Mechanisms of acrylamide formation: Maillard-induced transformation of asparagine.

The formation of acrylamide (AA) from L-asparagine was studied in Maillard model systems under pyrolysis conditions. While the early Maillard intermediate N-glucosylasparagine generated approximately 2.4 mmol/mol AA, the Amadori compound was a less efficient precursor (0.1 mmol/mol). Reaction with alpha-dicarbonyls resulted in relatively low AA amounts (0.2-0.5 mmol/mol), suggesting that the Strecker aldehyde pathway is of limited relevance. Similarly, the Strecker alcohol 3-hydroxypropanamide generated low amounts of AA (0.2 mmol/mol). On the other hand, hydroxyacetone afforded more than 4 mmol/mol AA, indicating that alpha-hydroxycarbonyls are more efficient than alpha-dicarbonyls in transforming asparagine into AA. The experimental results are consistent with the reaction mechanism proposed, i.e. (i) Strecker-type degradation of the Schiff base leading to azomethine ylides, followed by (ii) beta-elimination of the decarboxylated Amadori compound to release AA. The functional group in beta-position on both sides of the nitrogen atom is crucial. Rearrangement of the azomethine ylide to the decarboxylated Amadori compound is the key step, which is favored if the carbonyl moiety contains a hydroxyl group in beta-position to the N-atom. The beta-elimination step in the amino acid moiety was demonstrated by reacting under pyrolysis conditions decarboxylated model Amadori compounds obtained by synthesis.

Oxidation of caffeine and related methylxanthines in ascorbate and polyphenol-driven Fenton-type oxidations.

Caffeine and related methylxanthines were subjected to free radical mediated oxidation by incubation with Fe(3+)-EDTA/ascorbate and Fe(3+)-EDTA/polyphenolics. The reaction mixtures were analysed by reverse-phase HPLC, revealing the corresponding C-8 hydroxylated analogues as the major products of hydroxyl radical mediated attack. Further oxidation products of caffeine, analysed by liquid chromatography-mass spectrometry (LC-MS), were the N1-, N3- and N7-demethylated methylxanthine analogues theobromine, paraxanthine and theophylline, respectively. Isolable amounts of the imidazole ring operated 6-amino-5-(N-formylmethyl-amino)-1,3-dimethyl-uracil (1,3,7-DAU) derivative were also detected, which was characterised by 1H NMR and mass spectroscopy. The identified products indicate that the pertinent chemical reactions, i.e. C-8 hydroxylation, demethylations, and C8-N9 bond scission, are comparable to the primary metabolic pathways of caffeine in humans. The influence of pH, transition metals, hydrogen peroxide, free radical scavengers and metal chelators on caffeine oxidation was studied. This report illustrates that natural food-borne reactants can aid in identifying specific chemical markers of free radical induced damage. Furthermore, potentially anti-and pro-oxidative reactions can be elucidated which may be important in assessing the impact of nutrient additives and supplements on the shelf life and stability of foods and beverages.

Determination of the plant growth regulator chlormequat in food by liquid chromatography-electrospray ionisation tandem mass spectrometry.

A confirmatory method for the determination of trace levels of chlormequat in a variety of different food matrices was developed. It entails a single clean-up step over a solid-phase cation exchange resin and subsequent liquid chromatography-electrospray ionisation tandem mass spectrometry using a stable isotopically labelled internal standard. Mass spectral acquisition was done in selected reaction monitoring mode, selecting the transitions from both the 35Cl and the 37Cl isotope of chlormequat. Recoveries after extraction and clean-up, determined with radio-labelled chlormequat and averaged over the spiking range (16-65 microg kg(-1)) in four different commodities, were within 88-96%, with a coefficient of variation better than 8%. The method can be applied to pears, pear juice concentrates, fruit purées, and cereal products, with typical limits of detection for chlormequat estimated at 2-5 microg kg(-1). A survey of different food commodities revealed that chlormequat was detectable--albeit at very low levels--in many of the food samples analysed, with the highest concentration recorded in pears purchased in Switzerland and of South African origin (5.5 mg kg(-1)). Measurements were also conducted on two LC-MS instruments and demonstrate the versatility and robustness of the method and its applicability to instruments of different ion source design.

Application of liquid chromatography-electrospray ionization tandem mass spectrometry to the detection of 10 sulfonamides in honey.

