Proteomic analysis of the distribution of the major seed allergens in wild,landrace, ancestral,and modern soybean genotypes

BACKGROUND: A comparative analysis of seed allergens from various soybean genotypes is crucial for identifying and eliminating potential allergens. We have investigated the distribution of three major allergens (Gly m Bd 60K, Gly m Bd 30K and Gly m Bd 28K) in wild, landrace, ancestral and modern soybean genotypes. RESULTS: Gly m Bd 60K allergens consist of α subunits of β‐conglycinin and G2 subunits of glycinin. In wild genotypes, α subunits of β‐conglycinin separated into six to seven protein spots whereas five to seven spots were observed in the landraces. All genotypes of modern and ancestral groups showed 3–5 protein spots of α subunits of β‐conglycinin. All genotypes showed eight spots of glycinin G2 subunits except one ancestral genotype which had seven spots. Two protein spots were detected for Gly m Bd 30K in 14 genotypes but one spot was detected in two wild genotypes. Two protein spots were detected for Gly m Bd 28K in all genotypes. CONCLUSION: Considerable heterogeneity of the α subunit of β‐conglycinin distribution exists among these 16 soybean genotypes. Significant proteomic variation was observed between different soybean groups rather than among genotypes in the same group. This investigation would be valuable to researchers working with soybean and nutrition. Copyright © 2007 Society of Chemical Industry     

HPLC-DAD-ESI-MS analysis of flavonoid compounds in 5 seedless table grapes grown in Apulian Region

Flavonoids present in skin extracts of red seedless table grape varieties Summer Royal, Autumn Royal, and Crimson, and white seedless varieties Carati and Thompson were analyzed by HPLC-DAD-MS, in 3 y of study (2006 to 2008). The anthocyanins, delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside (with their corresponding p-coumaroyl derivatives), peonidin-3-O-glucoside, and malvidin-3-O-glucoside (with their corresponding acetyl, caffeoyl, and p-coumaroyl derivatives) were found. In addition the flavonols quercetin-3-O-glucuronide, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, kaempferol-3-O-galactoside, and the flavan-3-ols procyanidin B1, procyanidin B2, and catechin were also detected. Anthocyanins were the main flavonoids in red grapes ranging from 24 (Crimson) to 500 (Summer Royal) mg/kg fresh weight of grapes; consistent levels of flavonols and flavan-3-ols were also quantified in all varieties. To determine the effective climatic influence on flavonoids content in field conditions, viticultural practices have been developed, that could exclude the effects of direct solar radiation from confounding the assessment of those related to thermal conditions alone. A strong positive correlation was determined between flavonoids and temperature data that seem to be responsible for the difference of these metabolites along the years; furthermore, it has been possible to define a linear relationship (R(2) = 0.6871, P = 0.0057) between thermal amplitude and total flavonoids values in the red grapes. PRACTICAL APPLICATION: Grapes are economically the most important fruit species in the world and approximately 30% of its production is used as fresh fruit. Because of the very important role of flavonoids in food quality as well as their health-promoting properties, and considering that our experiments were performed along 3 consecutive years, gathered results in this research are quite promising to give a useful information on the flavonoid contents and their evolution in 5 seedless table grapes that are widespread in Mediterranean regions but also in California and South America, and are grown in a viticultural climate (Apulia, South Italy) very close to some regions of Spain, Turkey, Tunisia, and Israel.     

Isolation,Identification, and Quantification of Lichenysin,a Novel Nonvolatile Compound in Chinese Distilled Spirits

Nonvolatile components influence overall flavor volatility and perception in alcoholic beverages. However, little information has been reported about the nonvolatile compounds in Chinese distilled spirits. For the first time, we isolated and identified a cyclic lipopeptide “lichenysin” as a novel nonvolatile compound in Chinese liquors, using preparative high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometer and nuclear magnetic resonance. The concentrations of lichenysin were quantified in 14 main types of bottled Chinese liquors by using ultra performance liquid chromatography coupled with triple quadrupole mass spectrometer. The concentrations of lichenysin in Chinese liquor ranged from 0.01 to 111.74 μg/L, where the concentration was the highest in Dongjiu liquor. Furthermore, we found that lichenysin selectively affected aroma volatility in the Chinese liquor, using the headspace solid‐phase microextraction coupled with gas chromatography‐mass spectrometry. The headspace concentrations of 2‐phenylethanol, phenol, and 4‐methylphenol significantly decreased by respectively 58%, 76%, and 59% in 3 Chinese commercial liquors with an increase in lichenysin. These results indicated that lichenysin could be a novel nonvolatile compound that selectively affected aroma volatility in Chinese liquors.     

