）的检出限为0.01~0.20 ng/mL，定量限为0.03~0.67 ng/mL，方法的线性关系（R²>0.999）和重现性均良好，回收率范围为66%~125%. 对13种常见市售牛奶中全氟烷基酸进行分析，主要检出了7种全氟烷基酸，其牛奶质量浓度在2.43~12.00 ng/mL之间.
- 全氟羧酸 /
- 全氟磺酸 /
- 牛奶 /
- 液相色谱-串联质谱 /
- 固相萃取
Abstract: A simple method for the simultaneous quantification of 21 perfluoroalkyl acids in milk has been developed using solid-phase extraction (SPE) with anion-exchange resin and silica gel as the mixed packing material, combined with the liquid chromatography-mass spectrometry (LC-MS/MS) technique. A large amount of protein was precipitated by adding acetonitrile to the milk sample, and the extract solution was purified through the elution in a self-made solid phase extraction column using 1.5 mL of 80% acetonitrile water solution. The purified solution was analyzed using LC-MS under the following conditions: an XDB-C18 column with gradient elution using acetonitrile-5 mmol of aqueous ammonium acetate as the mobile phase, electrospray negative ionization mode ionization (ESI), the mass scan mode was multiple reaction monitoring (MRM) mode detection. The limits of detection (LOD) of perfluoroalkyl acids (PFAAs) were 0.01~0.20 ng/mL and the limits of quantification (LOQ) were 0.03~0.67 ng/mL. The linearity (R²>0.999) and reproducibility of the method were good, and the recoveries ranged from 66% to 125%. Thirteen common commercially available milk were analyzed for perfluoroalkyl acids, and seven perfluoroalkyl acids were mainly detected with milk mass concentrations ranging from 2.43 to 12.00 ng/mL.
- perfluorocarboxylic acid /
- perfluorosulfonic acid /
- milk /
- LC-MS/MS /
- SPE

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图 1 21种PFAAs的总离子流图

Figure 1. Total ion flow diagram of 21 PFAAs

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图 2 填料类型对萃取效率的影响

Figure 2. Effect of packing type on extraction efficiency

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图 3 填料比例对萃取效率的影响

（a）短链PFCAs（PFPeA，PFOA，PFHxA，PFNA，PFHpA，PFDA），（b）长链PFCAs（PFUdA，PFTeDA，PFDoA，PFHxDA，PFTrDA，PFODA），（c）短链PFSAs（PFBS，PFPeS），（d）长链PFSAs（PFHxS，PFNS，PFHpS，PFDoS）

Figure 3. Effect of packing ratio on extraction efficiency

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图 4 填料质量对萃取效率的影响

Figure 4. Effect of packing quality on extraction efficiency

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图 5 洗脱溶剂乙腈的体积分数对萃取效率的影响

Figure 5. Effect of volume fractions of eluting solvent acetonitrile on extraction efficiency

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图 6 洗脱溶剂体积对萃取效率的影响

Figure 6. Effect of volume of eluting solvent on extraction efficiency

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图 7 （a）WAX柱与自填柱的萃取效果对比，（b）WAX柱与自填柱洗脱液的紫外分析，（c）WAX柱洗脱液的maldi分析，（d）自填柱洗脱液的maldi分析

Figure 7. (a) Comparison of extraction effect between WAX column and self-filled column, (b) UV analysis of eluate of WAX column and self-filled column, (c) maldi analysis of eluate of WAX column, (d) maldi analysis of eluate of self-filled column

