An on-line push-pull sampling technique has been developed for continuous analysis of proteins of molecularweight from 5.7 to 67 kDa. The characteristics of the system include gradient elution with a total cycle time of 21 min, membrane stabihb,, unattended automatic operation, and adjustment of the sampling mode and extraction fraction (the ratio of the concentration of analyte in the clialysate to that in the sample) by varying the effective dialysis length. The push and pull flow rates were adjusted in a mannerwhich enabled three different modes of operation. When push-pull microclialysis was compared with conventional microclialysis sampling, significantly higher extraction fractions were obtained for all five model proteins studied. The technique has been applied to the quantification of proteins in cell samples. On-line fractionation enabled complementary MS identification of the proteins present.
Phenyl isocyanate is a highly reactive compound that is used as a reagent in organic synthesis and in the production of polyurethanes. The potential for extensive occupational exposure to this compound makes it important to elucidate its reactivity towards different nucleophiles and potential targets in the body. In vitro reactions between glutathione and phenyl isocyanate were studied. Three adducts of glutathione with phenyl isocyanate were identified using ultra-performance liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR). Mass spectrometric data for these adducts have not previously been reported. Nucleophilic attack on phenyl isocyanate occurred via either the cysteinyl thiol group or the glutamic acid α-amino group of glutathione. In addition, a double adduct was formed by the reaction of both these moieties. NMR analysis confirmed the proposed structure of the double adduct, which has not previously been described. These results suggest that phenyl isocyanate may react with free cysteines, the α-amino group and also with lysine residues whose side chain contains a primary amine.
Chemical attribution signatures (CAS) associated with different synthetic routes used for the production of Russian VX (VR) were identified. The goal of the study was to retrospectively determine the production method employed for an unknown VR sample. Six different production methods were evaluated, carefully chosen to include established synthetic routes used in the past for large scale production of the agent, routes involving general phosphorus-sulfur chemistry pathways leading to the agent, and routes whose main characteristic is their innate simplicity in execution. Two laboratories worked in parallel and synthesized a total of 37 batches of VR via the six synthetic routes following predefined synthesis protocols. The chemical composition of impurities and byproducts in each route was analyzed by GC/MS-EI and 49 potential CAS were recognized as important markers in distinguishing these routes using Principal Component Analysis (PCA). The 49 potential CAS included expected species based on knowledge of reaction conditions and pathways but also several novel compounds that were fully identified and characterized by a combined analysis that included MS-CI, MS-EI and HR-MS. The CAS profiles of the calibration set were then analyzed using partial least squares discriminant analysis (PLS-DA) and a cross validated model was constructed. The model allowed the correct classification of an external test set without any misclassifications, demonstrating the utility of this methodology for attributing VR samples to a particular production method. This work is part one of a three-part series in this Forensic VSI issue of a Sweden-United States collaborative effort towards the understanding of the CAS of VR in diverse batches and matrices. This part focuses on the CAS in synthesized batches of crude VR and in the following two parts of the series the influence of food matrices on the CAS profiles are investigated.
Position-specific
isotope analysis (PSIA) by NMR spectroscopy is
a technique that provides quantitative isotopic values for every site—a
so-called isotopic fingerprint—of a compound of interest. The
isotopic fingerprint can be used to link samples with a common origin
or to attribute a synthetic chemical to its precursor source. Despite
PSIA by NMR being a powerful tool in chemical forensics, it has not
yet been applied on chemical warfare agents (CWAs). In this study,
different batches of the CWA Soman were synthesized from three distinctive
pinacolyl alcohols (PinOHs). Prior to NMR analysis, the Soman samples
were hydrolyzed to the less toxic pinacolyl methylphosphonate (PMP),
which is a common degradation product. The PinOHs and PMPs were applied
to PSIA by
2
H NMR experiments to measure the isotopic distribution
of naturally abundant
2
H within the pinacolyl moiety. By
normalizing the
2
H NMR peak areas, we show that the different
PinOHs have unique intramolecular isotopic distributions. This normalization
method makes the study independent of references and sample concentration.
We also demonstrate, for the first time, that the isotopic fingerprint
retrieved from PSIA by NMR remains stable during the production and
degradation of the CWA. By comparing the intramolecular isotopic profiles
of the precursor PinOH with the degradation product PMP, it is possible
to attribute them to each other.
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