Development of a stable isotope dilution assay for tenuazonic acid

A stable isotope dilution assay (SIDA) for the Alternaria mycotoxin tenuazonic acid was developed. Therefore, [(13)C(6),(15)N]-tenuazonic acid was synthesized from [(13)C(6),(15)N]-isoleucine by Dieckmann intramolecular cyclization after acetoacetylation with diketene. The synthesized [(13)C(6),(15)N]-tenuazonic acid was used as the internal standard for determination of tenuazonic acid in tomato products by liquid chromatography tandem mass spectrometry after derivatization with 2,4-dinitrophenylhydrazine. Method validation revealed a limit of detection of 0.1 μg/kg and a limit of quantitation of 0.3 μg/kg. Recovery was close to 100% in the range of 3-300 μg/kg. Determination of tenuazonic acid in two samples of different tomato ketchups (naturally contaminated) was achieved with a coefficient of variation of 2.3% and 4.7%. Different tomato products (n = 16) were analyzed for their content of tenuazonic acid using the developed SIDA. Values were between 15 and 195 μg/kg (tomato ketchup, n = 9), 363 and 909 μg/kg (tomato paste, n = 2), and 8 and 247 μg/kg (pureed tomatoes and comparable products, n = 5).     

Development of stable isotope dilution assays for the simultaneous quantitation of biogenic amines and polyamines in foods by LC-MS/MS

Microbial amino acid metabolism may lead to substantial amounts of biogenic amines in either spontaneously fermented or spoiled foods. For products manufactured with starter cultures, it has been suggested that certain strains may produce higher amounts of such amines than others; however, to support efforts of food manufacturers in mitigating amine formation, reliable methods for amine quantitation are needed. Using 10 isotopically labeled biogenic amines as the internal standards, stable isotope dilution assays were developed for the quantitation of 12 biogenic amines and of the 2 polyamines, spermine and spermidine, in one LC-MS/MS run. Application of the method to several foods revealed high concentrations of, for example, tyramine and putrescine in salami and fermented cabbage, whereas histamine was highest in Parmesan cheese and fermented cabbage. On the other hand, ethanolamine was highest in red wine and Parmesan cheese. The results suggest that different amino acid decarboxylases are active in the respective foods depending on the microorganisms present. The polyamine spermine was highest in salami and tuna.     

Development of a stable isotope dilution assay for the quantitation of glycidamide and its application to foods and model systems

On the basis of a stable isotope dilution assay and derivatization with 2-mercaptobenzoic acid, the presence of the carcinogenic glycidamide ( 2) in processed foods was verified for the first time. Using (13)C-labeled 2 as the internal standard and the formation of the thioether derivatives, a new stable isotope dilution assay for the quantitation of 2 was developed. Application of the method on several potato samples revealed amounts between 0.3 and 1.5 mug/kg depending on the processing conditions. In a model experiment, the formation of 2 by an epoxidation of the double bond in acrylamide, that is, by a reaction with linoleic acid hydroperoxides, was established. This result was in good agreement with data showing that French fries processed in sunflower oil, which is high in linoleic acid, contained more 2 as compared to fries prepared in coconut oil. The derivatization procedure allows the simultaneous quantitation of acrylamide and glycidamide in foods.     

Quantitative determination of sulfur-containing wine odorants at sub parts per billion levels. 2. Development and application of a stable isotope dilution assay

