In-house validation of an LC-MS method for the multiplexed quantitative determination of total allergenic food in chocolate.

Mass spectrometry has been widely accepted as a confirmatory tool for the sensitive detection of undeclared presence of allergenic ingredients. Multiple methods have been developed so far, achieving different levels of sensitivity and robustness, still lacking harmonization of the analytical validation and impairing comparability of results. In this investigation, a quantitative method has been validated in-house for the determination of six allergenic ingredients (cow's milk, hen's egg, peanut, soybean, hazelnut, and almond) in a chocolate-based matrix. The latter has been produced in a food pilot plant to provide a real and well-characterized matrix for proper assessment of method performance characteristics according to official guidelines. In particular, recent considerations issued by the European Committee for Standardization have been followed to guide a rigorous single-laboratory validation and to feature the main method performance, such as selectivity, linearity, and sensitivity. Synthetic surrogates of the peptide markers have been used both in native and labelled forms in matrix-matched calibration curves as external calibrants and internal standards, respectively. A two-order of magnitude range was investigated, focusing on the low concentration range for proper assessment of the detection and quantification limits (LOD and LOQ) by rigorous calibration approach. Conversion factors for all six allergenic ingredients have been determined for the first time to report the final quantitative information as fraction of total allergenic food protein (TAFP) per mass of food (µgTAFP/gfood), since such a reporting unit is exploitable in allergenic risk assessment plans. The method achieved good sensitivity with LOD values ranging between 0.08 and 0.2 µgTAFP/gfood, for all ingredients besides egg and soybean, whose quantitative markers reported a slightly higher limit (1.1 and 1.2 µgTAFP/gfood, respectively). Different samples of chocolate bar incurred at four defined concentration levels close to the currently available threshold doses have been analyzed to test the quantitative performance of the analytical method, with a proper estimate of the measurement uncertainty from different sources of variability. The sensitivity achieved resulted in compliance with the various threshold doses issued or recommended worldwide.

Development of incurred chocolate bars and broth powder with six fully characterised food allergens as test materials for food allergen analysis.

The design and production of incurred test materials are critical for the development and validation of methods for food allergen analysis. This is because production and processing conditions, together with the food matrix, can modify allergens affecting their structure, extractability and detectability. For the ThRAll project, which aims to develop a mass spectrometry-based reference method for the simultaneous accurate quantification of six allergenic ingredients in two hard to analyse matrices. Two highly processed matrices, chocolate bars and broth powder, were selected to incur with six allergenic ingredients (egg, milk, peanut, soy, hazelnut and almond) at 2, 4, 10 and 40 mg total allergenic protein/kg food matrix using a pilot-scale food manufacturing plant. The allergenic activity of the ingredients incurred was verified using food-allergic patient serum/plasma IgE, the homogeneity of the incurred matrices verified and their stability at 4 °C assessed over at least 30-month storage using appropriate enzyme-linked immunosorbent assays (ELISA). Allergens were found at all levels from the chocolate bar and were homogenously distributed, apart from peanut and soy which could only be determined above 4 mg total allergenic ingredient protein/kg. The homogeneity assessment was restricted to analysis of soy, milk and peanut for the broth powder but nevertheless demonstrated that the allergens were homogeneously distributed. All the allergens tested were found to be stable in the incurred matrices for at least 30 months demonstrating they are suitable for method development.

Multi-Class UHPLC-MS/MS Method for Plant Toxins and Cyanotoxins in Food Supplements and Application for Belgian Market Samples.

