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Vegan and vegetarian diets are increasing in popularity. Consequently, the supply and demand of plant-based meat alternatives has increased steadily over the past few years. However, scientific research on spoilage processes for such products is still inadequate as compared to research on traditional meat products. In traditional meat products, biogenic amines are prominent spoilage markers and potential sources of food toxicity, especially for people sensitive to biogenic amines. Plant-based meat alternatives are manufactured to mimic the taste, look, texture, and nutritional value of meat, and they have a protein-rich basis. It is therefore hypothesized that biogenic amines could be markers for spoilage in such products as well. Further analysis of their presence and concentrations and comparison to conventional meat products is necessary. If biogenic amines are lower in plant-based meat alternatives, these products are possibly better suited for consumption by people with biogenic amine sensitivities. A simple and rapid extraction method, followed by HILIC-MS/MS separation and detection was therefore developed as a first step and validated for nine biogenic amines in plant-based meat alternatives. This method showed a strong linear correlation between amine concentration and detector response, high accuracy, and precision (< 12 %), as well as high sensitivity, as proven by the lowest limits of quantification (i.e., the lowest concentration within the calibration model) of 1 mg/kg for all analytes, which also compares well with other methods. Subsequently, as part of a pilot spoilage study, the method was applied to one vegetarian and nine vegan grill sausage alternatives during a period of 32 days of open-package storage at refrigerator temperature. Correlations with the results of microbiological testing of the same samples, as well as with the storage time were investigated. However, the results of the correlation analysis showed that biogenic amines are not suitable as spoilage indicators for plant-based meat alternatives, as almost no increase in biogenic amines was identified during the spoilage study for all the samples investigated. Differences in the microbiota of conventional meat products versus plant-based meat alternatives, as well as possibly lower concentrations of free amino acids, are proposed as reasons for biogenic amines not being similarly prevalent, and consequently, not being suitable spoilage markers in plant-based meat alternatives. However, as spoilage of the analyzed products was evident, both via sensory assessment and appearance of mold growth, further targeted and non-targeted research on potential spoilage markers for plant-based meat alternatives is required in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Highly polar low molecular weight organic molecules are still very challenging to analyze by liquid chromatography. Yet, with the steadily increasing application of metabolomics and similar approaches in chemical analysis, separating polar compounds might be even more important. However, almost all established liquid chromatography techniques (i.e., normal and reversed phase, hydrophilic interaction liquid chromatography (HILIC), ion chromatography) struggle with either carry-over, low sensitivity, or a lack of retention. For improving these shortcomings, electrostatic repulsion hydrophilic interaction chromatography (ERLIC) might be an alternative. By combining a HILIC mobile phase, that is highly organic with a low water content, and an ion exchange column, a distinct layer system develops. When the analyte's charge is of the same direction as the stationary phase, retention and elution are determined by two antagonistic forces: electrostatic repulsion and hydrophilicity. One prominent group of challenging polar analytes are the polyamines cadaverine, putrescine, spermidine, and spermine. Carrying charges from +2 to +4 at physiological pH, these compounds are essential cell constituents and found in all living organisms. However, they are still notoriously challenging to analyze via the established liquid chromatography methods. In the present work, an ERLIC tandem mass spectrometry method has been exemplarily developed, optimized, and validated for the quantitative determination of cadaverine, putrescine, spermidine, and spermine. This method enables symmetrical peak shapes and good separation of analytes with different charges while simultaneously selectively detecting the co-eluting diamines by MS/MS. Furthermore, high linearity (R > 0.998) and sensitivity (LODs ≤ 2 ng/mL) have been proven. Thus, ERLIC may be interesting for both targeted and untargeted analysis approaches of highly charged low molecular weight organic molecules., Competing Interests: Declaration of competing interest GALAB Laboratories GmbH is an independent service laboratory for external quality control, especially with regard to food analysis. Services include standard methods as well as methods tailored for individual specifications and requests. Part of daily business is the development of new methods for new target compounds. Authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

