TurboFlow在线净化-液相色谱-串联质谱法快速检测人尿中鹅膏肽类毒素

doi:10.3724/SP.J.1123.2020.06005

摘要/Abstract

摘要：

鹅膏肽类毒素是一类环状多肽类蘑菇毒素,中毒后会造成急性肝损伤,病死率非常高。我国因误食野生毒蘑菇导致的中毒事件常有发生,测定人尿中鹅膏肽类毒素的浓度,可为临床早期诊断和救治提供有价值的信息。该研究建立了TurboFlow (TF)在线净化-液相色谱-三重四极杆质谱快速定量检测尿液中5种鹅膏肽类毒素(α-鹅膏毒肽、β-鹅膏毒肽、γ-鹅膏毒肽、羧基二羟鬼笔毒肽和二羟鬼笔毒肽)的新方法。尿液样品经高速离心后,直接注入TurboFlow在线净化-液相色谱-串联质谱进行分析。对影响TF在线净化的参数如TF净化柱、上样溶剂、洗脱溶剂、转移流速、转移时间等进行了优化。在优化后的实验条件下,以TurboFlow^TMCyclone柱(50 mm×0.5 mm)为净化柱,Hypersil GOLD C₁₈柱(100 mm×2.1 mm)为分析柱,甲醇和4 mmol/L乙酸铵为流动相进行梯度洗脱,电喷雾正离子选择反应监测(SRM)模式下进行检测,基质匹配外标法定量。结果表明,鹅膏肽类毒素在1.0~50.0 μg/L范围内呈现良好的线性关系,相关系数均可达到0.997以上。方法的检出限为0.15~0.3 μg/L,定量限为0.5~1.0 μg/L。在2.0、5.0和10.0 μg/L的加标水平下,5种鹅膏肽类毒素的日内和日间回收率分别为87.0%~108.6%和86.8%~112.7%,日内、日间相对标准偏差(RSD)均小于14.5%。该检测方法准确、快速、灵敏度高、易操作,适用于公共卫生应急检测或临床中毒病因识别检测。

关键词: 在线净化, 液相色谱-串联质谱法, 鹅膏肽类毒素, 蘑菇毒素, 尿液

Abstract:

Amanita peptide toxins are cyclic polypeptide mushroom toxins that can cause acute liver damage. The fatality rate associated with these toxins is very high. Monitoring the concentration of amanita peptide toxins in human urine can provide valuable information for early clinical diagnosis and treatment. Therefore, a TurboFlow online clean-up-liquid chromatography-triple quadrupole mass spectrometry (TF-LC-MS/MS) method was established for the simultaneous quantitative determination of five amanita peptide toxins (α-amanitin, β-amanitin, γ-amanitin, phallacidin, and phalloidin) in human urine. After pre-treatment with high-speed centrifugation, urine samples were analyzed using TF-LC-MS/MS. The main factors influencing purification efficiency, including the TF column, loading solution, eluting solution, transfer flow, and transfer time, were optimized in this study. Under the optimized experimental conditions, the analytes were purified using a TurboFlow^TM Cyclone column (50 mm×0.5 mm) and separated on a Hypersil GOLD C₁₈ column (100 mm×2.1 mm) using the mobile phases of methanol and 4 mmol/L aqueous ammonium acetate solution with gradient elution. The analytes were detected in selected reaction monitoring (SRM) mode via positive electrospray ionization. Matrix-matched external standard calibration was used for quantitation. The linear range of the method ranged from 1.0 μg/L to 50.0 μg/L for all five amanita peptide toxins, with correlation coefficients (r²) higher than 0.997. The limits of detection were 0.15-0.3 μg/L and the limits of quantification (LOQs) were 0.5-1.0 μg/L for the five amanita peptide toxins in urine. The intra-day and inter-day recoveries of amanita peptide toxins were 87.0%-108.6% and 86.8%-112.7%, respectively, at the spiked levels of 2.0, 5.0, and 10.0 μg/L. The intra-day and inter-day relative standard deviations (RSDs) were less than 14.5%. The method is accurate, rapid, sensitive, easy to operate, and can satisfy the requirements of public health emergency testing or clinical poisoning testing.

Key words: online-cleanup, liquid chromatography-tandem mass spectrometry (LC-MS/MS), amanita peptide toxins, mushroom toxins, urine

中图分类号:

O658

方力, 邱凤梅, 余新威. TurboFlow在线净化-液相色谱-串联质谱法快速检测人尿中鹅膏肽类毒素[J]. 色谱, 2021, 39(3): 338-345.