The presence of plant toxins and/or cyanotoxins in food supplements implies consumer health risks. Therefore, a targeted ultra-high performance liquid chromatographic-tandem mass spectrometric method to detect/quantify 25 toxins simultaneously in food supplement formulations was developed and validated. Full validation for tablets/powders and secondary validation for a liquid and soft gel capsule indicated that most compounds were efficiently extracted (≥ 75%), while others were only partly extracted (18 - 61%). Trueness was fulfilled (70 - 120%), with some exceptions (mostly at the lowest validation level). Intralaboratory repeatability, intra- and interlaboratory reproducibility values of ≤ 20%, ≤ 25%, and ≤ 25% were obtained for most, respectively. Matrix effects were found to be significant for most compounds. Good sensitivity (µg/kg level) was observed for galegin(e), lycopsamine, lycorine, rubiadin, skimmiamine, and vascin(e), in contrast to helveticoside, lucidin, lucidin-3-primveroside, plumbagin(e), and thujone, which were detected at the mg/kg level. The other compounds were characterized by a sensitivity between 10 to 1000 µg/kg. The validated methodology was applied for 52 food supplements (tablets, capsules, liquids/syrup, etc.) purchased from the Belgian market. In more than 25% of the samples, one or more toxins were detected (concentrations determined using standard addition). Lycopsamine, microcystin LR, solamargine, thujone, and vasicin(e) were the most frequently detected toxins. A clear link between the toxins detected and the plant species on the food supplement ingredient list could not always be established. This generic "dilute-and-shoot" procedure can be used for further research on toxins in food supplements and by extension other plant/algae-based food/feed commodities (herbs, edible flowers, etc.).

Keeping mycotoxins away from the food: Does the existence of regulations have any impact in Africa?

Following the discovery of aflatoxins in the early 1960s, there have been many studies leading to the uncovering of many mycotoxins and the understanding of associated health effects in animals and humans. Consequently, there has been a global increase in the number of countries with mycotoxin regulations in foods. However, many African countries have only regulations for aflatoxins (or a few other mycotoxins) in specific foods, or no regulations at all. This paper critically reviews the challenges thwarting the establishment of mycotoxin regulations and their impacts on human dietary mycotoxin exposure in Africa. Mycotoxin regulatory limits for different countries are compared with mycotoxin tolerable daily intakes established by international food safety bodies taking into account consumption patterns. The agrarian setup, food insecurity, and mycotoxin analytical challenges in African countries are discussed; and more feasible mycotoxin dietary exposure reduction strategies are proposed.

Development of an LC-MS/MS method for the detection of traces of peanut allergens in chili pepper.

The recent detection of nuts (including peanut) in spices across the globe has led to enormous recalls of several spices and food products in the last two years. The lack of validated detection methods specific for spices makes it difficult to assess allergen presence at trace levels. Because of the urgent need for confirmation of possible peanut presence in chili peppers, an LC-MS/MS method was optimized and developed for this particular food matrix. Although several studies optimized LC-MS detection strategies specific for peanuts, the presence of complex components in the spices (e.g., phenolic components) makes method optimization and validation necessarily. Focus was laid on validation of the method with real incurred chili peppers (whereby a known amount of peanut is added) at low concentrations, to deal with possible matrix interferences. LC-MS/MS proves to be a good alternative to the currently most applied methods (ELISA and RT-PCR) and can be used as a complementary method of analysis when results are unclear. Peanut marker peptides were selected based on their abundancy in digested incurred chili peppers. The limit of detection was determined to be 24 ppm (mg peanut/kg), a level whereby the risk for potential allergic reactions is zero, considering the typical portion size of spices. The chili pepper powder under investigation proved to contain low levels of peanuts after LC-MS/MS, ELISA, and RT-PCR testing. Graphical abstract Standard curve of the detected peanuts in chili pepper samples using the novel LC-MS/MS method.

Virulence, Host-Selective Toxin Production, and Development of Three Cochliobolus Phytopathogens Lacking the Sfp-Type 4'-Phosphopantetheinyl Transferase Ppt1.

The Sfp-type 4'-phosphopantetheinyl transferase Ppt1 is required for activation of nonribosomal peptide synthetases, including α-aminoadipate reductase (AAR) for lysine biosynthesis and polyketide synthases, enzymes that biosynthesize peptide and polyketide secondary metabolites, respectively. Deletion of the PPT1 gene, from the maize pathogen Cochliobolus heterostrophus and the rice pathogen Cochliobolus miyabeanus, yielded strains that were significantly reduced in virulence to their hosts. In addition, ppt1 mutants of C. heterostrophus race T and Cochliobolus victoriae were unable to biosynthesize the host-selective toxins (HST) T-toxin and victorin, respectively, as judged by bioassays. Interestingly, ppt1 mutants of C. miyabeanus were shown to produce tenfold higher levels of the sesterterpene-type non-HST ophiobolin A, as compared with the wild-type strain. The ppt1 strains of all species were also reduced in tolerance to oxidative stress and iron depletion; both phenotypes are associated with inability to produce extracellular siderophores biosynthesized by the nonribosomal peptide synthetase Nps6. Colony surfaces were hydrophilic, a trait previously associated with absence of C. heterostrophus Nps4. Mutants were decreased in asexual sporulation and C. heterostrophus strains were female-sterile in sexual crosses; the latter phenotype was observed previously with mutants lacking Nps2, which produces an intracellular siderophore. As expected, mutants were albino, since they cannot produce the polyketide melanin and were auxotrophic for lysine because they lack an AAR.

