分散固相萃取-高效液相色谱法测定水产品中7种麻醉剂

doi:10.3724/SP.J.1123.2021.08002

色谱 ›› 2022, Vol. 40 ›› Issue (2): 139-147.DOI: 10.3724/SP.J.1123.2021.08002

分散固相萃取-高效液相色谱法测定水产品中7种麻醉剂

石芳¹, 寿旦², 金米聪³^,⁶, 王宏伟⁴^,^*(), 陈旭光⁵^,^*(), 朱岩¹^,⁶

1.浙江大学化学系, 浙江杭州 310028
2.浙江省中医药研究院中药研究中心, 浙江杭州 310007
3.宁波市疾病预防控制中心, 浙江宁波 315010
4.浙江省立同德医院, 浙江杭州 310012
5.浙江省冶金研究院有限公司, 浙江杭州 310007
6.浙江省微量有毒化学物健康风险评估技术研究重点实验室, 浙江杭州 310028

收稿日期:2021-08-04 出版日期:2022-02-08 发布日期:2022-01-26
通讯作者: 王宏伟,陈旭光
基金资助:
国家自然科学基金(21974121);浙江省公益技术应用研究项目(LGC20H300002);浙江省公益技术应用研究项目(LGC21B050006)

Dispersive solid-phase extraction combined with high-performance liquid chromatography for determination of seven anesthetics in aquatic products

SHI Fang¹, SHOU Dan², JIN Micong³^,⁶, WANG Hongwei⁴^,^*(), CHEN Xuguang⁵^,^*(), ZHU Yan¹^,⁶

1. Department of Chemistry, Zhejiang University, Hangzhou 310028, China
2. Department of Medicine, Zhejiang Academy of Traditional Chinese Medicine, Hangzhou 310007, China
3. Ningbo Municipal Center for Disease Control and Prevention, Ningbo 315010, China
4. Tongde Hospital of Zhejiang Province, Hangzhou 310012, China
5. Zhejiang Metallurgical Research Institute Co., LTD., Hangzhou 310007, China
6. Key Laboratory of Health Risk Appraisal for Trace Toxic Chemicals of Zhejiang Province, Zhejiang University, Hangzhou 310028, China

Received:2021-08-04 Online:2022-02-08 Published:2022-01-26
Contact: WANG Hongwei, CHEN Xuguang
Supported by:
National Natural Science Foundation of China(21974121);Zhejiang Provincial Program for Public Welfare of Technology Application Research Plan(LGC20H300002);Zhejiang Provincial Program for Public Welfare of Technology Application Research Plan(LGC21B050006)

摘要/Abstract

摘要：

随着麻醉剂广泛用于渔业生产过程和水产品运输等领域,建立水产品中麻醉剂残留的检测方法具有重要意义。由于水产品基质复杂且麻醉剂残留水平低,因此需要合适的前处理方法以提高检测灵敏度。该研究基于分散固相萃取-高效液相色谱,建立了一种同时检测水产品中普鲁卡因、丁氧卡因、三卡因、丁香酚、甲基丁香酚、异丁香酚、甲基异丁香酚7种麻醉剂的分析方法。前处理采用分散固相萃取;确定了1.0%甲酸乙腈为提取溶剂,20 mg苯乙烯-甲基丙烯酸缩水甘油酯聚合物微球(PS-GMA)、50 mg N-丙基乙二胺(PSA)和10 mg C18混合吸附剂为净化剂,二甲基亚砜(DMSO)辅助氮吹的前处理方法;优化了提取时间和DMSO用量等条件。7种麻醉剂采用Welch welchrom C18色谱柱(250 mm×4.6 mm, 5 μm)进行分离,以甲醇和0.05%甲酸-5 mmol/L乙酸铵水溶液为流动相进行梯度洗脱,检测波长为235、260和290 nm,以鱼肉和对虾两种基质匹配标准曲线进行定量分析。实验结果表明,在优化的实验条件下,7种目标麻醉剂在各自的浓度范围内具有良好的线性关系(相关系数R²>0.999),检出限(LOD)为0.011~0.043 mg/kg。在鱼肉样品中,3个水平的平均加标回收率为79.7%~109%,相对标准偏差(RSD)低于7.2%;在对虾样品中,平均回收率为78.0%~99.9%, RSD低于8.3%。该方法具有快捷简便、操作简单、灵敏度高等优点,可应用于水产品中3种氨基苯甲酸酯类和4种丁香酚类麻醉剂的检测。

关键词: 分散固相萃取, 高效液相色谱, 麻醉剂, 水产品

Abstract:

