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Widespread use of pesticides globally has led to serious concerns about environmental contamination, particularly with regard to aquatic and soil ecosystems. This work involved investigating concentrations of 64 pesticides in surface-water and soil samples collected in four provinces along the Mekong River in Cambodia during the dry and rainy seasons (276 samples in total), and conducting semi-structured interviews with local farmers about pesticide use. Furthermore, an ecological risk assessment of the detected pesticides was performed. In total, 56 pesticides were detected in surface water and 43 in soil, with individual pesticides reaching maximum concentrations of 1300 ng/L in the surface-water samples (tebufenozide) and 1100 ng/g dry weight in the soil samples (bromophos-ethyl). The semi-structured interviews made it quite evident that the instructions that farmers are provided regarding the use of pesticides are rudimentary, and that overuse is common. The perceived effect of pesticides was seen as an end-point, and there was a limited process of optimally matching pesticides to pests and crops. Several pesticides were used regularly on the same crop, and the period between application and harvest varied. Risk analysis showed that bromophos-ethyl, dichlorvos, and iprobenfos presented a very high risk to aquatic organisms in both the dry and rainy seasons, with risk quotient values of 850 for both seasons, and of 67 in the dry season and 78 in the rainy season for bromophos-ethyl, and 49 in the dry season and 16 in the rainy season for dichlorvos. Overall, this work highlights the occurrence of pesticide residues in surface water and soil along the Mekong River in Cambodia, and emphasizes the urgent need for monitoring and improving pesticide practices and regulations in the region.

Widespread use of pesticides globally has led to serious concerns about environmental contamination, particularly with regard to aquatic and soil ecosystems. This work involved investigating concentrations of 64 pesticides in surface-water and soil samples collected in four provinces along the Mekong River in Cambodia during the dry and rainy seasons (276 samples in total), and conducting semi-structured interviews with local farmers about pesticide use. Furthermore, an ecological risk assessment of the detected pesticides was performed. In total, 56 pesticides were detected in surface water and 43 in soil, with individual pesticides reaching maximum concentrations of 1300 ng/L in the surface-water samples (tebufenozide) and 1100 ng/g dry weight in the soil samples (bromophos-ethyl). The semi-structured interviews made it quite evident that the instructions that farmers are provided regarding the use of pesticides are rudimentary, and that overuse is common. The perceived effect of pesticides was seen as an end-point, and there was a limited process of optimally matching pesticides to pests and crops. Several pesticides were used regularly on the same crop, and the period between application and harvest varied. Risk analysis showed that bromophos-ethyl, dichlorvos, and iprobenfos presented a very high risk to aquatic organisms in both the dry and rainy seasons, with risk quotient values of 850 for both seasons, and of 67 in the dry season and 78 in the rainy season for bromophos-ethyl, and 49 in the dry season and 16 in the rainy season for dichlorvos. Overall, this work highlights the occurrence of pesticide residues in surface water and soil along the Mekong River in Cambodia, and emphasizes the urgent need for monitoring and improving pesticide practices and regulations in the region.

Opnurasib (JDQ-443) is a highly potent and promising KRASG12C inhibitor that is currently under clinical investigation. Results of the ongoing clinical research demonstrated the acceptable safety profile and clinical activity of this drug candidate as a single agent for patients with NSCLC harboring KRASG12C mutations. In this early stage of development, a deeper insight into pharmacokinetic properties in both preclinical and clinical investigations of this drug is very important. Thus, a reliable quantification method is required. To date, no quantitative bioanalytical assay of opnurasib was publicly available. In this study we present a validated assay to quantify opnurasib in mouse plasma and eight mouse tissue-related matrices utilizing liquid chromatography-tandem mass spectrometry. Erlotinib was used as internal standard and acetonitrile was utilized to treat 10 µl of the sample with protein precipitation in a 96-well plate format. Separation and detection were achieved using a BEH C18 column under basic chromatographic conditions and a triple quadrupole mass spectrometer, respectively. We have fully validated this assay for mouse plasma and partially for eight mouse tissue-related matrices over the range of 2–2000 ng/ml. The accuracy and precision of the assay fulfilled international guidelines (EMA & U.S. FDA) over the validated range. The method was proven selective and sensitive to quantify opnurasib down to 2 ng/ml in all investigated matrices. The recoveries of both analyte and internal standard in mouse plasma were ∼100 % with no significant matrix effect in any of the matrices. Opnurasib in mouse plasma was stable up to 12 h at room temperature, and up to 8 h at room temperature in tissue homogenates (except for kidney up to 4 h). This presented method has been successfully applied to quantify opnurasib in preclinical samples from a mouse study and demonstrated its usability to support preclinical pharmacokinetic studies.

Toxic cyanobacterial blooms impose a health risk to recreational users, and monitoring of cyanobacteria and associated toxins is required to assess this risk. Traditionally, monitoring for risk assessment is based on cyanobacterial biomass, which assumes that all cyanobacteria potentially produce toxins. While these methods may be cost effective, relatively fast, and more widely accessible, they often lead to an overestimation of the health risk induced by cyanotoxins. Monitoring methods that more directly target toxins, or toxin producing genes, may provide a better risk assessment, yet these methods may be more costly, usually take longer, or are not widely accessible. In this study, we compared six monitoring methods (fluorometry, microscopy, qPCR of 16S and mcyE, ELISA assays, and LC-MS/MS), of which the last three focussed on the most abundant cyanotoxin microcystins, across 11 lakes in the Netherlands during the bathing water season (May-October) of 2019. Results of all monitoring methods significantly correlated with LC-MS/MS obtained microcystin levels (the assumed ‘golden standard’), with stronger correlations for methods targeting microcystins (ELISA) and microcystin genes (mcyE). The estimated risk levels differed substantially between methods, with 78 % and 56 % of alert level exceedances in the total number of collected samples for fluorometry and microscopy-based methods, respectively, while this was only 16 % and 6 % when the risk assessment was based on ELISA and LC-MS/MS obtained toxin concentrations, respectively. Integrating our results with earlier findings confirmed a strong association between microcystin concentration and the biovolume of potential microcystin-producing genera. Moreover, using an extended database consisting of 4265 observations from 461 locations across the Netherlands in the bathing water seasons of 2015 – 2019, we showed a strong association between fluorescence and the biovolume of potentially toxin-producing genera. Ou

Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with diffe, Part of this work was granted by the European Commission – NextGenerationEU, Project “Strengthening the MIRRI Italian Research Infrastructure for Sustainable Bioscience and Bioeconomy”, code n. IR0000005. TG group acknowledges support from the Spanish Ministry of Science and Innovation for grants PID2021-126067NB-I00, CPP2021-008552, PCI2022-135066-2, and PDC2022-133266-I00, cofounded by ERDF “A way of making Europe”; from the Catalan Research Agency (AGAUR) SGR01551; from the European Union's Horizon 2020 research and innovation program (ERC-2016-724173); from the Gordon and Betty Moore Foundation (Grant GBMF9742); from the “La Caixa” foundation (Grant LCF/PR/HR21/00737), and from the Instituto de Salud Carlos III (IMPACT Grant IMP/00019 and CIBERINFEC CB21/13/00061- ISCIII-SGEFI/ERDF)., Peer Reviewed, Postprint (published version)

Alkaloids play an essential role in protecting plants against herbivores. Humans can also benefit from the pharmacological effects of these compounds. Plants produce an immense variety of structurally different alkaloids, including quinolizidine alkaloids, a group of bi-, tri-, and tetracyclic compounds produced by Lupinus species. Various lupin species produce different alkaloid profiles. To study the composition of quinolizidine alkaloids in lupin seeds, we collected 31 populations of two wild species native to Israel, L. pilosus and L. palaestinus, and analyzed their quinolizidine alkaloid contents. Our goal was to study the alkaloid profiles of these two wild species to better understand the challenges and prospective uses of wild lupins. We compared their profiles with those of other commercial and wild lupin species. To this end, a straightforward method for extracting alkaloids from seeds and determining the quinolizidine alkaloid profile by LC–MS/MS was developed and validated in-house. For the quantification of quinolizidine alkaloids, 15 analytical reference standards were used. We used GC–MS to verify and cross-reference the identity of certain alkaloids for which no analytical standards were available. The results enabled further exploration of quinolizidine alkaloid biosynthesis. We reviewed and re-analyzed the suggested quinolizidine alkaloid biosynthesis pathway, including the relationship between the amino acid precursor l-lysine and the different quinolizidine alkaloids occurring in seeds of lupin species. Revealing alkaloid compositions and highlighting some aspects of their formation pathway are important steps in evaluating the use of wild lupins as a novel legume crop.