An optimized protocol for pig intestinal mucosa proteomics.

The overall well-being of organisms is widely recognized to be closely intertwined with their intestinal health. The intestinal mucosal layer plays a pivotal role in ensuring the proper functioning of the intestine, a fact observed not only in humans but also in animals like pigs. Any alterations to the mucosal layer of a pig's intestine can potentially disrupt its functionality, thereby impacting the animal's health and productivity. Mass spectrometry-based proteome analysis serves as a valuable tool in investigating the intricate dynamics of the proteome within the intestinal mucosa. Such studies hold promise in uncovering causal relationships between mucosal changes and overall health outcomes in pigs. It is anticipated that insights gathered from proteome studies will inform future strategies aimed at enhancing the health and productivity of pigs. However, the research field lacks a standardized and detailed method to extract proteins from pig intestinal mucosa and prepare proteins for proteome analysis. In the present study, we evaluated three alternative S-Trap-based protocols for analyzing ileal mucosal scrapings from pigs. Samples were either freeze-dried and treated as solid samples or ground in liquid nitrogen, categorized as either solid or liquid samples. In our analysis, a total of 2840 proteins were identified across all samples. Through statistical analysis and gene ontology examinations, we investigated potential differences between the three approaches. Even though our findings revealed no significant differences among the three methods, we propose the use of the protocol wherein samples are freeze-dried and treated as solid for protein extraction. This protocol stands out as the most convenient and practical option, offering ease of use and ensuring consistent and reliable results. By establishing a standardized approach, we aim to advance research efforts in understanding pig intestinal health. SIGNIFICANCE: The development of an optimized protocol for protein extraction of intestinal mucosal scrapings in pigs addresses a gap in the field and enhances future research on pig intestinal health. By use of the protocol and mass spectrometry-based proteome analysis, valuable insights for improving the health and productivity of pigs can be presented. Studying the complex dynamics of the proteome within the intestinal mucosa, potentially identifying links between mucosal changes and health outcomes, provides us with information about the critical connection between intestinal health and the overall well-being and productivity of pigs. By creating a standardized approach, consistent, reliable, and reproducible results can be obtained for this type of research.

Chestnut Wood Tannins in Broiler Diets: Pharmacokinetics, Serum Levels during Rearing, and Intestinal Absorption Pattern of Gallic Acid.

Studies on the bioavailability, serum levels, and absorption of hydrolyzable tannin compounds are lacking. In this study, we performed a pharmacokinetic trial, measured the serum levels of compounds in broilers that were reared with different feed added or not with tannins, and tested the digestibility of tannins throughout the intestinal tract. Only gallic acid and 4-O-methyl gallic acid were found in the serum. Moreover, gallic acid showed a 41.8% absolute oral bioavailability and a 72.3% relative bioavailability of gallic acid from chestnut extract compared to the standard. The rapid metabolization caused alternating serum levels during the day and night. These patterns were not affected by the feed type or the previous addition of tannins in the feed. The absorption and metabolization in the intestines occurred gradually throughout the intestinal tract. The latter was true for gallic acid as well as ellagic acid, which was not found in the serum. We can conclude that components from chestnut tannins are absorbed throughout all components of the intestinal tract and are eliminated quickly with little interaction from the feed and previous addition of tannins. Moreover, ellagic acid seems to be absorbed but would remain accumulated in the intestinal tissue or be metabolized by the microbiome.

Stability and bioaccessibility of micronutrients and phytochemicals present in processed leek and Brussels sprouts during static in vitro digestion.