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表 1 全氟烷基酸的质谱定量参数

Table 1. Quantitative mass spectrometric parameters for perfluoroalkyl acid

分析物	分子式	分子量/ (g/mol)	前体离子/(m/z)	子离子/ (m/z)	碰撞能量/eV
PFBA	C₄HF₇O₂	214.0	213.0	168.8	5
PFPeA	C₅HF₉O₂	264.0	263.0	218.9	4
PFHxA	C₆HF₁₁O₂	314.0	313.0	269.0	4
PFHpA	C₇HF₁₃O₂	364.1	363.0	318.9	5
PFOA	C₈HF₁₅O₂	414.0	413.0	368.9	8
PFNA	C₉HF₁₇O₂	464.1	463.0	419.0	4
PFDA	C₁₀HF₁₉O₂	514.1	513.0	468.9	9
PFUdA	C₁₁HF₂₁O₂	564.1	563.0	518.9	9
PFDoA	C₁₂HF₂₃O₂	614.1	613.0	568.9	9
PFTrDA	C₁₃HF₂₅O₂	664.1	663.0	619.1	9
PFTeDA	C₁₄HF₂₇O₂	714.1	712.9	669.1	9
PFHxDA	C₁₆HF₃₁O₂	814.1	812.9	769.0	9
PFODA	C₁₈HF₃₅O₂	914.1	912.9	869.0	13
PFBS	C₄HF₉O₃S	300.1	298.9	298.9	26
PFPeS	C₅HF₁₁O₃S	350.1	348.9	348.9	21
PFHxS	C₆HF₁₃O₃S	400.1	398.9	398.9	17
PFHpS	C₇HF₁₅O₃S	450.1	448.9	448.9	21
PFOS	C₈HF₁₇O₃S	500.1	498.9	498.9	25
PFNS	C₉HF₁₉O₃S	550.1	548.9	548.9	25
PFDS	C₁₀HF₂₁O₃S	600.1	598.9	598.9	25
PFDoS	C₁₂HF₂₅O₃S	700.2	698.9	698.9	25

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表 2 PFAAs标准品的标准曲线、线性范围、重现性、检出限、定量限

Table 2. Standard curves, linear ranges, reproducibilities, detection limits, quantitative limits of PFAAs standards

分析物	标准曲线方程	R²	线性范围/（ng/mL）	重现性(0.5 ng/mL)		LOD/(ng/mL)	LOQ/(ng/mL)
分析物	标准曲线方程	R²	线性范围/（ng/mL）	日内/%	日间/%	LOD/(ng/mL)	LOQ/(ng/mL)
PFPeA	y=116.16x+3.53	0.999 8	0.1~10	3.12	3.15	0.05	0.17
PFHxA	y=434.18x−205.94	0.999 9	0.05~10	2.29	2.34	0.01	0.03
PFHpA	y=421.20x+7.16	0.999 1	0.5~50	4.21	3.17	0.15	0.50
PFOA	y=296.42x−127.38	0.999 3	0.1~10	3.56	2.13	0.05	0.17
PFNA	y=224.44x+15.86	0.999 5	0.1~11	1.04	2.11	0.05	0.17
PFDA	y=250.36x+9.61	0.999 1	0.1~12	3.93	2.45	0.01	0.03
PFUdA	y=279.18x−0.29	0.999 3	0.1~13	4.12	3.67	0.05	0.17
PFDoA	y=350.31x-29.64	0.999 3	0.05~10	1.69	3.62	0.02	0.07
PFTrDA	y=313.78x+6.43	0.999 7	0.05~10	1.75	4.15	0.02	0.07
PFTeDA	y=250.42x+8.91	0.999 9	0.5~50	2.94	4.26	0.20	0.67
PFHxDA	y=166.40x−60.46	0.999 8	0.1~50	1.34	4.21	0.01	0.03
PFODA	y=586.21x−58.74	0.999 6	0.05~10	2.87	2.13	0.01	0.03
PFBS	y=1 553.30x−6.86	0.999 8	0.1~10	2.62	1.13	0.05	0.17
PFPeS	y=934.53x−9.63	0.999 9	0.1~10	3.51	2.19	0.02	0.07
PFHxS	y=682.35x−17.01	0.999 5	0.05~10	3.42	2.57	0.01	0.03
PFHpS	y=638.18x−26.12	0.999 3	0.05~10	3.27	1.34	0.01	0.03
PFNS	y=983.64x−17.10	0.999 8	0.05~10	1.45	1.31	0.01	0.03
PFDS	y=1 098.70x−0.96	0.999 9	0.05~10	2.14	4.21	0.05	0.17
PFDoS	y=790.44x−3.30	0.999 1	0.05~5	2.99	2.92	0.01	0.03

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表 3 市售牛奶样品中PFAAs的含量测定

Table 3. Determination of PFAAs content in milk samples on market /(ng/mL)

