Development of Non-Targeted Mass Spectrometry Method for Distinguishing Spelt and Wheat.

Food fraud, even when not in the news, is ubiquitous and demands the development of innovative strategies to combat it. A new non-targeted method (NTM) for distinguishing spelt and wheat is described, which aids in food fraud detection and authenticity testing. A highly resolved fingerprint in the form of spectra is obtained for several cultivars of spelt and wheat using liquid chromatography coupled high-resolution mass spectrometry (LC-HRMS). Convolutional neural network (CNN) models are built using a nested cross validation (NCV) approach by appropriately training them using a calibration set comprising duplicate measurements of eleven cultivars of wheat and spelt, each. The results reveal that the CNNs automatically learn patterns and representations to best discriminate tested samples into spelt or wheat. This is further investigated using an external validation set comprising artificially mixed spectra, samples for processed goods (spelt bread and flour), eleven untypical spelt, and six old wheat cultivars. These cultivars were not part of model building. We introduce a metric called the D score to quantitatively evaluate and compare the classification decisions. Our results demonstrate that NTMs based on NCV and CNNs trained using appropriately chosen spectral data can be reliable enough to be used on a wider range of cultivars and their mixes.

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS.

The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0-15 kDa, 15-35 kDa, 35-70 kDa and 70-250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.

Effect of Cereal α-Amylase/Trypsin Inhibitors on Developmental Characteristics and Abundance of Digestive Enzymes of Mealworm Larvae (Tenebrio molitor L.).

The objective of this work was to investigate the potential effect of cereal α-amylase/trypsin inhibitors (ATIs) on growth parameters and selective digestive enzymes of Tenebrio molitor L. larvae. The approach consisted of feeding the larvae with wheat, sorghum and rice meals containing different levels and composition of α-amylase/trypsin inhibitors. The developmental and biochemical characteristics of the larvae were assessed over feeding periods of 5 h, 5 days and 10 days, and the relative abundance of α-amylase and selected proteases in larvae were determined using liquid chromatography tandem mass spectrometry. Overall, weight gains ranged from 21% to 42% after five days of feeding. The larval death rate significantly increased in all groups after 10 days of feeding (p < 0.05), whereas the pupation rate was about 25% among larvae fed with rice (Oryza sativa L.) and Siyazan/Esperya wheat meals, and only 8% and 14% among those fed with Damougari and S35 sorghum meals. As determined using the Lowry method, the protein contents of the sodium phosphate extracts ranged from 7.80 ± 0.09 to 9.42 ± 0.19 mg/mL and those of the ammonium bicarbonate/urea reached 19.78 ± 0.16 to 37.47 ± 1.38 mg/mL. The total protein contents of the larvae according to the Kjeldahl method ranged from 44.0 and 49.9 g/100 g. The relative abundance of α-amylase, CLIP domain-containing serine protease, modular serine protease zymogen and C1 family cathepsin significantly decreased in the larvae, whereas dipeptidylpeptidase I and chymotrypsin increased within the first hours after feeding (p < 0.05). Trypsin content was found to be constant independently of time or feed material. Finally, based on the results we obtained, it was difficult to substantively draw conclusions on the likely effects of meal ATI composition on larval developmental characteristics, but their effects on the digestive enzyme expression remain relevant.

Comparative quantification and differentiation of bracatinga (Mimosa scabrella Bentham) honeydew honey proteins using targeted peptide markers identified by high-resolution mass spectrometry.

Honey traceability is an important topic, especially for honeydew honeys, due to the increased incidence of adulteration. This study aimed to establish specific markers to quantify proteins in honey. A proteomics strategy to identify marker peptides from bracatinga honeydew honey was therefore developed. The proteomics approach was based on initial untargeted identification of honey proteins and peptides by LC-ESI-Triple-TOF-MS/MS, which identified the major royal jelly proteins (MRJP) presence. Afterwards, the peptides were selected by the in silico digestion. The marker peptides were quantified by the developed targeted LC-QqQ-MS/MS method, which provided good linearity and specificity, besides recoveries between 92 and 100% to quantify peptides from bracatinga honeydew honey. The uniqueness and high response in mass spectrometry were backed by further complementary protein analysis (SDS-PAGE). The selected marker peptides EALPHVPIFDR (MRJP 1), ILGANVK (MRJP 2), TFVTIER (MRJP 3), QNIDVVAR (MRJP 4), FINNDYNFNEVNFR (MRJP 5) and LLQPYPDWSWTK (MRJP 7), quantified by LC-QqQ-MS/MS, highlighted that the content of QNIDVVAR from MRJP 4 could be used to differentiate bracatinga honeydew honey from floral honeys (p < 0.05) as a potential marker for its authentication. Finally, principal components analysis highlighted the QNIDVVAR content as a good descriptor of the analyzed bracatinga honeydew honey samples.

