Gas chromatographic-mass spectrometric determination of british anti-lewisite in plasma.

British anti-Lewisite (BAL) (2,3-dimercapto-1-propanol) is a potential therapeutic compound when used against the effects of cutaneous sulfur mustard, and a method for its determination in plasma has been developed. BAL and the internal standard (IS) ethane dithiol were isolated from plasma samples through solid-phase extraction and then reacted with 1-pentafluoropropionylimidazole, forming stable pentafluoropropionyl derivates that are sensitive to gas chromatographic-mass spectrometric analysis. Examination of concentration versus peak-area ratios of the BAL and IS derivatives demonstrated the method to be linear over a concentration range of 0.48 to 124 ng/mL in plasma when fit to a weighted (1/y2) least-squares regression. Correlation coefficients were 0.9943 to 0.9995 for six runs, and coefficients of variation (CV) were 2.5 to 8.7% over the eight concentrations tested. The intra- and interday accuracy and precision of this method was measured by examining six groups of eight unknown test samples (n = 6). Intraday accuracy, as expressed by percent error, was found to range from -15.4 to 0.21%, whereas the precision, expressed as %CV, was less than 9.8% over all sample concentrations. Interday test unknown sample results were similar in that the accuracy was shown to be -7.1 to 0.4%, and precision was 4.7 to 9.5%. BAL levels in frozen plasma (-70 degrees C) remained constant for more than 14 days with a CV of less than 10% for the eight concentrations tested. The data indicate that the method will provide accurate and precise determination of BAL at concentrations down to approximately 1 ng/mL in plasma. This procedure has been applied to determine preliminary time-concentration profile studies of BAL in the hairless guinea pig.

The application of the fluoride reactivation process to the detection of sarin and soman nerve agent exposures in biological samples.

The fluoride reactivation process was evaluated for measuring the level of sarin or soman nerve agents reactivated from substrates in plasma and tissue from in vivo exposed guinea pigs (Cava porcellus), in blood from in vivo exposed rhesus monkeys (Macaca mulatta), and in spiked human plasma and purified human albumin. Guinea pig exposures ranged from 0.05 to 44 LD50, and reactivated nerve agent levels ranged from 1.0 ng/mL in plasma obtained from 0.05 LD50 sarin-exposed guinea pigs to an average of 147 ng/g in kidney tissue obtained from two 2.0 LD50 soman-exposed guinea pigs. Positive dose-response relationships were observed in all low-level, 0.05 to 0.4 LD50, exposure studies. An average value of 2.4 ng/mL for reactivated soman was determined in plasma obtained from two rhesus monkeys three days after a 2 LD50 exposure. Of the five types of guinea pig tissue studied, plasma, heart, liver, kidney and lung, the lung and kidney tissue yielded the highest amounts of reactivated agent. In similar tissue and with similar exposure procedures, reactivated soman levels were greater than reactivated sarin levels. Levels of reactivated agents decreased rapidly with time while the guinea pig was alive, but decreased much more slowly after death. This latter chemical stability should facilitate forensic retrospective identification. The high level of reactivated agents in guinea pig samples led to the hypothesis that the principal source of reactivated agent came from the agent-carboxylesterase adduct. However, there could be contributions from adducts of the cholinesterases, albumin and fibrous tissue, as well. Quantitative analysis was performed with a GC-MS system using selected ion monitoring of the 99 and 125 ions for sarin and the 99 and 126 ions for soman. Detection levels were as low as 0.5 ng/mL. The assay was precise and easy to perform, and has potential for exposure analysis from organophosphate nerve agents and pesticides in other animal species.

Analysis and stability of the candidate sulfur mustard decontaminant S-330.

The chloroamide compound 1,3,4,6-tetrachloro-7,8-diphenyl-2, 5-diiminoglycoluril (S-330) was found to be a strong reactant in dermal formulations for the decontamination of sulfur mustard (HD). In this report, we present analytical methodologies applicable to the characterization, purity determination and quantitation of S-330 in bulk material or formulations. High-performance liquid chromatography-mass spectrometry (LC-MS) coupled with atmospheric pressure chemical ionization (APCI) interface or ultraviolet detector and nuclear magnetic spectroscopy (NMR) were used to identify and characterize S-330 and impurities in the synthetic lots or degradation products in formulations. Bulk synthesis using a chlorination process has yielded a product of 90% purity. The major impurity has been separated and identified structurally as the trichloro analog of S-330. Higher purity S-330 can be made using column chromatography, but this does not appear to be economical for large-scale production. Factors affecting the stability of S-330 in topical formulations include water content, pH, alcohols and UV light. Chloroamide S-330 decomposes at 50-60 degrees C and is not amenable for GC analysis. The HPLC technique is superior to NMR or active chlorine assay in the purity determination for S-330 in bulk material or formulations. In topical formulations containing S-330, 5-10% of water can be tolerated, but alcohols and acidic and basic conditions should be avoided.

Reactions of sulfides with S-330, a potential decontaminant of sulfur mustard in formulations.

