A Simple and High-Throughput Analysis of Amatoxins and Phallotoxins in Human Plasma, Serum and Urine Using UPLC-MS/MS Combined with PRiME HLB μElution Platform

Abstract

Amatoxins and phallotoxins are toxic cyclopeptides found in the genus Amanita and are among the predominant causes of fatal food poisoning in China. In the treatment of Amanita mushroom poisoning, an early and definite diagnosis is necessary for a successful outcome, which has prompted the development of protocols for the fast and confirmatory determination of amatoxins and phallotoxins in human biological fluids. For this purpose, a simple, rapid and sensitive multiresidue UPLC-MS/MS method for the simultaneous determination of α-amanitin, β-amanitin, γ-amanitin, phalloidin (PHD) and phallacidin (PCD) in human plasma, serum and urine was developed and validated. The diluted plasma, serum and urine samples were directly purified with a novel PRiME technique on a 96-well μElution plate platform, which allowed high-throughput sample processing and low reagent consumption. After purification, a UPLC-MS/MS analysis was performed using positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. This method fulfilled the requirements of a validation test, with good results for the limit of detection (LOD), lower limit of quantification (LLOQ), accuracy, intra- and inter-assay precision, recovery and matrix effects. All of the analytes were confirmed and quantified in authentic plasma, serum and urine samples obtained from cases of poisoning using this method. Using the PRiME μElution technique for quantification reduces labor and time costs and represents a suitable method for routine toxicological and clinical emergency analysis.

Analysis of α- and β-amanitin in Human Plasma at Subnanogram per Milliliter Levels by Reversed Phase Ultra-High Performance Liquid Chromatography Coupled to Orbitrap Mass Spectrometry

“…Li et al provided the following stability data of α-amanitin in plasma: short-term, long-term, freeze-thaw (3 cycles), and postpreparative storage stability ( n = 6; 3 concentration levels evaluated for each experiment) [ 50 ]. In addition, Zhang et al also included β-amanitin in their stability studies in plasma, serum, and urine ( n = 3; after 1, 2, 4, and 8 weeks) [ 33 ]. Maurer et al provided most of the required stability data for the toxins and the IS γ-amanitin methyl ether as well as long-term stability in stock solutions for 6 months [ 35 ].…”

“…Urine is usually the matrix of choice for such analysis due to higher concentrations compared to other body fluids [26]. Several methods were published for their analysis in urine by using LC-MS, LC-(HR)MS/MS, capillary zone electrophoresis, HPLC, radioimmunoassay, ELISA, and recently a lateral flow immunoassay [27][28][29][30][31][32][33][34][35][36][37]. Also, blood plasma or serum can be used as demonstrated earlier [33,34,[38][39][40][41].…”

“…Several methods were published for their analysis in urine by using LC-MS, LC-(HR)MS/MS, capillary zone electrophoresis, HPLC, radioimmunoassay, ELISA, and recently a lateral flow immunoassay [ 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. Also, blood plasma or serum can be used as demonstrated earlier [ 33 , 34 , 38 , 39 , 40 , 41 ]. To our knowledge, only one study (in English) was published where the toxins were also found in human plasma or serum samples by using mass spectrometry [ 41 ].…”

Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine

“…Current methods of chemical detection of amatoxins in urine include liquid chromatography-mass spectrometry (LC-MS) methods [17][18][19][20][21][22][23] and antibody-based enzyme-linked immunosorbent assays (ELISAs) [24,25]. LC-MS methods require sample extraction and expensive equipment, while ELISA methods require specialized equipment.…”

Determination of a broad spectrum of endocrine-disrupting pesticides in fish samples by UHPLC-MS/MS using the pass-through cleanup approach

“…In the cleanup method, no cartridge conditioning is required or is performed, and the extracts are passed through the Oasis PriME HLB cartridge and collected. According to our knowledge, there is a little previous literature about the PRiME pass‐through cleanup approach except extraction of aflatoxins and amatoxins and phallotoxins in human serum. Therefore, we think that the PRiME pass‐through cleanup approach could be used for the simultaneous extraction of selected endocrine‐disrupting pesticides in fish samples.…”