Toxic cyanobacterial blooms impose a health risk to recreational users, and monitoring of cyanobacteria and associated toxins is required to assess this risk. Traditionally, monitoring for risk assessment is based on cyanobacterial biomass, which assumes that all cyanobacteria potentially produce toxins. While these methods may be cost effective, relatively fast, and more widely accessible, they often lead to an overestimation of the health risk induced by cyanotoxins. Monitoring methods that more directly target toxins, or toxin producing genes, may provide a better risk assessment, yet these methods may be more costly, usually take longer, or are not widely accessible. In this study, we compared six monitoring methods (fluorometry, microscopy, qPCR of 16S and mcyE, ELISA assays, and LC-MS/MS), of which the last three focussed on the most abundant cyanotoxin microcystins, across 11 lakes in the Netherlands during the bathing water season (May-October) of 2019. Results of all monitoring methods significantly correlated with LC-MS/MS obtained microcystin levels (the assumed ‘golden standard’), with stronger correlations for methods targeting microcystins (ELISA) and microcystin genes (mcyE). The estimated risk levels differed substantially between methods, with 78 % and 56 % of alert level exceedances in the total number of collected samples for fluorometry and microscopy-based methods, respectively, while this was only 16 % and 6 % when the risk assessment was based on ELISA and LC-MS/MS obtained toxin concentrations, respectively. Integrating our results with earlier findings confirmed a strong association between microcystin concentration and the biovolume of potential microcystin-producing genera. Moreover, using an extended database consisting of 4265 observations from 461 locations across the Netherlands in the bathing water seasons of 2015 – 2019, we showed a strong association between fluorescence and the biovolume of potentially toxin-producing genera. Our r

Introduction: The chemical classification of Cannabis is typically confined to the cannabinoid content, whilst Cannabis encompasses diverse chemical classes that vary in abundance among all its varieties. Hence, neglecting other chemical classes within Cannabis strains results in a restricted and biased comprehension of elements that may contribute to chemical intricacy and the resultant medicinal qualities of the plant. Objectives: Thus, herein, we report a computational metabolomics study to elucidate the Cannabis metabolic map beyond the cannabinoids. Methods: Mass spectrometry-based computational tools were used to mine and evaluate the methanolic leaf and flower extracts of two Cannabis cultivars: Amnesia haze (AMNH) and Royal dutch cheese (RDC). Results: The results revealed the presence of different chemical compound classes including cannabinoids, but extending it to flavonoids and phospholipids at varying distributions across the cultivar plant tissues, where the phenylpropnoid superclass was more abundant in the leaves than in the flowers. Therefore, the two cultivars were differentiated based on the overall chemical content of their plant tissues where AMNH was observed to be more dominant in the flavonoid content while RDC was more dominant in the lipid-like molecules. Additionally, in silico molecular docking studies in combination with biological assay studies indicated the potentially differing anti-cancer properties of the two cultivars resulting from the elucidated chemical profiles. Conclusion: These findings highlight distinctive chemical profiles beyond cannabinoids in Cannabis strains. This novel mapping of the metabolomic landscape of Cannabis provides actionable insights into plant biochemistry and justifies selecting certain varieties for medicinal use.

Chemically induced, targeted protein degradation with proteolysis targeting chimeras (PROTACs) has shown to be a promising pharmacological strategy to circumvent the poor "druggability" of intracellular targets. However, the favorable pharmacology comes with complex molecular properties limiting the oral bioavailability of these drugs. To foster the translation of PROTACs into the clinics it is of high importance to establish sensitive bioanalytical methods that enable the assessment of absorption, bioavailability, and disposition of PROTACs after oral dosing. In this study, two highly sensitive LC-MS/MS methods (LLOQ = 0.5 ng/mL) were developed and validated for the quantification of bavdeglutamide (ARV-110) and vepdegestrant (ARV-471) in rat plasma. Plasma samples were processed by protein precipitation and separated on a C18 column over a gradient of acetonitrile and water with 0.1 % formic acid. Selected reaction monitoring in positive ESI mode was applied to quantify ARV-110 and ARV-471. Both methods showed linearity, accuracy, and precision as well as matrix effects and carry-over within the predefined acceptance criteria. High stability of the compounds in plasma was demonstrated at long-term storage for seven weeks at-20°C, three freeze-thaw cycles, up to 20 min at room temperature, and as extracts in the autosampler. The plasma concentration-time curves after intravenous and intraduodenal bolus single-dose administrations in rats could be successfully quantified at clinically relevant doses per body weight. The highly sensitive bioanalytical assays presented in this work enable the application of a broad spectrum of in vivo studies to elucidate the oral absorption, bioavailability, and disposition of PROTACs.

Syftet med detta examensarbete var att validera en metod som kvantitativt bestämmer PFAS ijord, slam och sediment. Valideringen omfattade en del olika tester för att utvärdera metodensrapporteringsgräns, precision, riktighet, mätosäkerhet och specificitet. Excel har använts för attberäkna och sammanställa resultaten från de olika testerna. Blankprover och spikade prover haranalyserats för att undersöka rapporteringsgräns. Kontrollprover har analyserats för attundersöka precision samt specificitet. För att undersöka riktighet har en rad olika tester utförts;ISTD-matchning för några av PFAS-analyter som saknade en direkt ISTD-match, jämförelsermot certifierat referensmaterial, analys av tidigare utförda tester som redan hade analyserats iALS Prag och spikade provmatriser. Mätosäkerheten uppskattade och utvärderades i samrådmed kvalitetsansvarig. Resultatet från metodvalideringen bekräftar att metoden har visat sig vara väl validerad.Samtliga tester som utfördes under valideringsprocessen har konsekvent uppfyllt och överträffatde ställda acceptanskriterium för valideringen. Kriterierna inkluderade bland annat relativstandardavvikelse (RSD), utbyte, bias och halt för olika testparametrar. Den omfattandemetodvalideringen gav starka bevis att metoden är tillförlitlig och kan användas för att genererapålitliga resultat inom PFAS-analys., The purpose of this thesis was to validate a method for quantitatively determining PFAS in soil,sludge, and sediment. The validation involved a number of different tests to evaluate themethod's reporting limit, precision, accuracy, measurement uncertainty, and specificity. Excelwas used to calculate and summarize the results from the different tests. Blank and spikedsamples were analyzed to investigate the reporting limit. Control samples were analyzed toinvestigate precision and specificity. A variety of tests were performed to investigate accuracy;ISTD matching for some PFAS analytes that lacked a direct ISTD match, comparisons againstcertified reference material, analysis of previously performed tests that had already beenanalyzed at ALS Prague, and spiked sample matrices. The measurement uncertainty wasestimated and evaluated in consultation with the quality manager. The results of the method validation confirm that the method has been shown to be wellvalidated. All tests performed during the validation process have consistently met and exceededthe established acceptance criteria for the validation. Criteria included, among others, relativestandard deviation (RSD), recovery, bias, and concentration for various test parameters. Thecomprehensive method validation provided strong evidence that the method is reliable and canbe used to generate reliable results in PFAS analysis.

Chemically induced, targeted protein degradation with proteolysis targeting chimeras (PROTACs) has shown to be a promising pharmacological strategy to circumvent the poor "druggability" of intracellular targets. However, the favorable pharmacology comes with complex molecular properties limiting the oral bioavailability of these drugs. To foster the translation of PROTACs into the clinics it is of high importance to establish sensitive bioanalytical methods that enable the assessment of absorption, bioavailability, and disposition of PROTACs after oral dosing. In this study, two highly sensitive LC-MS/MS methods (LLOQ = 0.5 ng/mL) were developed and validated for the quantification of bavdeglutamide (ARV-110) and vepdegestrant (ARV-471) in rat plasma. Plasma samples were processed by protein precipitation and separated on a C18 column over a gradient of acetonitrile and water with 0.1 % formic acid. Selected reaction monitoring in positive ESI mode was applied to quantify ARV-110 and ARV-471. Both methods showed linearity, accuracy, and precision as well as matrix effects and carry-over within the predefined acceptance criteria. High stability of the compounds in plasma was demonstrated at long-term storage for seven weeks at-20°C, three freeze-thaw cycles, up to 20 min at room temperature, and as extracts in the autosampler. The plasma concentration-time curves after intravenous and intraduodenal bolus single-dose administrations in rats could be successfully quantified at clinically relevant doses per body weight. The highly sensitive bioanalytical assays presented in this work enable the application of a broad spectrum of in vivo studies to elucidate the oral absorption, bioavailability, and disposition of PROTACs.

Chemically induced, targeted protein degradation with proteolysis targeting chimeras (PROTACs) has shown to be a promising pharmacological strategy to circumvent the poor "druggability" of intracellular targets. However, the favorable pharmacology comes with complex molecular properties limiting the oral bioavailability of these drugs. To foster the translation of PROTACs into the clinics it is of high importance to establish sensitive bioanalytical methods that enable the assessment of absorption, bioavailability, and disposition of PROTACs after oral dosing. In this study, two highly sensitive LC-MS/MS methods (LLOQ = 0.5 ng/mL) were developed and validated for the quantification of bavdeglutamide (ARV-110) and vepdegestrant (ARV-471) in rat plasma. Plasma samples were processed by protein precipitation and separated on a C18 column over a gradient of acetonitrile and water with 0.1 % formic acid. Selected reaction monitoring in positive ESI mode was applied to quantify ARV-110 and ARV-471. Both methods showed linearity, accuracy, and precision as well as matrix effects and carry-over within the predefined acceptance criteria. High stability of the compounds in plasma was demonstrated at long-term storage for seven weeks at-20°C, three freeze-thaw cycles, up to 20 min at room temperature, and as extracts in the autosampler. The plasma concentration-time curves after intravenous and intraduodenal bolus single-dose administrations in rats could be successfully quantified at clinically relevant doses per body weight. The highly sensitive bioanalytical assays presented in this work enable the application of a broad spectrum of in vivo studies to elucidate the oral absorption, bioavailability, and disposition of PROTACs.

