Determination of chlormequat and mepiquat in pear, tomato, and wheat flour using on-line solid-phase extraction (Prospekt) coupled with liquid chromatography-electrospray ionization tandem mass spectrometry.

A sensitive and selective method is presented for the simultaneous analysis of the pesticides chlormequat and mepiquat at trace levels in tomato, pear, and wheat flour. The method entails direct injection of the food extract onto an on-line solid-phase extraction (SPE) instrument (Prospekt) using a strong cation-exchange resin. Analyte separation and detection is done by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). Surrogate standards (d9-chlormequat, d6-mepiquat) are employed to compensate for recovery losses and potential MS-MS signal suppression. The method achieves a limit of quantification for both cationic analytes at or below 5 microg/kg, and good intra- and inter-assay precision with mean variability values <7% over a concentration range up to 195 microg/kg. This study also addresses potential analyte carry-over in an SPE on-line system, as well as the robustness of the procedure and its applicability in routine quality control operations.

Alkylpyridiniums. 1. Formation in model systems via thermal degradation of trigonelline.

Trigonelline is a well-known precursor of flavor/aroma compounds in coffee and undergoes significant degradation during roasting. This study investigates the major nonvolatile products that are procured after trigonelline has been subjected to mild pyrolysis conditions (220-250 degrees C) under atmospheric pressure. Various salt forms of trigonelline were also prepared and the thermally produced nonvolatiles analyzed by thin layer chromatography, liquid chromatography-electrospray ionization tandem mass spectrometry, and (1)H and (13)C nuclear magnetic resonance. Results revealed the decarboxylated derivative 1-methylpyridinium as a major product of certain salts, the formation of which is positively correlated to temperature from 220 to 245 degrees C. Moreover, trigonelline hydrochloride afforded far greater amounts of 1-methylpyridinium compared to the monohydrate over the temperature range studied. Investigations into other potential quaternary amine products of trigonelline also indicate nucleophilic substitution reactions that lead to dialkylpyridiniums, albeit at concentration levels approximately 100-fold lower than those recorded for 1-methylpyridinium.

Alkylpyridiniums. 2. Isolation and quantification in roasted and ground coffees.

Recent model studies on trigonelline decomposition have identified nonvolatile alkylpyridiniums as major reaction products under certain physicochemical conditions. The quaternary base 1-methylpyridinium was isolated from roasted and ground coffee and purified by ion exchange and thin-layer chromatography. The compound was characterized by nuclear magnetic resonance spectroscopy ((1)H, (13)C) and mass spectrometry techniques. A liquid chromatography-electrospray ionization tandem mass spectrometry method was developed to quantify the alkaloid in coffee by isotope dilution mass spectrometry. The formation of alkylpyridiniums is positively correlated to the roasting degree in arabica coffee, and highest levels of 1-methylpyridinium, reaching up to 0.25% on a per weight basis, were found in dark roasted coffee beans. Analyses of coffee extracts also showed the presence of dimethylpyridinium, at concentrations ranging from 5 to 25 mg/kg. This is the first report on the isolation and quantification of alkylpyridiniums in coffee. These compounds, described here in detail for the first time, may have an impact on the flavor/aroma profile of coffee directly (e.g., bitterness), or indirectly as precursors, and potentially open new avenues in the flavor/aroma modulation of coffee.

In-depth mechanistic study on the formation of acrylamide and other vinylogous compounds by the maillard reaction.

The formation of acrylamide was studied in low-moisture Maillard model systems (180 degrees C, 5 min) based on asparagine, reducing sugars, Maillard intermediates, and sugar degradation products. We show evidence that certain glycoconjugates play a major role in acrylamide formation. The N-glycosyl of asparagine generated about 2.4 mmol/mol acrylamide, compared to 0.1-0.2 mmol/mol obtained with alpha-dicarbonyls and the Amadori compound of asparagine. 3-Hydroxypropanamide, the Strecker alcohol of asparagine, generated only low amounts of acrylamide ( approximately 0.23 mmol/mol), while hydroxyacetone increased the acrylamide yields to more than 4 mmol/mol, indicating that alpha-hydroxy carbonyls are much more efficient than alpha-dicarbonyls in converting asparagine into acrylamide. The experimental results are consistent with the reaction mechanism based on (i) a Strecker type degradation of the Schiff base leading to azomethine ylides, followed by (ii) a beta-elimination reaction of the decarboxylated Amadori compound to afford acrylamide. The 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 nitrogen atom. The beta-elimination step in the amino acid moiety was demonstrated by reacting under low moisture conditions decarboxylated model Amadori compounds obtained by synthesis. The corresponding vinylogous compounds were only generated if a beta-proton was available, for example, styrene from the decarboxylated Amadori compound of phenylalanine. Therefore, it is suggested that this thermal pathway may be common to other amino acids, resulting under certain conditions in their respective vinylogous reaction products.

