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.

A novel strategy for isolation and determination of sugars and sugar alcohols from conifers.

The ultrasound-assisted extraction method for isolation of 17 sugars and sugar alcohols from conifers with a subsequent hydrophilic interaction liquid chromatography-tandem mass spectrometry method for their determination is proposed. The optimization of extraction parameters was carried out using Taguchi - L9 (34) orthogonal array experimental design for the following parameters-a methanol concentration in the extraction solution, an extraction time, a type of plant sample and an extraction temperature. The optimal ultrasound-assisted extraction conditions were-MeOH concentration - 30% (water - 70%), extraction time - 30 min, type of plant sample - II (grinded leaves 2-4 mm long), extraction temperature - 60 °C. Pure water and acetonitrile were used as eluents in gradient elution mode to separate the analytes. Direct determination of multiple sugars and sugar alcohols was carried out using a mass spectrometric detector operated in a multiple reaction monitoring mode, providing detection limits in the range between 0.1 and 20 ng/mL and good analytical characteristics of the method without derivatization. The developed approach was validated by multiple successive extraction method applied to test its performance on a series of 10 samples, i.e. 2 samples per each of 5 genera: Abies, Larix, Picea, Pinus (Pinaceae) and Juniperus (Cupressaceae), widely distributed in the boreal conifer forests of Eurasia. The novel strategy can be used for profiling of sugars and sugar alcohols in a wide range of plant species.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry methylphosponic and alkyl methylphosphonic acids determination in environmental samples after pre-column derivatization with p-bromophenacyl bromide.

Once exposed to the environment organophosphate nerve agents readily degrade by rapid hydrolysis to the corresponding alkyl methylphosphonic acids which do not exist in nature. These alkyl methylphosphonic acids are finally slowly hydrolyzed to methylphosphonic acid. Methylphosphonic acid is the most stable hydrolysis product of organophosphate nerve agents, persisting in environment for a long time. A highly sensitive method of methylphosphonic acid and alkyl methylphosphonic acids detection in dust and ground mixed samples has been developed and validated. The fact that alkyl methylphosphonic acids unlike methylphosphonic acid did not react with p-bromophenacyl bromide under chosen conditions was discovered. This allowed simultaneous chromatographic separation and mass spectrometric detection of derivatized methylphosphonic acid and underivatized alkyl methylphosphonic acids using HILIC-MS/MS method. Very simple sample pretreatment with high recoveries for each analyte was developed. Methylphosphonic acid pre-column derivate and alkyl methylphosphonic acids were detected using tandem mass spectrometry with electrospray ionization after hydrophilic interaction liquid chromatography separation. The developed approach allows achieving ultra-low detection limits: 200 pg mL(-1) for methylphosphonic acid, 70 pg mL(-1) for ethyl methylphosphonic acid, 8 pg mL(-1) for i-propyl methylphosphonic acid, 8 pg mL(-1) for i-butyl methylphosphonic acid, 5 pg mL(-1) for pinacolyl methylphosphonic acid in the extracts of dust and ground mixed samples. This approach was successfully applied to the dust and ground mixed samples from decommissioned plant for the production of chemical weapons.