A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 microL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure.

Ricin is a toxalbumin derived from the castor bean plant, Ricinus communis. Ricinine is an alkaloid (3-cyano-4-methoxy-N-methyl-2-pyridone) that shares a common plant source with ricin, and its presence in urine infers ricin exposure. A new quantification method for ricinine was developed that uses solid-phase extraction to prepare 1-mL urine samples (81% recovery) for a 5-min, isocratic high-performance liquid chromatography method, followed by electrospray ionization tandem mass spectrometry. Protonated molecular ions were selected in the multiple reaction monitoring mode and quantified by isotope dilution with (13)C(6)-labelled ricinine as the internal reference. Urine pools enriched with ricinine at two concentrations were characterized as quality control materials and then used to validate the method. The method limit of quantification was 0.083 ng/mL, even with a confirmation ion of low relative abundance. Ricinine was stable in human urine when heated at 90 degrees C for 1 h, and during storage at 25 degrees C and 5 degrees C for 3 weeks. The method was applied to an animal exposure study, a crude ricin preparation scheme, and a forensic analysis. These studies show that ricinine can be measured in rat urine at least 48 h after exposure. Ricinine is present in crude preparations of ricin, and it can be found in human urine after a lethal exposure to ricin.

An automated high-throughput immunomagnetic separation (IMS) method for diagnosing exposure to the organophosphorus nerve agents (OPNAs) sarin (GB), cyclohexylsarin (GF), VX, and Russian VX (RVX) was developed to increase sample processing capacity for emergency response applications. Diagnosis of exposure to OPNAs was based on the formation of OPNA adducts to butyrylcholinesterase (BuChE). Data reported with this method represent a ratio of the agent-specific BuChE adduct concentration, relative to the total BuChE peptide concentration that provides a nonactivity measurement expressed as percent adducted. All magnetic bead transfer steps and washes were performed using instrumentation in a 96-well format allowing for simultaneous extraction of 86 clinical samples plus reference materials. Automating extractions increased sample throughput 50-fold, as compared to a previously reported manual method. The limits of detection, determined using synthetic peptides, were 1 ng/mL for unadducted BuChE and GB-, GF-, VX-, and RVX-adducted BuChE. The automated method was characterized using unexposed serum and serum pools exposed to GB, GF, VX, or RVX. Variation for the measurement of percent adducted was <12% for all characterized quality control serum pools. Twenty-six (26) serum samples from individuals asymptomatic for cholinesterase inhibitor exposure were analyzed using this method, and no background levels of OPNA exposure were observed. Unexposed BuChE serum concentrations measured using this method ranged from 2.8 μg/mL to 10.6 μg/mL, with an average concentration of 6.4 μg/mL.

]In July 2004, two individuals developed blisters after the destruction of a WWl-era munition. To determine the causative agent, urine samples were collected from both the highly blistered patient (patient 1; 6.5% of total body surface area) and patient 2, who had only one small blister. Their urine was analyzed for metabolites of known vesicants including sulfur mustard (HD), Lewisite (L1), and nitrogen mustards. The urine samples only tested positive for metabolites of HD. Additional metabolites were measured to confirm the exposure of sulfur mustard agent HD, including thiodiglycol (TDG), TDG-sulfoxide, and the bismercapturate of mustard sulfone. On day 2 after the exposure, patient I had a lS-lyase metabolite level of 41 ng/mL, and patient 2 had a level of 2.6 ng/mt. Detectable levels of the ~-Iyase metabolite were observed in patient 1 for 11 days and in patient 2 for 7 days. Levels of TDG and both TDG and its sulfoxide measured together in the urine of patient 1 were found to be 24 ng/mL and 50 ng/mL, respectively, on day 2. The bis-mercapturate of mustard sulfone was detected in patient 1 (3.1 ng/mL) on day 2 but was not detected in samples taken on subsequent days.The metabolism of HD has been studied extensively in rats (3,4). Research has identified several urinary metabolites, including the free and conjugated forms of the simple hydrolysis product thiodiglycol (TDG) and TDG-sulfoxide (TDG's oxidized form), the glutathione-derived metabolite 1,1'-sul- fonylbis[2-S-(N-acetylcysteinyl)ethane], and the I~-lyase metabolites 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio)-ethylsulfonyl]ethane (MSMTESE). Although 1,]'-sulfonylbis[2-(methylthio)ethane] (SBMTE)was not identified in rat urine, it is a likely precursor to the I~-lyase metabolites that were identified (3-8).Recently, two individuals were thought to have been exposed to HD accidentally during the destruction of a WWI-era shell (9,10). The extent of blistering between the two individuals varied. One individual (patient 1) had blisters on an estimated 6.5% of his body including his hands and forearms; the second individual (patient 2) had only one small blister (9,10). Urine samples from both individuals were analyzed to confirm that the symptoms were caused by HD exposure.

