Further development of a liquid chromatography–high‐resolution mass spectrometry/mass spectrometry‐based strategy for analyzing eight biomarkers in human urine indicating toxic mushroom or Ricinus communis ingestions

Abstract

Recently, we presented a strategy for analysis of eight biomarkers in human urine to verify toxic mushroom or Ricinus communis ingestions. However, screening for the full panel is not always necessary. Thus, we aimed to develop a strategy to reduce analysis time and by focusing on two sets of analytes. One set (A) for biomarkers of late-onset syndromes, such as phalloides syndrome or the syndrome after castor bean intake. Another set (B) for biomarkers of early-onset syndromes, such as pantherine-muscaria syndrome and muscarine syndrome. Both analyses should be based on hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry (MS)/MS (HILIC-HRMS/MS). For A, urine samples were prepared by liquid-liquid extraction using dichloromethane and subsequent solid-phase extraction of the aqueous supernatant. For B urine was precipitated using acetonitrile. Method A was validated for ricinine and αand β-amanitin and method B for muscarine, muscimol, and ibotenic acid according to the specifications for qualitative analytical methods. In addition, robustness of recovery and normalized matrix factors to matrix variability measured by urinary creatinine was tested. Moreover, applicability was tested using 10 urine samples from patients after suspected mushroom intoxication. The analytes αand β-amanitin, muscarine, muscimol, and ibotenic acid could be successfully identified. Finally, psilocin-O-glucuronide could be identified in two samples and unambiguously distinguished from bufotenine-O-glucuronide via their MS 2 patterns. In summary, the current workflow offers several advantages towards the previous method, particularly being more labor-, time-, and costefficient, more robust, and more sensitive.

Simultaneous Determination of Multi-Class Mushroom Toxins in Mushroom and Biological Liquid Samples Using LC-MS/MS

“…The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/separations11060183/s1, Table S1: Gradient mobile phase conditions of HSS T3 column; Table S2: Gradient mobile phase conditions of CSH PFP column; Table S3: Gradient mobile phase conditions of Trinity P1 column; Table S4: Gradient mobile phase conditions of ACE C18-Amide column; Table S5: Gradient mobile phase conditions of BEH Amide column; Table S6: Literature summary of mushroom toxins detection using LC-MS/MS [14][15][16]19,[35][36][37].…”

Application to several suspected poisoning cases of a validated analytical method for the determination of muscarine in human biological matrices using liquid chromatography with high‐resolution mass spectrometry detection

“…Some publications reported methods for the determination of muscarine in mushrooms using different technologies such as TLC‐MS, 11 LC‐UV detection, 11 and LC–MS/MS 11–13 . For assays developed from human biological matrices, five methods have been published for urinary assays using LC–MS, LC–MS/MS, LC‐Q‐TOF 8,14–18 or electrophoresis‐MS/MS 19 with a limit of detection (LOD) ranging from 0.09 to 5 μg/L. Only one qualitative method in a blood sample has been reported, based on an SPE and LC‐Q‐TOF leading to an LOD of 1.8 μg/L 16 …”