Untargeted and targeted analysis of sarin poisoning biomarkers in rat urine by liquid chromatography and tandem mass spectrometry.

Chemical warfare agents continue to pose a real threat to humanity, despite their prohibition under the Chemical Weapons Convention. Sarin is one of the most toxic and lethal representatives of nerve agents. The methodology for the targeted analysis of known sarin metabolites has reached great heights, but little attention has been paid to the untargeted analysis of biological samples of victims exposed to this deadly poisonous substance. At present, the development of computational and statistical methods of analysis offers great opportunities for finding new metabolites or understanding the mechanisms of action or effect of toxic substances on the organism. This study presents the targeted LC-MS/MS determination of methylphosphonic acid and isopropyl methylphosphonic acid in the urine of rats exposed to a non-lethal dose of sarin, as well as the untarget urine analysis by LC-HRMS. Targeted analysis of polar acidic sarin metabolites was performed on a mixed-mode reversed-phase anion-exchange column, and untargeted analysis on a conventional reversed-phase C18 column. Isopropyl methylphosphonic acid was detected and quantified within 5 days after subcutaneous injection of sarin at a dose of 1/4 LD50. A combination of generalized additive mixed models and dose-response analysis with database searches using accurate mass of precursor ions and corresponding MS/MS spectra enabled us to propose new six potential biomarkers of biological response to exposure. The results confirm the well-known fact that sarin poisoning has a significant impact on the victims' metabolome, with inhibition of acetylcholinesterase being just the first step and trigger of the complex toxicodynamic response.

pH-Dependent Piecewise Linear Correlation of 1H,31P Chemical Shifts: Application in NMR Identification of Nerve Agent Metabolites in Urine Samples.

The NMR-observable nuclei of the acidic and basic compounds experience pH dependence in chemical shift. This phenomenon can be exploited in NMR titrations to determine p Ka values of compounds, or in pH measurement of solutions using dedicated pH reference compounds. On the other hand, this sensitivity can also cause problems in, for example, metabolomics, where slight changes in pH result in significant difficulties for peak alignment between spectra of set of samples for comparative analysis. In worst case, the pH sensitivity of chemical shifts can prevent unambiguous identification of compounds. Here, we propose an alternative approach for NMR identification of pH-sensitive analytes. The 1H and X (13C, 15N, 31P, ...) chemical shifts in close proximity to the acidic or basic functional group should, when presented as ordered pairs, express piecewise linear correlation with distinct slope, intercept, and range. We have studied the pH dependence of 1H and 31P chemical shifts of the CH3-P moiety in urinary metabolites of nerve agents sarin, soman and VX using 2D 1H-31P fast-HMQC spectroscopy. The 1H and 31P chemical shifts of these chemicals appear in very narrow range, and due to subtle changes in sample pH the identification on either 1H or 31P chemical shift alone is uncertain. However, if the observed 1H and 31P chemical shifts of the CH3-P moiety of individual compounds are presented as ordered pairs, they fall into distinct linear spaces, thus, facilitating identification with high confidence.

Determination of nerve agent metabolites in human urine by isotope-dilution gas chromatography-tandem mass spectrometry after solid phase supported derivatization.

A simple and sensitive method has been developed and validated for determining ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA), isobutyl methylphosphonic acid (iBuMPA), and pinacolyl methylphosphonic acid (PMPA) in human urine using gas chromatography-tandem mass spectrometry (GC-MS/MS) coupled with solid phase derivatization (SPD). These four alkyl methylphosphonic acids (AMPAs) are specific hydrolysis products and biomarkers of exposure to classic organophosphorus (OP) nerve agents VX, sarin, RVX, and soman. The AMPAs in urine samples were directly derivatized with pentafluorobenzyl bromide on a solid support and then extracted by liquid-liquid extraction. The analytes were quantified with isotope-dilution by negative chemical ionization (NCI) GC-MS/MS in a selected reaction monitoring (SRM) mode. This method is highly sensitive, with the limits of detection of 0.02 ng/mL for each compound in a 0.2 mL sample of human urine, and an excellent linearity from 0.1 to 50 ng/mL. It is proven to be very suitable for the qualitative and quantitative analyses of degradation markers of OP nerve agents in biomedical samples.

