超高效液相色谱-串联质谱法测定血浆与尿液中14种麻痹性贝类毒素

doi:10.3724/SP.J.1123.2022.05030

摘要/Abstract

摘要：

人体生物基质中麻痹性贝类毒素的检测对其引起的食物中毒诊断和救治具有重要意义。研究建立了超高效液相色谱-串联质谱法测定血浆、尿液中14种麻痹性贝类毒素的分析方法。实验比较了不同固相萃取柱的影响,优化了前处理条件和色谱条件,血浆样品采用0.2 mL水、0.4 mL甲醇、0.6 mL乙腈提取后直接上机测定,尿液样品采用0.2 mL水、0.4 mL甲醇、0.6 mL乙腈提取,聚酰胺(PA)固相萃取柱净化后上机测定。采用Poroshell 120 HILIC-Z色谱柱(100 mm×2.1 mm, 2.7 μm)对14种贝类毒素进行分离,流动相为含0.1%(v/v)甲酸的5 mmoL/L甲酸铵缓冲溶液和0.1%(v/v)甲酸乙腈溶液,流速为0.50 mL/min。在电喷雾模式(ESI)下进行正负离子扫描,采用多反应监测(MRM)模式检测,外标法定量。结果表明,对于血浆和尿液样品,14种贝类毒素分别在0.24~84.06 ng/mL范围内线性关系良好,相关系数均大于0.995。尿液检测的定量限为4.80~34.40 ng/mL,血浆检测的定量限为1.68~12.04 ng/mL。尿液和血浆样品在1、2和10倍定量限加标水平下平均回收率为70.4%~123.4%,日内精密度为2.3%~19.1%,日间精密度为4.0%~16.2%。应用建立的方法对腹腔注射14种贝类毒素小鼠血浆和尿液进行测定,20份血浆样本中检出含量分别为19.40~55.60 μg/L和8.75~13.86 μg/L。该方法操作简便,样品取样量少,方法灵敏度高,适用于血浆和尿液中麻痹性贝类毒素的快速检测。

关键词: 超高效液相色谱-串联质谱, 麻痹性贝类毒素, 血浆, 尿液

Abstract:

The detection of paralytic shellfish toxins in human biological matrices is important for the diagnosis and treatment of food poisoning caused by them. An ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established for the determination of 14 paralytic shellfish toxins in plasma and urine. The effect of solid phase extraction (SPE) cartridges was also investigated and the pretreatment and chromatographic conditions were optimized. Under these optimal conditions, 0.2 mL water, 0.4 mL methanol, and 0.6 mL acetonitrile were successively added to plasma and urine samples for extraction. The supernatants from plasma extraction were subjected to an UHPLC-MS/MS analysis, whereas the supernatants from urine extraction were further purified using polyamide (PA) SPE cartridges and then analyzed by UHPLC-MS/MS. Chromatographic separation was conducted on a Poroshell 120 HILIC-Z column (100 mm×2.1 mm, 2.7 μm) with a flow rate of 0.5 mL/min. The mobile phase was 0.1% (v/v) formic acid aqueous solution containing 5 mmoL/L ammonium formate and acetonitrile containing 0.1% (v/v) formic acid. The analytes were detected in the multiple reaction monitoring (MRM) mode after being ionized by an electrospray ion (ESI) in positive and negative modes. Quantitation of the target compounds was performed using the external standard method. Under the optimal conditions, the method showed good linearity in the range of 0.24-84.06 μg/L, with correlation coefficients greater than 0.995. The limits of quantification (LOQs) for the plasma and urine samples were 1.68-12.04 ng/mL and 4.80-34.4 ng/mL, respectively. The average recoveries for all the compounds were 70.4%-123.4% at spiked levels of 1, 2, and 10 times the LOQs, the intra-day precisions were 2.3%-19.1% and the inter-day precisions were 5.0%-16.0%. The established method was used to determine the target compounds in the plasma and urine from mice intraperitoneally injected with 14 shellfish toxins. All 14 toxins were detected in the 20 urine and 20 plasma samples, with contents of 19.40-55.60 μg/L and 8.75-13.86 μg/L, respectively. The method is simple, sensitive, and only requires a small amount of sample. Therefore, it is highly suitable for the rapid detection of paralytic shellfish toxins in plasma and urine.

Key words: ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), paralytic shellfish toxins, plasma, urine

中图分类号:

O658

林强, 杨超, 李美丽, 王佳, 侯瀚然, 邵兵, 牛宇敏. 超高效液相色谱-串联质谱法测定血浆与尿液中14种麻痹性贝类毒素[J]. 色谱, 2023, 41(3): 274-280.

LIN Qiang, YANG Chao, LI Meili, WANG Jia, HOU Hanran, SHAO Bing, NIU Yumin. Determination of 14 paralytic shellfish toxins in plasma and urine by ultra-high performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2023, 41(3): 274-280.

