Identification of acidic degradation products of chemical warfare agents by methylation with trimethylsilyldiazomethane and gas chromatography-mass spectrometry

Sensitive and reliable analysis of alkylphosphonic acids (APAs) and 2-(N,N-dialkylamino)ethanesulfonic acids (SAs), the degradation products of chemical warfare agents (CWAs), is one of the most important tasks for verification of the Chemical Weapons Convention (CWC). Unambiguous identification of these chemicals is required in a variety of environmental matrices, including soil and water. These acids with low volatility are very polar, and efficient and reliable methylation methods for their derivatization are needed for analysis with gas chromatography-mass spectrometry (GC-MS). In this study, the derivatization conditions for trimethylsilyldiazomethane (TMSDAM) methylation were optimized for rapid GC-MS screening. Optimized methylation of APAs and SAs with TMSDAM was compared with methylation with diazomethane. The TMSDAM methylation of SAs and benzilic acid was further compared with silylation with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide. The significance and necessity of cation exchange prior to derivatization and analysis were tested on samples with a high inorganic background. A recommendation to use the method for methylation of water samples and aqueous extracts using TMSDAM is given. The robustness of the method was illustrated by the successful identification of APAs and SAs in aqueous samples from proficiency tests organized by the Organisation for the Prohibition of Chemical Weapons.Peer reviewe

Optimization of Sample Preparation for the Identification and Quantification of Saxitoxin in Proficiency Test Mussel Sample using Liquid Chromatography-Tandem Mass Spectrometry

Saxitoxin (STX) and some selected paralytic shellfish poisoning (PSP) analogues in mussel samples were identified and quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample extraction and purification methods of mussel sample were optimized for LC-MS/MS analysis. The developed method was applied to the analysis of the homogenized mussel samples in the proficiency test (PT) within the EQuATox project (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk). Ten laboratories from eight countries participated in the STX PT. Identification of PSP toxins in naturally contaminated mussel samples was performed by comparison of product ion spectra and retention times with those of reference standards. The quantitative results were obtained with LC-MS/MS by spiking reference standards in toxic mussel extracts. The results were within the z-score of +/- 1 when compared to the results measured with the official AOAC (Association of Official Analytical Chemists) method 2005.06, pre-column oxidation high-performance liquid chromatography with fluorescence detection (HPLC-FLD).Peer reviewe

Characterization of Ricin and R. communis Agglutinin Reference Materials

Ricinus communis intoxications have been known for centuries and were attributed to the toxic protein ricin. Due to its toxicity, availability, ease of preparation, and the lack of medical countermeasures, ricin attracted interest as a potential biological warfare agent. While different technologies for ricin analysis have been established, hardly any universally agreed-upon gold standards are available. Expert laboratories currently use differently purified in-house materials, making any comparison of accuracy and sensitivity of different methods nearly impossible. Technically challenging is the discrimination of ricin from R. communis agglutinin (RCA120), a less toxic but highly homologous protein also contained in R. communis. Here, we established both highly pure ricin and RCA120 reference materials which were extensively characterized by gel electrophoresis, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI MS/MS), and matrix-assisted laser desorption ionization-time of flight approaches as well as immunological and functional techniques. Purity reached >97% for ricin and >99% for RCA120. Different isoforms of ricin and RCA120 were identified unambiguously and distinguished by LC-ESI MS/MS. In terms of function, a real-time cytotoxicity assay showed that ricin is approximately 300-fold more toxic than RCA120. The highly pure ricin and RCA120 reference materials were used to conduct an international proficiency test.Peer reviewe

Results of a Saxitoxin Proficiency Test Including Characterization of Reference Material and Stability Studies