Chemically induced, targeted protein degradation with proteolysis targeting chimeras (PROTACs) has shown to be a promising pharmacological strategy to circumvent the poor "druggability" of intracellular targets. However, the favorable pharmacology comes with complex molecular properties limiting the oral bioavailability of these drugs. To foster the translation of PROTACs into the clinics it is of high importance to establish sensitive bioanalytical methods that enable the assessment of absorption, bioavailability, and disposition of PROTACs after oral dosing. In this study, two highly sensitive LC-MS/MS methods (LLOQ = 0.5 ng/mL) were developed and validated for the quantification of bavdeglutamide (ARV-110) and vepdegestrant (ARV-471) in rat plasma. Plasma samples were processed by protein precipitation and separated on a C18 column over a gradient of acetonitrile and water with 0.1 % formic acid. Selected reaction monitoring in positive ESI mode was applied to quantify ARV-110 and ARV-471. Both methods showed linearity, accuracy, and precision as well as matrix effects and carry-over within the predefined acceptance criteria. High stability of the compounds in plasma was demonstrated at long-term storage for seven weeks at-20°C, three freeze-thaw cycles, up to 20 min at room temperature, and as extracts in the autosampler. The plasma concentration-time curves after intravenous and intraduodenal bolus single-dose administrations in rats could be successfully quantified at clinically relevant doses per body weight. The highly sensitive bioanalytical assays presented in this work enable the application of a broad spectrum of in vivo studies to elucidate the oral absorption, bioavailability, and disposition of PROTACs.

Lupin is a dominant legume crop in Western Australia, with strong domestic and export markets. Due to its high protein and fibre content, coupled with low levels of carbohydrates and fat, lupin has garnered significant interest over other legumes. It is cultivated for a variety of food and animal feed applications. However, lupin products may pose health risks to consumers due to the presence of toxic quinolizidine alkaloids (QAs). Quinolizidine alkaloids serve as natural plant defence mechanisms, and they have wide industrial applications. However, they can be toxic to humans and animals if consumed above the industrial limits (200 mg/kg). The levels of alkaloids in sweet lupin species vary and can occasionally surpass safe consumption thresholds. An accurate and validated methodology for quantifying QAs in lupin seeds is important for monitoring their concentrations across different sweet lupin varieties. Furthermore, the factors contributing to variations in QA levels within lupin seeds demand additional exploration. Currently, most proteins implicated in the biosynthesis and transportation of QAs remain unidentified. This thesis comprises two parts: the first involves establishing a new method for QAs determination in lupin grain and tracking their concentrations across various lupin species and under different daylight durations. The second part explores proteins associated with alkaloid biosynthesis and transportation in lupin through proteomics analysis of lupin leaves, correlating with alkaloid levels. Chapter 1 of the thesis presents an extensive literature review on the occurrence of QAs in lupin seeds, highlighting the primary gaps in current knowledge. Alkaloids serve a crucial role in defending lupins against insect attacks and enabling plants to adapt to various environmental changes. However, when QAs are present in high concentrations in lupin seeds, they pose toxicity risks to both humans and animals. Since alkaloids are synthesized in lupin leaves

Syftet med detta examensarbete var att validera en metod som kvantitativt bestämmer PFAS ijord, slam och sediment. Valideringen omfattade en del olika tester för att utvärdera metodensrapporteringsgräns, precision, riktighet, mätosäkerhet och specificitet. Excel har använts för attberäkna och sammanställa resultaten från de olika testerna. Blankprover och spikade prover haranalyserats för att undersöka rapporteringsgräns. Kontrollprover har analyserats för attundersöka precision samt specificitet. För att undersöka riktighet har en rad olika tester utförts;ISTD-matchning för några av PFAS-analyter som saknade en direkt ISTD-match, jämförelsermot certifierat referensmaterial, analys av tidigare utförda tester som redan hade analyserats iALS Prag och spikade provmatriser. Mätosäkerheten uppskattade och utvärderades i samrådmed kvalitetsansvarig. Resultatet från metodvalideringen bekräftar att metoden har visat sig vara väl validerad.Samtliga tester som utfördes under valideringsprocessen har konsekvent uppfyllt och överträffatde ställda acceptanskriterium för valideringen. Kriterierna inkluderade bland annat relativstandardavvikelse (RSD), utbyte, bias och halt för olika testparametrar. Den omfattandemetodvalideringen gav starka bevis att metoden är tillförlitlig och kan användas för att genererapålitliga resultat inom PFAS-analys., The purpose of this thesis was to validate a method for quantitatively determining PFAS in soil,sludge, and sediment. The validation involved a number of different tests to evaluate themethod's reporting limit, precision, accuracy, measurement uncertainty, and specificity. Excelwas used to calculate and summarize the results from the different tests. Blank and spikedsamples were analyzed to investigate the reporting limit. Control samples were analyzed toinvestigate precision and specificity. A variety of tests were performed to investigate accuracy;ISTD matching for some PFAS analytes that lacked a direct ISTD match, comparisons againstcertified reference material, analysis of previously performed tests that had already beenanalyzed at ALS Prague, and spiked sample matrices. The measurement uncertainty wasestimated and evaluated in consultation with the quality manager. The results of the method validation confirm that the method has been shown to be wellvalidated. All tests performed during the validation process have consistently met and exceededthe established acceptance criteria for the validation. Criteria included, among others, relativestandard deviation (RSD), recovery, bias, and concentration for various test parameters. Thecomprehensive method validation provided strong evidence that the method is reliable and canbe used to generate reliable results in PFAS analysis.

Chemically induced, targeted protein degradation with proteolysis targeting chimeras (PROTACs) has shown to be a promising pharmacological strategy to circumvent the poor "druggability" of intracellular targets. However, the favorable pharmacology comes with complex molecular properties limiting the oral bioavailability of these drugs. To foster the translation of PROTACs into the clinics it is of high importance to establish sensitive bioanalytical methods that enable the assessment of absorption, bioavailability, and disposition of PROTACs after oral dosing. In this study, two highly sensitive LC-MS/MS methods (LLOQ = 0.5 ng/mL) were developed and validated for the quantification of bavdeglutamide (ARV-110) and vepdegestrant (ARV-471) in rat plasma. Plasma samples were processed by protein precipitation and separated on a C18 column over a gradient of acetonitrile and water with 0.1 % formic acid. Selected reaction monitoring in positive ESI mode was applied to quantify ARV-110 and ARV-471. Both methods showed linearity, accuracy, and precision as well as matrix effects and carry-over within the predefined acceptance criteria. High stability of the compounds in plasma was demonstrated at long-term storage for seven weeks at-20°C, three freeze-thaw cycles, up to 20 min at room temperature, and as extracts in the autosampler. The plasma concentration-time curves after intravenous and intraduodenal bolus single-dose administrations in rats could be successfully quantified at clinically relevant doses per body weight. The highly sensitive bioanalytical assays presented in this work enable the application of a broad spectrum of in vivo studies to elucidate the oral absorption, bioavailability, and disposition of PROTACs.

Widespread use of pesticides globally has led to serious concerns about environmental contamination, particularly with regard to aquatic and soil ecosystems. This work involved investigating concentrations of 64 pesticides in surface-water and soil samples collected in four provinces along the Mekong River in Cambodia during the dry and rainy seasons (276 samples in total), and conducting semi-structured interviews with local farmers about pesticide use. Furthermore, an ecological risk assessment of the detected pesticides was performed. In total, 56 pesticides were detected in surface water and 43 in soil, with individual pesticides reaching maximum concentrations of 1300 ng/L in the surface-water samples (tebufenozide) and 1100 ng/g dry weight in the soil samples (bromophos-ethyl). The semi-structured interviews made it quite evident that the instructions that farmers are provided regarding the use of pesticides are rudimentary, and that overuse is common. The perceived effect of pesticides was seen as an end-point, and there was a limited process of optimally matching pesticides to pests and crops. Several pesticides were used regularly on the same crop, and the period between application and harvest varied. Risk analysis showed that bromophos-ethyl, dichlorvos, and iprobenfos presented a very high risk to aquatic organisms in both the dry and rainy seasons, with risk quotient values of 850 for both seasons, and of 67 in the dry season and 78 in the rainy season for bromophos-ethyl, and 49 in the dry season and 16 in the rainy season for dichlorvos. Overall, this work highlights the occurrence of pesticide residues in surface water and soil along the Mekong River in Cambodia, and emphasizes the urgent need for monitoring and improving pesticide practices and regulations in the region.