[(2)H(10)]-4-Mercapto-4-methylpentan-2-one (d(10)-1), [(2)H(2)]-3-mercaptohexan-1-ol (d(2)-2), and [(2)H(5)]-3-mercaptohex-1-yl acetate (d(5)-3), deuterated analogues of impact odorants of wines, were used to determine quantitatively the natural compounds in white wines (Muscadet, Sauvignon, and Bacchus) with a stable isotope dilution assay using gas chromatography coupled either with ion trap tandem mass spectrometry (GC-ITMS-MS) or with atomic emission detection monitored on sulfur-selective acquisition (GC-AED). The thiol compounds were recovered from wines by liquid-liquid extraction, then purified from the wine extracts by covalent chromatography, and analyzed. The quantitative determination of 4-mercapto-4-methylpentan-2-one 1 in the wines that were analyzed was performed better with GC-AED than with GC-ITMS-MS under the conditions that were used. However, the detection limit of the method was higher than the odor threshold of 4-mercapto-4-methylpentan-2-one 1 in wine (5 vs 0.8 ng/L). The levels of this compound in the Sauvignon and Bacchus wines were much higher than its odor threshold, but it was not detectable in the Muscadet wines. On the contrary, GC-ITMS-MS was much more sensitive than GC-AED for detection of 3-mercaptohexan-1-ol 2 and 3-mercaptohex-1-yl acetate 3, and the detection limits were much lower than their odor thresholds in wine. The former compound was detected in all of the Muscadet wines that were analyzed at levels always higher than its odor detection threshold, while the latter occurred at levels higher than its odor threshold in only one Muscadet wine.     

Development of a stable isotope dilution assay for the quantification of 5-methyl-(E)-2-hepten-4-one: application to hazelnut oils and hazelnuts.

A stable isotope dilution assay was developed for the quantitation of the hazelnut odorant 5-methyl-(E)-2-hepten-4-one by mass chromatography using synthesized [(2)H](2)-5-methyl-(E)-2-hepten-4-one as the internal standard. Application of the method on two batches of commercial hazelnut oils, processed from either roasted or unroasted nuts, revealed 6.4 microg 5-methyl-(E)-2-hepten-4-one per kg of unroasted oil whereas 315.8 microg per kg was determined in the roasted nut oil. The about 50-fold higher amount of 5-methyl-(E)-2-hepten-4-one in roasted hazelnut oil suggested the necessity of a thermal treatment to generate the flavor compound. Pan frying of raw hazelnuts (9 to 15 min) or boiling of the crushed nut material for 1 h in water led to an increase of 5-methyl-(E)-2-hepten-4-one by factors of 600 and 800, respectively, thereby corroborating that the major part of the nut flavorant is formed during heat treatment from a yet unknown precursor in hazelnuts.     

A capillary electrophoresis laser-induced fluorescence method for analysis of potato glycoalkaloids based on a solution-phase immunoassay. 2. Performance evaluation

Glycoalkaloids (GAs) occur naturally in potatoes and are toxic to humans and animals. The objective of the present study was to evaluate the performance of a solution-phase immunoassay coupled to capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection for the determination of total glycoalkaloids in potatoes. The immunoassay was based on a competition between potato glycoalkaloids and fluorescently labeled solanidine. Reaction products were separated in the capillary zone electrophoresis mode. A calibration curve of signal vs log[GA] was linear from 50 to 400 nM. The CV for duplicate and day-to-day analyses averaged 5.7% and 12%, respectively. Spike recoveries ranged from 85 to 97% for spike levels ranging from 43 to 170 microg/g fresh potato. Potato samples with GA concentrations ranging from <40 to >200 microg/g were successfully analyzed, indicating that immuno-CE-LIF is a rapid alternative to traditional ELISA and HPLC methods.     

Identification of 9(E),11(E)-18:2 fatty acid methyl ester at trace level in thermal stressed olive oils by GC coupled to acetonitrile CI-MS and CI-MS/MS, a possible marker for adulteration by addition of deodorized olive oil

The olive oil market is suffering from sophisticated illegal treatments. One common adulteration process consists of the addition to virgin olive oil of lower quality oils, such as "lampante" oil, an inexpensive oil and with some organoleptic defects, which is then submitted to thermal deodorization under vacuum processes for removal of the undesired flavor components. Such a blending may not have a huge influence on the chemical composition and may not significantly affect the parameters usually checked as quality indicators, although the organoleptic properties may change. As a consequence, a major effort is being devoted to find reliable markers able to unmask such adulterations. We report here the complete characterization of a compound, detected at trace levels exclusively in thermal stressed oils, which could be a candidate marker for adulteration. The investigation, carried out by GC-MS and GC-MS/MS, provided its complete structure, including the stereochemistry, shown to be a 9(E),11(E)-18:2 fatty acid methyl ester. Experimental data also confirmed the influence of both temperature and heating time on formation and concentration of this compound.