Liquid chromatography (LC) in combination with tandem mass spectrometry (MS-MS) has been applied to the separation and detection of 10 different sulfonamides in honey. The methodology encompasses a simple hydrolysis of the honey sample to liberate sugar-bound sulfonamides followed by liquid-liquid extraction of the 10 analytes, filtration, and analysis by LC-MS-MS. Conditions for reversed-phase LC and electrospray ionization (ESI) MS-MS in the positive ion mode were optimized for the 10 compounds under study, monitoring two characteristic mass transitions simultaneously for each analyte. The procedure is a qualitative confirmatory method for 10 sulfonamides at the low microg/kg level in honey. Typical recoveries of the analytes in honey ranged from 44 to 73% at a fortification level of 50 microg/kg. This study also addresses the issue of matrix-induced suppression of ionization, an effect often encountered in trace residue analysis of food matrices using LC-ESI-MS-MS based methods.

The inhibitory effects of coffee on radical-mediated oxidation and mutagenicity.

Hydrogen peroxide (H2O2) has been implicated as a major contributor to coffee mutagenicity and genotoxicity in vitro. We have used three assays to show the gradual formation of H2O2 in freshly prepared roasted ground coffee and in instant coffees over time reaching levels of 400-450 microM after a 1-h incubation period. Formation of H2O2 occurs through an auto-oxidation process where polyphenolics, in the presence of transition metals, reduce atmospheric oxygen. However, because of these polyphenolics, coffee also possesses in vitro antioxidant activity as shown by its capacity to inhibit lipid peroxidation in Fenton-catalysed hydroxylation reactions. The pro- and antioxidative effects of coffee are also reflected in its mutagenic and antimutagenic activity in the Ames test. Coffee is directly mutagenic in strains TA100 and TA102 due to H2O2 formation. However, coffee is also an antioxidant and antimutagen. This beverage exerts a strong protective effect against the mutagenicity and cytotoxicity induced by the oxidant t-butylhydroperoxide (t-BOOH). Thus, coffee, like many antioxidants, exhibits dual effects in vitro which are highly dependent upon parameters such as dose, atmospheric oxygen, transition metals as well as the biological and chemical endpoints used for measurement. Consequently, the data obtained on the pro- and antioxidant properties of foods and beverages from in vitro bioassays must be interpreted with caution and the results are not easily extrapolated in vivo to assess the impact on human health.

Analysis of beta-lactam antibiotics in incurred raw milk by rapid test methods and liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A recently developed confirmatory LC-MS method has been applied to the quantification of five major beta-lactam antibiotics in suspect raw bovine milk samples that gave a positive response with receptor-based (BetaStar) and rapid microbial inhibitory screen tests (Delvotest SP). In total, 18 presumptive positive raw milk samples were reanalyzed; 16 samples showed traces of antibiotic residues that could be identified and quantified by the LC-MS method, ranging from the limits of confirmation up to 38 microg/kg. Of the positive samples, only five (approximately 30%) were found to be violative of EU maximum residue limits. The most frequently detected antibiotic residues were cloxacillin and penicillin G, the former often in combination with amoxicillin or ampicillin. This study compares the results obtained by the three methods on identical samples and addresses how these relate to certain criteria such as sensitivity and selectivity. Furthermore, the limitations of the LC-MS method and the potential impact of the presence of frequently more than one residue in the same milk sample on the response of the rapid test methods are discussed.

In vitro anti- and pro-oxidative effects of natural polyphenols.

The anti- and pro-oxidative effects of phenolic compounds and antioxidants were studied in two different in vitro model systems utilizing ethyl linoleate and 2'-deoxyguanosine (2'-dG) as oxidative substrates, and a Fenton reaction (H2O2, Fe2+) to initiate oxidation. Oxidation of the biomolecules in both model systems exhibited dose dependency. In the 2'-dG assay, oxidation was closely related to H2O2 generation, which occurred during autoxidation of the phenolics. Hydroxylating activity was greatly enhanced by Mn2+ and Cu2+, but not by Zn2+ or Co2+. Ethyl linoleate peroxidation was inhibited by low concentrations of catechol, quercitin, and instant coffee. However, peroxidation was promoted by high concentrations of the same compounds, probably by recycling of chelated inactive Fe3+ to the active Fe2+ state.

Esters of 3-chloro-1,2-propanediol (3-MCPD) in vegetable oils: significance in the formation of 3-MCPD.