Vegetables are frequently processed before consumption. However, vegetable functionalization continues beyond ingestion as the human digestive tract exposes vegetable products to various conditions (e.g. elevated temperature, pH alterations, enzymes, electrolytes, mechanical disintegration) which can affect the stability of micronutrients and phytochemicals. Besides the extent to which these compounds withstand the challenges posed by digestive conditions, it is equally important to consider their accessibility for potential absorption by the body. Therefore, this study investigated the impact of static in vitro digestion on the stability (i.e. concentration) and bioaccessibility of vitamin C, vitamin K1, glucosinolates, S-alk(en)yl-l-cysteine sulfoxides (ACSOs) and carotenoids in Brussels sprouts (Brassica oleracea var. gemmifera) and leek (Allium ampeloprasum var. porrum). Water-soluble compounds, glucosinolates and ACSOs, remained stable during digestion while vitamin C decreased by >48%. However, all water-soluble compounds were completely bioaccessible. Lipid-soluble compounds were also stable during digestion but were only bioaccessible for 26-81%.

Impact of refrigerated storage on (bio)chemical conversions of health-related compounds in pretreated, pasteurized Brussels sprouts and leek.

Vegetable processing often consists of multiple processing steps. Research mostly focused on the impact of individual processing steps on individual health-related compounds. However, there is a need for more holistic approaches to understand the overall impact of the processing chain on the health potential of vegetables. Therefore, this work studied the impact of pretreatment (relatively intact versus pureed vegetable systems), pasteurization and subsequent refrigerated storage (kinetic evaluation) on multiple health-related compounds (vitamin C, vitamin K1, carotenoids, glucosinolates and S-alk(en)yl-L-cysteine sulfoxides (ACSOs)) in Brussels sprouts and leek. It could be shown that differences introduced by different types of pretreatment were not nullified during pasteurization and refrigerated storage. Clearly, enzymatic conversions controlled during pretreatment resulted in different health-related compound profiles still observable after pasteurization. Moreover, about -42% and -100% relative concentration differences of ACSOs and dehydroascorbic acid, respectively, were detected immediately after pasteurization, while glucosinolates concentrations decreased by about 47% during refrigerated storage. All other compounds were stable during pasteurization and refrigerated storage.

Innovative analytical methodologies for characterizing chemical exposure with a view to next-generation risk assessment.

The chemical burden on the environment and human population is increasing. Consequently, regulatory risk assessment must keep pace to manage, reduce, and prevent adverse impacts on human and environmental health associated with hazardous chemicals. Surveillance of chemicals of known, emerging, or potential future concern, entering the environment-food-human continuum is needed to document the reality of risks posed by chemicals on ecosystem and human health from a one health perspective, feed into early warning systems and support public policies for exposure mitigation provisions and safe and sustainable by design strategies. The use of less-conventional sampling strategies and integration of full-scan, high-resolution mass spectrometry and effect-directed analysis in environmental and human monitoring programmes have the potential to enhance the screening and identification of a wider range of chemicals of known, emerging or potential future concern. Here, we outline the key needs and recommendations identified within the European Partnership for Assessment of Risks from Chemicals (PARC) project for leveraging these innovative methodologies to support the development of next-generation chemical risk assessment.

Nicotine in the button mushroom Agaricus bisporus, endogenous biosynthesis?

In early 2009 nicotine was unexpectedly detected in dried mushroom samples. As its origin has not yet been elucidated, this study addressed possible endogenous synthesis of nicotine. Therefore, Agaricus bisporus fruiting bodies were grown in a representative and controlled (nicotine-free) setup. Fruiting bodies (fresh versus stored, intact versus processed (sliced/cooked)) from different harvest days and flushes were analysed with a validated, sensitive dilute-and-shoot UHPLC-MS/MS methodology for nicotine and its precursors putrescine and nicotinic acid. Neither storage nor processing initiated any endogenous nicotine biosynthesis (detection limit 1.6 ng g-1 fresh weight). In contrast, putrescine and nicotinic acid were detected in all samples, with increasing amounts in the different treatments. In silico analysis of the fully sequenced genome of A. bisporus confirmed its inability to produce nicotine. The data obtained do not provide evidence for natural, endogenous presence of nicotine in mushrooms, indicating an exogenous contamination source (e.g. contamination during hand-picking, sample preparation/analysis).

