在线固相萃取-液相色谱-串联质谱法检测蘑菇中毒患者尿液中痕量α -鹅膏毒肽

doi:10.3724/SP.J.1123.2020.03010

摘要/Abstract

摘要：

建立了在线固相萃取-液相色谱-串联质谱（online SPE-LC-MS/MS）测定蘑菇中毒患者尿液中痕量α -鹅膏毒肽的分析方法。样品经甲酸酸化的乙腈-甲醇（5：1，v/v）沉淀蛋白质，反相液液微萃取去除样品提取液中的有机溶剂，毒素经ODS微柱（5 mm×2.1 mm，5 μm）在线SPE净化，XBridge^TM BEH C₁₈ 色谱柱（150 mm×3.0 mm，2.5 μm）分离，MS/MS测定。采用基于定量环的快速阀切换技术作为在线SPE和LC-MS/MS模块的接口，使得两个分离模块互相独立，无论是流动相还是压力，都不会互相干扰，保证了系统的稳定性；在线系统的精准净化，有效消除了后续质谱检测的基质效应，确保了尿液中痕量水平α -鹅膏毒肽的定性定量检测。尿液中α -鹅膏毒肽在0.1~50 μg/L范围内线性关系良好，相关系数（r ² ）为0.9983；检出限（LOD）为0.03 μg/L；α -鹅膏毒肽的加标（0.1、2.0和20 μg/L）平均回收率为84.3%~91.7%，相对标准偏差（RSD）为3.8%~7.2%。体内鹅膏毒肽代谢迅速，生物基质中痕量水平毒素的检测是其中毒实验室鉴定的主要难题，通过实际样品检测，证明该法操作简单，准确、灵敏；溶剂沉淀蛋白质和反相液液微萃取去除有机相和脂溶性基质的简单操作，可以作为水溶性毒素在线SPE-LC-MS/MS检测时快速且有效的配套前处理方法；基于在线SPE精准净化技术，可以实现尿液中α -鹅膏毒肽的高灵敏度测定（LOD为0.03 μg/L），解决了中毒时患者体内痕量水平α -鹅膏毒肽定性确证的难题，部分患者α -鹅膏毒肽中毒实验室鉴定的时间可以扩展到90 h以上；同时，痕量水平的定量检测技术，可以为中毒后迅速代谢的α -鹅膏毒肽在体内的剂量反应关系研究提供可靠的技术支撑。

关键词: 液相色谱-串联质谱, 在线固相萃取, α -鹅膏毒肽, 尿液, 痕量水平, 蘑菇中毒, 食源性疾病

Abstract:

An analytical method was established for the determination of trace α -amanitin in the urine of patients suffering from mushroom poisoning by online solid phase extraction-liquid chromatography-tandem mass spectrometry (online SPE-LC-MS/MS). The sample was protein precipitated with formic acid acidified acetonitrile-methanol (5:1, v/v). Reversed-phase liquid-liquid microextraction was used to remove the organic solvent from the sample extract. The toxin was purified by online SPE using an ODS micro column (5 mm×2.1 mm, 5 μm), and separated on an XBridge^TM BEH C₁₈ column (150 mm×3.0 mm, 2.5 μm). Finally, the toxin was measured by MS/MS in the negative electrospray ionization (ESI^- ) mode. Multiple reaction monitoring (MRM) was used, and the conditions were m /z 917.4>205.1 (quantitative ion transition) and m /z 917.4>257.1. Collision energy for both transitions was 55 eV. A fast valve-switching technique with a quantitative loop was used as an interface between the online SPE and LC-MS/MS modules. The two modules were independent, neither the mobile phase nor the pressure would interfere with each other, thus ensuring the stability of the system. Precise purification by the online system could effectively eliminate the matrix effects in the subsequent MS detection. Weak matrix suppression effects were found, with results of 88.7%-96.5%. The linear range of α -amanitin in urine was 0.1-50 μg/L with a correlation coefficient (r ² ) of 0.9983. The limit of detection (LOD) and limit of quantification (LOQ) in the sample matrix were 0.03 μg/L and 0.1 μg/L, respectively. The average recoveries at three spiked levels (0.1, 2.0 and 20 μg/L) were 84.3%-91.7% with relative standard deviations (RSDs) of 3.8%-7.2%. The accuracy and precision were evaluated using quality control samples with toxin contents of 0.1 μg/L (LOQ), 0.2 μg/L (2-fold LOQ), 2.0 μg/L (medium level), and 20 μg/L (high level). The calculated average intra-day accuracy was 85.1%-96.0% with the precision of 4.1%-7.8%. The inter-day accuracy was 82.9%-94.8% with the precision of 5.0%-9.5%. The specificity of the method was verified by negative samples derived from patients who suffered only gastroenteritis poisoning, without hepatotoxic symptoms. α -Amanitin was found in urine samples from nine mushroom poisoning patients with hepatotoxic symptoms. The sampling time ranged from 19 h to 92 h. The toxin contents were 0.11-53.1 μg/L. For patients with a high intake of poisonous mushrooms, the toxin content was 53.1 μg/L in a patient's urine sampled 19 h after accidental consumption and 0.19 μg/L in another patient's urine sampled 92 h after poisoning. The content of α -amanitin was only 0.53 μg/L in the urine sample obtained 23 h after consumption for a patient with low intake and 0.11 μg/L in the urine sampled from another patient 40 h after poisoning. Amatoxins can metabolize rapidly in vivo. The laboratory identification of amatoxin poisoning requires a method for trace-level analysis in the biological matrix. It is proved that this method is simple, accurate and sensitive by the application to the analysis of actual samples. The protein precipitation and reversed-phase liquid-liquid microextraction steps are fast and simple. Hence, they can be used as a rapid and effective pre-treatment method for online SPE-LC-MS/MS analysis of water-soluble toxins in biomaterial matrix. Highly sensitive analysis of α -amanitin in urine can be obtained using a precise purification technology via online SPE in this study. The problem of qualitative confirmation of the toxin at trace levels (0.03 μg/L) after poisoning can be solved. The laboratory identification time for amatoxin poisoning in some patients exceeds 90 h. The developed analytical method at trace level (0.1 μg/L of LOQ) can provide reliable technical support for establishing the dose-response relationship of α -amanitin in vivo. It can satisfy for the determination of trace α -amanitin in urine samples from patients with hepatotoxic mushroom poisoning.