Comparison of high-resolution and tandem mass spectrometry for the analysis of nerve agent metabolites in urine.

RATIONALE: Although use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. High-resolution mass spectrometry (HRMS) was compared to tandem mass spectrometry (MS/MS) analysis for the quantitation of five urinary metabolites specific to VX, Russian VX, soman, sarin and cyclosarin nerve agents. The HRMS method was further evaluated for qualitative screening of metabolites not included in the test panel. METHODS: Nerve agent metabolites were extracted from urine using solid-phase extraction, separated using hydrophilic interaction chromatography and analyzed using both tandem and high-resolution mass spectrometry. MS/MS results were obtained using selected reaction monitoring with unit resolution; HRMS results were obtained using a mass extraction window of 10 ppm at a mass resolution of 50 000. The benchtop Orbitrap HRMS instrument was operated in full scan mode, to measure the presence of unexpected nerve agent metabolites. RESULTS: The assessment of two quality control samples demonstrated high accuracy (99.5-104%) and high precision (2-9%) for both HRMS and MS/MS. Sensitivity, as described by the limit of detection, was overlapping for both detectors (0.2-0.7 ng/mL). Additionally, the HRMS method positively confirmed the presence of a nerve agent metabolite, not included in the test panel, using the accurate mass and relative retention time. CONCLUSIONS: The precision, accuracy, and sensitivity were comparable between the current MS/MS method and this newly developed HRMS analysis for five nerve agent metabolites. HRMS showed additional capabilities beyond the current method by confirming the presence of a metabolite not included in the test panel.

Quantification of organophosphorus nerve agent metabolites using a reduced-volume, high-throughput sample processing format and liquid chromatography-tandem mass spectrometry.

A reduced-volume, high-throughput analytical method has been developed for the quantification of organophosphorus (OP) nerve agent metabolites in human urine. Metabolites of soman, sarin, cyclohexyl-sarin, VX, and Russian-VX were quantified down to a lowest reportable limit of 1 ng/mL in human urine. One hundred microliter urine samples were preconcentrated using normal-phase 96-well solid-phase extraction silica sorbent beds. Dual-column hydrophilic interaction liquid chromatography was applied in a 2.5-min isocratic separation followed by negative electrospray isotope-dilution multiple-reaction-monitoring mass spectrometry. Method validation included the characterization of two synthetic urine pools, relative recovery experiments, and calculation of the method limit of detection. All liquid handling steps were processed in a high-density 96-well format, including sample aliquoting, extraction, dry-down, and reconstitution. This allows up to 3840 unknown samples, plus calibrators and quality control materials, to be prepared on a single liquid handler in a 24-h period. In a public health emergency involving OP-nerve agents, this method provides the sample preparation and analytical capacity to respond rapidly to a large number of patient samples.

Determination of alkylmethylphosphonic acids, the main metabolites of organophosphorus nerve agents, in biofluids by gas chromatography-mass spectrometry and liquid-liquid-solid-phase-transfer-catalyzed pentafluorobenzylation.

A simple gas chromatography-mass spectrometry (GC-MS) procedure has been developed for the main metabolites of organophosphorus nerve agents, alkylmethylphosphonic acids (AMPAs; alkyl = Et, i-Pr, and pinacolyl) in biofluids via extractive pentafluorobenzylation. The derivatization was carried out under liquid-liquid-solid-phase-transfer conditions using a polymer-bound tri-n-butylmethylphosphonium bromide as a catalyst. AMPAs in aqueous samples were semiquantitatively extracted into a small-volume organic layer as their pentafluorobenzyl derivatives at pH 4.5 (85 degrees C). Sample pretreatments for urine, serum, and saliva were each examined to minimize matrix interference. The detection limits of APMAs by electron-impact ionization GC-MS were around 50 ng/mL and 2.5-10 ng/mL in the full-scan and selected-ion monitoring modes, respectively. In order to detect trace-level AMPAs, negative-ion chemical ionization (NICI) was also employed to enhance sensitivity. The detection limits of AMPAs in biofluids were typically 60 pg/mL by GC-NICI-MS.