Evaluation of qualitative and quantitative immunoassays to detect barley contamination in gluten-free beer with confirmation using LC/MS/MS.

To meet the need for the detection and quantitation of barley gluten in beer, qualitative screening and quantitative immunoassays based on the monoclonal antigluten antibody 401/21 (Skerritt) were validated in a single laboratory. Sample replicates were tested at each stage of beer production using multiple yeast strains and methods of end-stage protein removal. Quantitation was performed using barley-specific standards based on barley flour extracts. Immunoassay results were confirmed using LC/MS/MS for barley-specific peptides. The LOD for the qualitative screening test was 5 mg/L barley gluten. Recovery for the barley-spiked worts ranged from 81 to 128% in the quantitative ELISA assay; the LOD was <1 mg/L, and the LOQ was 5 mg/L. Both screening and confirmation methods were found to be fit for the purposes of detection of low levels of barley gluten in beer.

CYP1A1 and CYP1B1-mediated biotransformation of the antitrypanosomal methamidoxime prodrug DB844 forms novel metabolites through intramolecular rearrangement.

DB844 (CPD-594-12), N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}-nicotinamidine, is an oral prodrug that has shown promising efficacy in both mouse and monkey models of second stage human African trypanosomiasis. However, gastrointestinal (GI) toxicity was observed with high doses in a vervet monkey safety study. In the current study, we compared the metabolism of DB844 by hepatic and extrahepatic cytochrome P450s to determine whether differences in metabolite formation underlie the observed GI toxicity. DB844 undergoes sequential O-demethylation and N-dehydroxylation in the liver to form the active compound DB820 (CPD-593-12). However, extrahepatic CYP1A1 and CYP1B1 produced two new metabolites, MX and MY. Accurate mass and collision-induced dissociation mass spectrometry analyses of the metabolites supported proposed structures of MX and MY. In addition, MY was confirmed with a synthetic standard and detection of nitric oxide (NO) release when DB844 was incubated with CYP1A1. Taken altogether, we propose that MX is formed by insertion of oxygen into the amidine CN to form an oxaziridine, which is followed by intramolecular rearrangement of the adjacent O-methyl group and subsequent release of NO. The resulting imine ester, MX, is further hydrolyzed to form MY. These findings may contribute to furthering the understanding of toxicities associated with benzamidoxime- and benzmethamidoxime-containing molecules.

Novel aspects of quantitation of immunogenic wheat gluten peptides by liquid chromatography-mass spectrometry/mass spectrometry.

A novel, specific and sensitive non-immunological liquid chromatography-mass spectrometry (LC-MS) based assay has been developed to detect and quantify trace levels of wheat gluten in food and consumer products. Detection and quantification of dietary gluten is important, because gluten is a principle trigger of a variety of immune diseases including food allergies and intolerances. One such disease, celiac sprue, can cause intestinal inflammation and enteropathy in patients who are exposed to dietary gluten. At present, immunochemistry is the leading analytical method for gluten detection in food. Consequently, enzyme-linked immunosorbent assays (ELISAs), such as the sandwich or competitive type assays, are the only commercially available methods to ensure that food and consumer products are accurately labeled as gluten-free. The availability of a comprehensive, fast and economic alternative to the immunological ELISA may also facilitate research towards the development of new drugs, therapies and food processing technologies to aid patients with gluten intolerances and for gluten-free labeling and certification purposes. LC-MS is an effective and efficient analytical technique for the study of cereal grain proteins and to quantify trace levels of targeted dietary gluten peptides in complex matrices. Initial efforts in this area afforded the unambiguous identification and structural characterization of six unique physiologically relevant wheat gluten peptides. This paper describes the development and optimization of an LC-MS/MS method that attempts to provide the best possible accuracy and sensitivity for the quantitative detection of trace levels of these six peptides in various food and consumer products. The overall performance of this method was evaluated using native cereal grains. Experimental results demonstrated that this method is capable of detecting and quantifying select target peptides in food over a range from 10pg/mg to 100ng/mg (corresponding to approximately 0.01-100ppm). Limits of detection (LOD) and quantification (LOQ) for the six target peptides were determined to range from 1 to 30pg/mg and 10-100pg/mg respectively. Reproducibility of the assay was demonstrated by evaluation of calibration data as well as data collected from the analysis of quality control standards over a period of four consecutive days. The average coefficient of determination (R(2)) for each peptide was consistently found to be >0.995 with residuals ranging from approximately 80% to 110%. Spike recovery data for each peptide in various matrices was evaluated at a concentration level near the approximate LOQ for each, as well as at higher concentration levels (30 and 60ng/mg). The average range of accuracy of detection for all peptides at the lower concentration level was determined to be 90% (+/-11), while accuracy at the 30 and 60ng/mg levels was 98% (+/-5%) and 98% (+/-3%), respectively. The usefulness and capabilities of this method are presented in a practical application to prospectively screen a variety of common commercially available (native and processed) gluten-containing and gluten-free foods and products.