Comparison of Procedures for Resveratrol Analysis in Beer: Assessment of Stilbenoids Stability through Wort Fermentation and Beer Aging

Three resveratrol extraction procedures were compared in beer. Two‐step pre‐cleaning with toluene and cyclohexane allows 76% trans‐resveratrol recovery by solid‐phase extraction (SPE) before RP‐HPLC‐MS/MS analysis. This procedure proved much more efficient than liquid/liquid extraction, which requires solvents that are too hydrophobic for stilbenoids. SPME‐GC‐MS, where the main limiting factor is non‐reproducibility from fibre to fibre, is an interesting alternative for resveratrol quantification in complex mixtures where pre‐cleaning is too difficult. With the optimized SPE procedure, 5 μg.L^?1 trans‐resveratrol was detected in four commercial beers. However, concentrations of stilbenoids in beer should be higher taking into account the presence of cis‐stilbenoids. During fermentation, trans‐resveratrol was partially regenerated from its glucoside, more stable through beer aging. Adding a stilbenoids‐enriched ethanolic hop extract after fermentation significantly increases the beer stilbenoids potential.     

Identification of desulphoglucosinolates in Brassicaceae by LC/MS/MS: comparison of ESI and atmospheric pressure chemical ionisation-MS

In order to develop a sensitive method for the detection of desulphoglucosinolates by HPLC-MS, the two most common interfaces for HPLC-MS, atmospheric pressure chemical ionisation (APCI) and ESI, were compared. While working with the APCI-interface the evaporation temperature and corona amperage were optimised. In doing so 300 degrees C and 6 muA proved to be most suitable for aliphatic and indole desulphoglucosinolates. The use of formic acid instead of water in the eluent in HPLC-ESI-MS measurements increased the sensitivity for the indole desulphoglucosinolates in the presence of 1 mM formic acid, while the sensitivity for the aliphatic desulphoglucosinolate desulphoglucoraphanin was substantially increased by the presence of 5 mM formic acid. Using an Agilent ion trap, two optimisation procedures for the MS parameters, smart and expert mode, were available. In smart mode the software optimises several parameters automatically, which is much more time efficient than expert mode, in which the optimisation is done manually. It turned out that ESI-MS is most sensitive in smart mode, while for APCI-MS a higher sensitivity could be gained using the expert mode. Comparing both interfaces, APCI-MS was more sensitive than ESI-MS. However, no additional information, in terms of structure determination, was obtained by APCI-MS.     

Determination of Xylazine and 2,6‐Xylidine in Animal Tissues by Liquid Chromatography—Tandem Mass Spectrometry

Xylazine is a potent α2‐adrenergic agonist used in veterinary medicine for sedation, analgesia, muscle relaxation, and so on. Its residue in animal‐derived food may cause the food safety problem. Moreover, the metabolite 2,6‐xylidine was reported to be a genotoxic and carcinogenic compound. Therefore, it is necessary to develop a high sensitive method for analyzing xylazine and metabolite residue in animal products. Here, we described a LC‐MS/MS method for simultaneous determination of xylazine and 2,6‐xylidine in 4 animal tissues: liver, meat, kidney, and fat. The samples were extracted by acetonitrile, and further clean up by hexane. The analysis was performed on a C18 reversed‐phase column and API 5000 Triple Quadrupole mass spectrometry with positive electrospray ionization interface operating in the multiple‐reaction monitoring mode. For all of the investigated sample matrix, the limit of detection (limit of quantitation) for xylazine and 2,6‐xylidine were 0.06 (0.2) and 1.5 (5) μg/kg, respectively, the recoveries were between 63.5% and 90.8%. The precision was within the range of required criteria for method development. The presented method is sensitive and reproducible, and thus suitable for accurate quantification of xylazine and metabolite residue in animal‐derived food products.     

