Determination of a bound musk xylene metabolite in carp hemoglobin as a biomarker of exposure by gas chromatography-mass spectrometry using selected ion monitoring.

Musk xylene (MX) is widely used as a fragrance ingredient in commercial toiletries. Identification and quantitation of a bound 4-amino-MX (AMX) metabolite was carried out by gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring (SIM). Detection of AMX occurred after the cysteine adducts in carp hemoglobin (Hb), derived from the nitroso metabolite, were released by alkaline hydrolysis. The released AMX metabolite was extracted into n-hexane. The extract was preconcentrated by evaporation and analyzed by GC-SIM-MS. The concentration of AMX metabolite was found to range from 6.0 to 30.6 ng/g in the carp Hb, collected from the Las Vegas Wash and Lake Mead, NV areas. The presence of an AMX metabolite in the carp Hb was confirmed when similar mass spectral features and the same retention time of the AMX metabolite were obtained for both standard AMX and carp Hb extract solutions. In the nonhydrolyzed and reagent blank extracts, the AMX metabolite was not detected.

Identification and characterization of a pro-tumor necrosis factor-alpha-processing enzyme from the ADAM family of zinc metalloproteases.

Tumor necrosis factor-alpha (TNF) is initially expressed as a 26-kDa membrane-bound precusor protein (pro-TNF) that is shed proteolytically from the cell surface, releasing soluble 17-kDa TNF. We have identified human ADAM 10 (HuAD10) from THP-1 membrane extracts as a metalloprotease that specifically clips a peptide substrate spanning the authentic cleavage site between Ala76 and Val77 in pro-TNF. To confirm that HuAD10 has TNF processing activity, we cloned, expressed, and purified an active, truncated form of HuAD10. Characterization of recombinant HuAD10 (rHuAD10) suggests that this enzyme has many of the properties (i.e. substrate specificity, metalloprotease activity, cellular location) expected for a physiologically relevant TNF-processing enzyme.

Volatile organic analysis by direct aqueous injection.

Gas chromatographic environmental analysis by direct aqueous injection (DAI) was studied for 24 volatile organic analytes (VOAs). Internal standardization was used to determine the precision of analysing these compounds by DAI. Aqueous samples were directly introduced to a gas chromatograph using fused-silica, mega-bore capillary column separation with subsequent full-scan ion trap mass spectral detection. Triplicate injections at seven levels of VOA standard solutions over a 10(3) concentration range were performed using an autosampler set up for on-column injection of 0.2 microl. Comparison of single-ion response curves to triple-ion response curves showed that triple-ion quantitation was more sensitive and precise than single-ion quantitation. Of the 24 VOAs determined at the 20 parts per billion (ppb) level, 19 and 20 were detected by the single-ion calibration and triple-ion calibration, respectively. The weighted and non-weighted regression correlation coefficients, r(2), for the 24 responses curves by the two methods, ranged from 0.910 to 0.998, with 76 of 96 being greater than 0.990. Precision, as measured by per cent relative standard deviation, was shown to be best for later eluting compounds and for higher concentrations. Analysis of an environmental sample by DAI was accomplished in 12 min and indicated the presence of benzene at 80 ppb and chlorobenzene at 2 ppm. This demonstrated the feasibility of applying this technique for screening. Several chlorinated benzenes were also detected, establishing the potential for expanding the method to include higher boiling compounds.

Supercritical fluid extraction of high sulfur soils, with use of a copper scavenger.

Supercritical fluid extraction (SFE) of sulfur-containing soils and sediments was hindered by deposition of elemental sulfur in the restrictor stopping the flow of carbon dioxide. It was found that a copper scavenger column placed after the sample cell removed the elemental sulfur, making SF extraction and quantification possible. Conditions are described for SFE and analysis of two standard reference materials (SRM), with use of a copper scavenger column. Quantification was performed by gas chromatography with mass spectrometric detection and the results were compared with the certified SRM values.

Environmental semivolatiles and nonvolatiles analysis by hyphenated spectral techniques.

Environmental sample extracts contain a variety of volatile and nonvolatile organic compounds exhibiting a range of polarities and concentrations. Although gas chromatography/mass spectrometry (GC/MS) is the method of choice thus far for such analyses, this technique used alone cannot adequately characterize the volatiles in such samples and is not amenable to environmental nonvolatiles. A more complete characterization of environmental and hazardous waste samples is required to assess the dangers posed to the nation's groundwater by hazardous waste dumps. Online spectral confirmation by directly linked GC/Fourier transform infrared (FTIR)/MS is shown to provide useful structural information on environmental volatiles in hazardous wastes, even when the analyte's spectrum is not in either spectral database. This information can lead to biological-hazard estimation. The diffuse reflectance Fourier transform infrared (DRIFT) technique, used in conjunction with thermospray MS or fast atom bombardment (FAB) MS, provides confirmed identifications or confirmed compound class assignments of organic nonvolatiles in solid wastes. This is believed to be the first report of spectral confirmation (identification or functionality) of organic volatiles and nonvolatiles in environmental samples.

Thermospray LC/MS/MS analysis of wastewater for disperse azo dyes.

