Improvements of the fluoride reactivation method for the verification of nerve agent exposure.

One of the most appropriate biomarkers for the verification of organophosphorus nerve agent exposure is the conjugate of the nerve agent to butyrylcholinesterase (BuChE). The phosphyl moiety of the nerve agent can be released from the BuChE enzyme by incubation with fluoride ions, after which the resulting organophosphonofluoridate can be analyzed with gas chromatography-mass spectrometry (GC-MS). This paper describes recent improvements of the fluoride-induced reactivation in human plasma or serum samples by enhancing the sample preparation with new solid-phase extraction cartridges and the MS analysis with large volume injections. Analysis is performed with thermal desorption GC with either mass selective detection with ammonia chemical ionization or high-resolution MS with electron impact ionization. The organophosphorus chemical warfare agents analyzed in this study are O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate, ethyl methylphosphonofluoridate, isopropyl methylphosphonofluoridate (sarin, GB), O-ethyl N,N-dimethylphosphoramidocyanidate, ethyl N,N-dimethylphosphoramidofluoridate, and cyclohexyl methylphosphonfluoridate. Detection limits of approximately 10 pg/mL plasma were achieved for all analytes, which corresponds to 0.09% inhibition with GB on a sample with normal BuChE levels.

Measurement of bisphenol A levels in human urine.

We report a new approach for assessing human exposure to bisphenol A (BPA) by measuring BPA in urine after enzymatic deglucuronidation. This method involves addition of (13)C(12)-labeled BPA, enzymatic deconjugation, solid-phase extraction, and derivatization with pentafluorobenzyl bromide. The product of the derivatization is separated by gas chromatography followed by mass spectrometric detection using negative chemical ionization and selected ion monitoring. Using this analysis method, urine samples fortified with both a constant level of labeled BPA and a range of unlabeled BPA levels (0.27-10.6 ng/ml) demonstrated constant percentage recovery. In addition, a range of urine sample volumes (0.25-10.0 ml) with constant amounts of added internal standard produced a linear response (r(2)=0.99). The method limit of detection was 0.12 ng/ml. This method was validated by duplicate analyses using gas chromatography coupled to a high-resolution mass spectrometer.

Levels of methyleugenol in a subset of adults in the general U.S. population as determined by high resolution mass spectrometry.

We developed a sensitive and accurate analytical method for quantifying methyleugenol (ME) in human serum. Our method uses a simple solid-phase extraction followed by a highly specific analysis using isotope dilution gas chromatography-high resolution mass spectrometry. Our method is very accurate; its limit of detection is 3.1 pg/g and its average coefficient of variation is 14% over a 200-pg/g range. We applied this method to measure serum ME concentrations in adults in the general U.S. population. ME was detected in 98% of our samples, with a mean ME concentration of 24 pg/g (range < 3.1-390 pg/g). Lipid adjustment of the data did not alter the distribution. Bivariate and multivariate analyses using selected demographic variables showed only marginal relationships between race/ethnicity and sex/fasting status with serum ME concentrations. Although no demographic variable was a good predictor of ME exposure or dose, our data indicate prevalent exposure of U.S. adults to ME. Detailed pharmacokinetic studies are required to determine the relationship between ME intake and human serum ME concentrations.

Isotope dilution--mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I.

An enzymatic hydrolysis isotope dilution-mass spectrometric method was developed for reference quantification of specific proteins. The analytical procedure involved measuring a reproducibly hydrolyzed peptide (serving as the primary standard) unique to a specific protein. This new mass spectrometric method was evaluated by assessing the concentration of apolipoprotein (apo) A-I in the European Community Bureau of Reference (BCR) lyophilized Certified Reference Material (CRM 393). We used the method to make 96 measurements (4 replicate analyses of 4 enzymatic digests of 6 vials of BCR-CRM 393), which gave an average total protein mass of 1.048 mg (+/- 1.0% at 99% confidence limits). The total overall analytical CV was 3.95%. The results of this evaluation of our model approach to determine the concentration of a specific protein in a purified preparation demonstrated that our new mass spectrometric method can be used to measure apolipoproteins and other specific proteins without the use of epitopic immunoassay methods.

Analytical characterization of peptide contaminants of L-tryptophan.

Reversed-phase liquid chromatographic fractions of extracts of 2 preparations of eosinophilia-myalgia syndrome (EMS)-associated L-tryptophan were analyzed by proton nuclear magnetic resonance spectrometry, mass spectrometry, microbial-growth inhibition, and amino acid residue analyses. Fraction components demonstrated properties of an antibiotic peptide resembling bacitracin. Many peptide antibiotics like bacitracin are secondary metabolites of Bacillus species, genus of the tryptophan producer organism for the implicated manufacturer. In order to determine whether a correlation exists between individual EMS cases and the concentration of peptides or bacitracin consumed, reliable methods must be developed for quantification of the total of isoforms.

An improved radioimmunoassay of C-peptide and its application in a multiyear study.

A commercial radioimmunoassay (RIA) for human proinsulin C-peptide was modified to improve its ruggedness and specificity, to decrease the influence of specimen matrix, and to shorten "hands-on" time. In the new protocol, we prepare calibrators in a C-peptide-free serum pool, prepared by treatment with activated charcoal (biological matrix), instead of in a defined matrix. This yielded essentially 100% analytical recoveries for C-peptide concentrations up to 300 pmol/L, a broader analytical range. We also corrected calibrators and unknown samples for nonspecific binding (NSB). Decreasing the concentration of ethanol (from 950 to 880 mL/L) for differential precipitation of the antigen-antibody complex resulted in an NSB of less than 10%, while maintaining high bound/total count percentages for samples and calibrators. C-peptide is thermally unstable without aprotinin at -20 degrees C and with or without aprotinin at 4 degrees C or above, but multiple freeze-thaw cycles do not affect C-peptide in serum. The modified C-peptide assay was applied to plasma from a multiyear study (fasting and post-carbohydrate-challenge subjects). During the four years of the study CVs ranged from 1.9% to 8.6% for replicate analyses of C-peptide in samples with concentrations less than or equal to 500 pmol/L. Between-run CVs were 3.8% to 8.2%, total CVs 3.8% to 10.7%.