Emerging pharmaceutical therapies of Ascidian-derived natural products and derivatives.

In a growing multidrug-resistant environment, the identification of potential new drug candidates with an acceptable safety profile is a substantial crux in pharmaceutical discovery. This review discusses several aspects and properties of approved marine natural products derived from ascidian sources (phylum Chordata, subphylum Tunicata) and/or their deduced analogues including their biosynthetic origin, (bio)chemical preclinical assessments and known efficacy-safety profiles, clinical status in trials, but also translational developments, opportunities and final conclusions. The review also describes the preclinical assessments of a large number of other ascidian compounds that have not been involved in clinical trials yet. Finally, the emerging research on the connectivity of the ascidian hosts and their independent or obligate symbiotic guests is discussed. The review covers the latest information on the topic of ascidian-derived marine natural products over the last two decades including 2022, with the majority of publications published in the last decade.

Assessment of transcriptional reprogramming of lettuce roots in response to chitin soil amendment.

Chitin soil amendment is known to improve soil quality, plant growth and stress resilience, but the underlying mechanisms are not well understood. In this study, we monitored chitin's effect on lettuce physiology every two weeks through an eight-week growth period, analyzed the early transcriptional reprogramming and related metabolomic changes of lettuce, in response to crab chitin treatment in peat-based potting soil. In commercial growth conditions, chitin amendment still promoted lettuce growth, increased chlorophyll content, the number of leaves and crop head weight from week six. The flavonoid content in lettuce leaves was altered as well, showing an increase at week two but a decrease from week six. Transcriptomic analysis showed that over 300 genes in lettuce root were significantly differentially expressed after chitin soil treatment. Gene Ontology-term (GO) enrichment analysis revealed statistical overrepresentation of GO terms linked to photosynthesis, pigment metabolic process and phenylpropanoid metabolic process. Further analysis of the differentially expressed genes (DEGs) showed that the flavonoid pathway was mostly upregulated whereas the bifurcation of upstream phenylpropanoid pathway towards lignin biosynthesis was mostly downregulated. Metabolomic analysis revealed the upregulation of salicylic acid, chlorogenic acid, ferulic acid, and p-coumaric acid in chitin-treated lettuce seedlings. These phenolic compounds (PCs) mainly influence the phenylpropanoid biosynthesis pathway and may play important roles in plant defense reactions. Our results suggest that chitin soil amendments might activate induced resistance by priming lettuce plants and promote lettuce growth via transcriptional changes.

Multi-Allergen Quantification in Food Using Concatemer-Based Isotope Dilution Mass Spectrometry: An Interlaboratory Study.

BACKGROUND: Food allergen analysis is essential for the development of a risk-based approach for allergen management and labeling. MS has become a method of choice for allergen analysis, even if quantification remains challenging. Moreover, harmonization is still lacking between laboratories, while interlaboratory validation of analytical methods is necessary for such harmonization. OBJECTIVE: This interlaboratory study aimed to evaluate the potential of MS for food allergen detection and quantification using a standard addition quantification strategy and a stable isotope-labeled (SIL) concatemer as an internal standard. METHODS: In-house-produced test material (cookies), blank and incurred with four allergens (egg, milk, peanut, and hazelnut), allergen standards, an internal standard, and the complete methodology (including sample preparation and ultra-HPLC-MS/MS method) were provided to nine laboratories involved in the study. Method sensitivity and selectivity were evaluated with incurred test material and accuracy with spiked test material. Quantification was based on the standard addition strategy using certified reference materials as allergen protein standards and a SIL concatemer as an internal standard. RESULTS: All laboratories were able to detect milk, hazelnut, and peanut in the incurred cookies with sufficient sensitivity to reach the AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR® 2016.002). Egg detection was more complicated due to food processing effects, yet five laboratories reached the sensitivity requirements. Recovery results were laboratory-dependent. Some milk and hazelnut peptides were quantified in agreement with SMPR 2016.002 by all participants. Furthermore, over 90% of the received quantification results agreed with SMPR 2016.002 for method precision. CONCLUSION: The encouraging results of this pioneering interlaboratory study represent an additional step towards harmonization among laboratories testing for allergens. HIGHLIGHTS: In this pioneering interlaboratory study, food allergens were analyzed by MS with characterized incurred and spiked test materials, calibrated with a certified reference material, and a single SIL concatemer used as an internal standard.