Formation of vinylogous compounds in model Maillard reaction systems.

The thermal degradation over temperature and time of selected amino acids (Asp, Gln, and Glu) in the presence of reducing sugars was investigated in low moisture model systems. Copyrolysis of glucose-Asp mixtures led to the release of acrylic acid, attaining >5 mmol/mol Asp at 230 degrees C after 5 min. Spurious amounts of 3-butenamide were detected upon heating Gln together with a carbonyl source. Apparently, intramolecular cyclization is favored to procure 2-pyrrolidinone, reaching levels >3 mmol/mol above 230 degrees C. 2-Pyrrolidinone was also formed in comparable amounts in pyrolyzed sugar-Glu mixtures, indicating that the Maillard reaction may be an important contributor to the formation of 2-pyrrolidinone in certain cooked foods. The chemical route to acrylic acid and 3-butenamide is probably analogous to that described for acrylamide recently. Evidence is also presented that acrylic acid may be an intermediate in the formation of acrylamide, and yields could be augmented by coincubation of fructose-Asp with certain amino acids such as Gln, reaching approximately 5% of the yield obtained by the Asn route. A computational study to determine the reactivity of the vinylogous products indicated a reduced ability of 3-butenamide as compared to acrylamide to form stable intermediates by Michael nucleophilic addition. Acrylamide and acrylic acid exhibited a similar theoretical reactivity potential toward nucleophiles. No information is as yet available on the occurrence of acrylic acid in cooked foods. Extensive toxicological evaluation indicates that acrylic acid is of no concern at the amounts to be expected in foods.

Semicarbazide is a minor thermal decomposition product of azodicarbonamide used in the gaskets of certain food jars.

Evidence is presented for the first time showing that semicarbazide (SEM) is a minor thermal decomposition product of the blowing agent azodicarbonamide (ADC). A novel direct analytical method based on liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESIMS/MS) has been developed to determine SEM in foamed polyvinyl chloride (PVC) seals of metal lids, as well as in commercially available ADC. The direct LC-MS/MS method for gaskets entails extraction of the gaskets in hot water, addition of ((15)N(2)(13)C)-SEM as internal standard, and injection of an aliquot directly into the LC-MS system, achieving good sensitivity (S/N = 348 for 2 ng injected on-column) and monitoring three characteristic mass transitions (m/z 76-->31; 76 -->44; 76-->59). Semicarbazide can be detected in thermally treated ADC, reaching up to 0.93 mmol mol(-1) at 220 degrees C, as determined by the direct LC-MS/MS method. This new method is also compared to the classical derivatization method using 2-nitrobenzaldehyde (2-NBA) that is routinely employed to determine SEM as an indicator of the usage of the antimicrobial drug nitrofurazone, the use of which is not authorized in the European Union (EU). Both methods revealed proportional results, with approx. 3-fold higher levels recorded by the direct SEM approach, probably due to differences in the extraction procedures used. A limited survey of plastic seals from used press twist and twist-off metal lids on food jars (non-foamed and foamed) revealed levels of SEM ranging from 2 to 8689 microg kg(-1)(average = 1593 microg kg(-1), n= 57 determinations).

Acrylamide from Maillard reaction products.

The discovery of the adventitious formation of the potential cancer-causing agent acrylamide in a variety of foods during cooking has raised much concern, but the chemical mechanism(s) governing its production are unclear. Here we show that acrylamide can be released by the thermal treatment of certain amino acids (asparagine, for example), particularly in combination with reducing sugars, and of early Maillard reaction products (N-glycosides). Our findings indicate that the Maillard-driven generation of flavour and colour in thermally processed foods can -- under particular conditions -- be linked to the formation of acrylamide.