[Determination of amanita peptide toxins in human urine by TurboFlow online clean-up-liquid chromatography-tandem mass spectrometry].

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, ß-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 TurboFlowTM 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.

Experimental study on acute hepatotoxicity in beagles induced by Amanita exitialis / 中华急诊医学杂志

Objective To establish acute hepatotoxic model induced by Amanita exitialis and to study the characteristics of acute toxic liver failure induced by mushrooms containing peptide toxins,in hope for providing some help to experimental research on poisoning induced by mushrooms containing peptide toxins.Methods UPLC-MS/MS (Ultra performance liquid chromatography-tandem mass spectrometry) method was used to detect peptide toxins in Amanita exitialis.To establish acute toxic liver hepatic failure model,the beagles were fed with 60 mg/kg of lyophilized powder of Amanita exitialis fungus which encapsulated in starch capsules.Toxic sighs were observed,coagulation function,hepatic and renal function,liver histopathological morphology,peptide toxin concentration in plasma and urine were detected during the experiment.Results Total peptide toxins in Amanita exitialis was (3 482.6 ± 124.94) mg/ kg.All the beagles had toxic signs including vomiting and diarrhea in 12-48 h after ingestion.On 24 h after ingestion,the beagles' ALT,AST,TBIL,ALP,PT and APTT levels increased obviously.On 36 h after ingestion,the beagles' ALT,AST,PT and APTT values reached their peaks (ALT:283.2 ± 112.9 Kallmann unit;AST:223.9 ±93.8 Kallmann units;PT:132.9 ± 152.6 s;APTT:131.4 ± 153.9 s).On 48 h after ingestion,the beagles' TBIL and ALP levels reached their peaks (TBIL:23.3 ± 14.6 mol/L;ALP:274.5 ± 115.5 U/L).The beagles' TBIL,TP and APTT returned to normal 1 week after ingestion,their ALT,AST and ALP levels returned to normal 3 weeks after ingestion.Three dogs died during 24-72 h after ingestion.Liver histopathological morphology study showed hemorrhagic necrosis of hepatocytes.Peptide toxins can be detected in plasma within 24 h after ingestion.Peptide toxins can be detected in urine within 96 h after ingestion.Conclusion Amanita peptide toxins can cause hemorrhagic necrosis of liver cells and lead to acute liver failure.This model is consistent with clinical pathophysiological process of acute toxic liver failure induced by mushrooms containing peptide toxins,and it can be applied to the study of diagnosis and treatment of poisoning induced by mushrooms containing peptide toxins.