Widespread use of pesticides globally has led to serious concerns about environmental contamination, particularly with regard to aquatic and soil ecosystems. This work involved investigating concentrations of 64 pesticides in surface-water and soil samples collected in four provinces along the Mekong River in Cambodia during the dry and rainy seasons (276 samples in total), and conducting semi-structured interviews with local farmers about pesticide use. Furthermore, an ecological risk assessment of the detected pesticides was performed. In total, 56 pesticides were detected in surface water and 43 in soil, with individual pesticides reaching maximum concentrations of 1300 ng/L in the surface-water samples (tebufenozide) and 1100 ng/g dry weight in the soil samples (bromophos-ethyl). The semi-structured interviews made it quite evident that the instructions that farmers are provided regarding the use of pesticides are rudimentary, and that overuse is common. The perceived effect of pesticides was seen as an end-point, and there was a limited process of optimally matching pesticides to pests and crops. Several pesticides were used regularly on the same crop, and the period between application and harvest varied. Risk analysis showed that bromophos-ethyl, dichlorvos, and iprobenfos presented a very high risk to aquatic organisms in both the dry and rainy seasons, with risk quotient values of 850 for both seasons, and of 67 in the dry season and 78 in the rainy season for bromophos-ethyl, and 49 in the dry season and 16 in the rainy season for dichlorvos. Overall, this work highlights the occurrence of pesticide residues in surface water and soil along the Mekong River in Cambodia, and emphasizes the urgent need for monitoring and improving pesticide practices and regulations in the region.

This article summarizes the characterization, by shotgun proteomics, of 11 bacterial strains identified as responsible for seafood spoilage. A total of 4455 peptide spectrum matches, corresponding to 4299 peptides and 3817 proteins were identified. Analyses of data determined the functional pathways they are involved in. The proteins identified were integrated into a protein-protein network that involves 371 nodes and 3016 edges. Those proteins are implicated in energy pathways, peptidoglycan biosynthesis, spermidine/putrescine metabolism. An additional 773 peptides were characterized as virulence factors, that participates in bacterial pathogenesis; while 14 peptides were defined as biomarkers, as they can be used to differentiate the bacterial species present. This report represents the most extensive proteomic repository available in the field of seafood spoilage bacteria; the data substantially advances the understanding of seafood decay, as well as provides fundamental bases for the recognition of the bacteria existent in seafood that cause spoilage during food processing/storage

Background: The increasing demand for food and feed products is stretching the capacity of the food value chain to its limits. A key step for ensuring food safety is checking for mycotoxin contamination of wheat. However, this analysis is typically performed by rather complex and expensive chromatographic methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). These costly methods require extensive sample preparation that is not easily carried out at different points along the food supply chain. To overcome such challenges in sample processing, an inexpensive and portable sample preparation device was needed, that required low skill, for rapid sample-to-result mycotoxin screening. Results: We describe 3D-printed and interconnectable modules for simple, integrated and on-site sample preparation, including grinding of wheat kernels, and solvent-based extraction. We characterized these 3D-printed modules for mycotoxin screening and benchmarked them against a laboratory mill using commercial lateral flow device(s) (LFD) and in-house validated LC-MS/MS analysis. Different integrated sieve configurations were compared based on grinding efficiency, and we selected a sieve size of 2 mm allowing grinding of 10 g of wheat within 5 min. Moreover, 10 first time-users were able to operate the grinder module with minimal instructions. Screening for deoxynivalenol (DON) in naturally contaminated samples at the regulatory/legal limit (1.25 mg kg−1) was demonstrated using the developed 3D-printed prototype. The whole process only takes 15 min, from sample preparation to screening result. The results showed a clear correlation (R2 = 0.96) between the LFD and LC-MS/MS. Significance: Our findings demonstrate the potential of 3D-printed sample handling equipment as a valuable extension of existing analytical procedures, facilitating the on-site implementation of rapid methods for the determination of mycotoxins in grains. The presented prototype is inexpensive with m

In their natural habitat, insects may bioaccumulate toxins from plants for defence against predators. When insects are accidently raised on feed that is contaminated with toxins from co-harvested herbs, this may pose a health risk when used for human or animal consumption. Plant toxins of particular relevance are the pyrrolizidine alkaloids (PAs), which are genotoxic carcinogens produced by a wide variety of plant species and the tropane alkaloids (TAs) which are produced by a number of Solanaceae species. This study aimed to investigate the transfer of these plant toxins from substrates to black soldier fly larvae (BSFL) and lesser mealworm (LMW). PAs and the TAs atropine and scopolamine were added to insect substrate simulating the presence of different PA- or TA-containing herbs, and BSFL and LMW were grown on these substrates. Bioaccumulation from substrate to insects varied widely among the different plant toxins. Highest bioaccumulation was observed for the PAs europine, rinderine and echinatine. For most PAs and for atropine and scopolamine, bioaccumulation was very low. In the substrate, PA N-oxides were quickly converted to the corresponding tertiary amines. More research is needed to verify the findings of this study at larger scale, and to determine the potential role of the insect and/or substrate microbiome in metabolizing these toxins.

Barley is commonly used in malting and brewing, and spent grain is repurposed for other foods. Barley contains gluten proteins called hordeins that cause intestinal damage and disease symptoms if eaten by people with celiac disease and related conditions. While the mashing process in brewing can partially hydrolyze immunogenic epitopes in hordeins, the immunogenic epitope load between the starting malt and spent grain has not been investigated. Herein, we quantified hordeins in commercially available spent grain and from matching malt. Liquid chromatography-mass spectrometry (LC-MS) and sandwich and competitive R5 ELISAs were used for quantification, revealing a higher abundance of gluten proteins in the spent grain product compared with the input malt. Certain hordein subtypes were enriched while others were depleted, and overall protein content was higher in spent grain. This suggests that the mashing process selectively extracts nonprotein components, leaving protein and hordein content elevated in spent grain. The spent grain products tested were not safe for consumers with celiac disease.

Due to methodological advances in the archaeological sciences, an increasing number of archaeological specimens undergo destructive sampling. However, the preservation of cultural heritage is a primary concern. This leads to a dilemma between accessing sample material and obtaining sufficient information for a meaningful analytical outcome. Ideally, sampling a specimen would preserve the object for further macro, micro, and molecular analyses. For palaeoproteomics, a number of minimally invasive sampling approaches have been proposed, representing different benefits and limitations. There have been studies comparing a selection of these protocols, however, these have focused on specimens from a homogenous preservation environment using Zooarchaeology by Mass Spectrometry (ZooMS). Here we expand on earlier work by extending the comparison to specimens from two highly different preservation environments through both ZooMS and liquid-chromatography tandem mass spectrometry (LC-MS/MS). We compare five sampling approaches and seven extraction protocols in total, on 10 Bos sp. humeri from the Early Neolithic site of La Draga, Spain, utilising MALDI-ToF MS and LC-MS/MS to generate proteomic output, while assessing protocol invasiveness using microscopy and 3D imaging. Five humeri originate from Sector A, which is mostly related to dry, terrestrial preservation conditions, while the other five humeri stem from Sector B, which is characterised by its phreatic/aquatic preservation conditions. We show that there is a significant difference in protein recovery and taxonomic specificity between the sampling techniques applied, as well as between burial conditions. Additionally, various surface modifications were observed depending on the specific sampling technique applied. It is therefore essential to assess protein preservation for each sedimentological context within an archaeological site before performing extensive sampling, as protein preservation can be highly inter- a

Electromembrane extraction (EME) is a miniaturized technique in which charged molecules are extracted from biological samples, through a porous membrane, into an acceptor solution. This process occurs in the presence of an electric field that enables increased analyte recovery in short extraction times. Studies are being carried out to establish recommended extraction conditions for different groups of compounds based on their physicochemical properties. In this work, pre-established generic liquid membranes for the extraction of basic compounds were tested using synthetic cathinones within a wide log P range as basic model analytes. First, recommended organic solvents for the chosen analytes were evaluated in terms of recovery. The best candidates were included in an optimization study with other relevant EME conditions (voltage, extraction time, and agitation rate). All analyses were performed using a UPLC-MS/MS with the multiple reaction monitoring mode. 2-Undecanone was chosen as the liquid membrane providing the best extraction efficiency in combination with 40 V, 35 min, and 725 rpm. Validation showed r2 > 0.99 in the 1–500 ng/mL range, accuracy of ±12%, imprecision of ≤19%, 1 ng/mL as limit of quantitation, 0.1–0.5 ng/mL as limit of detection, matrix effect 97–118%, and recovery 63–110%. Among the generic liquid membranes studied in this work, 2-Undecanone was the best choice for the target basic analytes. High recoveries and low matrix effects were achieved, thus demonstrating the applicability of recommended generic liquid membranes in the 96-well plate setup and with whole blood samples. Our findings contribute to further establishing EME as an actual sample preparation technique for routine analyses that could be further explored in a commercial format.

