Identification and in vitro cytotoxicity of ochratoxin A degradation products formed during coffee roasting

The mycotoxin ochratoxin A is degraded by up to 90% during coffee roasting. In order to investigate this degradation, model heating experiments with ochratoxin A were carried out, and the reaction products were analyzed by HPLC-DAD and HPLC-MS/MS. Two ochratoxin A degradation products were identified, and their structure and absolute configuration were determined. As degradation reactions, the isomerization to 14-(R)-ochratoxin A and the decarboxylation to 14-decarboxy-ochratoxin A were identified. Subsequently, an analytical method for the determination of these compounds in roasted coffee was developed. Quantification was carried out by HPLC-MS/MS and the use of stable isotope dilution analysis. By using this method for the analysis of 15 coffee samples from the German market, it could be shown that, during coffee roasting, the ochratoxin A diastereomer 14-(R)-ochratoxin A was formed in amounts of up to 25.6% relative to ochratoxin A. The decarboxylation product was formed only in traces. For toxicity evaluations, first preliminary cell culture assays were performed with the two new substances. Both degradation products exhibited higher IC50 values and caused apoptotic effects with higher concentrations than ochratoxin A in cultured human kidney epithelial cells. Thus, these cell culture data suggest that the degradation products are less cytotoxic than ochratoxin A.     

Comparison of proanthocyanidins in commercial antioxidants: grape seed and pine bark extracts

The major constituents in grape seed and pine bark extracts are proanthocyanidins. To evaluate material available to consumers, select lots were analyzed using high-performance liquid chromatography, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), gel permeation chromatography (GPC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Atmospheric pressure chemical ionization (APCI) LC/MS was used to identify monomers, dimers, and trimers present. GC/MS analyses led to the identification of ethyl esters of hexadecanoic acid, linoleic acid, and oleic acid, as well as smaller phenolic and terpene components. The GPC molecular weight (MW) distribution indicated components ranging from approximately 162 to approximately 5500 MW (pine bark less than 1180 MW and grape seed approximately 1180 to approximately 5000 MW). MALDI-TOF MS analyses showed that pine bark did not contain oligomers with odd numbers of gallate units and grape seed contained oligomers with both odd and even numbers of gallate. Reflectron MALDI-TOF MS identified oligomers up to a pentamer and heptamer, and linear MALDI-TOF MS showed a mass range nearly double that of reflectron analyses.     

Characterization of grape procyanidins using high-performance liquid Chromatography/Mass spectrometry and matrix-assisted laser Desorption/Ionization time-of-flight mass spectrometry

High-performance liquid chromatography/mass spectrometry (HPLC/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to characterize the procyanidin composition of the grape seed extract. The detection of the oligomers composed of (+)-catechin, (-)-epicatechin, and their galloylated derivatives in the grape seeds is demonstrated. With MALDI-TOF MS, oligomers up to nonamers were observed. The potential of the MALDI-TOF MS technique as a quantification tool is also discussed. The information presented in this study could lead to the determination of procyanidin content and their molecular weight distribution in grape seeds.     

NMR, ESI/MS, and MALDI-TOF/MS analysis of pear juice polymeric proanthocyanidins with potent free radical scavenging activity

The structure of a polymeric proanthocyanidin fraction isolated from pear juice was characterized by NMR, ESI/MS, and MALDI-TOF/MS analyses, and its antioxidant activity was investigated using the DPPH free radical scavenging method. The results obtained from 13C NMR analysis showed the predominance of signals representative of procyanidins. Typical signals in the chemical shift region between 70 and 90 ppm demonstrated the exclusive presence of epicatechin units. The results obtained through negative ESI/MS analysis showed singly and doubly charged ions corresponding to the molecular mass of procyanidins with a degree of polymerization up to 22. The spectra obtained through MALDI-TOF/MS analysis revealed the presence of two series of tannin oligomers. Supporting the observations from NMR spectroscopy, the first series consists of well-resolved tannin identified as procyanidin polymers units with chain lengths of up to 25. A second series of monogalloyl flavan-3-ols polymers with polymerization degree up to 25 were also detected. This is the first mass spectrometric evidence confirming the existence of galloylated procyanidin oligomers in pear fruits. Within each of these oligomers, various signals exist suggesting the presence of several oligomeric tannins. The antioxidant properties of the polymeric fraction were investigated through reduction of the DPPH free radical, and the results obtained showed that the polymeric fraction exhibited a higher antioxidant power compared to those of (+)-catechin and B3 procyanidin dimer.     

