High-sensitivity determination of the degradation products of chemical warfare agents by capillary electrophoresis-indirect UV absorbance detection.

Capillary electrophoresis coupled with indirect UV absorbance detection was employed for the determination of the chemical warfare agent degradation products: methylphosphonic acid, ethyl methylphosphonate, isopropyl methylphosphonate, and pinacolyl methylphosphonate. Glutamic acid was used as a buffering agent at its isoelectric point (pH 3.22). In its zwitterionic form, glutamic acid does not act as a competing co-anion in the system, thus providing buffering capacity while maintaining high sensitivity. The indirect probe (phenylphosphonic acid) concentration was lowered to 1 mM from the 10 mM in previous literature studies, further enhancing sensitivity. Detection limits of 2 microM were achieved with hydrodynamic injection and up to 100-fold lower using electrokinetic injection. The increased buffering capacity of this system over previous methods led to migration time reproducibility RSD values of 0.18 to 0.22%. This represents a 10-fold improvement in reproducibility over previous studies with comparable or improved sensitivity.

Indirect laser-induced fluorescence detection for capillary electrophoresis using a violet diode laser.

The violet (415 nm) diode laser is used for indirect laser-induced fluorescence detection in capillary electrophoretic separations of inorganic anions and chemical warfare agent degradation products. Inorganic anions were detected using 8-hydroxypyrene-1,3,6-trisulfonic acid as the indirect probe and achieved submicromolar (40-80 ppb) detection limits in a 2-min separation. The chemical warfare agent degradation products methylphosphonic acid, ethyl methylphosphonate, isopropyl methylphosphonate, and pinacolyl methylphosphonate were detected using the porphyrin tetrakis(4-sulfophenyl)porphine as the indirect probe and achieved detection limits of 0.1 microM (9 ppb), which are 1 order of magnitude better than that achieved using indirect UV detection. Baseline stability achieved with the violet diode laser was excellent, with dynamic reserve (DR) values of > 1000, which are 15 times better than that achieved using an unstabilized HeCd laser.

Competitive immunoassay for staphylococcal enterotoxin A using capillary electrophoresis with laser-induced fluorescence detection.

Staphylococcal enterotoxins are a family of toxic proteins secreted by S. aureus. Using capillary electrophoresis (CE) linked with laser-induced fluorescence, a highly sensitive and selective assay using antibody-antigen recognition was developed for the determination of Staphylococcal enterotoxin A. Staphylococcal enterotoxin A (SEA) was chemically labeled with fluorescein and the product was used as a fluorescent tracer. A competitive assay was developed to detect SEA at concentrations between 0.3 nM and 6.5 nM with standard deviations of less than 5%. The detection limit was found to be 3 amol with the potential improvement by further optimization of the assay. No cross-reactivity between staphylococcal enterotoxin B and the SEA antibody was found at the concentrations used for the CE immunoassay.

Elevated frequency of sister chromatid exchanges in lymphocytes of victims of the Tokyo sarin disaster and in experiments exposing lymphocytes to by-products of sarin synthesis.

More than 5000 passengers of Tokyo subway trains were injured with toxic chemicals including the nerve gas sarin. Most of the victims examined had marked miosis and decreased serum cholinesterase activity. To monitor the genetic aftereffects of sarin exposure, we measured sister chromatid exchanges (SCEs) of the victims using peripheral blood lymphocytes. The frequency of SCEs was significantly higher in the victims than in the control group. Analyzing results using samples of urine from the victims suggested that the victims were exposed to not only sarin per se, but by-products of sarin synthesis, i.e. diisopropyl methylphosphonate (DIMP), diethyl methylphosphonate (DEMP) and ethyl isopropyl methylphosphonate (EIMP). Thus, the in vitro SCE-inducing effect of DIMP, DEMP and EIMP was examined using human lymphocytes and we obtained positive results.

Biological monitoring of metabolites of sarin and its by-products in human urine samples.

More than 20,000 passengers of Tokyo underground trains were intoxicated with warfare toxic chemicals. Most of the patients examined had marked miosis and decreased serum cholinesterase activity. Transient increase of serum CPK activity after 3 days of the exposure was the another sign. We intensively analyzed the metabolites in the urine of 4 patients. The following analytic results indicated the exposure to sarin as well as contaminated compounds such as diisopropyl methylphosphonate (DIMP), ethyl methylphosphonate fluoridate (EMPF, or ethylsarin), diethyl methylphosphonate (DEMP), and ethyl isopropyl methylphosphonate (EIMP). (1) Isopropanol (IPA) and ethanol (EtOH) were detected of large quantities in the urine samples, and were thought to be derived from sarin and the sarin counterpart, EMPF, DIMP, DEMP and EIMP. (2) Monoalkyl methylphosphonic acids (isopropyl methylphosphonic acid (IMPA) and ethyl methylphosphonic acid (EMPA) also were excreted in large amounts with taking the similar excretion pattern of IPA and EtOH. (3) The metabolite only derived from sarin and ethylsarin is F anions whose integral output in the urine was less than the equimolar level of the excreted (IMPA + EMPA + IPA + EtOH). (4) Other corroborative findings were low lethality: of more than 5,510 patients treated, 11 were acutely dead. (5) Nine exposed males had higher sister chromatid exchange (SCE) rate (5.00 +/- 1.48/cell) than the control (3.81 +/- 0.697/cell), because dialkyl methylphosphonates seemed to have alkylating activity and producing DNA adducts. The SCE rate also increased after the in vitro exposure of lymphocytes to dialkyl methylphosphonates.