A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes.

The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.

N-Substituted amino acid N'-benzylamides: synthesis, anticonvulsant, and metabolic activities.

Amino acid amides (AAA) were prepared and evaluated in seizure models. The AAA displayed moderate-to-excellent activity in the maximal electroshock seizure (MES) test and were devoid of activity in the subcutaneous Metrazol-induced (scMet) seizure test. The AAA anticonvulsant activity was neither strongly influenced by the C(2) substituent nor by the degree of terminal amine substitution. An in vitro metabolism study suggested that the structure-activity relationship pattern was due, in part, to metabolic processes that occurred at the N-terminal amine unit.

Metabolites of an orally active antimicrobial prodrug, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime, identified by liquid chromatography/tandem mass spectrometry.

DB75 (2,5-bis(4-amidinophenyl)furan) is a promising antimicrobial agent against African trypanosomiasis and Pneumocystis carinii pneumonia. However, it suffers from poor oral activity in rodent models for both infections. In contrast, a novel prodrug of DB75, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), has excellent oral activity. DB289 is currently undergoing clinical investigation as a candidate drug to treat primary stage African trypanosomiasis and Pneumocystis carinii pneumonia. In this study, metabolites of DB289 formed after incubation with freshly isolated rat hepatocytes were characterized using liquid chromatography/ion trap mass spectrometry. Administration of DB289 and octadeuterated DB289 in a 1 : 1 mixture greatly facilitated metabolite identification by providing isotope patterns with twin ions separated by 8 m/z units in the ratio 1 : 1, in the extracted ion chromatograms of molecular ions and in the product ion mass spectra of metabolites. Ten metabolites were identified. Series of O-demethylations and N-dehydroxylations led to the metabolic activation of DB289 to DB75 with the production of four intermediate phase I metabolites. Phase II glucuronidation and sulfation led to the formation of four glucuronide and one sulfate metabolites.

Nitroxyl disulfides, novel intermediates in transnitrosation reactions.

A novel anionic RSN(O)SR species, the intermediate in transnitrosation reactions, was explored computationally with B3LYP and CBS-QB3 methods. The species resembles a nitroxyl coordinated to a highly distorted disulfide, and it differs significantly from intermediates in nucleophilic acyl substitution. Reactions of the following species were computed for comparison: MeS(-) + MeSNO; MeO(-) + MeONO; MeS(-) + MeSCHO; MeO(-) + MeOCHO. The last two have very different intermediates from the first two. Mass spectrometric experimental evidence is presented that is consistent with the formation of a nitroxyl disulfide in the gas phase. The calculated proton affinity and redox potentials of the intermediate are also reported.

Characterizing the fragmentation of 2,5-bis (4-amidinophenyl)furan-bis-O-methylamidoxime and selected metabolites using ion trap mass spectrometry.

A novel prodrug [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289)] of the promising antimicrobial agent, 2,5-bis(4-amidinophenyl)furan (DB75), has excellent oral activity. It is currently undergoing phase II clinical evaluation as an orally administered drug candidate against African trypanosomiasis and Pneumocystis carinii pneumonia. The sequential product ion (MS(n)) fragmentations of DB289 and selected metabolites were characterized using ion trap mass spectrometry with electrospray ionization. An unusual homolytic bond cleavage, formation of an odd-electron ion from an even-electron ion with the loss of a radical, was commonly seen in the fragmentation patterns of DB289 and its metabolites. Both O-ethyl and N-methyl homologues of DB289 were utilized to confirm this fragmentation pathway. The labile hydrogen atoms in DB289 are readily exchanged with deuterium atoms in the solvent containing deuterium oxide (D2O) instead of water. The mass shift patterns displayed in the product ion spectra of DB289 in D2O proved useful in verifying the fragmentation pathway. Octadeuterated DB289 and DB75 (d-labeling on the diphenyl rings) showed unequivocally that the diphenylfuran moiety is not involved in the fragmentation. The fragmentation pathways uncovered in this work will facilitate structural characterization of all the metabolites produced in the metabolic activation of DB289.