Relative Abundance of Alpha-Amylase/Trypsin Inhibitors in Selected Sorghum Cultivars.

Sorghum is of growing interest and considered as a safe food for wheat related disorders. Besides the gluten, α-amylase/trypsin-inhibitors (ATIs) have been identified as probable candidates for these disorders. Several studies focused on wheat-ATIs although there is still a lack of data referring to the relative abundance of sorghum-ATIs. The objective of this work was therefore to contribute to the characterization of sorghum ATI profiles by targeted proteomics tools. Fifteen sorghum cultivars from different regions were investigated with raw proteins ranging from 7.9 to 17.0 g/100 g. Ammonium bicarbonate buffer in combination with urea was applied for protein extraction, with concentration from 0.588 ± 0.047 to 4.140 ± 0.066 mg/mL. Corresponding electrophoresis data showed different protein profiles. UniProtKB data base research reveals two sorghum ATIs, P81367 and P81368; both reviewed and a targeted LC-MS/MS method was developed to analyze these. Quantifier peptides ELAAVPSR (P81367) and TYMVR (P81368) were identified and retained as biomarkers for relative quantification. Different reducing and alkylating agents were assessed and combination of tris (2 carboxyethyl) phosphine/iodoacetamide gave the best response. Linearity was demonstrated for the quantifier peptides with standard recovery between 92.2 and 107.6%. Nine sorghum cultivars presented up to 60 times lower ATI contents as compared to wheat samples. This data suggests that sorghum can effectively be considered as a good alternative to wheat.

Comprehensive Characterization and Relative Quantification of α-Amylase/Trypsin Inhibitors from Wheat Cultivars by Targeted HPLC-MS/MS.

The α-amylase/trypsin inhibitors (ATIs) are discussed as being responsible for non-celiac wheat sensitivity (NCWS), besides being known as allergenic components for baker's asthma. Different approaches for characterization and quantification including proteomics-based methods for wheat ATIs have been documented. In these studies generally the major ATIs have been addressed. The challenge of current study was then to develop a more comprehensive workflow encompassing all reviewed wheat-ATI entries in UniProt database. To substantially test proof of concept, 46 German and Turkish wheat samples were used. Two extractions systems based on chloroform/methanol mixture (CM) and under buffered denaturing conditions were evaluated. Three aspects were optimized, tryptic digestion, chromatographic separation, and targeted tandem mass spectrometric analysis (HPLC-MS/MS). Preliminary characterization with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) documented the purity of the extracted ATIs with CM mixture and the amylase (60-80%)/trypsin (10-20%) inhibition demonstrated the bifunctional activity of ATIs. Thirteen (individual/common) biomarkers were established. Major ATIs (7-34%) were differently represented in samples. Finally, to our knowledge, the proposed HPLC-MS/MS method allowed for the first time so far the analysis of all 14 reviewed wheat ATI entries reported.

A New Approach of Extraction of α-Amylase/trypsin Inhibitors from Wheat (Triticum aestivum L.), Based on Optimization Using Plackett-Burman and Box-Behnken Designs.

Wheat is one of the most consumed foods in the world and unfortunately causes allergic reactions which have important health effects. The α-amylase/trypsin inhibitors (ATIs) have been identified as potentially allergen components of wheat. Due to a lack of data on optimization of ATI extraction, a new wheat ATIs extraction approach combining solvent extraction and selective precipitation is proposed in this work. Two types of wheat cultivars (Triticum aestivum L.), Julius and Ponticus were used and parameters such as solvent type, extraction time, temperature, stirring speed, salt type, salt concentration, buffer pH and centrifugation speed were analyzed using the Plackett-Burman design. Salt concentration, extraction time and pH appeared to have significant effects on the recovery of ATIs (p < 0.01). In both wheat cultivars, Julius and Ponticus, ammonium sulfate substantially reduced protein concentration and inhibition of amylase activity (IAA) compared to sodium chloride. The optimal conditions with desirability levels of 0.94 and 0.91 according to the Doehlert design were: salt concentrations of 1.67 and 1.22 M, extraction times of 53 and 118 min, and pHs of 7.1 and 7.9 for Julius and Ponticus, respectively. The corresponding responses were: protein concentrations of 0.31 and 0.35 mg and IAAs of 91.6 and 83.3%. Electrophoresis and MALDI-TOF/MS analysis showed that the extracted ATIs masses were between 10 and 20 kDa. Based on the initial LC-MS/MS analysis, up to 10 individual ATIs were identified in the extracted proteins under the optimal conditions. The positive implication of the present study lies in the quick assessment of their content in different varieties especially while considering their allergenic potential.