Key words: liquid chromatography-tandem mass spectrometry (LC-MS/MS), online solid phase extraction (online SPE), α -amanitin, urine, trace level, mushroom poisoning, foodborne disease

徐小民, 张京顺, 蔡增轩, 孟真, 黄百芬, 陈苘. 在线固相萃取-液相色谱-串联质谱法检测蘑菇中毒患者尿液中痕量α -鹅膏毒肽[J]. 色谱, 2020, 38(11): 1281-1287.

Xiaomin XU, Jingshun ZHANG, Zengxuan CAI, Zhen MENG, Baifen HUANG, Qing CHEN. Determination of trace α -amanitin in urine of mushroom poisoning patient by online solid phase extraction-liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2020, 38(11): 1281-1287.

图/表 7

图1 在线SPE-LC-MS/MS系统流路切换示意图

Fig. 1 Diagram of the flow path switching in the online SPE-LC-MS/MS system

图2 在线SPE洗脱梯度程序中2.0~3.0 min时流动相中甲醇体积分数分别为30%、35%和40%时α -鹅膏毒肽的洗脱曲线

Fig. 2 Elution curves of α -amanitin with volume fractions of methanol of 30%, 35%, and 40% at 2.0-3.0 min in the online SPE elution gradient procedure

图3 在线和离线SPE净化后尿液中α -鹅膏毒肽的色谱图

Fig. 3 Chromatograms of α -amanitin cleaned by online SPE and offline SPE a. blank matrix; b. positive sample (0.53 μg/L); c. positive sample (0.53 μg/L).

图4 α -鹅膏毒肽的全扫描质谱图和二级质谱图

Fig. 4 Full scan mass and MS/MS spectra of α -amanitin

表1 α -鹅膏毒肽在6种空白尿液中的基质效应(n =6)

Table 1 Matrix effects (MEs) of α -amanitin in the six blank urine samples (n =6)

Source of the blank matrix	0.1 μg/L		50 μg/L
Source of the blank matrix	ME/%	RSD/%	ME/%	RSD/%
* Mushroom poisoning patient only with normal gastroenteritis symptoms.
Female volunteer 1	96.5	3.6	92.9	3.8
Female volunteer 2	91.5	4.1	91.5	3.4
Male volunteer 1	93.4	5.6	88.7	4.8
Male volunteer 2	89.2	4.3	94.0	3.7
Patient 1^*	96.1	4.7	91.5	2.7
Patient 2^*	89.0	5.0	95.4	3.9

表2 α -鹅膏毒肽的日内和日间准确度和精密度

Table 2 Intra- and inter-day accuracies and precisions of α -amanitin

Level/(μg/L)	Intra-day (n =6)		Inter-day (n =3)
Level/(μg/L)	Accuracy/%	Precision/%	Accuracy/%	Precision/%
0.1	85.1	7.8	82.9	9.5
0.2	88.2	7.0	86.5	8.3
2.0	96.0	4.7	94.8	5.5
20	90.3	4.1	90.1	5.0

图5 9名中毒患者尿液中α -鹅膏毒肽的含量(n =3)

Fig. 5 Contents of α -amanitin in the urines of the nine poisoning patients (n =3)

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