High-performance thin-layer chromatography (HPTLC) is a suitable method for the analysis of peptides and proteins due to a wide selection of stationary and mobile phases and various detection options. Especially, two-dimensional HPTLC (2D-HPTLC) enables a higher resolution compared to one-dimensional HPTLC in the separation of complex peptide mixtures. Similar to 2D electrophoresis, characteristic peptide patterns can be obtained, allowing a differentiation of ingredients based on varying protein origins. The aim of this study was to evaluate 2D-HPTLC with regard to its suitability for the characterization of proteins/peptides and to verify whether it is possible to predict the retention behavior of peptides based on their properties. As models, the five most abundant milk proteins α-lactalbumin, β-lactoglobulin, α-, β-, and κ-Casein were used. In order to determine the repeatability of the peptide separation by 2D-HPTLC, each tryptic protein hydrolyzate was separated eight times. The standard deviations of the retardation factors for the separated peptides varied between 1.0 and 11.1 mm for the x-coordinate and 0.5-7.3 mm for the y-coordinate. It was also shown that after the chromatographic separation, peptides of the individual protein hydrolyzates were located in specific areas on the HPTLC plate, so that a clustering could be obtained for the whey proteins' as well as the caseins' hydrolyzates. For establishing correlations between the properties of the peptides and their retardation factors, 51 of 85 selected peptides were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). On this basis, statistically significant correlations (α = 0.05) between the retardation factors of the peptides and their isoelectric points, as well as the percentage of anionic and non-polar amino acids in the peptides were established. Finally, it was investigated, whether the retardation factors for peptides can be predicted on the basis of a linear regression of the percentage of non-polar amino acids in a peptide. For this purpose, a mixture of artifical (synthetic) peptides (n = 14) was separated by 2D-HPTLC and the measured retardation factors were compared with the corresponding retardation factors calculated. Absolute deviations of 0.3-17.9 mm were obtained. In addition, the universal applicability of the method to other protein sources other than milk proteins (animal protein) was tested using a mixture of pea peptides (plant protein, n = 3) resulting in absolute deviations of 0.7-8.6 mm., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

The use of macro- and microalgae, as well as cyanobacteria, becomes increasingly important for human nutrition, even in Western diets. Health effects, positive as well as negative, are believed to result mainly from minor components in the food. In macro- and microalgae as well as in certain cyanobacteria, one class of such minor compounds is sulfolipids, more precisely sulfoquinovosylmonoacylglycerol (SQMG) and sulfoquinovosyldiacylglycerol (SQDG) derivatives. SQMGs and SQDGs consist of a diacylglycerol esterified with varying fatty acid combinations and a sulfoquinovose moiety. Sulfoquinovose is a sulfonated hexose analogous to D-glucose, but featuring a stable carbon-sulfur bond. With regard to their chemical structure, SQDGs can be distinguished according to their sn1- and sn2-bound fatty acids. Although there is great interest in SQDGs, because of their controversially discussed bioactivities, only a negligible number of comprehensive methods for identification and quantification has been published, so far. Within this work, a sample preparation including a quantitative isolation of SQDGs from selected raw materials, a clean-up with solid-phase extraction (SPE), and a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous identification and quantitation of different, intact SQMGs and SQDGs were developed and validated. The applicability of the method was further demonstrated by comparing a prominent cyanobacterium (Arthrospira sp.) with a microalgae preparation (Chlorella vulgaris), and selected leafy vegetables (spinach, basil).

In this study, a clean-up approach using a two-dimensional liquid chromatography (2D-LC) consisting of a hydrophilic interaction liquid chromatography column and a reversed phase column was investigated. A fully automated 2D-LC system was used and compared with a traditional quick, easy, cheap, effective, rugged, and safe (QuEChERS) liquid chromatography (QuE-LC) method. The comparison was based on the results of a validation of selected analytes. It was investigated whether the detectability of analytes could be improved by the use of the 2D-LC. On the basis of these results, the relative detection rates were determined for every matrix. By means of those detection rates, the matrices were categorized regarding their complexity. Furthermore, the applicability of the 2D-LC was tested by participation in the European Proficiency Test in Fruits and Vegetables Screening Methods. In order to evaluate the separation and the elution profile of matrix components, multivariate data analysis was applied. For this purpose, ten matrices were processed in accordance to a QuEChERS protocol and the protocol for 2D-LC analysis. Moreover, the reagent blanks of the corresponding matrix were processed and analyzed by QuE-LC and 2D-LC. The results allowed evaluating the number of detected compounds for both methods. Additionally, the influence of compounds originating from reagent blanks can be estimated. In general, less compounds could be detected when 2D-LC was used. Especially, these were very polar compounds and compounds with m/z values >1500. These compounds seem to originate primarily from the used reagents especially from the citrate salts. However, the most of these compounds could be separated and were not detectable any more when 2D-LC was used. The results of the comparison based on validation and participation in the European Proficiency Test also show a better detectability for the most analytes with 2D-LC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A new sensitive, fast, and robust method using ion chromatography tandem mass spectrometry (IC-MS/MS) for the determination of fosetyl and phosphonic acid in plant-derived matrices was developed. For compensation of matrix effects and differences in recovery rates the isotopically labeled internal standard (ILIS) 18 O 3 -labeled phosphonic acid was added to the samples prior to the extraction of the target compounds. The validation of the method for the matrices tomato, apple, lemon, sultana, avocado, and wheat was performed according to the actual EU guidance document SANTE/11945/2015. The precision and accuracy were determined in five replicates at spiking levels of 0.010 and 0.100 mg/kg with recovery rates between 76 and 105% and RSDs between 1.2 and 17.8%. In this paper, it was achieved for the first time to detect both fosetyl and phosphonic acid at the reporting level of 0.010 mg/kg most relevant for organic plant food commodities.

