High-Confidence Qualitative Identification of Organophosphorus Nerve Agent Adducts to Human Butyrylcholinesterase

In this study, a data-dependent, high-resolution tandem mass spectrometry (ddHRMS/MS) method capable of detecting all organophosphorus nerve agent (OPNA) adducts to human butyrylcholinesterase (BChE) was developed. After an exposure event, immunoprecipitation from blood with a BChE-specific antibody and digestion with pepsin produces a nine amino acid peptide containing the OPNA adduct. Signature product ions of this peptic BChE nonapeptide (FGES*AGAAS) offer a route to broadly screen for OPNA exposure. Taking this approach on an HRMS instrument identifies biomarkers, including unknowns, with high mass accuracy. Using a set of pooled human sera exposed to OPNAs as quality control (QC) materials, the developed method successfully identified precursor ions with <1 ppm and tied them to signature product ions with <5 ppm deviation from their chemical formulas. This high mass accuracy data from precursor and product ions, collected over 23 independent immunoprecipitation preparations, established method operating limits. QC data and experiments with 14 synthetic reference peptides indicated that reliable qualitative identification of biomarkers was possible for analytes >15 ng/mL. The developed method was applied to a convenience set of 96 unexposed serum samples and a blinded set of 80 samples treated with OPNAs. OPNA biomarkers were not observed in convenience set samples and no false positive or negative identifications were observed in blinded samples. All biomarkers in the blinded serum set >15 ng/mL were correctly identified. For the first time, this study reports a ddHRMS/MS method capable of complementing existing quantitative methodologies and suitable for identifying exposure to unknown organophosphorus agents

Quantification of Metabolites for Assessing Human Exposure to Soapberry Toxins Hypoglycin A and Methylenecyclopropylglycine

Ingestion of soapberry fruit toxins hypoglycin A and methylenecyclopropylglycine has been linked to public health challenges worldwide. In 1976, over 100 years after Jamaican vomiting sickness (JVS) was first reported, the cause of JVS was linked to the ingestion of the toxin hypoglycin A produced by ackee fruit. A structural analogue of hypoglycin A, methylenecyclopropylglycine (MCPG), was implicated as the cause of an acute encephalitis syndrome (AES). Much of the evidence linking hypoglycin A and MCPG to these diseases has been largely circumstantial due to the lack of an analytical method for specific metabolites. This study presents an analytical approach to identify and quantify specific urine metabolites for exposure to hypoglycin A and MCPG. The metabolites are excreted in urine as glycine adducts methylenecyclopropylacetyl-glycine (MCPA-Gly) and methylenecyclopropylformyl-glycine (MCPF-Gly). These metabolites were processed by isotope dilution, separated by reverse-phase liquid chromatography, and monitored by electrospray ionization tandem mass spectrometry. The analytical response ratio was linearly proportional to the concentration of MCPF-Gly and MCPA-Gly in urine from 0.10 to 20 μg/mL with a correlation coefficient of <i>r</i> > 0.99. The assay demonstrated accuracy ≥80% and precision ≤20% RSD across the calibration range. This method has been applied to assess exposure to hypoglycin A and MCPG as part of a larger public health initiative and was used to provide the first reported identification of MCPF-Gly and MCPA-Gly in human urine

Quantification of Toxins in Soapberry (Sapindaceae) Arils: Hypoglycin A and Methylenecyclopropylglycine

Methylenecyclopropylglycine (MCPG) and hypoglycin A (HGA) are naturally occurring amino acids found in some soapberry fruits. Fatalities have been reported worldwide as a result of HGA ingestion, and exposure to MCPG has been implicated recently in the Asian outbreaks of hypoglycemic encephalopathy. In response to an outbreak linked to soapberry ingestion, the authors developed the first method to simultaneously quantify MCPG and HGA in soapberry fruits from 1 to 10 000 ppm of both toxins in dried fruit aril. Further, this is the first report of HGA in litchi, longan, and mamoncillo arils. This method is presented to specifically address the laboratory needs of public-health investigators in the hypoglycemic encephalitis outbreaks linked to soapberry fruit ingestion