Precision diabetes medicine (PDM) aims to reduce errors in prevention programmes, diagnosis thresholds, prognosis prediction and treatment strategies. However, its advancement and implementation are difficult due to the heterogeneity of complex molecular processes and environmental exposures that influence an individual’s disease trajectory. To address this challenge, it is imperative to develop robust screening methods for all areas of PDM. Innovative proteomic technologies, alongside genomics, have proven effective in precision cancer medicine and are showing promise in diabetes research for potential translation. This narrative review highlights how proteomics is well-positioned to help improve PDM. Specifically, a critical assessment of widely adopted affinity-based proteomic technologies in large-scale clinical studies and evidence of the benefits and feasibility of using MS-based plasma proteomics is presented. We also present a case for the use of proteomics to identify predictive protein panels for type 2 diabetes subtyping and the development of clinical prediction models for prevention, diagnosis, prognosis and treatment strategies. Lastly, we discuss the importance of plasma and tissue proteomics and its integration with genomics (proteogenomics) for identifying unique type 2 diabetes intra- and inter-subtype aetiology. We conclude with a call for action formed on advancing proteomics technologies, benchmarking their performance and standardisation across sites, with an emphasis on data sharing and the inclusion of diverse ancestries in large cohort studies. These efforts should foster collaboration with key stakeholders and align with ongoing academic programmes such as the Precision Medicine in Diabetes Initiative consortium. Graphical Abstract: (Figure presented.).

Electromembrane extraction (EME) is a miniaturized technique in which charged molecules are extracted from biological samples, through a porous membrane, into an acceptor solution. This process occurs in the presence of an electric field that enables increased analyte recovery in short extraction times. Studies are being carried out to establish recommended extraction conditions for different groups of compounds based on their physicochemical properties. In this work, pre-established generic liquid membranes for the extraction of basic compounds were tested using synthetic cathinones within a wide log P range as basic model analytes. First, recommended organic solvents for the chosen analytes were evaluated in terms of recovery. The best candidates were included in an optimization study with other relevant EME conditions (voltage, extraction time, and agitation rate). All analyses were performed using a UPLC-MS/MS with the multiple reaction monitoring mode. 2-Undecanone was chosen as the liquid membrane providing the best extraction efficiency in combination with 40 V, 35 min, and 725 rpm. Validation showed r2 > 0.99 in the 1–500 ng/mL range, accuracy of ±12%, imprecision of ≤19%, 1 ng/mL as limit of quantitation, 0.1–0.5 ng/mL as limit of detection, matrix effect 97–118%, and recovery 63–110%. Among the generic liquid membranes studied in this work, 2-Undecanone was the best choice for the target basic analytes. High recoveries and low matrix effects were achieved, thus demonstrating the applicability of recommended generic liquid membranes in the 96-well plate setup and with whole blood samples. Our findings contribute to further establishing EME as an actual sample preparation technique for routine analyses that could be further explored in a commercial format.

A solid-liquid extraction method using ethanol-water mixtures was combined with cLC-DAD, LC-MS/MS and chemometric analyses for establishing the optimum extraction conditions of valuable polyphenols from spent coffee grounds. Chlorogenic and p-coumaric acids were the most abundant polyphenols found, ranging from 0.02 to 4.8 mg g−1 and 0.173–0.50 mg g−1, respectively. In addition, total polyphenol content (9–29 mg GAE g−1 DW), total flavonoid content (11–27 mg QE g−1 DW), total antioxidant activity (0.3–7 mg GAE g−1 DW) and free radical scavenging ability (DPPH assay, 64–927 µg extract g−1 at EC50) of obtained extracts were determined. Response surface methodology allowed obtaining predictive models for the extraction of each individual polyphenol. On the other hand, multifactorial ANOVA was used to establish differences between coffee and spent coffee ground extracts. Principal component analysis was also employed to relate antioxidant activities, total polyphenol and total flavonoid contents with both the polyphenols extracted and the residue coffee type. The overall results suggested that spent coffee grounds could be reused as a promising, inexpensive and natural source of bioactive polyphenols with potential industrial applications, thus minimizing the waste disposal and environmental impact., Comunidad de Madrid Programa FEDER, Ministerio de Economía y Competitividad, Universidad Complutense de Madrid, Depto. de Química Analítica, Fac. de Ciencias Químicas, TRUE, pub

A simple and efficient low-cost matrix solid-phase dispersion (MSPD) extraction based on TiO2nanopar-ticles (NPs) and diatomaceous earth has been developed for the recovery of phenolic compounds fromresidual brewing yeast. Experimental conditions for MSPD extraction were optimized by an experimen-tal design approach. A screening factorial design plus replicates at the center point, followed by surfaceresponse analysis were used. The simultaneous identification and quantification of eleven main nat-ural polyphenols: caffeic, chlorogenic, p-coumaric, 3,4-dihydroxibenzoic, trans-ferulic and gallic acids,kaempferol, myricetin, naringin, quercetin and rutin, was possible by combining MSPD and capillaryliquid chromatography couple to a diode array detection system (cLC-DAD) and liquid chromatogra-phy couple to a triple quadrupole analyzer (LC–MS/MS). Moreover, residual brewing yeast extracts wereevaluated in terms of DPPH (1,1-diphenyl–2 picrylhydrazyl) free radical scavenging activity. Polyphenol-nanoparticle interaction was studied by UV–vis spectroscopy and electron transmission microscopy(TEM), pointing out a stable interplay that assists phenolic isolation. The extracted polyphenol quan-tities were within the 3.2-1,500 g g−1range, and the high antioxidant activity estimated suggested thatdeveloped MSPD is a successful, simple, efficient and rapid method for the extraction and recovery of bioactive phenolic compounds, which promotes the reuse and re-evaluation of brewing yeast agri-foodby-products., Community of Madrid/FEDERprogram, Ministry ofEconomy and Competitiveness, Complutense University, Depto. de Química Analítica, Fac. de Ciencias Químicas, TRUE, pub

Objectives: Salivary cortisol and cortisone at late night and after dexamethasone suppression test (DST) are increasingly used for screening of Cushing's syndrome (CS). We aimed to establish reference intervals for salivary cortisol and cortisone with three liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques and for salivary cortisol with three immunoassays (IAs), and evaluate their diagnostic accuracy for CS.Methods: Salivary samples at 08:00 h, 23:00 h and 08:00 h after a 1-mg DST were collected from a reference population (n=155) and patients with CS (n=22). Sample aliquots were analyzed by three LC-MS/MS and three IA methods. After establishing reference intervals, the upper reference limit (URL) for each method was used to calculate sensitivity and specificity for CS. Diagnostic accuracy was evaluated by comparing ROC curves.Results: URLs for salivary cortisol at 23:00 h were similar for the LC-MS/MS methods (3.4-3.9 nmol/L), but varied between IAs: Roche (5.8 nmol/L), Salimetrics (4.3 nmol/L), Cisbio (21.6 nmol/L). Corresponding URLs after DST were 0.7-1.0, and 2.4, 4.0 and 5.4 nmol/L, respectively. Salivary cortisone URLs were 13.5-16.6 nmol/L at 23:00 h and 3.0-3.5 nmol/L at 08:00 h after DST. All methods had ROC AUCs =0.96.Conclusions: We present robust reference intervals for salivary cortisol and cortisone at 08:00 h, 23:00 h and 08:00 h after DST for several clinically used methods. The similarities between LC-MS/MS methods allows for direct comparison of absolute values. Diagnostic accuracy for CS was high for all salivary cortisol and cortisone LC-MS/MS methods and salivary cortisol IAs evaluated.

We developed and validated an assay utilizing a liquid chromatography-tandem mass spectrometry technique to quantify the KRAS inhibitor adagrasib in mouse plasma and seven tissue-related matrices. The straightforward protein precipitation technique was selected to extract adagrasib and the internal standard salinomycin from the matrices. Gradient elution of acetonitrile and water modified with 0.5% (v/v) ammonium hydroxide and 0.02% (v/v) acetic acid on a C 18 column at a flow rate of 0.6 ml/min was applied to separate the analytes. Both adagrasib and salinomycin were detected with a triple quadrupole mass spectrometer with positive electrospray ionization in a selected reaction monitoring mode. A linear calibration range of 2-2,000 ng/ml of adagrasib was demonstrated during the validation. In addition, the reported precision values (intra- and inter-day) were between 3.5 and 14.9%, while the accuracy values were 85.5-111.0% for all tested levels in all investigated matrices. Adagrasib in mouse plasma was reported to have good stability at room temperature, while adagrasib in tissue-related matrices was stable on ice for up to 4 h (matrix dependent). Finally, this method was successfully applied to determine the pharmacokinetic profile and tissue distribution of adagrasib in wild-type mice.

We have successfully developed and validated a bioanalytical assay using liquid chromatography tandem mass spectrometry to simultaneously quantify the first approved KRAS G12C inhibitor sotorasib and its major circulating metabolite (M24) in various mouse matrices. M24 was synthesized in-house via low-pH hydrolysis. We utilized a fast and efficient protein precipitation method in a 96-well plate format to extract both analytes from biological matrices. Erlotinib was selected as the internal standard in this assay. Gradient elution using methanol and 0.1 % formic acid in water (v/v) was applied on an Acquity UPLC BEH C18 column to separate all analytes. Sotorasib, M24, and erlotinib were detected with a triple quadrupole mass spectrometer in positive electrospray ionization in multiple reaction monitoring mode. During the validation and sample quantification, a linear calibration range was observed for both sotorasib and M24 in a range of 4 - 4000 nM and 1 - 1000 nM, respectively. The %bias and %CV (both intra- and inter-day) for all tested levels in all investigated matrices were lower than 15 % as required by the guidelines. Sotorasib had a rather short room temperature stability in mouse plasma for up to 8 h compared to M24 which was stable up to 16 h at room temperature. This method has been successfully applied to measure sotorasib and M24 in several mouse matrices from three different mouse strains. We can conclude that the plasma exposure of sotorasib in mice is limited via human CYP3A4- and mouse Cyp3a-mediated metabolism of sotorasib into M24.

