title: Formation of pyrophosphate-like adducts from nerve agents sarin, soman and cyclosarin in phosphate buffer: implications for analytical and toxicological investigations.
abstract: Phosphate buffer is frequently used in biological, biochemical and biomedical applications especially when pH is to be controlled around the physiological value of 7.4. One of the prerequisites of a buffer compound among good buffering capacity and pH stability over time is its non-reactivity with other constituents of the solution. This is especially important for quantitative analytical or toxicological assays. Previous work has identified a number of amino alcohol buffers like TRIS to react with G-type nerve agents sarin, soman and cyclosarin to form stable phosphonic diesters. In case of phosphate buffer we were able to confirm not only the rapid hydrolysis of these agents to the respective alkyl methylphosphonates but also the formation of substantial amounts of pyrophosphate-like adducts (phosphorylated methylphosphonates), which very slowly hydrolyzed following zero-order kinetics. This led to a complex mixture of phosphorus containing species with changing concentrations over time. We identified the molecular structure of these buffer adducts using 1D ¹H-³¹P HSQC NMR and LC-ESI-MS/MS techniques. Reaction rates of adduct formation are fast enough to compete with hydrolysis in aqueous solution and to yield substantial amounts of buffer adduct over the course of just a couple of minutes. Possible reaction mechanisms are discussed with respect to the formation and subsequent hydrolysis of the pyrophosphate-like compounds as well as the increased rate of hydrolysis of the nerve agent to the corresponding alkyl methylphosphonates. In summary, the use of phosphate buffer for the development of new assays with sarin, soman and cyclosarin is discouraged. Already existing protocols should be carefully reexamined on an individual basis.
authors.affiliation: Blum-Scientific Services, Ledererstrasse 23, 80331 Munich, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Gäb
authors.forename: Harald
authors.initials: H
authors.lastname: John
authors.forename: Marc-Michael
authors.initials: MM
authors.lastname: Blum
mesh_terms: D002021:Buffers; D002619:Chemical Warfare Agents / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D011756:Diphosphates / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009943:Organophosphorus Compounds / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry*; D012524:Sarin / Q000737:chemistry*; D012999:Soman / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D002021:Buffers; D002619:Chemical Warfare Agents; D011756:Diphosphates; D009943:Organophosphorus Compounds; D010710:Phosphates; D012999:Soman; D012524:Sarin; C053914:cyclohexyl methylphosphonofluoridate