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Background: Short-chain fatty acids (SCFAs), including propionic acid (PA), are key in immunological research. Supplementing PA has shown benefits for autoimmune diseases. A comprehensive understanding of the PA pharmacokinetics is essential for the optimal design and execution of studies utilizing orally administered PA. Objective: We propose two methods of measuring PA in serum, carried out by different laboratories. Design: Blood samples from 20 volunteers were collected hourly following PA supplementation. Methods: Serum propionate quantification was performed with two independent mass spectrometry-based (MS) analyses, including liquid-chromatography (LC)-MS and direct-infusion (DI)-MS. Results: PA levels increased within 1 h of ingestion of 500 mg PA. Serum concentrations ranged from 1.3 to 4.5 µmol/L, rising significantly after 1 h (p < 0.05). Serum levels returned to baseline within 2 h. No significant differences were found regarding sex or diet. Conclusion: The shown pharmacokinetics can be used in future PA research. [ABSTRACT FROM AUTHOR]

In order to investigate the key nodes affecting the changes of Yixian dark tea flavor and quality during the Pile fermentation, in this study, the sensory quality and non-volatile profiles of Yixian dark tea samples at different pile fermentation periods (0, 3, 6, 9, 12, 15, 18 h) were investigated by sensory evaluation, amino acids analyzer, liquid chromatography and liquid chromatography mass spectrometry. The results showed that the intensity of bitterness in dark tea infusions declined after 9 h of pile fermentation, along with the increasing intensities of astringency, thickness and stale-mellow. Total catechins showed a significant decrease in concentrations during pile fermentation. In contrast, the concentration of gallic acid increased. Concentration of caffeine reduced by 14.7% compared to the original concentration after 18 h of pile fermentation. Total amino acids concentration showed an increasing tendency in the later period of pile fermentation. Concentrations of flavonoids and its mono-glycosides increased after 9-12 hours, while its di-glycosides showed an increasing tendency after 12 hours of pile fermentation. The analysis of key taste substances (VIP>1, DOT>1) showed that myricetin-3-O-galactoside, astragalin, isoquercitrin, quercetin-3-O-rutinose, epigallocatechin, epicatechin, epigallocatechin gallate, and caffeine were the most important flavor presenting substances affecting the quality changes of Yixian dark tea. Among them, the content of the first three glycosides increased significantly from 9-12 hours of stacking; The contents of epigallocatechin and epicatechin were significantly reduced during 9-12 hours. Epigallocatechin gallate and caffeine showed a significant decrease after pile fermentation; Quercetin-3-O-rutinoside decreased significantly in the middle stage (9-12 hours) and increased significantly after 15 hours. It was an important factor in the increase of astringency of tea broth in the late stage of pile fermentation. This study give a theoretical basis for improving the tea processing of Yixian dark tea and understanding the formation mechanism of its key flavor. [ABSTRACT FROM AUTHOR]

This document presents the results of a study comparing the accuracy of a cortisol immunoassay to LC-MS/MS. The study found a significant positive bias in the cortisol immunoassay compared to LC-MS/MS, but could not identify the cause of this difference. However, the study suggests that despite the bias, the cortisol immunoassay may still be considered for adrenal vein sampling (AVS) due to the minimal impact on clinical decision making. The study recommends careful consideration of the positive bias for other indications. The document concludes with acknowledgments to the individuals who contributed to the analysis of the samples. [Extracted from the article]

Structure identification of drug degradation products is one of the key components of any pharmaceutical product development. Knowledge-based in-silico tools are widely used to predict the probable degradation and excipient interaction products. However, multiple structures of the same masses are predicted through this software, which makes identification of the correct/exact structure of the degradation product difficult. In this study, the utilization of a simple yet powerful analytical tool, LC-MS HDX was explored for improving the predictive capability of the in-silico tool. For the same, 5 drugs were selected as representative of pharmaceutical development workflow and subjected to a stress degradation study as per ICH regulatory requirements. There were in total 55 degradation products, for which all possible structures were predicted through Zeneth® software. The number of labile hydrogens in each of the 55 products was obtained through a simple LC-MS HDX method. While scrutinizing predicted structures with the number of labile hydrogens, many false positive predictions could be ruled out. Out of 55 degradation products, this approach has helped in improving the predictability of 45 products corresponding to ∼82% cases. It is simple to adopt for the industry by simply amalgamating analytical practices with AI prediction. [ABSTRACT FROM AUTHOR]