图/表 8

参考文献

[1]	Wang D Z, Zhang S F, Zhang Y, et al. J Proteomics, 2016(135): 132
[2]	Tian J J, Han G, Liu H T, et al. Ocean Environment Science, 2019, 38(3): 464
[2]	田娟娟, 韩刚, 刘海棠, 等. 海洋环境科学, 2019, 38(3): 464
[3]	Peake R W A, Zhang V Y, Azcue N, et al. J Chromatogr B, 2016(1036/1037): 42
[4]	Eangoor P, Indapurkar A S, Vakkalanka M D, et al. Analyst, 2019(144): 4702
[5]	Bragg W A, Lemire S W, Coleman R M, et al. Toxicon, 2015(99): 118
[6]	Coleman R M, Ojeda-Torres G, Bragg W, et al. J Anal Toxicol, 2018(42): 61
[7]	D’Agostino P M, Boundy M J, Harwood T D, et al. Toxicon, 2019(158): 1
[8]	Wu Y N, Shao B, Chen D W, et al. Practical Detection Technology of Common Chemical Food Poisoning Substances. Beijing: Science Press, 2021
[8]	吴永宁, 邵兵, 陈达炜, 等. 常见化学性食物中毒物质实用检测技术. 北京: 科学出版社, 2021
[9]	Degrasse S, Rivera V, Roach J, et al. Deep-Sea RES PT II, 2014(103): 368
[10]	Xu X M, Huang B F, Xu J J, et al. J Chromatogr B, 2018 (1072): 267
[11]	He M Z, Yuan G X, Qin X Y, et al. Journal of Food Safety & Quality, 2021, 12(9): 3465
[11]	何明珠, 袁冠湘, 秦逍云, 等. 食品安全质量检测学报, 2021, 12(9): 3465
[12]	Yang Y H. Journal of Fisheries Research, 2020, 42(5): 473
[12]	杨云辉. 渔业研究, 2020, 42(5): 473
[13]	Fu Z J, Zhao Y G. Chinese Journal of Health Laboratory Technology, 2021, 31(21): 2581
[13]	付振军, 赵永纲. 中国卫生检验杂志, 2021, 31(21): 2581
[14]	Tang Y Y, Huang D M, Cai Y Q. Quality and Safety of Agro-Products, 2020(6): 29
[14]	汤云瑜, 黄冬梅, 蔡友琼. 农产品质量与安全, 2020(6): 29
[15]	Liu B, Zhao H Q, Liu B, et al. Journal of Food Safety and Quality, 2021, 12(5): 1746
[15]	刘斌, 赵慧琴, 刘波, 等. 食品安全质量检测学报, 2021, 12(5): 1746
[16]	Zhang Y Y, Yan G W, Wu H Y, et al. Oceanologia et Limnologia Sinica, 2020, 51(2): 298
[16]	张亚亚, 闫国旺, 吴海燕, 等. 海洋与湖沼, 2020, 51 (2): 298

Analyte	Precursor ion (m/z)	Product ions (m/z)	Collision energies/eV	Declustering potential/V	Retention time/min
Gonyautoxin 1 (GTX1)	410.1	367.1^*, 349.1	-28, -22	-100	5.66
Gonyautoxin 2 (GTX2)	394.1	333.1^*, 351.1	-30, -22	-130	5.74
Gonyautoxin 3 (GTX3)	396.1	298.2^*, 315.9	29, 16	50	6.60
Gonyautoxin 4 (GTX4)	412.1	332.1^*, 314.1	19, 27	20	6.57
Gonyautoxin 5 (GTX5)	380.2	300.2^*, 282.2	21, 29	20	6.76
Gonyautoxin 6 (GTX6)	396.1	316.2^*, 298.0	20, 32	30	6.94
Decarbamoyl-gonyautoxin 2 (DCGTX2)	353.3	255.1^*, 273.0	27, 13	60	5.95
Decarbamoyl-gonyautoxin 3 (DCGTX3)	353.3	255.1^*, 273.0	27, 13	60	6.65
N-Sulfocarbamoyl-gonyautoxin 1 (C1)	473.9	122.0^*, 333.0	-30, -43	-40	6.60
N-Sulfocarbamoyl-gonyautoxin 2 (C2)	473.9	122.0^*, 333.0	-30, -43	-40	6.93
Saxitoxin (STX)	300.1	204.2^*, 138.0	30, 35	140	6.68
Decarbamoyl-saxitoxin (DCSTX)	257.1	126.3^*, 222.3	25, 29	100	6.70
Neosaxitoxin (NEO)	316.1	220.1^*, 126.1	34, 31	80	6.68
Decarbamoyl-neosaxitoxin (DCNEO)	273.1	225.2^*, 126.1	31, 23	30	6.68

