Identification of 2-(diethylamino)ethylthiol dipeptide (Cys-Pro) adduct as biomarker of nerve agents VR and CVX in human plasma using liquid chromatography-high-resolution tandem mass spectrometry.

Organophosphorus nerve agents pose a significant threat to human health. The most toxic compounds in this class include V-type poisonous substances such as VX, CVX, and VR. Although all stockpiles of this type of substance are subject to destruction under the Chemical Weapons Convention (CWC), there is still a risk that they could be used for criminal and terrorist purposes. The latter determines the relevance of studies aimed at identification of biomarkers that may indicate the exposure of these group substances to the organism. A liquid chromatography mass spectrometry/high-resolution mass spectrometry (LC-MS/HR MS) method for determination of trace amounts of nerve agents such as VR and CVX in human plasma was proposed. The method is based on enzymatic plasma hydrolysis with the use of pronase to form a stable adduct of 2-(diethylamino)ethylthiol with dipeptide cysteine-proline (DEAET-CP) with its subsequent determination by LC-MS/HR MS. Synthesis of DEAET-CP as reference compound was conducted using non-toxic precursors. Sample preparation of human blood plasma samples exposed to VR was carried out with the use of solid-phase extraction (SPE). Liquid chromatography (LC) separation on the reversed-phase column and mass spectrometric detection (selection of optimal transitions and detection modes) were performed. The achieved limit of detection (LOD) of VR (in the form of DEAET-CP) in human blood plasma was 0.05 ng mL-1. The proposed approach was developed using plasma samples exposed to VR and CVX obtained in the frame of the Fifth Official Biomedical Test of the Organization for the Prohibition of Chemical Weapons (OPCW) and showed good specificity of detection.

Rapid IC-MS/MS determination of methylphosphonic acid in urine of rats exposed to organophosphorus nerve agents.

A direct approach for the determination of a specific hydrolysis product of organophosphorus nerve agents such as methylphosphonic acid (MPA) in urine by ion chromatography and tandem mass spectrometry (IC-MS/MS) has been developed. The first advantage of the proposed approach is a rapid and simple sample preparation, which does not require a large sample volume, complicated and laborious preconcentration and derivatization steps, and takes less than 7min per sample. The second advantage is the fast and selective IC determination of MPA carried out on a noncommercial anion exchanger based on a poly(styrene-co-divinylbenzene) (PS-DVB) substrate with a high degree of crosslinking and a covalently-bonded branched functional layer, which enables complete resolution of MPA from major urine matrix components and allows one to overcome matrix effects. Hyphenation of IC with tandem mass spectrometry results in highly sensitive and reliable MPA determination with the lowest detection limit (4ngmL-1) reported so far for HPLC determination of MPA in urine. The proposed approach is successfully applied for the analysis of urine from rats exposed to nonlethal doses of organophosphorus nerve agents such as sarin, soman, and VR in up to 13days after initial exposure, which shows the possibility to verify the nerve agent exposure after a long period of time.

Single-run HPLC/ESI-LITMS profiling of ginsenosides in plant extracts and ginseng based products.

A rapid single-run analytical approach suitable to achieve a comprehensive characterization of ginsenosides - the main bioactive compounds present in plant materials from Panax species and ginseng-based products - was developed. The method is based on high-performance liquid chromatography coupled with electrospray positive ionization linear ion trap mass spectrometry (HPLC/ESI-LITMS). The main ions in the ESI-LITMS spectra were attributed to molecular adducts with sodium and potassium and fragments corresponding to cleavage of the glycosidic bonds. The simplicity of the approach allows laborious sample preparation and sophisticated spectral information-dependent acquisition to be avoided, and provides an opportunity for rapid screening. The method may replace existing HPLC-DAD profiling approaches. The results of this study indicate that HPLC/ESI-LITMS is applicable for quality control purposes on processed products and allows the rapid and direct identification of ginsenosides in crude plant extracts.

A validated LC-MS/MS method for rapid determination of methotrexate in human saliva and its application to an excretion evaluation study.

A sensitive and simple method for the methotrexate quantification was developed using aminopterin as internal standard. Methotrexate is an anticancer agent that is widely used in a variety of human cancers including primary central nervous system lymphoma. The compound was quantified by liquid-chromatography coupled to electrospray ionization (positive ion-mode) low-energy collision dissociation-tandem mass spectrometry. Quantitative detection was by multiple reaction monitoring of the transitions of the [M+H]+ ion of MTX to its common product ion at m/z 308.4 and of aminopterin at m/z 441.2→m/z 294.0. The method demonstrated linearity over at least three orders of magnitude and had a detection limit of 1ng/ml for methotrexate. A run time of less than 8.0min for each sample made it possible to analyze a large number of human saliva samples per day. Application of this procedure was demonstrated to a saliva excretion study of methotrexate on the samples obtained after an intravenously administration of 1mg/kg/dose of methotrexate to six patients with acute lymphoblastic leukemia.

The use of linear ion trap for qualitative analysis of phytochemicals in Korean ginseng tea.

A new approach to qualitative analysis of ginsenosides in challenging matrices was developed on the basis of high-performance liquid chromatography/tandem mass spectrometry. Using the extracts from samples of ginseng tea, the approach was validated. Analysis of extracts was carried out using a reversed-phase chromatography with SB-C18 sorbent. For compound identification, electrospray ionization and a quadrupole/linear ion trap mass-spectrometer in different modes were used. A meticulous study of the fragmentation of ginsenosides in the linear ion trap and its application for analysis of these compounds was performed in this work. The accuracy of the identification was proven with standards of ginsenosides Rb1, Rg1, Re, Rf, Rd, Rb2, Rb3 and Rc.

Lewisite metabolites detection in urine by liquid chromatography-tandem mass spectrometry.

A sensitive and simple method for the quantification and for the detection of 2-chlorovinylarsonous (CVAA) and 2-chlorovinylarsonic (CVAOA) acids was developed. CVAA and CVOA are important biological markers in human and rat urine specific to lewisite (chlorovinylarsonous chloride compounds) exposure. The developed assay was based on the use of solid-phase extraction (SPE) followed by liquid-chromatography coupled to electrospray ionization (negative ion-mode) low-energy collision dissociation-tandem mass spectrometry (ESI-CID-MS/MS). The method demonstrated linearity over at least three orders of magnitude and had a detection limit (LOD) of 0.5 ng/ml for CVAA and 3 ng/ml for CVAOA. The relative standard deviations 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 no lethal doses of lewisite and markers levels in urine samples were analyzed for 21 days post-exposure.