3-Mono-chloropropane-1,2-diol (3-MCPD) is a contaminant that occurs in food in its free (diol) form as well as in an esterified (with fatty acids) form. Using a simple intestinal model, it was demonstrated that 3-MCPD monoesters and 3-MCPD diesters are accepted by intestinal lipase as substrates in vitro. Under the chosen conditions, the yield of 3-MCPD from a 3-MCPD monoester was greater than 95% in approximately 1 min. Release from the diesters was slower, reaching about 45, 65 and 95% of 3-MCPD after 1, 5 and 90 min of incubation, respectively. However, in human, the hydrolysis of 3-MCPD esters is unlikely to release 100% as 3-MCPD, as triglycerides and phospholipids are hydrolysed in the intestine liberating 2-monoglycerides. Assuming a similar metabolism for 3-MCPD esters as that known for acylglycerols in humans in vivo, the de-esterification in positions 1 and 3 would thus be favoured by pancreatic lipases. Therefore, 3-MCPD, and 3-MCPD-2 monoesters would be released, respectively, from the 1-/3-monoesters, and the diesters potentially present in food. Hence, information on the exact amounts of the partial fatty acid chloroesters, i.e. 3-MCPD mono- and diesters, is important to assess the contribution of foods to the bioavailability of 3-MCPD. Therefore, a rapid method for the determination of the ratio of 3-MCPD monoesters to diesters in fats and oils was developed using gas chromatography-mass spectrometry (GC-MS) and isotopically labelled 3-MCPD esters as internal standards. The analysis of 11 different samples of fat mixes typically employed in food manufacturing demonstrated that a maximum of about 15% of the total amount of 3-MCPD bound in esters is present in the monoesterified form. The potentially slower release of 3-MCPD from 3-MCPD diesters, and the mono- to diesters ratio suggest that 3-MCPD esters may in fact contribute only marginally to the overall dietary exposure to 3-MCPD. Further work on the bioavailability, metabolism and possible toxicity of chloroesters per se is warranted.

Impact of extraction conditions on the content of acrylamide in model systems and food.

It has recently been suggested that the analytical methods that have been developed to date for the determination of acrylamide (AA) may underestimate the concentration of AA in certain foods, because significantly higher results were obtained upon extraction of the food matrix under alkaline conditions. The present study employs food (potato, rye) and chemical model systems to better understand the tentative release of AA under high pH extraction conditions. The experimental design is based on the generation of AA in an environment containing an AA-isotopomer, and by comparing the ratio of AA, respectively the AA-isotopomer, after extraction at pHs 7 and 12. The results show that the additional AA released is not due to improved extractability of AA from the food matrix, and should therefore be regarded as an extraction artefact. Strongly alkaline conditions seem to induce net formation of AA from water-soluble precursors formed during thermolysis.

Simultaneous determination of five beta-lactam antibiotics in bovine milk using liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A multiresidue method for the detection of five important beta-lactam antibiotics (amoxicillin, ampicillin, cloxacillin, oxacillin, penicillin G) in fresh milk is presented that allows quantitation of the analytes well below established legislative limits. The method avoids the use of acid during the extraction procedure and entails a cleanup step over a C18 cartridge. The analytes are separated and detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) using a stable isotope-labeled internal standard. Mass spectral acquisition is done in the positive ion mode by applying selected reaction monitoring of two or more fragmentation transitions per analyte to provide a high degree of sensitivity and specificity. The typical recoveries for all five beta-lactams in fresh milk ranged from 76 to 94% at a fortification level of 4 microg/kg. This study also addresses common problems encountered in the stability of penicillins during sample preparation as well as the employment of postcolumn infusion of a standard compound to verify potential matrix-induced signal suppression in ESI-MS.

Analysis of chloramphenicol in honeys of different geographical origin by liquid chromatography coupled to electrospray ionization tandem mass spectrometry.

A sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to detect trace amounts of the antibiotic chloramphenicol (CAP) in honey. The methodology entailed a solid-phase extraction of aqueous honey solutions followed by liquid-liquid partitioning, filtration and direct injection onto the LC-MS/MS system. Honey extracts were spiked with an isotopically labelled internal standard (d(5)-CAP) to compensate for analyte loss and potential ion suppression during the MS stage. Detection of the analyte was achieved by negative ionization electrospray in the selected reaction monitoring (SAM) mode. For confirmation, four characteristic mass transitions were monitored each for the analyte and the surrogate standard. The method was validated according to the latest European Union criteria for the analyses of veterinary drug residues in food. At all three fortification levels studied (0.1, 0.2, 0.5 microg kg(-1)) the method was accurate to within 15%. The repeatability and within-laboratory reproducibilities were <12 and 18%, respectively. The decision limit (CC alpha) and detection capability (CC beta) were both <0.1 microg kg(-1). The procedure provides a sensitive and reliable method for the determination of residues of chloramphenicol in honey. Numerous raw honeys of various geographical origins were analysed, showing extensive contamination particularly those of Chinese origin.

Antimutagenicity and catechin content of soluble instant teas.