The study focused on the examination of the different fungal species isolated from commercial rice samples, applying conventional culture techniques, as well as different molecular and phylogenic analyses to confirm phenotypic identification. Additionally, the mycotoxin production and contamination were analyzed using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). In total, 40 rice samples were obtained covering rice berry, red jasmine rice, brown rice, germinated brown rice, and white rice. The blotting paper technique applied on the 5 different types of rice samples detected 4285 seed-borne fungal infections (26.8%) for 16,000 rice grains. Gross morphological data revealed that 19 fungal isolates belonged to the genera Penicillium/Talaromyces (18 of 90 isolates; 20%) and Aspergillus (72 of 90 isolates; 80%). To check their morphologies, molecular data (fungal sequence-based BLAST results and a phylogenetic tree of the combined ITS, BenA, CaM, and RPB2 datasets) confirmed the initial classification. The phylogenic analysis revealed that eight isolates belonged to P. citrinum and, additionally, one isolate each belonged to P. chermesinum, A. niger, A. fumigatus, and A. tubingensis. Furthermore, four isolates of T. pinophilus and one isolate of each taxon were identified as Talaromyces ( T. radicus, T. purpureogenum, and T. islandicus). The results showed that A. niger and T. pinophilus were two commonly occurring fungal species in rice samples. After subculturing, ochratoxin A (OTA), generated by T. pinophilus code W3-04, was discovered using LC-MS/MS. In addition, the Fusarium toxin beauvericin was detected in one of the samples. Aflatoxin B1 or other mycotoxins, such as citrinin, trichothecenes, and fumonisins, were detected. These preliminary findings should provide valuable guidance for hazard analysis critical control point concepts used by commercial food suppliers, including the analysis of multiple mycotoxins. Based on the current fi

Objectives: Salivary cortisol and cortisone at late night and after dexamethasone suppression test (DST) are increasingly used for screening of Cushing’s syndrome (CS). We aimed to establish reference intervals for salivary cortisol and cortisone with three liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques and for salivary cortisol with three immunoassays (IAs), and evaluate their diagnostic accuracy for CS. Methods: Salivary samples at 08:00 h, 23:00 h and 08:00 h after a 1-mg DST were collected from a reference population (n=155) and patients with CS (n=22). Sample aliquots were analyzed by three LC-MS/MS and three IA methods. After establishing reference intervals, the upper reference limit (URL) for each method was used to calculate sensitivity and specificity for CS. Diagnostic accuracy was evaluated by comparing ROC curves. Results: URLs for salivary cortisol at 23:00 h were similar for the LC-MS/MS methods (3.4–3.9 nmol/L), but varied between IAs: Roche (5.8 nmol/L), Salimetrics (4.3 nmol/L), Cisbio (21.6 nmol/L). Corresponding URLs after DST were 0.7–1.0, and 2.4, 4.0 and 5.4 nmol/L, respectively. Salivary cortisone URLs were 13.5–16.6 nmol/L at 23:00 h and 3.0–3.5 nmol/L at 08:00 h after DST. All methods had ROC AUCs ≥0.96. Conclusions: We present robust reference intervals for salivary cortisol and cortisone at 08:00 h, 23:00 h and 08:00 h after DST for several clinically used methods. The similarities between LC-MS/MS methods allows for direct comparison of absolute values. Diagnostic accuracy for CS was high for all salivary cortisol and cortisone LC-MS/MS methods and salivary cortisol IAs evaluated.

Objectives: Salivary cortisol and cortisone at late night and after dexamethasone suppression test (DST) are increasingly used for screening of Cushing’s syndrome (CS). We aimed to establish reference intervals for salivary cortisol and cortisone with three liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques and for salivary cortisol with three immunoassays (IAs), and evaluate their diagnostic accuracy for CS. Methods: Salivary samples at 08:00 h, 23:00 h and 08:00 h after a 1-mg DST were collected from a reference population (n=155) and patients with CS (n=22). Sample aliquots were analyzed by three LC-MS/MS and three IA methods. After establishing reference intervals, the upper reference limit (URL) for each method was used to calculate sensitivity and specificity for CS. Diagnostic accuracy was evaluated by comparing ROC curves. Results: URLs for salivary cortisol at 23:00 h were similar for the LC-MS/MS methods (3.4–3.9 nmol/L), but varied between IAs: Roche (5.8 nmol/L), Salimetrics (4.3 nmol/L), Cisbio (21.6 nmol/L). Corresponding URLs after DST were 0.7–1.0, and 2.4, 4.0 and 5.4 nmol/L, respectively. Salivary cortisone URLs were 13.5–16.6 nmol/L at 23:00 h and 3.0–3.5 nmol/L at 08:00 h after DST. All methods had ROC AUCs ≥0.96. Conclusions: We present robust reference intervals for salivary cortisol and cortisone at 08:00 h, 23:00 h and 08:00 h after DST for several clinically used methods. The similarities between LC-MS/MS methods allows for direct comparison of absolute values. Diagnostic accuracy for CS was high for all salivary cortisol and cortisone LC-MS/MS methods and salivary cortisol IAs evaluated.

En el presente estudio se analizaron 40 muestras de pan, tanto de trigo blanco como integral, procedentes de elaboración industrial y artesanal disponibles en el mercado de Castilla y León (España). El objetivo principal fue identificar la presencia de diferentes micotoxinas: deoxinivalenol (DON), ocratoxina A (OTA), aflatoxina B1 (AFB1) y aflatoxina B2 (AFB2). Para lograr este objetivo, se utilizó la técnica de cromatografía líquida acoplada a espectrometría de masas en tándem (LC-MS/MS). Las micotoxinas detectadas fueron DON y OTA, mientras que AFB2 fue hallada por debajo del límite de detección, y AFB1 no pudo ser identificada. Tanto DON como OTA, se observaron con mayor frecuencia en las muestras de pan integral en comparación con las de trigo blanco, así como en las muestras de elaboración industrial en contraste con las artesanales. Destacar, que todas las muestras analizadas se encontraron dentro de los límites establecidos por las regulaciones alimentarias, lo que sugiere que los niveles de micotoxinas detectados no presentan un riesgo inmediato para la seguridad alimentaria en relación con los estándares vigentes., Ingeniería Agrícola y Forestal, Máster en Calidad, Desarrollo e Innovación de Alimentos

BackgroundThe VALID Act is a legislative effort that, if enacted, would alter the regulatory requirements of laboratory developed tests (LDTs) used for clinical testing in the United States. Benzodiazepines, which are primarily excreted into urine as glucuronidated metabolites such as lorazepam, cross-react poorly with FDA-cleared immunoassays, leading to false-negatives. This shortfall can be addressed with LDTs created by adding glucuronidase to the immunoassay reagents producing "high sensitivity" assays that detect glucuronidated metabolites.MethodsPrecision and stability of two high-sensitivity (HS) benzodiazepine immunoassays from Roche and Thermo Scientific were evaluated using manufacturer-supplied quality control (QC) material and glucuronidated QC material. The immunoassays were directly compared to an LC-MS/MS LDT benzodiazepine assay to determine clinical sensitivity/specificity using urine specimens (n = 82 for Thermo Scientific; n = 265 for Roche). The clinical impact of the HS LDT immunoassay was determined by analyzing clinical testing results 60 days before and after its implementation.ResultsThe precision and clinical sensitivity/specificity of the HS-Thermo Scientific and HS-Roche benzodiazepine assays were acceptable. The reagent stability of the HS-Thermo Scientific immunoassay was poor, whereas the HS-Roche immunoassay was stable. After implementation of the HS-Roche benzodiazepine immunoassay as an LDT, there was a 30-fold increase (p-value: < 0.00001) in the percentage of lorazepam confirmations.ConclusionsWe demonstrate the development and validation of an immunoassay LDT with improved sensitivity for glucuronidated benzodiazepines. This LDT can detect glucuronidated benzodiazepines in clinical urine specimens and is stable for 60 days. Importantly, we were able to validate the immunoassay as an LDT by utilizing an LC-MS/MS LDT.