Development and Application of a Method for the Analysis of 9 Mycotoxins in Maize by HPLC‐MS/MS

A reliable and sensitive liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous determination of aflatoxins (AFB₁, AFB₂, AFG₁, and AFG₂), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEA), fumonisin B₁ (FB₁), and T2‐toxin in maize. The samples were first extracted using acetonitrile: water: acetic acid (79 : 20 : 1), and then further cleaned‐up using OASIS HLB cartridge. Optimum conditions for the extraction and chromatographic separation were investigated. The mean recoveries of mycotoxins in spiked maize ranged from 68.3% to 94.3%. Limits of detection and quantification ranged from 0.01 to 0.64 μg/kg and from 0.03 to 2.12 μg/kg, respectively. The LC‐MS/MS method has also been successfully applied to 60 maize samples, which were collected from Shaanxi Province of China. Twenty‐four of the total 60 samples (40%) were contaminated with at least 1 of these 9 mycotoxins. Occurrence of mycotoxins were 6.7%, 1.7%, 3.3%, 6.7%, 1.7%, 23.3%, and 3.3% for AFB₁, AFB₂, OTA, ZEA, DON, FB₁, and T2‐toxin, respectively. The results demonstrated that the procedure was suitable for the simultaneous determination of these mycotoxins in maize matrix.     

Polyphenol compounds in artichoke plant tissues and varieties

Polyphenol compounds, particularly caffeoylquinic acids and flavonoids, were measured in different tissues and developmental stages of 6 artichoke varietal types diffused in the Mediterranean region. Flower heads were subdivided into external, intermediate, internal bracts, and receptacle, while leaves were collected at the vegetative and productive stages. The main polyphenols detected were chlorogenic acid, cynarin, luteolin 7-O-rutinoside, and luteolin 7-O-glucoside. "Violet de Provence" artichoke proved to retain the highest content of total phenols. Single polyphenols accumulated preferentially in specific parts of capitula. In leaves, most polyphenols were detected in the productive stage of the plant. Altogether, results provide useful indications for the promotion of artichoke as nutraceutical food and for the extraction of health-promoting substances in particular tissues/stages of the artichoke plant. PRACTICAL APPLICATION: We describe the content of phenolic compounds in various artichoke tissues, developmental stages, and varieties. Results indicate that artichoke leaves represent an important source of these health-promoting compounds, also useful for phytopharmaceutical applications. A wider utilization of specific artichoke types should be strongly encouraged not only as a food for the fresh market, as they are now used, but also for the pharmaceutical industry, since the content of phenolic compounds is abundant both in the heads and in the leaves.     

Detection of melamine and cyanuric acid in vegetable protein products used in food production

The multitude of food recalls in 2007 clearly demonstrated that total nitrogen-content (ΣN) determination by means of near-infrared spectroscopy (NIRS) and Kjeldahl-based measurements can be deceived, and should no longer be regarded as a complete quality assurance program for nutritive-protein evaluations. Furthermore, contemporary Canadian-employed analytical tools are precariously limited in their ability to effectively assure a product where there is no a priori knowledge of the environmental toxin(s) involved. In light of these challenges, this study explored a number of analytical techniques used to assess and furthermore assure the quality of vegetable protein products (VPPs). Using liquid chromatography with tandem mass spectrometry (LC/MS/MS) technologies, a combination of VPP-based samples was analyzed for the presence of nitrogen-bearing environmental toxicants. Of the 52 samples tested, involving an assortment of matrices, melamine and cyanuric acid were positively identified (>1 ng/mL) in 22 and 17 samples, respectively. Subsequent high pressure liquid chromatography with ultraviolet/visible (HPLC-UV) amino acid profiling further confirmed the adulteration of those materials contaminated with melamine and melamine-related compounds. Based on the evidence presented herein, LC/MS/MS in combination with HPLC-UV provides for a reliable food safety detection system as applied to VPPs. Moreover, HPLC-UV is indispensable as a stand-alone 1st level of screening to assess the integrity of a VPP or any nutritive protein-based sample.     

