MSACL 2017 EU Abstract

Novel LC-MS Approaches to Determination of Chemical Warfare Agents and Related Compounds in Biomedical Samples

Igor Rodin (Presenter)
Moscow State University

Bio: Igor A. Rodin 21.11.1983 Education and degrees 06.2006 – M.Sc. in analytical chemistry, Moscow State University 04.2009 – Ph.D. in analytical chemistry, Moscow State University 02.2017 – Doctor of Science (Dr. Habil.), in analytical chemistry, Moscow State University Positions 04.2005-02.2007 – Junior Researcher, analytical chemistry division, Moscow State University 02.2007-12.2011 - Researcher, analytical chemistry division, Moscow State University 12.2011 – present - Senior Researcher, analytical chemistry division, Moscow State University 10.2010 – present - LCMS Research Group Head, analytical chemistry division, Moscow State University Area of interests Liquid Chromatography Mass Spectrometry, Analytical Methods Development, Bioanalytical Analysis, Forensics Analysis, Environmental Analysis. Teaching of analytical chemistry.

Authorship: T.M. Baygildiev1, I.A. Rodin1*, O.A. Shpigun1, I.V. Rybalchenko2
1. Moscow State University, Russia 2. Kostroma State University, Russia

Short Abstract A summary of the author’s approaches for investigation of the mass spectral behavior of some chemical warfare agents (CWAs), their degradation products and metabolites, as well as the results of development of analytical methods for confirmation of nerve and blister agents application in biomedical samples are presented. Hydrolysis and oxidation metabolites of nerve agents, sulfur mustard and lewisite were used as biomarkers of the exposure. Sensitive analytical methods have been developed for their detection, based mainly on tandem mass spectrometry coupled with liquid chromatography. Several techniques for fast screening of CWAs degradation products based on capillary electrophoresis were also proposed. Some of developed approaches were successfully applied in the frame of the proficiency testing system of the Organization for the Prohibition of Chemical Weapons.

Long Abstract

Introduction

Chemical warfare agents (CWAs) were employed several times in conflicts around the world. Commonly CWAs are divided into several categories according to their physiological effect. The first group, the vesicants or blister agents that affect the eyes, lungs and skin. Sulphur mustard (bis(2-chloroethyl)sulphide) was used in World War I and in the Iran–Iraq war. The second group, nerve agents, which cause the continuous cholinergic stimulation of the nervous system by inhibition of acetylcholinesterase. The first organophosphorus nerve agents, tabun (GA) and sarin (GB), were developed in the 1930s. Together with soman (GD) and VX these compounds are recognized as the major produced and weaponized nerve agents. Also several analogues of VX, such as VR (Russian VX), VE, VG, VM and VP are described by military science. The third group, blood agents, interfere with blood oxygen transport and may cause death by suffocation. The fourth group, incapacitating agents which have non-lethal physiological effects. Apart from this categorization there is a large group of possible chemical or biological warfare agents (BWAs), including bioregulators and toxins such as botulinium toxin, saxitoxin, abrin and ricin.

When the Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on their Destruction (the Chemical Weapons Convention; CWC) entered into force in 1997 the toxic chemicals, their precursors and degradation products essential for verification measures were listed in Schedules 1–3 of the Annex on Chemicals of the CWC [1]. These compounds have different chemical and physical properties: polar and less polar, neutral chemicals, acids, bases, volatiles and non-volatiles, with phosphorus, sulphur and arsenic heteroatoms as a part of the molecule.

Methods

Determination of analytes related to the OPCW convention was performed using various LC-MS and LC-MS/MS systems: Dionex Ultimate 3000 coupled with the AB Sciex 3200 Qtrap tandem mass-spectrometer, Agilent 1200 coupled with Bruker Amazon and LC-MS/MS Varian 1200.

