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

doi:10.3724/SP.J.1123.2021.08002

Abstract

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

CLC Number:

O658

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.

Figures/Tables 10

References

[1]	Li J, Zhang J, Liu Y. Anal Bioanal Chem, 2015, 407(21):6563
[2]	Becker A G, Parodi T V, Heldwein C G, et al. Fish Physiol Biochem, 2012, 38(3):789
[3]	Wang W, Dong H, Sun Y, et al. J Appl Ichthyol, 2019, 35(2):551
[4]	Lü S W, Lei H T, Sun Y M. Food Science, 2012, 33(1):267
[4]	吕书为, 雷红涛, 孙远明. 食品科学, 2012, 33(1):267
[5]	Zahl I H, Samuelsen O, Kiessling A. Fish Physiol Biochem, 2012, 38(1):201
[6]	Popovic N, Strunjak-Perovic I, Coz-Rakovac R, et al. J Appl Ichthyol, 2012, 28(4):553
[7]	Sun H, Lai J P, Chen F, et al. Anal Bioanal Chem, 2015, 407(6):1745
[8]	Huang W, Xu J L, Liu J F, et al. Journal of Food Safety and Quality, 2018, 9(1):103
[8]	黄武, 徐金龙, 刘建芳, 等. 食品安全质量检测学报, 2018, 9(1):103
[9]	Wang C X, Xiong G Q, Bai C, et al. Journal of Food Safety and Quality, 2019, 10(8):2195
[9]	王彩霞, 熊光权, 白婵, 等. 食品安全质量检测学报, 2019, 10(8):2195
[10]	Scherpenisse P, Bergwerff A A. Anal Chim Acta, 2007, 586(1/2):407
[11]	Xie C, Li Q, Han G, et al. Biomed Chromatogr, 2019, 33(5):e4512
[12]	Li J, Liu H, Wang C, et al. Anal Bioanal Chem, 2016, 408(24):6537
[13]	Huang S, Xu J, Wu J, et al. Talanta, 2017, 168:263
[14]	Zheng X H, Sun T, Chen Y, et al. Food Science and Technology, 2020, 45(4):333
[14]	郑向华, 孙婷, 陈燕, 等. 食品科技, 2020, 45(4):333
[15]	Ke C, Liu Q, Li L, et al. J Chromatogr B Analyt Technol Biomed Life Sci, 2016, 1031:189
[16]	Botrel B M C, Abreu D C P, Bazana M J F, et al. Food Anal Methods, 2019, 12(6):1390
[17]	Li J, Liu H, Yu M, et al. Anal Methods, 2014, 6(22):9124
[18]	Xuan K Z, Liang Z G, Chen W H, et al. Physical Testing and Chemical Analysis Part B: Chemical Analysis, 2018, 54(12):1405
[18]	禤开智, 梁振纲, 陈文慧, 等. 理化检验-化学分册, 2018, 54(12):1405
[19]	Tan X R, Zhao B, Lu J W, et al. Chinese Journal of Chromatography, 2022, 40(1):57
[19]	谭学蓉, 赵斌, 陆建伟, 等. 色谱, 2022, 40(1):57
[20]	Sun P, Gao Y, Lian Y. Food Anal Methods, 2017, 10(10):3217
[21]	Aladaghlo Z, Fakhari A, Behbahani M. J Sep Sci, 2016, 39(19):3798
[22]	Yu J, Di S, Ning T, et al. Microchim Acta, 2020, 187(9):531
[23]	Lou C, Wu C, Zhang K, et al. J Chromatogr A, 2018, 1550:45
[24]	Ji X F, Li S, Wu G G, et al. Chinese Journal of Chromatography, 2021, 39(8):896
[24]	纪雪峰, 李爽, 吴阁格, 等. 色谱, 2021, 39(8):896
[25]	Xu X, Feng T, Zhang J, et al. Anal Methods, 2018, 10(42):5091
[26]	Li S, Liang Q, Ahmed S A H, et al. Food Anal Methods, 2020, 13(5):1111
[27]	Cen J B, Liang Z S, Ou S J, et al. Chinese Journal of Chromatography, 2020, 38(6):672
[27]	岑建斌, 梁志森, 区硕俊, 等. 色谱, 2020, 38(6):672
[28]	Zhong Y, Zhou W, Zhu H, et al. Anal Chim Acta, 2011, 686(1/2):1
[29]	Guo D, Lou C, Wang N, et al. Talanta, 2017, 168:188
[30]	Gao P, Chen R M, Zeng D D, et al. Chinese Journal of Analysis Laboratory, 2018, 37(1):88
[30]	高平, 陈日檬, 曾丹丹, 等. 分析试验室, 2018, 37(1):88

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