PMID- 34213098 OWN - NLM STAT- MEDLINE DCOM- 20210727 LR - 20210727 IS - 1872-2059 (Electronic) IS - 1000-8713 (Linking) VI - 38 IP - 11 DP - 2020 Nov 8 TI - [Determination of trace alpha-amanitin in urine of mushroom poisoning patient by online solid phase extraction-liquid chromatography-tandem mass spectrometry]. PG - 1281-1287 LID - 10.3724/SP.J.1123.2020.03010 [doi] AB - An analytical method was established for the determination of trace alpha-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 mmx2.1 mm, 5 mum), and separated on an XBridge(TM) BEH C(18) column (150 mmx3.0 mm, 2.5 mum). 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 alpha-amanitin in urine was 0.1-50 mug/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 mug/L and 0.1 mug/L, respectively. The average recoveries at three spiked levels (0.1, 2.0 and 20 mug/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 mug/L (LOQ), 0.2 mug/L (2-fold LOQ), 2.0 mug/L (medium level), and 20 mug/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. alpha-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 mug/L. For patients with a high intake of poisonous mushrooms, the toxin content was 53.1 mug/L in a patient's urine sampled 19 h after accidental consumption and 0.19 mug/L in another patient's urine sampled 92 h after poisoning. The content of alpha-amanitin was only 0.53 mug/L in the urine sample obtained 23 h after consumption for a patient with low intake and 0.11 mug/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 alpha-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 mug/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 mug/L of LOQ) can provide reliable technical support for establishing the dose-response relationship of alpha-amanitin in vivo. It can satisfy for the determination of trace alpha-amanitin in urine samples from patients with hepatotoxic mushroom poisoning. FAU - Xu, Xiaomin AU - Xu X AD - Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China. FAU - Zhang, Jingshun AU - Zhang J AD - Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China. FAU - Cai, Zengxuan AU - Cai Z AD - Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China. FAU - Meng, Zhen AU - Meng Z AD - Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China. FAU - Huang, Baifen AU - Huang B AD - Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China. FAU - Chen, Qing AU - Chen Q AD - Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China. LA - chi PT - Journal Article PL - China TA - Se Pu JT - Se pu = Chinese journal of chromatography JID - 9424804 RN - 0 (Alpha-Amanitin) SB - IM MH - Alpha-Amanitin/*urine MH - Chromatography, High Pressure Liquid MH - Humans MH - Mushroom Poisoning/*urine MH - Solid Phase Extraction MH - Tandem Mass Spectrometry OTO - NOTNLM OT - foodborne disease OT - liquid chromatography-tandem mass spectrometry (LC-MS/MS) OT - mushroom poisoning OT - online solid phase extraction (online SPE) OT - trace level OT - urine OT - alpha-amanitin EDAT- 2021/07/03 06:00 MHDA- 2021/07/28 06:00 CRDT- 2021/07/02 09:12 PHST- 2021/07/02 09:12 [entrez] PHST- 2021/07/03 06:00 [pubmed] PHST- 2021/07/28 06:00 [medline] AID - 10.3724/SP.J.1123.2020.03010 [doi] PST - ppublish SO - Se Pu. 2020 Nov 8;38(11):1281-1287. doi: 10.3724/SP.J.1123.2020.03010.