The impact of skin decontamination on the time window for effective treatment of percutaneous VX exposure.

The main goal of the present study was to obtain insight into depot formation and penetration following percutaneous VX poisoning, in order to identify an appropriate decontamination window that can enhance or support medical countermeasures. The study was executed in two phases, using the hairless guinea pig as an animal model. In the first phase the effect of various decontamination regimens on levels of free VX in skin and plasma were studied as well as on blood cholinesterase levels. Animals were exposed to 0.5 mg/kg VX and were not decontaminated (control), decontaminated with RSDL once at 15 or 90 min after exposure or three times at 15, 25 and 35 (10-min interval) or 15, 45 and 75 min after exposure (30-min interval). There was no significant effect of any of the decontamination regimens on the 6-h survival rate of the animals. However, all animals that had been decontaminated 15 min after exposure, showed a survival rate of more than 90%, compared to 50-60% in animals that were not decontaminated or decontaminated at 90 min after exposure. In the second phase of the study, hairless guinea pigs were exposed to 1 mg/kg VX on the shoulder, followed either by decontamination with RSDL (10 min interval), conventional treatment on indication of clinical signs or a combination thereof. It appeared that a thorough, repeated decontamination alone could not save the majority of the animals. A 100% survival rate was observed in the group that received a combination of decontamination and treatment. In conclusion, the effects of VX exposure could be influenced by various RSDL decontamination regimens. The results in freely moving animals showed that skin decontamination, although not fully effective in removing all VX from the skin and skin depot is crucial to support pharmacological intervention.

New tools in diagnosis and biomonitoring of intoxications with organophosphorothioates: case studies with chlorpyrifos and diazinon.

Organophosphate (OP) pesticides are neurotoxic compounds that are widely used in agriculture. Classical methods for monitoring OP exposure comprise the measurement of intact OP, its metabolites or cholinesterase activity. Newly developed methods focus on the analysis of the OP adduct bound to proteins such as butyrylcholinesterase (BuChE) and albumin. These adducts can be analyzed by means of fluoride reactivation or by analysis with LC-MS/MS of the pepsin or pronase digest of butyrylcholinesterase and albumin, respectively. The utility of these methods is illustrated through the analysis of plasma samples obtained from patients taken 1-49 days after ingestion of the organophosphate pesticides chlorpyrifos and/or diazinon. Thus, in this particular case several independent methodologies were applied to the biomedical samples, all pointing to the same exposure.

Comprehensive gas chromatography with Time of Flight MS and large volume introduction for the detection of fluoride-induced regenerated nerve agent in biological samples.

Recently, several methods have been developed to verify exposure to nerve agents. Most of these methods, such as the fluoride reactivation technique and the analysis of inhibited phosphonylated butyrylcholinesterase (BuChE), are based on mass spectrometry. The high specificity of the mass spectrometer might also imply a disadvantage, because the acquisition mass, i.e. the identity of the analyte must be known beforehand in order to direct the MS analysis in the most sensitive mode. In real cases, the identity of the nerve agent is not always known beforehand and the mass spectrometer should be operated in a scanning mode, with the consequence that sensitivity of the method will be lower. Comprehensive GC, or GC x GC, is a technique which offers enhanced separation. The implied larger selectivity of the GC separation allows mass spectrometry to be conducted in a less specific, scanning, mode. By the use of this configuration, the identity of the nerve agent does not have to be known beforehand but can be traced. In order to be able to detect lower concentrations and assess lower exposure levels, a large volume injection technique was developed allowing sample sizes up to 100 microL. The technique was tested with plasma samples that had been inhibited with various nerve agents. Subsequently, the cholinesterase-bound nerve agent was regenerated by the fluoride reactivation technique. Using the newly developed comprehensive GC-MS method it was possible to detect nerve agent at an exposure level of 1% BuChE inhibition, which is approximately 70 pg nerve agent/mL. These low exposure levels cannot be verified with a cholinesterase (ChE) activity assay. Moreover, the identity of the regenerated nerve agent was verified by the mass spectrum that was generated by the TOF mass spectrometer. This paper presents a technique able to deliver full-scan data on the analysis of nerve agents in biomedical samples at relevant exposure levels (1% BuChE inhibition). This full-scan data meets for a large part the forensic requirements that are in place for the analysis of biomedical samples in the context of alleged use of Chemical Warfare Agents.

Covalent binding of organophosphorothioates to albumin: a new perspective for OP-pesticide biomonitoring?

