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Direct derivatization and trace determination of nerve agent biomarkers in urine samples
Umeå University, Faculty of Science and Technology, Department of Chemistry.
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Abstract [en]

A rapid direct derivatization and gas chromatography mass spectrometry (GC-MS) method has been developed for the determination of alkylphosphonic acids (APAs) in urine, which act as biomarkers of organophosphorus nerve agents. The method involves rapid (5 min) and direct derivatization of acidified urine samples (25 μL) using the highly fluorinated phenyldiazomethane reagent [1-(diazomethyl)-3,5-bis(trifluoromethyl)benzene]. The APA derivatives are then trapped on reversed-phase C18 bonded silica to remove any underivatized APAs and HCl in the sample solution. After elution, the APA derivatives are then determined by low resolution mass spectrometry using negative ion chemical ionization single ion monitoring.The selectivity of the method was further improved by using high resolution negative ion chemical ionization selected ion monitoring on a double focusing magnetic sector mass spectrometer. High sensitivity was obtained with limits of detection ranging from 0.1 to 10 ng/mL of the APAs (in urine) for both techniques. The conditions for the derivatization were optimized using statistical experimental design and multivariate data analysis. Based on the current results and earlier work on APAs in water using the direct derivatization approach, we conclude that the method offers excellent performance and is viable for the simultaneous trace determination of a wide range of nerve agent markers.

Keyword [en]
Biomarkers, nerve agents
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-54641OAI: oai:DiVA.org:umu-54641DiVA: diva2:524692
Available from: 2012-05-03 Created: 2012-05-03 Last updated: 2012-05-04Bibliographically approved
In thesis
1. Simplified Routines for Sample Preparation and Analysis of Chemical Warfare Agent Degradation Products
Open this publication in new window or tab >>Simplified Routines for Sample Preparation and Analysis of Chemical Warfare Agent Degradation Products
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The thesis describes the development of new and improved methods for analyzing degradation markers from organophosphorus Chemical Warfare Agents (CWAs).

Paper I and II describes an innovative and significantly improved method for the enrichment, derivatization (trimethysilylation) and GC-MS analysis of a broad range of organophosphorus CWAs degradation markers, namely the alkylphosphonic acids and a zwitterionic compound. That was achieved using solid phase disc extraction in combination with solid phase derivatization. The new method overcomes most limitations observed with existing techniques: it offers almost 100 % recoveries, requires no elution or evaporation steps, facilitates miniaturization of the solid sorbent and reagent, is compatible with in-vial derivatization, and minimizes the chromatographic background due to the use of a highly selective anion exchange sorbent disc.

Paper III describes the development of new fluorinated diazomethane derivatization reagents and their evaluation for rapid and high sensitivity screening and identification of nerve agent degradation markers. The reagents are water-tolerant to some extent, which simplifies the derivatization step. The best reagent identified was 3,5-bis(trifluoromethyl)benzyl diazomethane, which outperformed the other reagent isomers tested and also the established commercial alternative, pentafluorobenzylbromide, allowing for the rapid (5 min) and direct derivatization of a 25 μL aqueous sample in acetonitrile. The spectra of the formed derivatives (high-energy collision induced fragmentation MS/MS) were used to construct a database (Paper IV) that proved to be superior in terms of match factor and probability compared to EI data gathered for trimethylsilyl derivatives. The study also focused on efforts towards achieving detailed structure information on the alkyl chains of the compounds in question using diagnostic ion interpretation.

The final paper (paper V) describes the first rapid direct derivatization method for analyzing nerve agent metabolites in urine at trace levels. The method is based on the derivative from the paper III and the unambiguous identification was proven using a combination of low resolution and high resolution negative ion chemical ionization selected ion monitoring techniques.

Novel results presented in these papers include: the first in-situ derivatization of alkylphosphonic acids on an SPE disc; the first direct derivatization of nerve agent markers in water and biomedical samples; the first high sensitivity GC-MS screening for these markers; and the first highly reproducible high-energy isomer specific CID MS/MS library. Overall, the results presented in this thesis represent significant contributions to the analysis of nerve agent degradation products.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2012. 58 p.
Keyword
Nerve agents, Alkyl alkylphosphonic acids, Solid phase derivatization, Fluorinated diazomethane reagent, High-energy collision induced dissociation, CID MS/MS library
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:umu:diva-54639 (URN)978-91-7459-440-9 (ISBN)
Public defence
2012-05-25, KB3B1, KBC-huset, Umeå University, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-05-04 Created: 2012-05-03 Last updated: 2012-05-04Bibliographically approved

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Subramaniam, Raja

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