Introduction: Doping and steroid use represent a serious threat to animal health and can even lead to their untimely and painful death. However, doping is an acute problem in today's animal racing world, particularly in camel racing. Testosterone and its ten esters (benzoate, valerate, isocaproate, hexahydrobenzoate, decanoate, undecanoate, laurate, enanthate, cypionate, and caproate) are of utmost importance, because when they are administered to animals it is difficult to measure them efficiently. The levels of testosterone and its esters in camels and other animals are typically determined using urine and blood tests. The aim of this study was to develop and validate a liquid chromatographic–mass spectrometric (LC-MS/MS) method to determine testosterone esters in camel hair, and to apply the validated method to determine testosterone esters in collected samples. To our knowledge, this is the first report of such research. Results and Discussion: The levels of testosterone and its ten derivatives, along with the cortisol-D4 internal standard, were optimised for LC–MS/MS analysis; however, only testosterone along with its seven esters (namely benzoate, valerate, isocaproate, hexahydrobenzoate, decanoate, undecanoate and laurate) could be validated in camel hair. Only five testosterone esters could be determined in camel hair samples; the concentrations were obtained as 10.5–14.9 pg/mg for valerate (in three camels), 12.5–151.6 pg/mg for hexahydrobenzoate (in six camels), 4.8–32.1 pg/mg for laurate (in five camels), 5.1 pg/mg decanoate (in one camel), and 8.35–169 pg/mg for testosterone (in all 24 camels). Interestingly, the three racing camels displayed high concentrations of testosterone (59.2–169 pg/mg, all three camels), laurate (4.8–14.5 pg/mg, two camels), hexahydrobenzoate (116 pg/mg, one camel), decanoate (5.1 pg/mg, one camel), and valerate (11.7 pg/mg, one camel). Methods: Camel hair samples were collected from 21 non-racing dromedary camels along with three racing camels in Al Ain, UAE; these were decontaminated, pulverised, sonicated, and extracted prior to analysis. An LC–MS/MS method was employed to determine the levels of testosterone esters in the hair samples. Conclusions: This novel camel-hair test procedure is accurate, sensitive, rapid, and robust. The findings reported in this study could be significant to evaluate racing camels for suspected doping offenses. Further controlled testosterone supplementation studies are required to evaluate individual esters' effects on camel health and diseases and on performance enhancement levels. This new hair test could promote further studies in doping control, toxicology, and pharmacology, as well as having other clinical applications relating to camel health, injury, and disease. [ABSTRACT FROM AUTHOR]

This study aimed to determine whether leaf extracts from seven Eruca vesicaria subsp. sativa cultivars and their biochemically active compounds (glucosinolates and downstream-derived products) inhibit mycelia growth of three well-known pathogenic oomycetes, Phytopythium chamaehyphon, Phytopythium vexans and Phytophthora citrophthora; being the most significant in the development of Kiwifruit Vine Decline Syndrome (KVDS). Leaf extract quantity of 10, 20 and 30 mgwereinoculated in Petridish (90mmØ, each 22mLof liquidmedium-Potato Dextrose Agar), for in vitro bioassays. A pathogen plug was placed in the centre of each plate and the Oomycota colony perimeter was marked 5 days after inoculation. Radial colony growth was measured from 4 marks per plate 5, 10, and 15 days after inoculation, further elaborated with Image J software image analysis. Growthrates forall strainswereinhibitedbyaround67%after15days. This wasmostpronouncedwhenapplying thehighest concentrationof leaf extract. By using Liquid Chromatography Mass Spectrometry (LC-MS) and Gas Chromatography Mass Spectrometry (GC-MS), fifteen glucosinolate compounds, of which glucosativin was found in the highest quantity, were identified. Concentrations of hydrolysis products produced by leaves (erucin and sativin) were also investigated, and were significantly associated with colony radial growth, especially towards Pp. chamaehyphon and Pp. vexans. Three downstream products of glucosinolates (two pure isothiocyanates, AITC and PEITC; and one indole I3C; all commonly present in Brassicaceae) were also tested, and a statistically significant inhibition of growth was observed at the highest concentration (0.6 µL). [ABSTRACT FROM AUTHOR]