Isopropyl methylphosphonic acid (IMPA), isobutyl methylphosphonic acid (IBMPA), pinacolyl methylphosphonic acid (PMPA), 1,1'-sulphonylbis[2-S (N-acetylcysteinyl)ethane] (SBSNAE), 1,1'-sulphonylbis[2-methylsulphinyl)ethane] (SBMSE), 1-methylsulphinyl-2-[2-(methylthio ethylsulphonyl]ethane (MSMTESE), thiodiglycolic acid (TDGA), 2-chlorovinylarsonous acid (CVAA) and 2-chlorovinylarsonic (CVAOA) were obtained from Sigma Aldrich. HPLC-grade acetonitrile was purchased from Panreac (Barcelona, Spain) and deionized water was prepared by a MilliQ purification system (Millipore Corp., Bedford, MA). Formic acid for mass spectrometry was obtained from Fluka (Milwaukee, WI, USA) Ammonium acetate of high purity was purchased from Roth (Germany).

Results

An approach to the detection of organophosphorus nerve agents, such as IMPA, PMPA and IBMPA in plasma samples using liquid chromatography and mass spectrometry was applied in our laboratory [2]. Mass spectrometric detection was performed with electrospray ionization (ESI) in the negative ion mode, using deprotonated molecules of AMPAs. Chromatographic separation was conducted by reversed phase chromatography using hydrophilic endcapped adsorbents. Solid phase extraction (SPE) on reversed phase adsorption cartridges containing a copolymer of styrene and divinylbenzene was proposed for sample preparation. Sufficiently low LODs (4, 0.6 and 1 ng/mL) were achieved for IMPA, PMPA and IBMPA respectively. The curves of nerve agent metabolites excretion were obtained for plasma samples of rats exposed by toxic substances. Application of tandem mass spectrometry and ultra-performance liquid chromatography allowed to lower LODs for the respective metabolites in human urine samples analyzed [3]. For EMPA, IMPA, PMPA and IBMPA LODs of 0.8, 0.5, 0.1 and 0.4 ng/mL respectively were achieved. Compounds were determined using ESI in negative ion mode and detected as deprotonated molecules. Analysis of urine samples was carried out using reversed-phase chromatography with C18 sorbent. SPE based on reversed-phase adsorption cartridges containing copolymer of sterol and divinylbenzene was employed in sample preparation procedure. The developed approach was applied to the analysis of human urine spiked with 5-50 ng/mL of the AMPAs during the world test of the Organization for the Prohibition of Chemical Weapons (OPCW).

An approach for the SBSNAE determination in human plasma and rat urine for quantification mustard exposure was proposed by the authors [4]. It employs solid-phase extraction sample preparation, followed by LC separation with negative ion electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The method LOD for SBSNAE determination in rat urine samples is 0.05 ng/mL with relative standard deviation (RSD) value less than 10%. Application of this procedure was demonstrated in the mustard animal exposure model. Rats were exposed intravenously with 5 mg/kg HD, and SBSNAE on different concentration levels in urine samples was observed for 22 days after exposure.

A sensitive rapid separation LC–MS/MS screening method for the simultaneous determination of another transformation products of mustard in urine - SBMSE and MSMTESE and degradation products of nerve agents - EMPA, IMPA and PMPA has been proposed by our group [5]. The analysis of these compounds is of interest because they are specific metabolites of different of CWAs: sulfur mustard, sarin, soman, VX and Russian VX. Application of “dilute-and-shoot” sample preparation and rapid separation LC and tandem mass spectrometry allowed to develop rapid, direct and sensitive method for determining CW exposure. Chromatographic separation of the metabolites was performed using a reverse phase column with gradient mobile phases consisting of 0.5% formic acid in water and acetonitrile. Identification and quantification of species were achieved using ESI-MS/MS monitoring two precursor-to-product ion transitions for each compound. The method demonstrated linearity over at least two orders of magnitude and had detection limits of 0.5 ng/mL in urine.

A sensitive fast LC-MS/MS approach for TDGA determination in urine has been established in the authors laboratory [6]. The use of "dilute-and-shoot" method helps to shorten sample preparation stage and provides a sensitive and direct approach for TDGA determination in aqueous samples and urine. Chromatographic separation of the analyte and other urine compounds was achieved using a reverse phase LC column with mobile phases consisting of 0.1% formic acid in water and acetonitrile in gradient elution mode. For identification and quantification of TDGA ESI-MS/MS was used. The method demonstrates good linearity and has detection limit of 50 ng/mL in urine.

