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.

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 α- and ß-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 α- and ß-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 MS2 patterns. In summary, the current workflow offers several advantages towards the previous method, particularly being more labor-, time-, and cost-efficient, more robust, and more sensitive.

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

Amatoxins are known to be one of the main causes of serious to fatal mushroom intoxication. Thorough treatment, analytical confirmation, or exclusion of amatoxin intake is crucial in the case of any suspected mushroom poisoning. Urine is often the preferred matrix due to its higher concentrations compared to other body fluids. If urine is not available, analysis of human blood plasma is a valuable alternative for assessing the severity of intoxications. The aim of this study was to develop and validate a liquid chromatography (LC)-high resolution tandem mass spectrometry (HRMS/MS) method for confirmation and quantitation of α- and ß-amanitin in human plasma at subnanogram per milliliter levels. Plasma samples of humans after suspected intake of amatoxin-containing mushrooms should be analyzed and amounts of toxins compared with already published data as well as with matched urine samples. Sample preparation consisted of protein precipitation, aqueous liquid-liquid extraction, and solid-phase extraction. Full chromatographical separation of analytes was achieved using reversed-phase chromatography. Orbitrap-based MS allowed for sufficiently sensitive identification and quantification. Validation was successfully carried out, including analytical selectivity, carry-over, matrix effects, accuracy, precision, and dilution integrity. Limits of identification were 20 pg/mL and calibration ranged from 20 pg/mL to 2000 pg/mL. The method was applied to analyze nine human plasma samples that were submitted along with urine samples tested positive for amatoxins. α-Amanitin could be identified in each plasma sample at a range from 37-2890 pg/mL, and ß-amanitin was found in seven plasma samples ranging from <20-7520 pg/mL. A LC-HRMS/MS method for the quantitation of amatoxins in human blood plasma at subnanogram per milliliter levels was developed, validated, and used for the analysis of plasma samples. The method provides a valuable alternative to urine analysis, allowing thorough patient treatment but also further study the toxicokinetics of amatoxins.

Development and application of a strategy for analyzing eight biomarkers in human urine to verify toxic mushroom or ricinus communis ingestions by means of hydrophilic interaction LC coupled to HRMS/MS.

The analytical proof of a toxic mushroom and/or plant ingestion at an early stage of a suspected intoxication can be crucial for fast therapeutic decision making. Therefore, comprehensive analytical procedures need to be available. This study aimed to develop a strategy for the qualitative analysis of α- and ß-amanitin, psilocin, bufotenine, muscarine, muscimol, ibotenic acid, and ricinine in human urine by means of hydrophilic interaction liquid chromatography-high resolution MS/MS (HILIC-HRMS/MS). Urine samples were prepared by hydrophilic-phase liquid-liquid extraction using dichloromethane and subsequent solid-phase extraction and precipitation, performed in parallel. Separation and identification of the biomarkers were achieved by HILIC using acetonitrile and methanol as main eluents and Orbitrap-based mass spectrometry, respectively. The method was validated as recommended for qualitative procedures and tests for selectivity, carryover, and extraction recoveries were included to also estimate the robustness and reproducibility of the sample preparation. Limits of identification were 1 ng/mL for α- and ß-amanitin, 5 ng/mL for psilocin, bufotenine, muscarine, and ricinine, and 1500 ng/mL and 2000 ng/mL for ibotenic acid and muscimol, respectively. Using γ-amanitin, l-tryptophan-d5, and psilocin-d10 as internal standards, compensation for variations of matrix effects was shown to be acceptable for most of the toxins. In eight urine samples obtained from intoxicated individuals, α- and ß-amanitin, psilocin, psilocin-O-glucuronide, muscimol, ibotenic acid, and muscarine could be identified. Moreover, psilocin-O-glucuronide and bufotenine-O-glucuronide were found to be suitable additional targets. The analytical strategy developed was thus well suited for analyzing several biomarkers of toxic mushrooms and plants in human urine to support therapeutic decision making in a clinical toxicology setting. To our knowledge, the presented method is by far the most comprehensive approach for identification of the included biomarkers in a human matrix.

Toxicokinetics and Analytical Toxicology of Flualprazolam: Metabolic Fate, Isozyme Mapping, Human Plasma Concentration and Main Urinary Excretion Products.

An increasing number of benzodiazepine-type compounds are appearing on the new psychoactive substances market. 8-Chloro-6-(2-fluorophenyl)-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine (well known as flualprazolam) represents a potent 'designer benzodiazepine' that has been associated with sedation, loss of consciousness, memory loss and disinhibition. The aims of the present study were to tentatively identify flualprazolam metabolites using in vitro incubations with pooled human liver S9 fraction or HepaRG cells by means of liquid-chromatography-high resolution tandem mass spectrometry. Isozymes involved in phase I and II biotransformation were identified in vitro. Results were then confirmed using human biosamples of an 18-year old male who was admitted to the emergency department after suspected flualprazolam ingestion. Furthermore, the plasma concentration was determined using the standard addition method. Seven flualprazolam metabolites were tentatively identified. Several cytochrome P450 and UDP-glucuronosyltransferase isozymes, amongst them CYP3A4 and UGT1A4, were shown to be involved in flualprazolam biotransformation reactions, and an influence of polymorphisms as well as drug-drug or drug-food interactions cannot be excluded. Alpha-hydroxy flualprazolam glucuronide, 4-hydroxy flualprazolam glucuronide and the parent glucuronide were identified as most abundant signals in urine, far more abundant than the parent compound flualprazolam. These metabolites are thus recommended as urine-screening targets. If conjugate cleavage was performed during sample preparation, the corresponding phase I metabolites should be added as targets. Both hydroxy metabolites can also be recommended for blood screening. The flualprazolam plasma concentration determined in the intoxication case was as low as 8 µg/L underlining the need of analytical methods with sufficient sensitivity for blood-screening purposes.

Evaluation of novel organosilane modifications of paper spray mass spectrometry substrates for analyzing polar compounds.

Paper spray mass spectrometry (PSMS) is currently used in different analytical fields, but less effort has been made so far to use PSMS for highly polar compounds. Such analytes usually show poor performance in PSMS due to their high affinity for common paper substrates in addition to high matrix effects. In this study, strategies for hydrophobic modifications of commercially available paper substrates using ten different organosilanes were developed. The modified substrates were generated, characterized, and applied for PSMS analysis of polar toxins. By using the modified paper, PSMS performance of some of the toxins could be considerably increased, especially for orellanine, showing a more than 80-fold signal enhancement when substrates modified with chlorotrimethylsilane were used. For other toxins like ricinine, only small beneficial effects could be shown on PSMS performance when using modified substrates. Statistical equivalence tests showed sufficient ruggedness of the developed procedures also compared to conventional substrates. Thus, further systematic development of paper substrates modified with organosilane derivatives based on the presented study for application in PSMS should be encouraged.