Cocoa Bean Proteins-Characterization, Changes and Modifications due to Ripening and Post-Harvest Processing.

The protein fractions of cocoa have been implicated influencing both the bioactive potential and sensory properties of cocoa and cocoa products. The objective of the present review is to show the impact of different stages of cultivation and processing with regard to the changes induced in the protein fractions. Special focus has been laid on the major seed storage proteins throughout the different stages of processing. The study starts with classical introduction of the extraction and the characterization methods used, while addressing classification approaches of cocoa proteins evolved during the timeline. The changes in protein composition during ripening and maturation of cocoa seeds, together with the possible modifications during the post-harvest processing (fermentation, drying, and roasting), have been documented. Finally, the bioactive potential arising directly or indirectly from cocoa proteins has been elucidated. The "state of the art" suggests that exploration of other potentially bioactive components in cocoa needs to be undertaken, while considering the complexity of reaction products occurring during the roasting phase of the post-harvest processing. Finally, the utilization of partially processed cocoa beans (e.g., fermented, conciliatory thermal treatment) can be recommended, providing a large reservoir of bioactive potentials arising from the protein components that could be instrumented in functionalizing foods.

Selected Plant Metabolites Involved in Oxidation-Reduction Processes during Bud Dormancy and Ontogenetic Development in Sweet Cherry Buds (Prunus avium L.).

Many biochemical processes are involved in regulating the consecutive transition of different phases of dormancy in sweet cherry buds. An evaluation based on a metabolic approach has, as yet, only been partly addressed. The aim of this work, therefore, was to determine which plant metabolites could serve as biomarkers for the different transitions in sweet cherry buds. The focus here was on those metabolites involved in oxidation-reduction processes during bud dormancy, as determined by targeted and untargeted mass spectrometry-based methods. The metabolites addressed included phenolic compounds, ascorbate/dehydroascorbate, reducing sugars, carotenoids and chlorophylls. The results demonstrate that the content of phenolic compounds decrease until the end of endodormancy. After a long period of constancy until the end of ecodormancy, a final phase of further decrease followed up to the phenophase open cluster. The main phenolic compounds were caffeoylquinic acids, coumaroylquinic acids and catechins, as well as quercetin and kaempferol derivatives. The data also support the protective role of ascorbate and glutathione in the para- and endodormancy phases. Consistent trends in the content of reducing sugars can be elucidated for the different phenophases of dormancy, too. The untargeted approach with principle component analysis (PCA) clearly differentiates the different timings of dormancy giving further valuable information.

Transport of pharmaceuticals in columns of a wastewater-irrigated Mexican clay soil.

The irrigation or agricultural land with wastewater is increasingly practiced in many parts of the world as a consequence of growing populations and urbanization. The risks emerging from pharmaceuticals that are contained in wastewater for soils and groundwater have hardly been investigated. We studied leaching and effects of naproxen, ibuprofen, bezafibrate, diclofenac, gemfibrocil, clarithromycin, trimethoprim, clindamycin, erythromycin, and metoprolol in a soil column experiment simulating an irrigation event with 8.6 cm of wastewater containing 20 microg L(-1) or 2000 microg L(-1) of each compound or of erythromycin alone. The leached fraction of applied pharmaceuticals ranged from 0.1 +/- 0.1% (clarithromycin, 2000 microg L(-1)) to 130 +/- 41% (naproxen, 20 microg L(-1)) and tended to increase with decreasing K(d) or K(oc). Naproxen transport was similar to that of the tracer chloride. Ibuprofen was also hardly retarded (R = 1.20 +/- 0.18), but showed a higher degradation rate of 0.02 +/- 0.004 h(-1) (2000 microg L(-1)) than naproxen. The transport of a pulse of 2000 microg L(-1) of bezafibrate could be described with a retardation factor of 1.5 and a degradation rate of 0.033 h(-1). The application of erythromycin alone or of a cocktail of all pharmaceuticals significantly increased soil CO2 emissions by 50% 1 d after the application. There is a considerable risk that pharmaceuticals are leached to groundwater during wastewater irrigation.

Re-evaluation of the conformational structure of sulfadiazine species using NMR and ab initio DFT studies and its implication on sorption and degradation.