样品	PFAAs质量浓度
样品	PFPeA	PFHxA	PFHpA	PFOA	PFDA	PFHpS	PFDoA	∑
牛奶1	0.91±0.05	2.04±0.02	0.31±0.02	8.22±0.05	0.41±0.02	0.15±0.00	N.D.	12.00±0.11
牛奶2	1.81±0.07	N.D	0.37±0.02	7.24±0.11	0.62±0.04	N.D.	0.66±0.06	10.70±0.18
牛奶3	1.45±0.09	N.D.	0.30±0.01	4.18±0.05	N.D.	N.D.	N.D.	5.93±0.15
牛奶4	0.62±0.05	N.D.	0.36±0.03	4.55±0.06	N.D.	N.D.	N.D.	5.53±0.09
牛奶5	0.78±0.06	2.22±0.02	0.21±0.03	5.38±0.03	N.D.	N.D.	N.D.	8.59±0.03
牛奶6	1.18±0.07	0.70±0.12	0.22±0.01	2.45±0.18	N.D.	N.D.	N.D.	4.55±0.32
牛奶7	1.42±0.09	1.04±0.05	0.35±0.03	2.54±0.11	N.D.	N.D.	N.D.	5.35±0.26
牛奶8	1.43±0.21	0.83±0.08	0.25±0.03	2.53±0.10	N.D.	N.D.	N.D.	5.04±0.22
牛奶9	1.56±0.13	0.62±0.04	0.25±0.02	N.D.	N.D.	N.D.	N.D.	2.43±0.16
牛奶10	2.11±0.18	0.66±0.05	0.47±0.03	2.92±0.18	N.D.	N.D.	N.D.	6.16±0.18
牛奶11	2.57±0.10	0.49±0.03	0.46±0.05	2.43±0.11	N.D.	N.D.	N.D.	5.95±0.05
牛奶12	0.74±0.15	0.42±0.02	0.36±0.03	2.45±0.08	N.D.	N.D.	N.D.	3.97±0.13
牛奶13	0.88±0.09	N.D.	0.21±0.02	2.10±0.11	N.D.	N.D.	N.D.	3.19±0.18

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表 4 13种牛奶的加标回收率

Table 4. Spiked recovery of 13 milks /%

样品	PFPeA	PFHxA	PFHpA	PFBS	PFOA	PFPeS	PFNA	PFHxS	PFDA
牛奶1	96	69	115	87	66	105	87	103	88
牛奶2	67	118	110	93	116	108	93	105	76
牛奶3	76	113	76	68	71	78	72	77	72
牛奶4	79	110	70	76	95	75	82	79	73
牛奶5	82	72	80	69	70	88	76	74	66
牛奶6	117	119	101	100	67	93	72	92	81
牛奶7	88	98	102	100	87	91	75	88	85
牛奶8	107	89	91	85	96	80	72	83	77
牛奶9	94	102	99	92	120	76	67	90	79
牛奶10	101	113	100	100	100	96	96	97	116
牛奶11	68	122	106	103	94	99	79	90	119
牛奶12	78	117	105	92	114	79	88	92	95
牛奶13	100	118	125	104	106	92	79	91	121

样品	PFHpS	PFUdA	PFDoA	PFNS	PFTrDA	PFTeDA	PFDoS	PFHxDA	PFODA
牛奶1	93	72	102	102	95	100	101	118	121
牛奶2	88	81	86	96	90	88	95	112	115
牛奶3	73	95	74	77	84	83	79	108	105
牛奶4	68	85	70	71	76	76	70	114	85
牛奶5	67	68	84	75	75	75	71	89	88
牛奶6	103	104	77	98	106	100	99	117	107
牛奶7	106	118	86	106	102	106	101	119	101
牛奶8	86	99	73	90	92	97	84	109	81
牛奶9	97	112	77	93	101	99	92	107	69
牛奶10	114	119	97	101	103	101	110	116	101
牛奶11	103	123	118	107	96	89	98	96	92
牛奶12	107	106	79	102	101	99	95	120	67
牛奶13	111	121	109	111	104	107	115	109	93

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