Identification of Adducts of O-Isopropylmethylphosphonic and O-Cyclohexylmethylphosphonic Acids with a Tripeptide (Tyr-Thr-Lys) in Human Plasma by Liquid Chromatography–Mass Spectrometry

Abstract

We proposed a method for determining the presence of trace amounts of adducts of isopropylmethylphosphonic acid (IMPA) and cyclohexylmethylphosphonic acid (CMPA), biomarkers of exposure to sarin and cyclosarin in a body, with a tripeptide (Tyr-Thr-Lys). The method is based on plasma trypsinolysis with the formation of stable adducts IMPA–Tyr-Thr-Lys and СMPA–Tyr-Thr-Lys and their detection by high-performance liquid chromatography (HPLC) with high-resolution tandem mass spectrometry detection. The following conditions for the sample preparation of human blood plasma exposed to sarin and cyclosarin were optimized: aliquot portion volume, enzymatic hydrolysis conditions, mass spectrometric detection (selection of optimal pairs of ion reactions and detection modes), and gradient elution programs in the HPLC separation of the test mixture. The limit of detection for sarin and cyclosarin in human blood plasma was 5 ng mL–1. The proposed approach was tested in the analysis of blood plasma samples submitted to analysis in the framework of the Fourth Official Biomedical Test of the Organization for the Prohibition of Chemical Weapons; the results showed good determination specificity.