Analyte	Precursor ion (m/z)	Product ions (m/z)	Collision energies/eV	Declustering potential/V	Retention time/min
Gonyautoxin 1 (GTX1)	410.1	367.1^*, 349.1	-28, -22	-100	5.66
Gonyautoxin 2 (GTX2)	394.1	333.1^*, 351.1	-30, -22	-130	5.74
Gonyautoxin 3 (GTX3)	396.1	298.2^*, 315.9	29, 16	50	6.60
Gonyautoxin 4 (GTX4)	412.1	332.1^*, 314.1	19, 27	20	6.57
Gonyautoxin 5 (GTX5)	380.2	300.2^*, 282.2	21, 29	20	6.76
Gonyautoxin 6 (GTX6)	396.1	316.2^*, 298.0	20, 32	30	6.94
Decarbamoyl-gonyautoxin 2 (DCGTX2)	353.3	255.1^*, 273.0	27, 13	60	5.95
Decarbamoyl-gonyautoxin 3 (DCGTX3)	353.3	255.1^*, 273.0	27, 13	60	6.65
N-Sulfocarbamoyl-gonyautoxin 1 (C1)	473.9	122.0^*, 333.0	-30, -43	-40	6.60
N-Sulfocarbamoyl-gonyautoxin 2 (C2)	473.9	122.0^*, 333.0	-30, -43	-40	6.93
Saxitoxin (STX)	300.1	204.2^*, 138.0	30, 35	140	6.68
Decarbamoyl-saxitoxin (DCSTX)	257.1	126.3^*, 222.3	25, 29	100	6.70
Neosaxitoxin (NEO)	316.1	220.1^*, 126.1	34, 31	80	6.68
Decarbamoyl-neosaxitoxin (DCNEO)	273.1	225.2^*, 126.1	31, 23	30	6.68

Analyte	Linear range/(μg/L)	r²	LODs/(μg/L)		LOQs/(μg/L)
Analyte	Linear range/(μg/L)	r²	Urine	Plasma	Urine	Plasma
GTX1	0.29-36.72	0.996	1.93	0.68	5.80	2.03
GTX2	0.50-63.44	0.995	3.33	1.17	10.00	3.50
GTX3	0.42-26.88	0.995	2.80	0.98	8.40	2.94
GTX4	0.36-11.56	0.996	2.40	0.84	7.20	2.52
GTX5	1.03-32.97	0.997	6.87	2.40	20.60	7.21
GTX6	0.24-7.72	0.995	1.60	0.56	4.80	1.68
DCGTX2	1.72-55.16	0.996	11.47	4.01	34.40	12.04
DCGTX3	0.51-16.25	0.997	3.33	1.17	10.00	3.50
C1	0.66-84.06	0.998	4.40	1.54	13.20	4.62
C2	0.39-25.16	0.996	2.60	0.91	7.80	2.73
STX	0.60-38.59	0.995	4.00	1.40	12.00	4.20
DCSTX	0.26-33.44	0.996	1.73	0.61	5.20	1.82
NEO	0.25-32.05	0.997	1.67	0.58	5.00	1.75
DCNEO	0.49-15.78	0.996	3.27	1.14	9.80	3.43

Analyte	Linear range/(μg/L)	r²	LODs/(μg/L)		LOQs/(μg/L)
Analyte	Linear range/(μg/L)	r²	Urine	Plasma	Urine	Plasma
GTX1	0.29-36.72	0.996	1.93	0.68	5.80	2.03
GTX2	0.50-63.44	0.995	3.33	1.17	10.00	3.50
GTX3	0.42-26.88	0.995	2.80	0.98	8.40	2.94
GTX4	0.36-11.56	0.996	2.40	0.84	7.20	2.52
GTX5	1.03-32.97	0.997	6.87	2.40	20.60	7.21
GTX6	0.24-7.72	0.995	1.60	0.56	4.80	1.68
DCGTX2	1.72-55.16	0.996	11.47	4.01	34.40	12.04
DCGTX3	0.51-16.25	0.997	3.33	1.17	10.00	3.50
C1	0.66-84.06	0.998	4.40	1.54	13.20	4.62
C2	0.39-25.16	0.996	2.60	0.91	7.80	2.73
STX	0.60-38.59	0.995	4.00	1.40	12.00	4.20
DCSTX	0.26-33.44	0.996	1.73	0.61	5.20	1.82
NEO	0.25-32.05	0.997	1.67	0.58	5.00	1.75
DCNEO	0.49-15.78	0.996	3.27	1.14	9.80	3.43

Analytes	Matrices	Sample volume/mL	Pre-processing method	LOQs/(μg/L)	Ref.
STX, NEO	urine	0.45	Online SPE (Oasis WCX exchange columns)	STX: 5.10, NEO: 2.62	[5]
NEO	plasma	1.00	SPE (Enviro-Clean CCX carboxylic acid cation exchange columns)	0.40	[3]
STX, NEO	urine	0.20	SPE (PA exchange columns)	STX: 1.40, NEO: 1.00	[10]
14 paralytic shellfish toxins	urine, plasma	0.20	urine: liquid-liquid extraction plasma: SPE (PA exchange columns)	urine: 4.80-34.40, plasma: 1.68-12.04	this research