Method for the analysis of the methylphosphonic acid metabolites of sarin and its ethanol-substituted analogue in urine as applied to the victims of the Tokyo sarin disaster.

An analysis method for the methylphosphonic acid metabolites of sarin in urine using trimethylsilyl derivatization and flame photometric detection is described in this report. Authentic reference standards of isopropyl methylphosphonic acid (IMPA) and ethyl methylphosphonic acid (EMPA) as well as methylphosphonic acid were employed to estimate the concentration in human urine. A sample pretreatment procedure was developed for urine using a column of cation-loaded ion-exchange resins (Ag+ -, Ba2+ - or H+ -Dowex) and adjusting the pH of the eluate from the column to 3.75-3.85 improved recovery of the target compounds. The eluate was evaporated to dryness under vacuum prior to trimethylsilylation, to remove water and any hydroxy- or amino-carrying volatile substances. The sarin metabolites, because of their low volatility, were concentrated and could be derivatized for analysis. The use of synthesized authentic sarin and ethylsarin metabolites, i.e., IMPA and EMPA, made it possible to establish the necessary sample pretreatment procedures for derivatization and gas chromatography-flame photometric detection (GC-FPD) analysis. The detection limits were 0.025 ppm both for EMPA and [MPA, and 0.625 microM for MPA, respectively. This method can be useful for estimating the exposure level to sarin by assaying the metabolites in urine and it is applicable to a large numbers of samples.

Picomolar assay of native proteins by capillary electrophoresis precolumn labeling, submicellar separation, and laser-induced fluorescence detection.

We report a method for the assay of proteins at concentrations lower than 10(-)(10) M with as little as 200 amol of protein. High sensitivity is accomplished by derivatizing the ε-amino group of the protein's lysine residues with the fluorogenic dye 5-furoylquinoline-3-carboxaldehyde and use of a sheath flow cuvette fluorescence detector. Most proteins have a large number of lysine residues; therefore, a large number of fluorescent molecules can be attached to each protein molecule. In general, precolumn labeling improves sensitivity but degrades resolution due to the inhomogeneity of the reaction products from multiple labeling. However, we demonstrate that, through careful manipulation of the separation and reaction conditions, high sensitivity can be obtained without excessive loss in separation efficiency. Over 190 000 theoretical plates are obtained for fluorescently labeled ovalbumin.

Efficacy of HLö-7 and pyrimidoxime as antidotes of nerve agent poisoning in mice.

The toxicity and efficacy of two oximes, HLö-7 and pyrimidoxime, were evaluated in mice and compared to those obtained with HI-6. HLö-7 and pyrimidoxime produced 24 h LD50 values of 356 and 291 mg/kg (i.p.), respectively. In combination with atropine (17.4 mg/kg, i.p.), HLö-7 was a very efficient therapy against poisoning by 3 x LD50 dose of soman, sarin and GF and 2 x LD50 dose of tabun with ED50 values of 12.4, 0.31, 0.32 and 25.2 mg/kg, respectively. In contrast, pyrimidoxime was a relatively poor therapy which resulted in ED50 values of greater than 150, 5.88, 100 and 71 mg/kg against poisoning by soman, sarin, GF and tabun, respectively. HLö-7 produced significant (p less than 0.05) reactivation of phosphorylated acetylcholinesterase, in vivo, resulting in 47, 38, 27 and 10% reactivation of sarin, GF, soman and tabun inhibited mouse diaphragm acetylcholinesterase, respectively. HLö-7 also antagonized sarin-induced hypothermia in mice suggesting that it reactivated central acetylcholinesterase. The potential of HLö-7 as a replacement oxime for the treatment of nerve agent poisoning is discussed.

Comparison of several oximes against poisoning by soman, tabun and GF.

Three oximes currently being evaluated for adoption as replacement nerve agent therapy by various countries were compared for therapeutic efficacy against the toxic organophosphate inhibitors soman and tabun under a standard set of conditions. These oximes together with PAM-Cl and toxogonin, were also compared for efficacy against GF, an agent weaponized by Iraq. The order of effectiveness against soman was HI-6 greater than HLö-7 greater than pyrimidoxime. HLö-7 was very effective against tabun poisoning while HI-6 and pyrimidoxime were of moderate value. Against GF, HI-6 and HLö-7 were extremely effective, toxogonin was moderately effective, and PAM-Cl and pyrimidoxime were the least effective. HI-6 provided a high level of protection against all of the agents tested as did HLö-7 to a slightly lesser degree. The other oximes suffered from their lack of effects against one or more of the organophosphates.

Toxicity of organophosphate nerve agents and related phosphonylated oximes compared to their anticholinesterase activity in neuron cultures.

Oximes, such as pralidoxime and toxogonin, are important therapeutic agents for the treatment of organophosphate (OP) nerve agent poisoning. Oximes can react with these nerve agents to give intermediates, phosphonylated oximes, which may be equally toxic to the parent OP. The sc LD50s of a series of phosphonylated 2-butanone and 2,3-butanedione monoximes were compared to the sc LD50s of their parent OPs (tabun, sarin, and VX) in CD-1 mice. In every case the derivatives were significantly less toxic than their parent nerve agents. Times to death, and to signs of poisoning, were inversely proportional to the dose of test compound, and in all mortalities, blood serum acetylcholinesterase (AChE) was severely inhibited. The relative potencies of these compounds, as well as soman, cyclohexyl methylphosphonofluoridate, and diisopropyl fluorophosphate, as inhibitors of AChE in primary cultures of mouse embryo neurons, correlated with their in vivo toxicities. The results indicate that mouse embryo neuron cultures may be a useful model with which to study this class of compounds.