Abrin is a toxic protein found in the jequirity seed. L-Abrine (N-methyl-tryptophan) is also found in the jequirity seed and can be used as a biomarker for abrin exposure. Analysis of L-abrine was added to an existing method for quantifying ricinine as a marker for ricin exposure in human urine and analytically validated. Accuracy and reproducibility were enhanced by including a newly synthesized (13)C(1)(2)H(3)-L-abrine internal standard. One-milliliter urine samples were processed using solid-phase extraction prior to a 6-min high-performance liquid chromatography separation. Protonated molecular ions were formed via electrospray ionization in a triple-quadrupole mass spectrometer and quantified via multiple reaction monitoring. Method validation included the characterization of two enriched urine pools, which were used as quality control materials. Endogenous levels of L-abrine were quantified in a reference range of 113 random urine samples at 0.72 +/- 0.51 ng/mL. Urinary concentrations of L-abrine were monitored in an intentional rat exposure study for up to 48 h. Comparing the results from the human reference range and the animal exposure study indicates that this method is suitable for quantifying L-abrine within 24 h post-exposure. Quantification of L-abrine beyond 24 h is limited by rapid excretion of the biomarker and the level of the L-abrine dose.

Benzene, an important component in gasoline, is a widely distributed environmental contaminant that has been linked to known health effects in animals and humans, including leukemia. In Mexico City, environmental benzene levels, which may be elevated because of the heavy traffic and the poor emission control devices of older vehicles, may pose a health risk to the population. To assess the potential risk, portable passive monitors and blood concentrations were used to survey three different occupational groups in Mexico City. Passive monitors measured the personal exposure of 45 workers to benzene, ethylbenzene, toluene, o-xylene and m-/p-xylene during a work shift. Blood concentrations of the above volatile organic compounds (VOCs), methyl tert-butyl ether, and styrene were measured at the beginning and the end of a work shift. Passive monitors showed significantly higher (p > 0.0001) benzene exposure levels among service station attendants (median = 330 microg/m3; range 130-770) as compared to street vendors (median = 62 microg/m3; range 49-180) and office workers (median = 44 microg/m3, range 32-67). Baseline blood benzene levels (BBLs) for these groups were higher than those reported for similar populations from Western countries (median = 0.63 microg/L, n = 24 for service station attendants; median = 0.30 microg/L, n = 6 for street vendors; and median = 0.17 microgr;g/L, n = 7 for office workers). Nonsmoking office workers who were nonoccupationally exposed to VOCs had BBLs that were more than five times higher than those observed in a nonsmoking U.S. population. BBLs of participants did not increase during the work shift, suggesting that because the participants were chronically exposed to benzene, complex pharmacokinetic mechanisms were involved. Our results highlight the need for more complete studies to assess the potential benefits of setting environmental standards for benzene and other VOCs in Mexico.ImagesFigure 1

The analysis of volatile organic compounds (VOCs) in whole human blood at the low parts-per-trillion level has until recently required the use of a high-resolution mass spectrometer to obtain the specificity and detection limits required for epidemiological studies of VOC exposure in the general public. Because of the expense and expertise required to operate and maintain a high-resolution instrument, the applicability of this method has been limited. These limitations are overcome in a new method using automated headspace solid-phase microextraction (SPME) in conjunction with a gas chromatograph and a benchtop quadrupole mass spectrometer. A combination of SPME and multiple single-ion monitoring minimizes the interferences and chemical noise associated with whole blood samples. This method permits the analysis of 10 VOCs in human blood while simplifying the sample preparation and reducing the possible exposure of the analyst to blood aerosols. Twelve samples can be run successively in a fully automated mode, thus eliminating the need for operator attention. Detection limits are below 50 ppt (pg/mL) for a majority of the VOCs tested with a 5-mL sample.

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