The antimutagenic properties of soluble instant teas were examined using the bacterial Ames assay. Inhibition of the numbers of revertants induced from a number of known mutagens indicates that aqueous extracts of instant teas have antimutagenic activity and antioxidative properties, and can inhibit nitrosation reactions. Despite a significant reduction in the amounts of major green tea catechins, quantified using reversed-phase HPLC with electro-chemical detection, no differences in antimutagenicity were observed between the instant teas, a black fermented tea and a green tea. Oxidation of polyphenolic compounds which occurs during the production of instant tea does not therefore decrease the antioxidant, free radical scavenging and antimutagenic properties. This suggests that catechins are not the only compounds responsible for the protective effects of teas.

Quantitative analysis of clenbuterol in meat products using liquid chromatography-electrospray ionisation tandem mass spectrometry.

A method is presented that allows quantitation of clenbuterol in meat and liver products at the ng/kg level by liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESIMS-MS) using a stable isotopically labeled internal standard. The practical procedure involves acid extraction followed by two solid-phase clean-up steps with C18 and strong cation-exchange (SCX) resins. The typical recovery of the analyte spiked at 0.4 microg/kg in meat and liver samples was at 63+/-7%. Mass spectral acquisition was done in multiple reaction monitoring (MRM) to provide a high degree of sensitivity, achieving a limit of detection and quantitation at 10 and 15 ng/kg, respectively. Two precursor ions at m/z 277 and 279, corresponding to the characteristic isotopic cluster of the two chlorine atoms of clenbuterol, were monitored by LC-ESIMS-MS to provide unambiguous identity of the analyte. Samples of meat and liver of various origins with either incurred residues or spiked with known amounts of clenbuterol were used to validate the method.

Determination of 8-oxoguanine in DNA by gas chromatography--mass spectrometry and HPLC--electrochemical detection: overestimation of the background level of the oxidized base by the gas chromatography--mass spectrometry assay.

Two analytical methods, one involving the combined use of reverse-phase HPLC and electrochemical detection (HPLC-EC) and one involving a mass spectrometric detection after gas chromatography separation (GC/MS), were developed for the detection of 8-oxoguanine in DNA. In order to obtain quantitative results, 2,6-diamino-8-oxopurine, whose chemical structure and electrochemical response are very similar to 8-oxoguanine, has been employed as an internal standard in the HPLC-EC assay. In the case of the GC/MS method, an isotopically stable (M + 4) 8-oxoguanine has been employed as an internal standard. Both methods are able to detect approximately 1 modification per 10(6) DNA bases. The background level of 8-oxoguanine in DNA as determined by GC/MS is approximately 50-fold higher than that determined by the HPLC-EC assay. The discrepancy between the two methods is due to an artifactual oxidation of guanine during the derivatization reaction as demonstrated by using pure guanine. The amount of 8-oxoguanine in guanine, determined by GC/MS, increases linearly with the time of derivatization, indicating that an oxidation occurs during the silylation reaction. Derivatization under nitrogen atmosphere reduces but does not suppress the artifactual oxidation. The amount of 8-oxoguanine in DNA, quantified by GC/MS, is comparable to that obtained by HPLC-EC when 8-oxoguanine is prepurified by HPLC or by immunoaffinity chromatography, prior to the silylation reaction. The artifactual formation of 8-oxoguanine during the derivatization reaction may explain, at least in part, why the values reported for 8-oxoguanine determination by GC/MS are generally about 1 order of magnitude higher than that determined by HPLC-EC. Prepurification of 8-oxoguanine from guanine is recommended in order to obtain reliable results by GC/MS which may be compared to HPLC-EC.

Synthesis of multiply-labeled [15N3,13C1]-8-oxo-substituted purine bases and their corresponding 2'-deoxynucleosides.

Stable isotope-labeled analogues of oxidatively modified purine bases are required as internal standards for accurate quantitation of free radical induced damage in DNA using the isotope-dilution GC/MS technique. For this reason, we report on a facile and expedient method to synthesize the isotope-labeled oxidized DNA bases 8-oxoguanine (8-oxo-Gua, 5a) and 8-oxo-adenine (8-oxo-Ade, 5b). Both routes have in common the introduction of two exocyclic 15N isotopes simultaneously by halogen displacement of chlorine-substituted pyrimidines with [15N]-benzylamine. Debenzylation is achieved by either catalytic hydrogenation or treatment with aluminium chloride in benzene. An additional isotope is incorporated by nitrosation with 15N-labeled sodium nitrite. Cyclocondensation of the triamines with 13C-labeled urea then affords 5a and 5b in overall yields of 34% and 27%, respectively, and each with four isotope labels and at least 99 atom % excess. A further one-step enzyme catalyzed coupling of the C8 adducted purines with 2'-deoxyribose furnishes the isotope-labeled 2'-deoxynucleosides 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxo-dGuo) and 2'-deoxy-7,8-dihydro-8-oxoadenosine (8-oxo-dAdo).