Aripiprazol är den aktiva substansen i läkemedel för bipolaritet och schizofreni och metaboliseras av två enzym till den aktiva metaboliten dehydroaripiprazol. Till följd av interindividuella skillnader i aktiviteten hos enzymen samt att koncentrationen in vivo kan påverkas av andra läkemedel rekommenderas terapeutisk läkemedelsövervakning (TDM). Därmed har en selektiv och känslig vätskekromatografi- tandem masspektrometri (LC-MS/MS) validerats för kvantifiering av aripiprazol och dess aktiva metabolit dehydroaripiprazol i humant serum vid Specialkemi, Klinisk kemi och farmakologi vid Lunds universitetssjukhus utifrån interna kriterier byggda på riktlinjer från European Medicine Agency (EMA). LC-MS/MS analys utfördes på Tripple Quad 6500+ från AB Sciex med joniserande elektrospray (ESI) och multi reaction monitoring (MRM). Valideringen fastslog metodens mätområde till 4 – 2 500 nmol/L och kvantifieringsgräns till 4 nmol/L för respektive analyt. Metodens inomdags- och mellandags noggrannhet (variationskoefficient (CV%)) och riktighet (nominell differens) för kontrollprover (10 och 1 000 nmol/L) var mellan 3,5 – 9,1 % och mellan -6,8 – -13,0 för respektive analyt vilket var inom godkända kriterier. Innan metoden implementeras på kliniska prover bör framtida utvärdering undersöka om minimering av provsmittan är möjlig samt utvärdera långtidsstabiliteten av analyterna.

Aripiprazol är den aktiva substansen i läkemedel för bipolaritet och schizofreni och metaboliseras av två enzym till den aktiva metaboliten dehydroaripiprazol. Till följd av interindividuella skillnader i aktiviteten hos enzymen samt att koncentrationen in vivo kan påverkas av andra läkemedel rekommenderas terapeutisk läkemedelsövervakning (TDM). Därmed har en selektiv och känslig vätskekromatografi- tandem masspektrometri (LC-MS/MS) validerats för kvantifiering av aripiprazol och dess aktiva metabolit dehydroaripiprazol i humant serum vid Specialkemi, Klinisk kemi och farmakologi vid Lunds universitetssjukhus utifrån interna kriterier byggda på riktlinjer från European Medicine Agency (EMA). LC-MS/MS analys utfördes på Tripple Quad 6500+ från AB Sciex med joniserande elektrospray (ESI) och multi reaction monitoring (MRM). Valideringen fastslog metodens mätområde till 4 – 2 500 nmol/L och kvantifieringsgräns till 4 nmol/L för respektive analyt. Metodens inomdags- och mellandags noggrannhet (variationskoefficient (CV%)) och riktighet (nominell differens) för kontrollprover (10 och 1 000 nmol/L) var mellan 3,5 – 9,1 % och mellan -6,8 – -13,0 för respektive analyt vilket var inom godkända kriterier. Innan metoden implementeras på kliniska prover bör framtida utvärdering undersöka om minimering av provsmittan är möjlig samt utvärdera långtidsstabiliteten av analyterna.

In this paper, an LC-MS/MS method for the simultaneous identification and quantification of cyanotoxins with hydrophilic and lipophilic properties in edible bivalves is presented. The method includes 17 cyanotoxins comprising 13 microcystins (MCs), nodularin (NOD), anatoxin-a (ATX-a), homoanatoxin (h-ATX) and cylindrospermopsin (CYN). A benefit to the presented method is the possibility for the MS detection of MC-LR-[Dha7] and MC-LR-[Asp3] as separately identified and MS-resolved MRM signals, two congeners which were earlier detected together. The performance of the method was evaluated by in-house validation using spiked mussel samples in the quantification range of 3.12-200 mu g/kg. The method was found to be linear over the full calibration range for all included cyanotoxins except CYN for which a quadratic regression was used. The method showed limitations for MC-LF (R-2 = 0.94), MC-LA (R-2 <= 0.98) and MC-LW (R-2 <= 0.98). The recoveries for ATX-a, h-ATX, CYN, NOD, MC-LF and MC-LW were lower than desired (<70%), but stable. Despite the given limitations, the validation results showed that the method was specific and robust for the investigated parameters. The results demonstrate the suitability of the method to be applied as a reliable monitoring tool for the presented group of cyanotoxins, as well as highlight the compromises that need to be included if multi-toxin methods are to be used for the analysis of cyanotoxins with a broader range of chemical properties. Furthermore, the method was used to analyze 13 samples of mussels (Mytilus edulis) and oysters (Magallana gigas) collected in the 2020-2022 summers along the coast of Bohuslan (Sweden). A complementary qualitative analysis for the presence of cyanotoxins in phytoplankton samples collected from marine waters around southern Sweden was performed with the method. Nodularin was identified in all samples and quantified in bivalve samples in the range of 7-397 <= g/kg. Toxins produced by cyanoba

In this paper, an LC-MS/MS method for the simultaneous identification and quantification of cyanotoxins with hydrophilic and lipophilic properties in edible bivalves is presented. The method includes 17 cyanotoxins comprising 13 microcystins (MCs), nodularin (NOD), anatoxin-a (ATX-a), homoanatoxin (h-ATX) and cylindrospermopsin (CYN). A benefit to the presented method is the possibility for the MS detection of MC-LR-[Dha7] and MC-LR-[Asp3] as separately identified and MS-resolved MRM signals, two congeners which were earlier detected together. The performance of the method was evaluated by in-house validation using spiked mussel samples in the quantification range of 3.12-200 mu g/kg. The method was found to be linear over the full calibration range for all included cyanotoxins except CYN for which a quadratic regression was used. The method showed limitations for MC-LF (R-2 = 0.94), MC-LA (R-2 <= 0.98) and MC-LW (R-2 <= 0.98). The recoveries for ATX-a, h-ATX, CYN, NOD, MC-LF and MC-LW were lower than desired (<70%), but stable. Despite the given limitations, the validation results showed that the method was specific and robust for the investigated parameters. The results demonstrate the suitability of the method to be applied as a reliable monitoring tool for the presented group of cyanotoxins, as well as highlight the compromises that need to be included if multi-toxin methods are to be used for the analysis of cyanotoxins with a broader range of chemical properties. Furthermore, the method was used to analyze 13 samples of mussels (Mytilus edulis) and oysters (Magallana gigas) collected in the 2020-2022 summers along the coast of Bohuslan (Sweden). A complementary qualitative analysis for the presence of cyanotoxins in phytoplankton samples collected from marine waters around southern Sweden was performed with the method. Nodularin was identified in all samples and quantified in bivalve samples in the range of 7-397 <= g/kg. Toxins produced by cyanoba

Pesticides are extensively used in the cultivation and postharvest protection of citrus fruits, therefore continuous monitoring and health risk assessments of their residues are required. This study aimed to investigate the occurrence of pesticide residues on citrus fruits and to evaluate the acute and chronic risk for adults and children. The risk ranking of twenty-three detected pesticides was carried out according to a matrix ranking scheme. Multiple residues were detected in 83% of 76 analyzed samples. In addition, 28% contained pesticides at or above maximum residue levels (MRLs). The most frequently detected pesticides were imazalil, azoxystrobin, and dimethomorph. According to the risk ranking method, imazalil was classified in the high-risk group, followed by prochloraz, chlorpyrifos, azinphos-methyl, tebufenpyrad, and fenpiroximate, which were considered to pose a medium risk. The majority of detected pesticides (74%) posed a low risk. The health risk assessment indicated that imazalil and thiabendazole contribute to acute (HQa) and chronic (HQc) dietary risk, respectively. The HQc was negligible for the general population, while the HQa of imazalil and thiabendazole exceeded the acceptable level in the worst-case scenario. Cumulative chronic/acute risk (HIc/HIa) assessment showed that chronic risk was acceptable in all samples for children and adults, while the acute risk was unacceptable in 5.3% of citrus fruits for adults and 26% of citrus fruits for children. Sensitivity analyses indicated that the ingestion rate and individual body weight were the most influential risk factors.

In this paper, an LC-MS/MS method for the simultaneous identification and quantification of cyanotoxins with hydrophilic and lipophilic properties in edible bivalves is presented. The method includes 17 cyanotoxins comprising 13 microcystins (MCs), nodularin (NOD), anatoxin-a (ATX-a), homoanatoxin (h-ATX) and cylindrospermopsin (CYN). A benefit to the presented method is the possibility for the MS detection of MC-LR-[Dha7] and MC-LR-[Asp3] as separately identified and MS-resolved MRM signals, two congeners which were earlier detected together. The performance of the method was evaluated by in-house validation using spiked mussel samples in the quantification range of 3.12-200 mu g/kg. The method was found to be linear over the full calibration range for all included cyanotoxins except CYN for which a quadratic regression was used. The method showed limitations for MC-LF (R-2 = 0.94), MC-LA (R-2 <= 0.98) and MC-LW (R-2 <= 0.98). The recoveries for ATX-a, h-ATX, CYN, NOD, MC-LF and MC-LW were lower than desired (<70%), but stable. Despite the given limitations, the validation results showed that the method was specific and robust for the investigated parameters. The results demonstrate the suitability of the method to be applied as a reliable monitoring tool for the presented group of cyanotoxins, as well as highlight the compromises that need to be included if multi-toxin methods are to be used for the analysis of cyanotoxins with a broader range of chemical properties. Furthermore, the method was used to analyze 13 samples of mussels (Mytilus edulis) and oysters (Magallana gigas) collected in the 2020-2022 summers along the coast of Bohuslan (Sweden). A complementary qualitative analysis for the presence of cyanotoxins in phytoplankton samples collected from marine waters around southern Sweden was performed with the method. Nodularin was identified in all samples and quantified in bivalve samples in the range of 7-397 <= g/kg. Toxins produced by cyanoba

Warfarin is a vitamin K antagonist (VKAs) anticoagulant associated with interindividual patients’ response to therapy, narrow therapeutic index, and serious adverse drug events. Monitoring ensures efficacy and safety by measuring drug concentration in the bloodstream, which demands blood or plasma samples from patients through conventional sampling. Conventional sampling often requires invasive methods that may be uncomfortable for patients. Microsampling offers an opportunity to reduce the burden of multiple blood sampling on patients and simplifies the process of sample transportation and storage. This systematic review describes conventional and microsampling techniques for quantifying warfarin in human blood samples and the bioanalytical method employed to perform the analysis. Related studies (537) were screened from several databases and narrowed down into 12 eligible articles, which were then used to fulfill the purpose of this review in a narrative form. It is concluded that while there are still limited studies regarding the application of microsampling for warfarin quantification in patients, there has been evidence of comparable plasma and micro samples results, highlighting the potential for future development of routine monitoring using the microsampling technique.