Nowadays, anesthetics are widely used in fishery production processes, such as fish breeding, surgery, and fresh aquatic product transportation. Because of the widespread application of anesthetic drugs in aquatic products, there is an increasing demand for the rapid and sensitive detection of anesthetic drugs in aquatic products. The complex aquatic product matrix contains a variety of interfering substances, such as proteins, fats, and phospholipids, along with anesthetic drug residues at very low concentrations; therefore, it is necessary to adopt appropriate pretreatment methods for improving the sensitivity of detection. In this study, a dispersive solid-phase extraction (DSPE) method, combined with high-performance liquid chromatography, was established for the simultaneous detection of seven anesthetic drugs in aquatic products, viz. procaine, oxybuprocaine, tricaine, eugenol, methyl eugenol, isoeugenol, and methyl isoeugenol. For the DSPE step, pretreatment conditions, such as extraction solvent, extraction time, adsorbent amount, and DMSO dosage, were optimized. Sample pretreatment is a three-step process. First, in ultrasound-assisted extraction, 2.0 g samples were extracted using 10.0 mL 1.0% formic acid in acetonitrile under ultrasound conditions for 10 min. Then, DSPE was performed with mixed adsorbents: the solvent extracts were cleaned using 20 mg poly(styrene-glycidylmethacrylate) microspheres (PS-GMA), 50 mg primary secondary amines (PSA), and 10 mg C18, followed by separation by centrifugation. Finally, DMSO-assisted concentration was applied: the organic layer was collected and was dried at 40 ℃ in a N₂ stream with 100 μL DMSO. Water was added to the residue to obtain a final volume of 1.0 mL for HPLC analysis. The seven anesthetic drugs were separated on a Welch welchrom C18 column (250 mm×4.6 mm, 5 μm) by gradient elution using methanol and 0.05% formic acid in 5 mmol/L ammonium acetate aqueous solution as mobile phases. The detection wavelengths were 235, 260, and 290 nm. Two matrix matching standard curves for fish and shrimp were applied for quantitative analysis. Under optimized conditions, the seven target anesthetics showed good linear relationships in their respective concentration ranges (R²>0.999), with the limit of detection (LOD) ranging from 0.011 to 0.043 mg/kg. In fish samples, the mean recoveries obtained at three concentration levels were between 79.7% and 109%, with relative standard deviations (RSDs) being less than 7.2%. In shrimp samples, mean recoveries were 78.0%-99.9%, with RSDs being less than 8.3%. This simple, rapid, accurate, and sensitive method can be applied to the detection of three kinds of aminobenzoic acid esters and four kinds of eugenol anesthetic drugs in aquatic products.

Key words: dispersive solid-phase extraction (DSPE), high-performance liquid chromatography (HPLC), anesthetic drugs, aquatic products

中图分类号:

O658

石芳, 寿旦, 金米聪, 王宏伟, 陈旭光, 朱岩. 分散固相萃取-高效液相色谱法测定水产品中7种麻醉剂[J]. 色谱, 2022, 40(2): 139-147.

SHI Fang, SHOU Dan, JIN Micong, WANG Hongwei, CHEN Xuguang, ZHU Yan. Dispersive solid-phase extraction combined with high-performance liquid chromatography for determination of seven anesthetics in aquatic products[J]. Chinese Journal of Chromatography, 2022, 40(2): 139-147.

图/表 10

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Analyte	Linear range/(mg/L)	Matrix	Linear equation	R²	LOD/(mg/kg)	LOQ/(mg/kg)	ME/%
Procaine	0.05-10.0	fish	Y=0.9383X+0.0028	0.9999	0.011	0.036	-2.04
		shrimp	Y=0.9332X-0.0097	0.9997	0.011	0.037	-2.57
Oxybuprocaine	0.10-10.0	fish	Y=0.4977X-0.0106	0.9998	0.024	0.082	+0.24
		shrimp	Y=0.4930X-0.0249	0.9991	0.025	0.084	-0.70
Tricaine	0.10-10.0	fish	Y=0.5702X+0.0085	0.9999	0.023	0.077	-0.75
		shrimp	Y=0.5681X+0.0144	0.9992	0.024	0.080	-1.12
Eugenol	0.10-10.0	fish	Y=0.4011X-0.0052	0.9998	0.033	0.109	-3.02
		shrimp	Y=0.4036X-0.0253	0.9996	0.034	0.113	-2.42
Isoeugenol	0.05-10.0	fish	Y=1.0784X+0.0371	0.9995	0.013	0.044	-8.97
		shrimp	Y=1.1077X+0.0393	0.9994	0.013	0.043	-6.50
Methyl eugenol	0.20-10.0	fish	Y=0.4086X-0.0265	0.9998	0.043	0.142	-7.96
		shrimp	Y=0.4147X-0.0224	0.9997	0.043	0.144	-6.18
Methyl isoeugenol	0.10-10.0	fish	Y=0.9359X-0.0715	0.9993	0.021	0.070	-11.3
		shrimp	Y=0.9569X-0.0586	0.9992	0.021	0.071	-9.32