A saxitoxin (STX) proficiency test (PT) was organized as part of the Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk (EQuATox) project. The aim of this PT was to provide an evaluation of existing methods and the European laboratories' capabilities for the analysis of STX and some of its analogues in real samples. Homogenized mussel material and algal cell materials containing paralytic shellfish poisoning (PSP) toxins were produced as reference sample matrices. The reference material was characterized using various analytical methods. Acidified algal extract samples at two concentration levels were prepared from a bulk culture of PSP toxins producing dinoflagellate Alexandrium ostenfeldii. The homogeneity and stability of the prepared PT samples were studied and found to be fit-for-purpose. Thereafter, eight STX PT samples were sent to ten participating laboratories from eight countries. The PT offered the participating laboratories the possibility to assess their performance regarding the qualitative and quantitative detection of PSP toxins. Various techniques such as official Association of Official Analytical Chemists (AOAC) methods, immunoassays, and liquid chromatography-mass spectrometry were used for sample analyses.Peer reviewe

pH-Dependent Piecewise Linear Correlation of H-1, P-31 Chemical Shifts : Application in NMR Identification of Nerve Agent Metabolites in Urine Samples

The NMR-observable nuclei of the acidic and basic compounds experience pH dependence in chemical shift. This phenomenon can be exploited in NMR titrations to deter- mine pK a values of compounds, or in pH measurement of solutions using dedicated pH reference compounds. On the other hand, this sensitivity can also cause problems in, for example, metabolomics, where slight changes in pH result in significant difficulties for peak alignment between spectra of set of samples for comparative analysis. In worst case, the pH sensitivity of chemical shifts can prevent unambiguous identification of compounds. Here, we propose an alternative approach for NMR identification of pH-sensitive analytes. The H-1 and X (C-13, N-15, P-31, ...) chemical shifts in close proximity to the acidic or basic functional group should, when presented as ordered pairs, express piecewise linear correlation with distinct slope, intercept, and range. We have studied the pH dependence of H-1 and P-31 chemical shifts of the CH3-P moiety in urinary metabolites of nerve agents sarin, soman and VX using 2D H-1-P-31 fast-HMQC spectroscopy. The H-1 and P-31 chemical shifts of these chemicals appear in very narrow range, and due to subtle changes in sample pH the identification on either H-1 or P-31 chemical shift alone is uncertain. However, if the observed H-1 and P-31 chemical shifts of the CH3-P moiety of individual compounds are presented as ordered pairs, they fall into distinct linear spaces, thus, facilitating identification with high confidence.Peer reviewe

Genetic Polymorphisms and Recurrent Spontaneous Abortions: An Overview of Current Knowledge

The relevance of gene polymorphisms in the development of unexplained recurrent spontaneous abortion is still unclear. Cytokines, angiogenic mediators, and hormones are involved in all stages of reproduction and pregnancy outcome. Impaired production and/or unbalanced ratios of these mediators have been implicated in the pathogenesis of unexplained recurrent spontaneous abortion. Functional polymorphism influence gene activity and therefore can interfere with the expression of mediators. Several studies have been carried out to evaluate the relationship between cytokines, angiogenic mediators, and hormones gene polymorphisms and unexplained recurrent spontaneous abortion. the results of these studies are mostly contradictory, and few significant associations have been identified. Up to present time, the evidence is insufficient to support the evaluation of cytokines, angiogenic mediators, and hormones gene polymorphism in routine workup in all cases of recurrent pregnancy loss, and these tests are not included in any of the major obstetric guidelines.Universidade Federal de São Paulo, Dept Obstet, BR-01415002 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Obstet, BR-01415002 São Paulo, BrazilWeb of Scienc

Results of a saxitoxin proficiency test including characterization of reference material and stability studies

A saxitoxin (STX) proficiency test (PT) was organized as part of the Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk (EQuATox) project. The aim of this PT was to provide an evaluation of existing methods and the European laboratories\u27 capabilities for the analysis of STX and some of its analogues in real samples. Homogenized mussel material and algal cell materials containing paralytic shellfish poisoning (PSP) toxins were produced as reference sample matrices. The reference material was characterized using various analytical methods. Acidified algal extract samples at two concentration levels were prepared from a bulk culture of PSP toxins producing dinoflagellate Alexandrium ostenfeldii. The homogeneity and stability of the prepared PT samples were studied and found to be fit-for-purpose. Thereafter, eight STX PT samples were sent to ten participating laboratories from eight countries. The PT offered the participating laboratories the possibility to assess their performance regarding the qualitative and quantitative detection of PSP toxins. Various techniques such as official Association of Official Analytical Chemists (AOAC) methods, immunoassays, and liquid chromatography-mass spectrometry were used for sample analyses