Identification and formation pathway of laccase-mediated oxidation products formed from hydroxyphenylureas

Hydroxyphenylureas are the first main metabolites formed in the environment from pesticide and biocide urea compounds. Because fungi release potent exocellular oxidases, we studied the ability of laccases produced by the white rot fungus, T. versicolor, to catalyze in vitro the transformation of five hydroxyphenylureas, to identify transformation pathways and mechanisms. Our results establish that the pH of the reaction has a strong influence on both the kinetics of the reaction and the nature of the transformation products. Structural characterization by spectroscopic methods (NMR, mass spectrometry) of eleven transformation products shows that laccase oxidizes the substrates to quinones or to polyaromatic oligomers. Slightly acidic conditions favor the formation of quinones as final transformation products. In contrast, at pH 5-6, the quinones further react with the remaining substrate in solution to give hetero-oligomers via carbon-carbon or carbon-oxygen bond formation. A reaction pathway is proposed for each of the identified products. These results demonstrate that fungal laccases could assist the transformation of hydroxyphenylureas.     

Quantification of 4-methylimidazole in class III and IV caramel colors: validation of a new method based on heart-cutting two-dimensional liquid chromatography (LC-LC)

4-Methylimidazole (4MeI) is a nitrogen compound formed during the manufacture of class III and IV caramel colors. The European Commission has limited its content to 250 ppm. Two methods were compared to perform 4MeI quantification in caramels. The first one, currently used and considered to be the reference method, consists of a hot extraction of caramel color with dichloromethane and an analysis of the acetyl derivative of the extract by gas chromatography coupled to mass spectrometry (GC-MS). The second method is based on the heart-cutting two-dimensional liquid chromatography technique (LC-LC) to directly separate 4MeI from the other components present in caramel color sample (diluted in water) in <30 min. The accuracy profile validation method and the comparison between the results obtained with the two methods show that the new and completely automated LC-LC method is usable to quantify 4MeI in caramels.     

Determination of 4(5)-methylimidazole in soy sauce and other foods by LC-MS/MS after solid-phase extraction

A method for the determination of 4(5)-methylimidazole (4MeI) in naturally brewed soy sauce was developed for the first time using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). SPE on silica-based reversed-phase cartridges with heptafluorobutyric acid as an ion-pairing reagent was used for the efficient cleanup of 4MeI. A multimode ODS column was employed for the chromatographic separation. To subtract the matrix effect during LC-MS/MS analysis, a standard addition method was used. The levels of 4MeI found in naturally brewed soy sauce were extremely low (ranging from <0.002 to 0.023 μg/g), whereas those in soy sauces containing caramel color were generally high (ranging from 0.43 to 4.8 μg/g). The method proved to be useful for the analysis of 4MeI in other foods such as caramel colors, drinks, and Worcestershire sauce.     