LC-MS approach for lewisite metabolite detection in human and rat urine samples: CVAA (detection limit - 10 ng/mL) was developed in the authors laboratory [7]. The validation of the approach on human urine samples spiked with 2-chlorovinylarsonous acid and urine samples from rats intoxicated with lewisite was carried out. Compounds determination using atmospheric pressure chemical ionization in negative ion mode and applying deprotonated molecules as detecting ions was carried out. Biological samples analysis was carried out by reversed-phase chromatography using hydrophilic endcapped sorbent. It was shown that CVAA may be detected in rat urine during 13 days after intoxication. SPE based on reversed-phase adsorption cartridges containing copolymer of styrene and divinylbenzene was suggested as sample preparation procedure. To increase reliability of the approach, product of CVAA oxidation – 2-chlorovinylarsonic acid was determined simultaneously with CVAA. To lower LODs tandem mass-spectrometry was applied. A sensitive and simple method for the quantification and for the detection of CVAA and CVAOA was developed [8]. The developed assay was based on the use of SPE followed by LC coupled to negative ion-mode low-energy collision dissociation ESI-MS/MS. The method demonstrated linearity over at least three orders of magnitude and had LOD of 0.5 ng/mL for CVAA and 3 ng/ml for CVAOA. The RSD values for the quality control samples ranged from 6 to 11%. Application of this procedure was demonstrated in the lewisite animals exposure model. Rats were exposed intravenously by non-lethal doses of lewisite and marker levels in urine samples were analyzed for 21 days post-exposure.

Conclusions & Discussion

A wide range of approaches, which allow comprehensive solution of the problem for establishing the fact of using nerve and blister agents, has been developed. Sensitivity of developed LC-MS approaches were comparable with the most sensitive existing GC-MS approaches and did not require derivatization, reducing the time of the analysis and the number of false negative results. The developed complex of approaches was designed for sensitive LC-MS determination of the majority of known chemical weapon metabolites on the same equipment and has been tested in the frame of OPCW proficiency testing system.

References & Acknowledgements:

References

[1] Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on their Destruction, Technical Secretariat of the Organisation for Prohibition of Chemical Weapons, The Hague, 1997, accessible through internet http: / /www.opcw.nl.

[2] I. A. Rodin, A. V. Braun, I. A. Anan’eva, O. A. Shpigun, E. I. Savel’eva, I. V. Rybal’chenko, S. L. Bolotov, G. M. Rodchenkov. J. Anal. Chem., 66, 1417 (2011).

[3] I. A. Rodin, A.V. Braun, A.N. Stavrianidi, O.A. Shpigun, I.V. Ribalshenko. Analitika i kontrol (in Russian), 16, 254 (2012).

[4] I. Rodin, A. Braun, E. Savelieva Elena, I. Rybalсhenko, I. Ananieva, O. Shpigun. J. Liq. Chromatogr. Relat. Technol., 34, 1676 (2011).

[5] I. Rodin, A. Braun, A. Stavrianidi, T. Baygildiev, O. Shpigun, D. Oreshkin, I. Rybalchenko. J. Anal. Toxicol., 39, 69 (2014).

[6] I. Rodin, A. Braun, A. Stavrianidi, T. Baygildiev, I. Rybalchenko, O. Shpigun. Int. J. Environ. Anal. Chem., 96, 436 (2016).

[7] I.A. Rodin, A.V. Braun, A.N. Stavrianidi, O.A. Shpigun, I.V. Rybalchenko. Mass-spektrometria (in Russian), 8, 217 (2011).

[8] A. Braun, I. Rodin, A. Stavrianidi, O. Shpigun, I. Rybalchenko. J. Chromatogr. B., 879, 3370 (2011)

Acknowledgments

This work was supported by Russian Science Foundation (Grant No. 15-13-10005) for Kostroma State University.

Financial Disclosure

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

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