Identification and characterization of CYP71 subclade cytochrome P450 enzymes involved in the biosynthesis of bitterness compounds in Cichorium intybus.

Industrial chicory (Cichorium intybus var. sativum) and witloof (C. intybus var. foliosum) are crops with an important economic value, mainly cultivated for inulin production and as a leafy vegetable, respectively. Both crops are rich in nutritionally relevant specialized metabolites with beneficial effects for human health. However, their bitter taste, caused by the sesquiterpene lactones (SLs) produced in leaves and taproot, limits wider applications in the food industry. Changing the bitterness would thus create new opportunities with a great economic impact. Known genes encoding enzymes involved in the SL biosynthetic pathway are GERMACRENE A SYNTHASE (GAS), GERMACRENE A OXIDASE (GAO), COSTUNOLIDE SYNTHASE (COS) and KAUNIOLIDE SYNTHASE (KLS). In this study, we integrated genome and transcriptome mining to further unravel SL biosynthesis. We found that C. intybus SL biosynthesis is controlled by the phytohormone methyl jasmonate (MeJA). Gene family annotation and MeJA inducibility enabled the pinpointing of candidate genes related with the SL biosynthetic pathway. We specifically focused on members of subclade CYP71 of the cytochrome P450 family. We verified the biochemical activity of 14 C. intybus CYP71 enzymes transiently produced in Nicotiana benthamiana and identified several functional paralogs for each of the GAO, COS and KLS genes, pointing to redundancy in and robustness of the SL biosynthetic pathway. Gene functionality was further analyzed using CRISPR/Cas9 genome editing in C. intybus. Metabolite profiling of mutant C. intybus lines demonstrated a successful reduction in SL metabolite production. Together, this study increases our insights into the C. intybus SL biosynthetic pathway and paves the way for the engineering of C. intybus bitterness.

Development and Validation of a Quantitative Method for Multiple Allergen Detection in Food Using Concatemer-Based Isotope Dilution Mass Spectrometry.

BACKGROUND: Accurate food labeling is essential to protect allergic consumers. However, allergen contaminations may occur during the whole food production process. Reliable, sensitive, and robust methods for detecting multiple allergens in food are needed. OBJECTIVE: This work aims to develop and validate an LC coupled to tandem mass spectrometry (MS/MS) method for the detection and quantification of hazelnuts, peanuts, milk, and eggs in processed food products. METHODS: In-house-produced incurred test materials, cookies and chocolates, were used for the method development and validation. The quantification was based on the standard addition strategy using qualified reference materials as allergen protein standards and an innovative stable isotope-labeled concatemer as an internal standard. RESULTS: A method targeting 19 allergen-specific peptides was developed and validated in two laboratories, which strengthens its robustness. The AOAC INTERNATIONAL performance requirements for repeatability, intermediate precision, reproducibility, and recovery were reached for at least one peptide per allergen across both matrixes, and quantification limits complied with the action levels of the Food Industry Guide to the Voluntary Incidental Trace Allergen Labelling (VITAL®) Program Version 3.0. CONCLUSION: The combination of incurred test materials, standard addition strategy, and stable isotope-labeled concatemer as an internal standard allowed us to develop and validate a robust method for detecting and quantifying multiple allergens in food with sufficient sensitivity to protect allergic consumers. HIGHLIGHTS: The combination of characterized incurred test material, calibration with certified reference material, a single stable isotope labelled concatemer and cross-lab validation result in the required standardization and harmonization in food allergen detection according to the stakeholders' group to assess the robustness of our method.

Selecting Processing Robust Markers Using High-Resolution Mass Spectrometry for the Detection of Milk in Food Products.