Characterization and estimation of proanthocyanidins and other phenolics in coffee pulp (Coffea arabica) by thiolysis-high-performance liquid chromatography

Fresh and 3-day-old coffee pulp of the Arabica variety were analyzed for polyphenol composition followed by characterization by two different methods. The first method consisted in subjecting coffee pulp powder to direct thiolysis. For the second method, coffee pulp was subjected to successive solvent extractions, followed by thiolysis. Quantification of phenolic compounds was then achieved by high-performance liquid chromatography (HPLC) analysis of thiolysis products. Four major classes of polyphenols were identified: flavan-3-ols (monomers and procyanidins), hydroxycinnamic acids, flavonols, and anthocyanidins. Differences in concentration of procyanidins were observed between fresh and 3-day-old coffee pulp. Constitutive units were mainly epicatechin, representing more than 90% of the proanthocyanidin units, with average degrees of polymerization in the range of 3.8-9.1. Monomer to hexamer units of flavan-3-ols from fresh coffee pulp were separated by normal-phase HPLC. Molecular size of oligomeric proanthocyanidins was obtained by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results obtained confirm the presence of oligomers of the flavan-3-ol (-)-epicatechin.     

Role of neck region in the thermal aggregation of myosin.

Carp dorsal myosin formed oligomers that retained ATPase activity upon heating. Cleavage of the oligomeric myosin at subfragment-1 (S-1)/rod junction released monomeric S-1 and rod, indicating that ATPase retaining myosin associated near the S-1/rod junction. The digest also contained rod oligomers. Heating a mixture of S-1 and rod generated neither ATPase retaining S-1 oligomers nor rod oligomers. Electron microscopic observation of the heated myosin revealed that some oligomers were formed by associating at the S-1/rod joining region, exhibiting a recognized double head, probably ATPase retaining oligomers. No myosin oligomers associated at the tail region were observed, thus, rod aggregation would be formed at its very restricted region near the S-1/rod junction. Based on the findings, we proposed that the neck structure is important in the thermal oligomerization process of myosin.     

Mass spectrometric evidence for the existence of oligomeric anthocyanins in grape skins

The fractionation of a grape skin extract by multilayer countercurrent chromatography coupled with step gradient elution allowed the preparation of a fraction almost devoid of free anthocyanins. This fraction appeared to be almost exclusively polymeric, as judged by liquid chromatographic-mass spectrometric (LC-MS) analysis, color-bleaching tests with sulfur dioxide, and thiolysis. Electrospray mass spectrometric analysis indicated that the pigmented material in this fraction was chiefly composed of direct condensation products of anthocyanin extending up to trimers. With regard to their linkages, the anthocyanin units in the oligomers were possibly linked by either an A-type (by both carbon-carbon and ether bonds) or B-type (by carbon-carbon bond) linkage, like proanthocyanidins. The terminal anthocyanin unit of the oligomers is consistently in the flavylium form but the extension units are in the flavan form for the A-type oligomers and in the flavene form for the B-type oligomers. Although their linkages still need to be defined rigorously, this is the first mass spectrometric evidence confirming the existence of anthocyanin oligomers in the grape skin extract.     

Effects of Frying and Microwave Heating on Decomposition of Benzoyl Peroxide in Flour

Effects of frying and microwave heating on the decomposition of benzoyl peroxides (BPO) in flour were studied. First, BPO and its main decomposition product, benzoic acid (BA), were extracted from different flour products by ultrasonication in ethanol. Next, three samples of commercial flour, two fried foods, and two microwave foods were analyzed by high performance liquid chromatography (HPLC) and mass spectrum (MS). Results revealed that the BPO content decreased after frying and microwave heating treatments. Furthermore, the existence of biphenyl in fried food was successfully verified by HPLC‐MS. Biphenyl that decomposed from BPO may be one source of toxicity in fried foods. In contrast to fried food, new components made through the special heating module were not found in microwave food.     

Kapillar-GC — MS von Huminsäureabbauprodukten eines Podsols

Capillary GC — MS of degradation products obtained from podzol humic acids Humic acids' degradation products, formed by permanganate oxidation, mostly consist of aliphatic und aromatic dicarboxylic acids as well as fatty acids, whose distribution downwards a podzol profile will be discussed. Because of improved chromatographic techniques it is possible to detect 200–300 compounds. Their identification is based on the interpretation of mass spectra, high resolution mass spectrometry, and the use of N-, P- and S-spezific gaschromatographic detectors.     