Warfarin is a vitamin K antagonist (VKAs) anticoagulant associated with interindividual patients’ response to therapy, narrow therapeutic index, and serious adverse drug events. Monitoring ensures efficacy and safety by measuring drug concentration in the bloodstream, which demands blood or plasma samples from patients through conventional sampling. Conventional sampling often requires invasive methods that may be uncomfortable for patients. Microsampling offers an opportunity to reduce the burden of multiple blood sampling on patients and simplifies the process of sample transportation and storage. This systematic review describes conventional and microsampling techniques for quantifying warfarin in human blood samples and the bioanalytical method employed to perform the analysis. Related studies (537) were screened from several databases and narrowed down into 12 eligible articles, which were then used to fulfill the purpose of this review in a narrative form. It is concluded that while there are still limited studies regarding the application of microsampling for warfarin quantification in patients, there has been evidence of comparable plasma and micro samples results, highlighting the potential for future development of routine monitoring using the microsampling technique.

A genome-wide association study (GWAS), which uses information on single nucleotide polymorphisms (SNPs) from many accessions, has become a powerful approach to gene identification. A metabolome GWAS (mGWAS), which relies on phenotypic information based on metabolite accumulation, can identify genes that contribute to primary and secondary metabolite contents. In this study, we carried out a mGWAS using seed metabolomic data from Arabidopsis thaliana accessions obtained by liquid chromatography–mass spectrometry to identify SNPs highly associated with the contents of metabolites such as glucosinolates. These SNPs were present in genes known to be involved in glucosinolate biosynthesis, thus confirming the effectiveness of our analysis. We subsequently focused on SNPs detected in an unknown methyltransferase gene associated with N-methylhistidine content. Knockout and overexpression of A. thaliana lines of this gene had significantly decreased and increased Nmethylhistidine contents, respectively. We confirmed that the overexpressing line exclusively accumulated histidine methylated at the pi position, not at the tau position. Our findings suggest that the identified methyltransferase gene encodes a key enzyme for N-methylhistidine biosynthesis in A. thaliana., Frontiers in Plant Science, 14, art. no. 1201129; 2023

Objectives: Salivary cortisol and cortisone at late night and after dexamethasone suppression test (DST) are increasingly used for screening of Cushing’s syndrome (CS). We aimed to establish reference intervals for salivary cortisol and cortisone with three liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques and for salivary cortisol with three immunoassays (IAs), and evaluate their diagnostic accuracy for CS. Methods: Salivary samples at 08:00 h, 23:00 h and 08:00 h after a 1-mg DST were collected from a reference population (n=155) and patients with CS (n=22). Sample aliquots were analyzed by three LC-MS/MS and three IA methods. After establishing reference intervals, the upper reference limit (URL) for each method was used to calculate sensitivity and specificity for CS. Diagnostic accuracy was evaluated by comparing ROC curves. Results: URLs for salivary cortisol at 23:00 h were similar for the LC-MS/MS methods (3.4–3.9 nmol/L), but varied between IAs: Roche (5.8 nmol/L), Salimetrics (4.3 nmol/L), Cisbio (21.6 nmol/L). Corresponding URLs after DST were 0.7–1.0, and 2.4, 4.0 and 5.4 nmol/L, respectively. Salivary cortisone URLs were 13.5–16.6 nmol/L at 23:00 h and 3.0–3.5 nmol/L at 08:00 h after DST. All methods had ROC AUCs ≥0.96. Conclusions: We present robust reference intervals for salivary cortisol and cortisone at 08:00 h, 23:00 h and 08:00 h after DST for several clinically used methods. The similarities between LC-MS/MS methods allows for direct comparison of absolute values. Diagnostic accuracy for CS was high for all salivary cortisol and cortisone LC-MS/MS methods and salivary cortisol IAs evaluated.

The biogenic methylation of inorganic, divalent mercury (Hg(II)) by methylating microorganisms leads to formation and bioaccumulation of monomethyl mercury (MeHg) in the environment and can cause severe damage to ecosystems and human health. Diverse microorganisms carry the gene sequence hgcAB and are able to methylate Hg(II) intracellularly. The interplay of biological, chemical and physical parameters is driving mercury (Hg) transformation by microorganisms. The chemical speciation of Hg(II) with thiol compounds, both with dissolved low molecular mass (LMM) thiols and thiols present on microbial membrane surfaces, is one key factor for Hg availability and transformation. In this work the role of thiol compounds with respect to Hg speciation, uptake and transformation was studied by the iron-reducing model organisms Geobacter sulfurreducens. The turnover of dissolved thiols and the role of outer and inner membrane thiols was studied with novel experimental strategies. In Paper I and II the formation of thiol compounds was studied under varying nutrient conditions. It was shown that the formation of LMM-thiol compounds was impacted by divalent iron, Fe(II). Furthermore, we showed the turnover of the small LMM-thiol cysteine to the branched LMM-thiol penicillamine, which was further amplified by the addition of exogenous cysteine or nutrients. This turnover of small to branched LMM-thiols impacted the Hg(II) speciation in methylation assays and the relative contribution between cysteine and penicillamine was important for Hg(II) availability, uptake and methylation. In addition, the partition of Hg(II) between the cell-adsorbed and dissolved phase was shifted towards the latter at higher LMM-thiol concentrations. Nutrient concentrations impacted cell physiology due to a shift to an active metabolism and a faster metabolization of LMM-thiols. We concluded that the interplay between thiol metabolism, Hg(II) speciation and cell physiology are key parameters for Hg(II) m

In this paper, an LC-MS/MS method for the simultaneous identification and quantification of cyanotoxins with hydrophilic and lipophilic properties in edible bivalves is presented. The method includes 17 cyanotoxins comprising 13 microcystins (MCs), nodularin (NOD), anatoxin-a (ATX-a), homoanatoxin (h-ATX) and cylindrospermopsin (CYN). A benefit to the presented method is the possibility for the MS detection of MC-LR-[Dha7] and MC-LR-[Asp3] as separately identified and MS-resolved MRM signals, two congeners which were earlier detected together. The performance of the method was evaluated by in-house validation using spiked mussel samples in the quantification range of 3.12-200 mu g/kg. The method was found to be linear over the full calibration range for all included cyanotoxins except CYN for which a quadratic regression was used. The method showed limitations for MC-LF (R-2 = 0.94), MC-LA (R-2 <= 0.98) and MC-LW (R-2 <= 0.98). The recoveries for ATX-a, h-ATX, CYN, NOD, MC-LF and MC-LW were lower than desired (<70%), but stable. Despite the given limitations, the validation results showed that the method was specific and robust for the investigated parameters. The results demonstrate the suitability of the method to be applied as a reliable monitoring tool for the presented group of cyanotoxins, as well as highlight the compromises that need to be included if multi-toxin methods are to be used for the analysis of cyanotoxins with a broader range of chemical properties. Furthermore, the method was used to analyze 13 samples of mussels (Mytilus edulis) and oysters (Magallana gigas) collected in the 2020-2022 summers along the coast of Bohuslan (Sweden). A complementary qualitative analysis for the presence of cyanotoxins in phytoplankton samples collected from marine waters around southern Sweden was performed with the method. Nodularin was identified in all samples and quantified in bivalve samples in the range of 7-397 <= g/kg. Toxins produced by cyanoba

Warfarin is a vitamin K antagonist (VKAs) anticoagulant associated with interindividual patients’ response to therapy, narrow therapeutic index, and serious adverse drug events. Monitoring ensures efficacy and safety by measuring drug concentration in the bloodstream, which demands blood or plasma samples from patients through conventional sampling. Conventional sampling often requires invasive methods that may be uncomfortable for patients. Microsampling offers an opportunity to reduce the burden of multiple blood sampling on patients and simplifies the process of sample transportation and storage. This systematic review describes conventional and microsampling techniques for quantifying warfarin in human blood samples and the bioanalytical method employed to perform the analysis. Related studies (537) were screened from several databases and narrowed down into 12 eligible articles, which were then used to fulfill the purpose of this review in a narrative form. It is concluded that while there are still limited studies regarding the application of microsampling for warfarin quantification in patients, there has been evidence of comparable plasma and micro samples results, highlighting the potential for future development of routine monitoring using the microsampling technique.