Primary effluent from a municipal wastewater treatment plant was used as the feed in bench scale activated sludge systems. These systems were spiked with disperse azo dyes at 1 mg 1-1 and 5 mg 1-1 levels and were sampled at various points in the process. Samples were analysed by high performance liquid chromatography with UV = visible detection and by thermospray ionization MS and tandem mass spectrometry (MS/MS) using direct injection or via column chromatography. The tandem mass spectrometry techniques were used both for method development purposes and for the specificity and extra information these techniques can provide. The investigation of the fate of disperse azo dyes in the activated sludge process was a major feature of this study. Major degradation products have been identified by tandem mass spectrometry analyses of these wastewaters. Precision and accuracy data generated by the thermospray tandem mass spectrometry technique are compared to those derived from the high performance liquid chromatography/UV-visible method.

Quantitation of chemically induced DNA strand breaks in human cells via an alkaline unwinding assay.

DNA strand breaks induced in human CCRF-CEM cells by electrophilic chemicals (carcinogens/mutagens) can be readily quantitated via a facile alkaline unwinding assay. This procedure estimates the number of chemically induced DNA strand breaks on the basis of the percentage DNA converted from double-stranded to single-stranded form during an exposure to the alkaline unwinding conditions. The assay is based on the assumption that each strand break serves as a strand unwinding point during the alkaline denaturation. The extent of strand separation can be standardized with respect to the initial level of induced strand breaks by the use of X-rays, which produce known levels of DNA strand breaks per rad in mammalian cells. Subsequent to the alkaline exposure, the single- and double-stranded DNA were separated by use of thermostated hydroxylapatite columns (60 degrees C), and the DNA was quantitated via a fluorescence assay (Hoechst 33258 compound). A correlation was shown between mammalian DNA strand-breaking potential (as measured in this procedure) and the propensity of these chemicals to revert Salmonella typhimurium TA100.

Comparative metabolism of 1,2,4-trichlorobenzene in the rat and rhesus monkey.

1,2,4-Trichloro[14C]benzene (TCB) was administered po (10 mg/kg) and iv (10 mg/kg) to rats and rhesus monkeys. Urine was collected at 24 hr and the major urinary metabolites were quantified and identified. By 24 hr, the monkey had excreted 22% of the iv dose and roughly 40% of the po dose in the urine. Less than 1% of the radioactivity was found in the monkey's feces. An isomeric pair of 3,4,6-trichloro-3,5-cyclohexadiene-1,2-diol glucuronides accounted for between 48 and 61% of the urinary metabolites. Glucuronides of 2,4,5- and 2,3,5-trichlorophenol (TCP) accounted for 14 to 37%, and unconjugated TCP's accounted for 1-37% of the monkey's urinary metabolites. For the rat, 84% of the po dose and 78% of the iv dose were collected in the urine by 24 hr; 11% and 7%, respectively, were the amounts collected in the feces. Two isomers, 2,4,5- and 2,3,5-, of N-acetyl-S-(trichlorophenyl)-L-cysteine accounted for 60-62% of the rat's urinary metabolites. Free 2,4,5- and 2,3,5-isomers of trichlorothiophenol amounted to 33% of the urinary metabolites in the po dosed rats and 28% in the iv dosed rats; free 2,4,5- and 2,3,5-TCP's amounted to 1% and 10%, respectively. These results show that there is a sharp division in the types of conjugates formed in the metabolism of 1,2,4-TCB by the rat and rhesus monkey.

Metabolism of bis(2-chloroethyl)ether and bis(2-chloroisopropyl)ether in the rat.

Male rats were given single peroral doses of bis(1-14C-2-chloroethyl)ether ([1-14C]BCEE) (40 mg/kg) and of bis(1-14C-2-chloroisopropyl)ether ([1-14C]BCIE) (90 mg/kg). Excretion of 14CO2 and urinary 14C was followed for 48 hr. The time required to eliminate one half of the dose was 12 hr for [1-14C]BCEE and 19 hr for [1-14C]BCIE. In the case of [1-14C]BCEE, expired 14CO2 accounted for 11.5 +/- 5.6(SD)% of the dose, urinary 14C accounted for 64.7 +/- 14.8%, and 2.4 +/- 1.3% was found in the feces. The figures for [1-14C]BCIE were 20.3 +/- 9.4% expired as 14CO2, 47.5 +/- 8.1% as urinary 14C, and 3.8 +/- 0.3% as fecal 14C. Thiodiglycolic acid (TDGA) accounted for roughly 75% of the total urinary 14C collected after the [1-14C]BCEE dose. Lesser metabolites of BCEE were 2-chloroethoxyacetic acid (CEAA) (5%), and N-acetyl-S-[2-(2-chloroethoxy)ethyl]-L-cysteine (ACEEC) (7%). Metabolites of [1-14C]BCIE identified in rat urine were 2-(2-chloro-1-methylethoxy)propanoic acid (CMEPA), roughly 36% of the total urinary 14C, and N-acetyl-S-(2-hydroxypropyl)-L-cysteine (AHPC) at 19%.