The flavonoid glycosides and procyanidin composition of Deglet Noor dates (Phoenix dactylifera)

The fruits of the date palm (Phoenix dactylifera) are consumed throughout the world and are an important part of the diet in the Middle East. Dates at the rutab and tamar maturity and ripening stages contain a wide array of phenolic antioxidants, but little is known about the composition of phenolic compounds in dates at the khalal stage of ripening. In the current study, the flavonoid glycoside and procyanidin compositions of dates of the cultivar Deglet Noor harvested at the khalal stage of maturity were characterized using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI/MS/MS). Procyanidin oligomers through decamers were identified in extracts of these dates. Higher molecular weight polymers, undecamers through heptadecamers, were also apparent from mass spectra. Thirteen flavonoid glycosides of luteolin, quercetin, and apigenin, 19 when considering isomeric forms, were also identified. Mass spectra indicate that both methylated and sulfated forms of luteolin and quercetin are present as mono-, di-, and triglycosylated conjugates whereas apigenin is present as only the diglycoside. LC-ESI/MS/MS spectra indicate that quercetin and luteolin formed primarily O-glycosidic linkages whereas apigenin is present as the C-glycoside.     

Identification of procyanidins in cocoa (Theobroma cacao) and chocolate using high-performance liquid chromatography/mass spectrometry

Monomeric and oligomeric procyanidins present in cocoa and chocolate were separated and identified using a modified normal-phase high-performance liquid chromatography (HPLC) method coupled with on-line mass spectrometry (MS) analysis using an atmospheric pressure ionization electrospray chamber. The chromatographic separation was achieved using a silica stationary phase in combination with a gradient ascending in polarity. This qualitative report confirms the presence of a complex series of procyanidins in raw cocoa and certain chocolates using HPLC/MS techniques. Although both cocoa and chocolate contained monomeric and oligomeric procyanidin units 2-10, only use of negative mode provided MS data for the higher oligomers (i.e., >pentamer). Application of this method for qualitative analysis of proanthocyanidins in other food products and confirmation of this method as a reliable quantitative tool for determining levels of procyanidins in cocoa, chocolate, and other food products are currently being investigated.     

Improved method to track chlorophyll degradation

An analytical method capable of identifying >30 chlorophyll-related compounds in plant extracts has been developed. The method employs liquid chromatography coupled to UV-vis, MS, and MS/MS detection. It can be applied without modification to analyze natural chlorophyll degradation products and other metalloporphyrines. It was successfully applied to identify chlorophyll derivatives found in rehydrated spinach powder and conventionally canned and Veri-Green-processed beans. In the Veri-Green-processed beans several degradation products were identified that are zinc-containing analogues to the chlorophyll derivatives found in vegetables after conventional canning. They have been characterized by liquid chromatography and mass spectrometry.     

Analysis of illegally distributed anabolic steroid products by liquid chromatography with identity confirmation by mass spectrometry or infrared spectrophotometry

Anabolic steroid products found in the illegal market are primarily oil-based injectables or tablets and often do not contain the ingredients declared on the label. An analytical scheme based on a reverse-phase liquid chromatographic (LC) system for screening, tentative identification, and quantitation is presented. Methanolic sample extracts are chromatographed on an octadecyl column using 2 mobile phases (methanol and (75 + 25) methanol-water) and tracked at 3 wavelengths (240, 210, and 280 nm) or with a photodiode array UV detector. Retention time ratios (RR) relative to testosterone and UV data are used for tentative identification. The same LC system serves as a cleanup and isolation step for identity confirmation by direct insertion probe mass spectrometry (MS) or Fourier transform infrared spectrophotometry (FTIR). Recoveries range from 96.2 to 100.2% for 11 different steroids extracted from sesame oil. LC RR values for over 40 steroids, analytical results for typical products, and MS and FTIR spectra for selected compounds are presented.     