Comparison and Characterization of Compounds with Antioxidant Activity in Lycium barbarum Using High‐Performance Thin Layer Chromatography Coupled with DPPH Bioautography and Tandem Mass Spectrometry

Methanol extracts from 50 batches of Lycium barbarum (L. barbarum, wolfberry) in China were compared and characterized using high‐performance thin‐layer chromatography coupled with 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) bioautography (HPTLC‐DPPH) and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (ESI‐Q‐TOF‐MS/MS), respectively. Results showed that similar components occupying the major antioxidant activity existed in L. barbarum collected from different origins. However, the average antioxidant capacities of methanol extracts of L. barbarum collected in Ningxia were significantly higher than those of Qinghai, Xinjiang, Inner Mongolia, and Gansu, which may contribute to rational use of L. barbarum in China. Furthermore, the chemical structure of compound with the highest antioxidant capacity was tentatively identified as 2‐O‐β‐d ‐glucopyranosyl‐l ‐ascorbic acid using ESI‐Q‐TOF‐MS/MS analysis, which possessed high potentials to be used as an antioxidant biomarker for the quality control of L. barbarum. Results are helpful for the bioactivity‐based quality control of L. barbarum, and beneficial for the improvement of their performance in functional/health foods area, suggesting that HPTLC‐DPPH bioautography with ESI‐Q‐TOF‐MS/MS could be used as a routine approach for quality control of antioxidant components in L. barbarum.     

Enrichment and Cytotoxic Activity of Curcuminoids from Turmeric Using Macroporous Resins

Curcuminoids are functional secondary metabolites abundant in turmeric. In the present study, a simple and efficient method for enrichment of curcuminoids from turmeric was developed using macroporous resin. Eight different types of macroporous resins were examined by static adsorption/desorption properties and the type of XDA‐7 was selected as the optimum one. Under the optimized conditions, the final contents of refined extract excluded 84.2% of impurities, by comparison with crude extract in a scale‐up experiment. Meanwhile, 8 representative curcuminoids including 4 dihydro‐ and tetrahydro‐curcuminoids were enriched, isolated, and identified by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and nuclear magnetic resonance (NMR) data. In addition, the individual curcuminoids were prepared to evaluate their cytotoxic activity toward HeLa tumor cell lines. All compounds, especially the trace amount of curcuminoids, demonstrated notable cytotoxic activity. The results supported that those trace amount of curcuminoids can be good candidates for drug development as anticancer agents. The purification process was simple and efficient, which could afford a potential method to enrich and concentrate not only the major curcuminoids, but also the trace amount of ones from turmeric raw materials for industrial applications.     

Determination of naturally occurring progestogens in bovine milk as their oxime derivatives using high performance liquid chromatography–electrospray ionization–tandem mass spectrometry

BACKGROUND: Hormones and hormone‐like substances which are present in the environment have been repeatedly accused of being the cause of most endocrine disruption. However, the possible role of endogenous hormones in food of animal origin deserves to be discussed as well. The relation between steroid hormones and several human health problems has been previously reported, such as prostate and breast cancer, perturbation of human reproduction and endocrine disruption on humans and wildlife. This research is particularly concerned with cow's milk, which contains a considerable amount of sex hormones. RESULTS: A liquid chromatography–tandem mass spectrometry method has been developed for the simultaneous detection and quantification of four naturally occurring steroid hormones in commercial bovine milk (pregnenolone (P₅), progesterone (P₄), 17‐hydroxypregnenolone (17‐OHP₅), 17‐hydroxyprogesterone (P₄)). Oxime derivatives of steroids were analyzed in positive ionization and multiple reaction monitoring mode. Methodology has been validated according to Decision 2002/657/EC criteria. CONCLUSION: This method has been successfully used in real samples. It is fast and easy‐handling and provides a useful tool for the assessment of progestogens in bovine milk. Copyright © 2010 Society of Chemical Industry