Because the vesicant sulfur mustard (HD) remains a major chemical threat from either domestic terrorists or countries in conflict, topical preparations are being evaluated as protectants from HD exposure. The objective of this study was to evaluate the effectiveness of chloroamide S-330 as a potential reactive component in topical formulations. Therefore, the rate, mechanism and by-products of the oxidation reactions of sulfides by S-330 in solvent media or specific formulation vehicles were investigated. Using NMR, LC, LC-MS and GC-MS, the reactions of S-330 with HD, dibutyl sulfide (DBS) and methyl phenyl sulfide (MPS) were studied in acetonitrile, chloroform and perfluoropolyether (PFPE) oil. The oxidation of the three sulfides with S-330 was very rapid and completed in <4 min in acetonitrile-water or PFPE oil, but the rates of reaction in chloroform were significantly slower. In a large excess of S-330, the major products resulted from chlorination of the side chains. At a high HD/S-330 ratio, the major product was HD sulfoxide. Under both conditions, only a trace of HD sulfone, also a blistering agent, was observed. Reactions with DBS and MPS primarily gave sulfoxides and sulfones, with less side-chain chlorination. The chloroamide S-330 appeared to be a rapid and effective decontaminant of HD in either polar media or in a PFPE oil. The two alkyl and aryl sulfides are suitable simulants of HD for the initial screening and evaluation of S-330 or other similar oxidizing agents.

Multicomponent spectroscopic assay for hemoglobin and ferrihemoglobin species in methemoglobin treatment of cyanide poisoning.

Monitoring the concentrations of various hemoglobin and ferrihemoglobin species and their cyanide complexes is important in the study of the efficacy of methemoglobin-forming agents for the treatment of cyanide toxicity. In this study, the visible absorption spectra of three hemoglobin intermediates were experimentally determined and compared with computer-generated spectra. The data supported the assumption that the molar absorptivities of the intermediates are equivalent to the combined absorptivities of the component subunits. A multicomponent Fourier transform (FT)-aided full spectrum quantitation system (FSQ) to simultaneously measure hemoglobin (Hb), hemimethemoglobin (hemiMetHb), and the cyanide complex dicyanohemimethemoglobin (dicyhemiMetHb) was also evaluated. It was found that FSQ had satisfactory accuracy and precision to quantitate hemiMetHb and dicyhemiMetHb at concentrations from 2 to 20% of the total Hb, a range commonly encountered in the treatment of cyanide poisoning using methemoglobin-forming agents. The simplicity and rapid throughput of the method make it suitable for clinical evaluation studies and form the basis for the design of a portable instrument for field analysis of these species.

Analysis of hemoglobin derivatives by capillary isoelectric focusing and its application in the antidotal research of cyanide poisoning.

Cyanide toxicity can be reduced by the use of methemoglobin (MetHb) formers, and antidotal dosage is based on the extent of MetHb formation. Hemoglobin and ferrihemoglobin (MetHb, hemimethemoglobins alpha3+beta2+ and alpha2+beta3+, tetracyanmethemoglobin, and dicyanmethemoglobin) concentrations in human, pig, and mouse blood were determined after separation by isoelectric focusing with an octyl-bonded capillary. The predominant species formed in blood when MetHb formers, such as potassium ferricyanide, hydroxylamine, sodium nitrite, and 4-dimethylaminophenol (DMAP), added at molar ratios ranging from 1:10 to 1:1 to hemoglobin, are the valency hybrid intermediates alpha3+beta2+ and alpha2+beta3+. In the detoxication of cyanide with methemoglobin, an intermediate dicyanhemimethemoglobin was demonstrated to be the predominant species in the formation of tetracyanmethemoglobin. Complex mixtures of hemoglobin derivatives were observed with DMAP at 1:1 or greater molar ratio to hemoglobin. Comparison of the MetHb values obtained with a hemoxometer indicated that the valency hybrids were measured as MetHb and the values of oxidized hemoglobin were overestimated. In cyanide poisoning, incorrect dosages of MetHb formers could be calculated, and misinterpretation of MetHb data would result from methods that fail to discriminate among the various species of MetHb.

Degradation of three related bis(pyridinium)aldoximes in aqueous solutions at high concentrations: examples of unexpectedly rapid amide group hydrolysis.

The principal initial degradation products of two bis(pyridinium)aldoxime organophosphate-inhibited acetylcholinesterase reactivators, 1 (HI-6) and 3 (HS-6), in concentrated nonbuffered aqueous solutions approximating potential therapeutic dosage concentrations were found to be the carboxylic acid derivatives 2 and 4 formed from the hydrolysis of the amide functional group. Compounds 2 and 4 were prepared by heating 1 and 3 in the presence of high concentrations of hydroxylamine hydrochloride and characterized by 1H and 13C NMR, IR, and UV analyses. Estimates of the rates of hydrolysis of the amide groups in 1 and 3 and in model compounds 5, 7, and 8 under similar conditions were determined. The unexpectedly rapid hydrolysis of the amide groups in 1 and 3 was attributed to both the hydrogen ion catalysis of the concentrated aqueous solutions of the unusually acidic bis(pyridinium)aldoximes 1 and 3 and general acid catalysis by the aldoxime group.