BACKGROUND: Cow's milk allergy is one of the most reported food allergies in Europe. To help patients suffering from food allergies it is important to be able to detect milk in different foods. An analytical method that is gaining interest in the field of allergen detection is ultrahigh performance liquid chromatography-tandem mass spectrometry, where the analyte is a target peptide. When these peptide biomarkers are selected, the effect of food processing should be taken into account to allow a robust detection method. OBJECTIVE: This work aims at identifying such processing stable peptide markers for milk for the ultrahigh performance liquid chromatography-tandem mass spectrometry based detection of food allergens in different food products. METHOD: Milk-incurred food materials that underwent several processing techniques were produced. This was followed by establishing tryptic peptide profiles from each matrix using ultrahigh performance liquid chromatography-high resolution mass spectrometry. RESULTS: A careful comparison of peptide profiles/intensities and the use of specific exclusion criteria resulted in the selection of eight peptide biomarkers suitable for application in ultrahigh performance liquid chromatography-tandem mass spectrometry based milk detection methods. One of these markers is an α-lactalbumin specific peptide, which has been determined to be stable in different incurred materials for the first time. CONCLUSIONS: To our knowledge, this is the first systematic and experimentally based approach for the selection of suitable milk peptide biomarkers robust toward multiple, often applied food processing techniques for milk. Ensuring the exact knowledge of the food processing circumstances by starting from well-defined raw material and using fully controlled settings to produce incurred test material allowed the construction of a peptide database with robust markers. These robust markers can be used for the development of a robust detection method for milk in different food matrixes. HIGHLIGHTS: To facilitate food allergen detection in processed food, processing stable peptide markers for the detection of milk in food products were determined using Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry on well-defined raw materials which were processed in accordance with often used processing techniques.

Sunflower Bark Extract as a Biostimulant Suppresses Reactive Oxygen Species in Salt-Stressed Arabidopsis.

A survey of plant-based wastes identified sunflower (Helianthus annuus) bark extract (SBE), produced via twin-screw extrusion, as a potential biostimulant. The addition of SBE to Arabidopsis (Arabidopsis thaliana) seedlings cultured in vitro showed a dose-dependent response, with high concentrations causing severe growth inhibition. However, when priming seeds with SBE, a small but significant increase in leaf area was observed at a dose of 0.5 g of lyophilized powder per liter. This optimal concentration of SBE in the culturing medium alleviated the growth inhibition caused by 100 mM NaCl. The recovery in shoot growth was accompanied by a pronounced increase in photosynthetic pigment levels and a stabilization of osmotic homeostasis. SBE-primed leaf discs also showed a similar protective effect. SBE mitigated salt stress by reducing the production of reactive oxygen species (ROS) (e.g., hydrogen peroxide) by about 30% and developing more expanded true leaves. This reduction in ROS levels was due to the presence of antioxidative agents in SBE and by activating ROS-eliminating enzymes. Polyphenols, carbohydrates, proteins, and other bioactive compounds detected in SBE may have contributed to the cellular redox homeostasis in salt-stressed plants, thus promoting early leaf development by relieving shoot apical meristem arrest. Sunflower stalks from which SBE is prepared can therefore potentially be valorized as a source to produce biostimulants for improving salt stress tolerance in crops.

The use of library identification and common fragments for the identification of corticosteroids in forensic samples.

The detection of corticosteroids and sex steroids in samples with no content indication, which are confiscated for forensic investigation, is a challenge in doping analysis. A screening method based on the identification of androgens, estrogens, gestagens, and their esters by means of a mass spectral library, along with a fast ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method, was recently developed in our lab for the analysis of dietary supplements. However, for forensic investigations, it is important to extend the scope of the method to corticosteroids in various matrices. Therefore, 36 corticosteroids were added to the mass spectral library, and the sample preparation step was modified so that androgens, gestagens, corticosteroids, and their esters could be analyzed with only one injection with the UPLC-MS method. A complementary tool to the existing library identification was found in the extraction of common fragment ions out of the full scan data obtained for the library search. The fragment ion with m/z 147 was found to be a good marker for the detection of steroids. Extra confirmation was obtained from the fragment ions with m/z 135 (for all steroids) and 237 (specific for corticosteroids) or from the fragment ions with m/z 77, 91, and 105. The effectiveness of this approach was evaluated on some samples previously screened for forensic investigation with thin-layer chromatography and confirmed with a targeted gas chromatography-mass spectrometry method. This study shows that the combination of the library identification and the common fragment ions approach can be a valuable tool in the detection of steroids without defining any target at the start of the analysis.