Bile acids are important signaling molecules that regulate cholesterol, glucose, and energy homoeostasis and have thus been implicated in the development of metabolic disorders. Their bioavailability is strongly modulated by the gut microbiota, which contributes to generation of complex individual-specific bile acid profiles. Hence, it is important to have accurate methods at hand for precise measurement of these important metabolites. Here, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous identification and quantitation of primary and secondary bile acids as well as their taurine and glycine conjugates was developed and validated. Applicability of the method was demonstrated for mammalian tissues, biofluids, and cell culture media. The analytical approach mainly consists of a simple and rapid liquid-liquid extraction procedure in presence of deuterium-labeled internal standards. Baseline separation of all isobaric bile acid species was achieved and a linear correlation over a broad concentration range was observed. The method showed acceptable accuracy and precision on intra-day (1.42-11.07 %) and inter-day (2.11-12.71 %) analyses and achieved good recovery rates for representative analytes (83.7-107.1 %). As a proof of concept, the analytical method was applied to mouse tissues and biofluids, but especially to samples from in vitro fermentations with gut bacteria of the family Coriobacteriaceae. The developed method revealed that the species Eggerthella lenta and Collinsella aerofaciens possess bile salt hydrolase activity, and for the first time that the species Enterorhabdus mucosicola is able to deconjugate and dehydrogenate primary bile acids in vitro.

A novel method for the determination of phomopsin A (1) in lupin flour, pea flour, and bean flour as well as whole lupin plants was established based on stable isotope dilution assay (SIDA) LC-MS/MS using (15)N6-1 as an isotopically labeled internal standard. Artificially infected samples were used to develop an optimized extraction procedure and sample pretreatment. The limits of detection were 0.5-1 μg/kg for all matrices. The limits of quantitation were 2-4 μg/kg. The method was used to analyze flour samples generated from selected legume seeds and lupin plant samples that had been inoculated with Diaporthe toxica and two further fungal strains. Finally, growing lupin plants infected with D. toxica were investigated to simulate a naturally in-field mycotoxicosis. Toxin levels of up to 10.1 μg/kg of 1 were found in the pods and 7.2 μg/kg in the stems and leaves.

Recently, hydroxy sterols and bile acids have gained growing interest as they are important regulators of energy homoeostasis and inflammation. The high number of different hydroxy sterols and bile acid species requires powerful analytical tools to quantify these structurally and chemically similar analytes. Here, we introduce a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for rapid quantification of 34 sterols (hydroxy sterols, primary, secondary bile acids as well as their taurine and glycine conjugates). Chromatographic baseline separation of isomeric hydroxy sterols and bile acids is obtained using a rugged amide embedded C18 (polar embedded) stationary phase. The current method features a simple extraction protocol validated for blood plasma, urine, gall bladder, liver, feces, and adipose tissue avoiding solid phase extraction as well as derivatization procedures. The total extraction recovery for representative analytes ranged between 58-86% in plasma, 85% in urine, 79-92% in liver, 76-98% in adipose tissue, 93-104% in feces and 62-79% in gall bladder. The validation procedure demonstrated that the calibration curves were linear over the selected concentration ranges for 97% of the analytes, with calculated coefficients of determination (R2) of greater than 0.99. A feeding study in wild type mice with a standard chow and a cholesterol-enriched Western type diet illustrated that the protocol described here provides a powerful tool to simultaneously quantify cholesterol derivatives and bile acids in metabolically active tissues and to follow the enterohepatic circulation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cylindrospermopsin (CYN) is a cyanobacterial toxin associated with human and animal poisonings. Due to its toxicity in combination with its widespread occurrence, the development of reliable methods for selective, sensitive detection and accurate quantification is mandatory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using stable isotope dilution analysis (SIDA) represents an ideal tool for this purpose. U-[(15)N5]-CYN was synthesized by culturing Aphanizomenon flos-aquae in Na(15)NO3-containing cyanobacteria growth medium followed by a cleanup using graphitized carbon black columns and mass spectrometric characterization. Subsequently, a SIDA-LC-MS/MS method for the quantification of CYN in freshwater and Brassica matrices was developed showing satisfactory performance data. The recovery ranged between 98 and 103 %; the limit of quantification was 15 ng/L in freshwater and 50 μg/kg dry weight in Brassica samples. The novel SIDA was applied for CYN determination in real freshwater samples as well as in kale and in vegetable mustard exposed to toxin-containing irrigation water. Two of the freshwater samples taken from German lakes were found to be CYN-contaminated above limit of quantification (17.9 and 60.8 ng/L). CYN is systemically available to the examined vegetable species after exposure of the rootstock leading to CYN mass fractions in kale and vegetable mustard leaves of 15.0 μg/kg fresh weight and 23.9 μg/kg fresh weight, respectively. CYN measurements in both matrices are exemplary for the versatile applicability of the developed method in environmental analysis.