Influence of processing parameters on acrylamide formation during frying of potatoes

Consistent evidence suggests that the probable human carcinogen acrylamide is formed in starch-rich foodstuffs through heat-induced interaction of asparagine and reducing sugars during Maillard browning. However, information regarding the influence of processing parameters on acrylamide formation is scarce. We investigated the impact of temperature, heating time, browning level, and surface-to-volume ratio (SVR) on acrylamide generation in fried potatoes. Acrylamide content was determined by liquid chromatography (LC) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). In potato shapes with low SVR, acrylamide content consistently increased with increasing temperature and processing times. By contrast, in shapes with intermediate to high SVR, maximal acrylamide formation occurred at 160-180 degrees C, while higher temperatures or prolonged processing times caused a decrease of acrylamide levels. Moreover, browning levels were not a reliable measure of acrylamide content in large-surface products.     

Acrylamide in foods: occurrence,sources, and modeling

Acrylamide in food products-chiefly in commercially available potato chips, potato fries, cereals, and bread-was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were homogenized with water/dichloromethane, centrifuged, and filtered through a 5 kDa filter. The filtrate was cleaned up on mixed mode, anion and cation exchange (Oasis MAX and MCX) and carbon (Envirocarb) cartridges. Analysis was done by isotope dilution ([D(3)]- or [(13)C(3)]acrylamide) electrospray LC-MS/MS using a 2 x 150 mm (or 2 x 100 mm) Thermo HyperCarb column eluted with 1 mM ammonium formate in 15% (or 10% for the 2 x 100 mm column) methanol. Thirty samples of foods were analyzed. Concentrations of acrylamide varied from 14 ng/g (bread) to 3700 ng/g (potato chips). Acrylamide was formed during model reactions involving heating of mixtures of amino acids and glucose in ratios similar to those found in potatoes. In model reactions between amino acids and glucose, asparagine was found to be the main precursor of acrylamide. Thus, in the reaction between nitrogen-15 (amido)-labeled asparagine and glucose, corresponding (15)N-labeled acrylamide was formed. The yield of the model reaction is approximately 0.1%.     

Characterization of cigarette tobacco by direct electrospray ionization-ion trap mass spectrometry (ESI-ITMS) analysis of the aqueous extract--a novel and simple approach

In support of the efforts to combat smuggling, as well as illegal sale and distribution of cigarettes, an analytical approach for the characterization of tobacco has been proposed and evaluated. It involves aqueous extraction of the filler tobaccos followed by direct analysis of the extracts by electrospray ionization-ion trap mass spectrometry (ESI-ITMS) in the negative mode. Typically, the deprotonated ions, [M - H](-), of organic acids (malic, citric, caffeic, quinic acid) and polyphenols (chlorogenic acid, rutin, scopoletin) were detected. MS/MS spectra of the ion at m/z 191, which is the [M - H](-) of quinic acid, citric acid, and scopoletin, and a fragment ion of chlorogenic acid were acquired. Significant differences in the MS and MS/MS spectra were observed between counterfeit samples and the corresponding authentic brand name cigarettes. Analysis of 25 commercial cigarettes showed that straight Virginia blends were readily distinguished from the blended products containing different tobacco types (Virginia, burley, and Oriental). The former exhibited consistently higher relative abundances of m/z 353 (chlorogenic acid) to m/z 133 (malic acid) in the MS spectra (0.9-1.2 vs 0.4-0.6) and higher intensity ratios of m/z 176 (scopoletin) to m/z 173 (0.4-0.8 vs 0.1-0.3) and of m/z 127 (quinic acid) to m/z 173 (0.7-1.0 vs 0.3-0.5) in the MS/MS spectra. Evidence is presented to demonstrate that the spectral differences were related not only to the tobacco type (Virginia, burley and Oriental) but also to the tobacco part (stem, lamina) used in the manufacture of the cigarettes.