We here report on the covalent binding of various organophosphorothioate (OPT) pesticides to albumin at in vitro exposure levels that did not give rise to butyrylcholinesterase inhibition. Adduct formation occurred at the Tyr-411 residue of albumin, as was firmly corroborated by LC-tandem MS analysis of a pepsin digest of OPT-modified albumin. It cannot be excluded that other (tyrosine) residues become modified as well. A convenient method for mass spectrometric determination of the OPT tyrosine adduct has also been developed based on the pronase digestion of albumin and subsequent LC-tandem MS analysis of the digest. The resulting tyrosine phosphorothioate ester displayed favorable chromatographic and mass spectrometric properties for sensitive analysis. In vitro exposure levels of parathion and chlorpyrifos down to 1 microM could readily be assessed. The remarkable affinity of OPTs for albumin opens the way for a more complete assessment of OP pesticide exposure.

Verification of exposure to cholinesterase inhibitors: generic detection of OPCW Schedule 1 nerve agent adducts to human butyrylcholinesterase.

Phosphylated butyrylcholinesterase is one of the most important biomarkers to verify an exposure to nerve agents, and it can be analyzed with liquid chromatography-tandem mass spectrometry (LC-MS-MS) by detection of a phosphylated nonapeptide that results after digestion of butyrylcholinesterase (BuChE) with pepsin. For a sensitive analysis (low degree of BuChE inhibition), the identity of the cholinesterase inhibitor has to be known in order to use the LC-MS-MS instrument in the most sensitive selected reaction monitoring mode. In practice, the identity of the cholinesterase inhibitor will not be known beforehand, and the number of possible organophosphates is greater than 1000. However, the number of possible molecular masses of organophosphates is approximately 170. A method for which only 34 transitions in the multiple reaction monitoring mode have to be acquired in order to screen for an exposure to all Organization for the Prohibition of Chemical Weapons Schedule 1 nerve agents was developed.

Verification of exposure to organophosphates: Generic mass spectrometric method for detection of human butyrylcholinesterase adducts.

We present a generic mass spectrometric method to verify exposure to organophosphates, based on the chemical conversion of the phosphylated peptides obtained after pepsin digestion of human butyrylcholinesterase (HuBuChE) to a common precursor peptide. After exposure of plasma to various organophosphates (nerve agents, pesticides), HuBuChE was isolated from plasma by procainamide affinity-based solid-phase extraction. Upon subsequent pepsin digestion, the respective phosphylated nonapeptides could be identified in the digests. After treatment of the pepsin digests with Ba(OH)2 in the presence of a nucleophilic tag (a thiol or amine), the phosphylated nonapeptides were transformed into a common tagged nonapeptide that could be analyzed sensitively by means of LC tandem MS. So far, best results were obtained with 2-(3-aminopropylamino)ethanol as nucleophilic tag. By applying the presented method, HuBuChE inhibition can now be monitored accurately by mass spectrometry, without advance knowledge of the structure of the inhibitor.

Retrospective detection of exposure to organophosphorus anti-cholinesterases: mass spectrometric analysis of phosphylated human butyrylcholinesterase.

In this paper a novel and general procedure is presented for detection of organophosphate-inhibited human butyrylcholinesterase (HuBuChE), which is based on electrospray tandem mass spectrometric analysis of phosphylated nonapeptides obtained after pepsin digestion of the enzyme. The utility of this method is exemplified by the positive analysis of serum samples from Japanese victims of the terrorist attack with sarin in the Tokyo subway in 1995.

Quality control of histamine and methacholine in diagnostic solutions with capillary electrophoresis.

Solutions of histamine and methacholine bromide in different matrices for diagnostic purposes were analyzed for stability and quality control using capillary electrophoresis. Histamine (2,[4-imidazolyl]ethylamine) [CAS No. 51-45-6] was determined using a 0.1 M Tris-borate buffer of pH 8.3 with 5.10(-5) M cetyltrimethylammonium bromide (CTAB) and 0.005% poly(vinyl alcohol) (PVA) and detected at 214 nm using clenbuterol [4-amino-alpha-(tert.-butylaminomethyl)-3,5-dichlorobenzyl alcohol] [37148-27-9] as an internal standard. Metacholine bromide (acetyl-beta-methacholine bromide) [333-31-3] was determined with a 0.01 M creatinine-chloride buffer of pH 4.85 and detected with indirect UV at 230 nm using potassium as an internal standard. Histamine solutions were stable for a prolonged period of time, whereas under enforced degradation conditions methacholine was hydrolyzed, yielding acetic acid and (tentatively) beta-methylcholine as reaction products.

Detection of point mutations in DNA using capillary electrophoresis in a polymer network.

The use of capillary electrophoresis (CE) in a polymer network for single-strand conformation polymorphism (SSCP) is investigated. SSCP is a method to detect DNA point mutations, essential in the diagnosis of several diseases. The PCR (polymerase chain reaction) amplified p53 gene, a tumour suppressor gene known to be frequently mutated in malignant cells, was subjected to CE analysis. Two single-strand DNA fragments of 372 bp in length differing in only one nucleotide could be separated. We conclude that SSCP using CE in a polymer network is a powerful method for the detection of point mutations in DNA sequences.