Analyte	Linear range/(mg/L)	Matrix	Linear equation	R²	LOD/(mg/kg)	LOQ/(mg/kg)	ME/%
Procaine	0.05-10.0	fish	Y=0.9383X+0.0028	0.9999	0.011	0.036	-2.04
		shrimp	Y=0.9332X-0.0097	0.9997	0.011	0.037	-2.57
Oxybuprocaine	0.10-10.0	fish	Y=0.4977X-0.0106	0.9998	0.024	0.082	+0.24
		shrimp	Y=0.4930X-0.0249	0.9991	0.025	0.084	-0.70
Tricaine	0.10-10.0	fish	Y=0.5702X+0.0085	0.9999	0.023	0.077	-0.75
		shrimp	Y=0.5681X+0.0144	0.9992	0.024	0.080	-1.12
Eugenol	0.10-10.0	fish	Y=0.4011X-0.0052	0.9998	0.033	0.109	-3.02
		shrimp	Y=0.4036X-0.0253	0.9996	0.034	0.113	-2.42
Isoeugenol	0.05-10.0	fish	Y=1.0784X+0.0371	0.9995	0.013	0.044	-8.97
		shrimp	Y=1.1077X+0.0393	0.9994	0.013	0.043	-6.50
Methyl eugenol	0.20-10.0	fish	Y=0.4086X-0.0265	0.9998	0.043	0.142	-7.96
		shrimp	Y=0.4147X-0.0224	0.9997	0.043	0.144	-6.18
Methyl isoeugenol	0.10-10.0	fish	Y=0.9359X-0.0715	0.9993	0.021	0.070	-11.3
		shrimp	Y=0.9569X-0.0586	0.9992	0.021	0.071	-9.32

Sample	Analyte	Analytical method	Preparation method	Preparation time/min	Preparation steps	V(organic solvent)/mL	LOD/ (μg/kg)	Reference
Tilapia	eugenol	HPLC-UV	LLE	51	12	26	30	[8]
Ietalurus punetaus	eugenol	HPLC-UV	lyophilization	>63	18	16	45	[9]
Aquatic products	six anesthetics	HPLC-UV	QuEChERS-SPE	>31	19	45	60	[30]
Fish and shrimp	seven anesthetics	HPLC-UV	DSPE	24	8	10	11-43	this work

Sample	Analyte	Analytical method	Preparation method	Preparation time/min	Preparation steps	V(organic solvent)/mL	LOD/ (μg/kg)	Reference
Tilapia	eugenol	HPLC-UV	LLE	51	12	26	30	[8]
Ietalurus punetaus	eugenol	HPLC-UV	lyophilization	>63	18	16	45	[9]
Aquatic products	six anesthetics	HPLC-UV	QuEChERS-SPE	>31	19	45	60	[30]
Fish and shrimp	seven anesthetics	HPLC-UV	DSPE	24	8	10	11-43	this work

Analyte	Spiked/ (mg/kg)	Fish			Shrimp			Batch-to-batch RSD/%
Analyte	Spiked/ (mg/kg)	Recovery/ %	Intra-day RSD/%	Inter-day RSD/%	Recovery/ %	Intra-day RSD/%	Inter-day RSD/%	Batch-to-batch RSD/%
Procaine	0.2	79.7	6.2	7.2	82.0	3.1	5.5	6.3
	0.5	89.7	3.4	3.6	87.2	2.3	2.6
	1.0	89.1	3.4	2.7	86.9	2.7	3.0
Oxybuprocaine	0.2	89.8	2.8	2.3	93.9	2.2	6.0	2.1
	0.5	91.5	5.2	4.2	90.2	3.3	5.0
	1.0	89.6	4.1	4.0	89.0	3.9	4.4
Tricaine	0.2	83.5	5.1	2.6	78.0	5.5	2.7	5.0
	0.5	88.3	4.0	1.9	84.2	2.6	6.3
	1.0	85.4	2.2	3.5	86.7	3.5	6.0
Eugenol	0.2	91.2	6.8	2.7	83.2	3.3	5.0	6.7
	0.5	89.1	2.8	3.5	85.4	2.6	5.4
	1.0	89.2	2.0	2.3	91.2	1.7	4.6
Isoeugenol	0.2	84.7	4.2	7.2	94.8	3.2	8.2	4.3
	0.5	82.1	3.1	5.6	85.8	3.2	5.4
	1.0	84.5	1.9	6.1	96.5	2.0	7.0
Methyl eugenol	0.2	109.0	2.8	1.8	99.9	6.0	8.1	6.2
	0.5	101.0	4.6	3.8	98.0	2.5	3.8
	1.0	94.1	2.3	3.8	95.0	1.3	5.1
Methyl isoeugenol	0.2	106.0	4.1	3.3	93.0	6.7	3.1	5.3
	0.5	96.4	4.1	4.5	96.6	2.9	8.3
	1.0	92.7	3.9	5.3	93.8	1.2	5.3

分散固相萃取-高效液相色谱法测定水产品中7种麻醉剂

Dispersive solid-phase extraction combined with high-performance liquid chromatography for determination of seven anesthetics in aquatic products

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