In the environment, the sorption and the degradation of organic pollutants are of increasing interest. The investigation of the chemical structures provides a basis for the development of a suitable binding model approach and for the mechanistic understanding of the chemical fate processes. The aim of this study was the identification of different species of the antibiotic compound sulfadiazine (SDZ) using (1)H and (13)C NMR experiments and ab initio density functional theory (DFT) calculations. In the neutral, aprotic solvent dimethylsulfoxide-d(6) (DMSO-d(6)), a new sulfadiazine structure containing an O-H-N hydrogen bond was identified. In the protic solvent water-d(2) and in dependence on pH and the position of the amidogen hydrogen atom nine possible SDZ conformations were analyzed and five structures were identified. Good conformity between theory and calculation of (1)H NMR was observed. Unfortunately, (13)C NMR is not sensitive enough for comparison and differentiation. In order to verify the identified structures, additional NBO/NLMO (natural localized molecular orbital) analyses were conducted (calculation of net atomic charges, bond polarity, atomic valence, and electron delocalization). Finally, conformation optimizations were performed in order to investigate the stability of the SDZ species. We showed that SDZ contains no S=O double bond, but that it has two S-O single bonds. Surprisingly, negative charges were observed at the pyrimidine nitrogen atom. With these results, the known structure of SDZ was revised. Studies of the geometrical structure and the torsion angles showed that SDZ is very flexible and can be easily fitted to the sorbent. These observations would explain the strong sorbance and hence the rapid formation of non-extractable residues in the environment because SDZ acts as a strong ligand. These results show that that the sulfonamide hydrogen is important for the biological activity but the pyrimidine nitrogen and the sulfonamide oxygen is responsible for the sorbance in environment.

Ecotoxicological classification of the Berlin river system using bioassays in respect to the European Water Framework Directive.

Bioassays as well as biochemical responses (biomarkers) in ecosystems due to environmental stress provide us with signals (environmentally signalling) of potential damage in the environment. If these responses are perceived in this early stage in ecosystems, the eventual damage can be prevented. Once ecosystem damage has occurred, the remedial action processes for recovery could be expensive and pose certain logistical problems. Ideally, "early warning signals" in ecosystems using sensing systems of biochemical responses (biomarkers) would not only tell us the initial levels of damage, but these signals will also provide us with answers by the development of control strategies and precautionary measures in respect to the European Water Framework Directive (WFD). Clear technical guidelines or technical specifications on monitoring are necessary to establish and characterise reference conditions for use in an ecological status classification system for surface water bodies. For the Ecotoxicological Risk Assessment (ERA) of endocrine effects we used an approach of the exposure - dose - response concept. Based on the "Ecototoxicological Classification System of Sediments" that uses pT-values to classify effects in different river systems, we transferred the bio-monitoring data to the five-level ecological system of the WFD. To understand the complexity of the structure of populations and processes behind the health of populations, communities and ecosystems an ERA should establish links between natural factors, chemicals, and biological responses so as to assess causality. So, our ecological monitoring assessment has incorporated exposure & effects data.

Environmental risk assessment of medicinal products for human use according to European Commission recommendations.

Presented here, based on new recommendations of the European Commission, is an environmental risk assessment (ERA) of a selected group of pharmaceuticals for Phase I, environmental exposure assessment, and Phase II Tier A, initial environmental fate and effect analysis. This pharmaceutical group is composed of the 111 highest-selling human drug substances that have annual sales in Germany of more than 5,000 kg. The data required for this ERA came from analyzing: (1) sales annually (in kg or IU) of the 2671 active pharmaceutical drug substances (2001) on the German market in all medicinal products sold by pharmacies (with and without prescriptions) and used in hospitals in 1996-2001; (2) the use pattern of drug substances as categorized according to Anatomical Therapeutic Chemical (ATC) classification indexes ATC3 and ATC7; (3) data for excretion, toxicity, and metabolites of the 111 selected human drug substances; (4) the physicochemical properties of these substances; and (5) the degradability of selected drug substances in sewage treatment plants (STPs) by using a validated and accredited liquid chromatography-electrospray ionization tandem mass spectrometry method. A correction factor for the pharmaceutical therapeutic (PT) activity of metabolites, the PT(Index) (excretion rate/100) for drug substances and PT active metabolites was established to refine the predicted environmental concentration (PEC(SURFACEWATER)). A refinement of the PEC(SURFACEWATER) was carried out with the market penetration factor of the human drug substances in Germany. In addition, for effect analysis the predicted no-effects concentration (PNEC) was calculated using assessment factors. The estimated PEC results were validated with the exposure results of effluents of the STPs. All results on ERA of drug substances have been documented in a Microsoft Access 2000 database.