FANG Li, QIU Fengmei, YU Xinwei. Determination of amanita peptide toxins in human urine by TurboFlow online clean-up-liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2021, 39(3): 338-345.

图/表 7

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Step No.	Start time/ min	Step time/ min	Function	Loading pump					Eluting pump
Step No.	Start time/ min	Step time/ min	Function	Flow rate/ (mL/min)	φ(0.1% FA)/%	φ(MeOH)/ %	φ(H₂O)/ %	φ(ACN)/ %	Flow rate/ (mL/min)	φ(4 mmol/L CH₃COONH₄)/%	φ(MeOH)/ %
1	0.0	1.0	loading	1.5	100	0	0	0	0.3	95	5
2	1.0	1.0	transferring	0.1	80	0	0	20	0.2	95	5
3	2.0	2.0	washing	1.5	0	0	90	10	0.3	80	20
4	4.0	1.5	washing	1.5	0	0	0	100	0.3	60	40
5	5.5	1.0	washing	1.5	0	100	0	0	0.3	40	60
6	6.5	0.5	washing	1.5	0	0	0	100	0.3	10	90
7	7.0	2.0	filling the loop	1.5	80	0	0	20	0.3	10	90
8	9.0	0.2	equilibrating	1.5	100	0	0	0	0.3	95	5
9	9.2	1.8	equilibrating	1.5	100	0	0	0	0.3	95	5

Step No.	Start time/ min	Step time/ min	Function	Loading pump					Eluting pump
Step No.	Start time/ min	Step time/ min	Function	Flow rate/ (mL/min)	φ(0.1% FA)/%	φ(MeOH)/ %	φ(H₂O)/ %	φ(ACN)/ %	Flow rate/ (mL/min)	φ(4 mmol/L CH₃COONH₄)/%	φ(MeOH)/ %
1	0.0	1.0	loading	1.5	100	0	0	0	0.3	95	5
2	1.0	1.0	transferring	0.1	80	0	0	20	0.2	95	5
3	2.0	2.0	washing	1.5	0	0	90	10	0.3	80	20
4	4.0	1.5	washing	1.5	0	0	0	100	0.3	60	40
5	5.5	1.0	washing	1.5	0	100	0	0	0.3	40	60
6	6.5	0.5	washing	1.5	0	0	0	100	0.3	10	90
7	7.0	2.0	filling the loop	1.5	80	0	0	20	0.3	10	90
8	9.0	0.2	equilibrating	1.5	100	0	0	0	0.3	95	5
9	9.2	1.8	equilibrating	1.5	100	0	0	0	0.3	95	5

Analyte	Scan mode	Precursor ion (m/z)	Product ion (m/z)	S-lens/ V	CE/ eV
α-Amanitin	ESI⁺	919.6	259.0^*/86.0	180	33/48
β-Amanitin	ESI⁺	920.6	259.0^*/86.0	180	32/46
γ-Amanitin	ESI⁺	903.6	243.0^*/86.0	180	37/54
Phallacidin	ESI⁺	847.6	157.0^*/329.9	160	50/28
Phalloidin	ESI⁺	789.6	157.0^*/330.0	160	45/33

Analyte	Scan mode	Precursor ion (m/z)	Product ion (m/z)	S-lens/ V	CE/ eV
α-Amanitin	ESI⁺	919.6	259.0^*/86.0	180	33/48
β-Amanitin	ESI⁺	920.6	259.0^*/86.0	180	32/46
γ-Amanitin	ESI⁺	903.6	243.0^*/86.0	180	37/54
Phallacidin	ESI⁺	847.6	157.0^*/329.9	160	50/28
Phalloidin	ESI⁺	789.6	157.0^*/330.0	160	45/33

Toxin	Linear range/(μg/L)	Solvent		Urine
Toxin	Linear range/(μg/L)	Curve	R²	Curve	R²	ME/%	LOD/(μg/L)	LOQ/(μg/L)
α-Amanitin	1.0-50.0	y=918.615x -191.188	0.9994	y=578.167x-298.264	0.9990	-37.1	0.3	1.0
β-Amanitin	1.0-50.0	y=1018.51x-34.4314	0.9970	y=481.957x-61.4861	0.9998	-52.7	0.3	1.0
γ-Amanitin	1.0-50.0	y=1371.64x -126.402	0.9996	y=915.107x-172.278	0.9990	-33.3	0.3	1.0
Phallacidin	1.0-50.0	y=1794.66x-587.165	0.9994	y=942.94x-126.634	0.9999	-47.5	0.3	1.0
Phalloidin	1.0-50.0	y=4240.93x+3.11143	0.9992	y=2899.95x+548.874	0.9995	-31.6	0.15	0.5