Background: The inclusion of dexmedetomidine (DEX) within a balanced general anaesthesia protocol is effective in improving the clinical outcome and recovery quality of anaesthesia in horses. This study aimed to determine the pharmacokinetic profile of DEX following repeated subcutaneous (SC) administration at 2 µg/kg every 60 min till the end of the procedure in comparison to intravenous constant rate infusion (CRI) at 1 µg/kg/h in anaesthetized horses undergoing diagnostic procedures up to the end of the diagnostic procedure. Results: In the CRI and SC groups DEX maximum concentrations (Cmax) were 0.83 ± 0.27 ng/mL and 1.14 ± 0.71 ng/mL, respectively, reached at a time (Tmax) of 57.0 ± 13.4 min and 105.5 ± 29.9 min. Mean residence time to the last measurable concentration (MRTlast) was 11.7 ± 6.2 and 55.8 ± 19.7 min for the CRI group and SC groups, respectively. The apparent elimination half-life was 18.0 ± 10.0 min in the CRI group and 94.8 ± 69.8 min for the SC group, whereas the area under the curve (AUC0-last) resulted 67.7 ± 29.3 and 83.2 ± 60.5 min*ng/mL for CRI and SC group, respectively. Clearance was 16.26 ± 8.07 mL/min/kg for the CRI group. No signs of adverse effects were recorded in both groups. Conclusions: The pharmacokinetic profile of DEX following repeated SC administration in anaesthetized horses was comparable to intravenous CRI administration during the intranaesthetic period and beneficial during the recovery phase from general anaesthesia. The SC route could be considered as an alternative to CRI for improving the recovery quality of equine patients undergoing general anaesthesia. [ABSTRACT FROM AUTHOR]

[Display omitted] Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under the good manufacturing practice (GMP) regime, due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. This article is the second part of a two-tiered publication aiming at providing guidance for implementation of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) in a QC laboratory. The first part [1] focuses on technical considerations, while this second part provides considerations related to GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG). [ABSTRACT FROM AUTHOR]

Filipendula ulmaria, commonly known as meadowsweet, is a wild herbaceous flowering plant that is widely distributed in Europe. A range of salicylic acid derivatives and flavonol glycosides have been previously associated with the antirheumatic and diuretic properties of F. ulmaria. In the present work, a hydroalcoholic extract from F. ulmaria aerial parts was extensively profiled using an efficient NMR-based dereplication strategy. The approach involves the fractionation of the crude extract by centrifugal partition chromatography (CPC), 13C NMR analysis of the fractions, 2D-cluster mapping of the entire NMR dataset, and, finally, structure elucidation using a natural metabolite database, validated by 2D NMR data interpretation and liquid chromatography coupled with mass spectrometry. The chemodiversity of the aerial parts was extensive, with 28 compounds unambiguously identified, spanning various biosynthetic classes. The F. ulmaria extract and CPC fractions were screened for their potential to enhance skin epidermal barrier function and skin renewal properties using in vitro assays performed on Normal Human Epidermal Keratinocytes. Fractions containing quercetin, kaempferol glycosides, ursolic acid, pomolic acid, naringenin, β-sitosterol, and Tellimagrandins I and II were found to upregulate genes related to skin barrier function, epidermal renewal, and stress responses. This research is significant as it could provide a natural solution for improving hydration and skin renewal properties. [ABSTRACT FROM AUTHOR]

In clinical practice, steroid measurements are performed mainly by direct, non-extraction immunoassays adapted to high throughput, automated immunoassay platforms and employing secondary calibrators. The accuracy of such steroid immunoassays is limited by cross-reactivity with structurally related steroids and nonspecific matrix interference as well as the metrological traceability of manufacturer supplied calibrators. The accuracy of steroid immunoassay calibrators has been little investigated by independent chemical methods. Steroid concentrations of 41 calibrators (4–6 replicates per calibrator) supplied by four manufacturers for use in testosterone (T), estradiol (E2), and progesterone (P4) commercial immunoassays were measured by ultra-pressure liquid chromatography-mass spectrometry (UPLC-MS). Among 14 non-zero T calibrators, six (43 %) deviated significantly from the label concentration with 29 % outside 20 % of it. Among 14 E2 calibrators, eight (57 %) deviated significantly, whereas seven (50 %) were outside 20 % of the label concentration. Among 11 P4 calibrators, eight (73 %) deviated significantly whereas four (36 %) were outside within 20 % of the label concentration. We conclude that inaccurate calibration of manufacturer's supplied standards may contribute to inaccuracy of commercial direct steroid immunoassays. [ABSTRACT FROM AUTHOR]

[Display omitted] Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under good manufacturing practice (GMP) due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. Here, current literature related to the development and application of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) is compiled with the aim of providing guidance for the implementation of MAM in a QC laboratory. This article, focusing on technical considerations, is the first part of a two-tiered publication, whereby the second part will focus on GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG). [ABSTRACT FROM AUTHOR]

Despite recent developments in managing metastatic melanomas, patients' overall survival remains low. Therefore, the current study aims to understand better the proteome‐wide changes associated with melanoma metastasis that will assist with identifying targeted therapies. The latest development in mass spectrometry‐based proteomics, together with extensive bioinformatics analysis, was used to investigate the molecular changes in 60 formalin‐fixed and paraffin‐embedded samples of primary and lymph nodes (LN) and distant organ metastatic melanomas. A total of 4631 proteins were identified, of which 72 and 453 were significantly changed between the LN and distant organ metastatic melanomas compared to the primary lesions (adj. p‐value <0.05). An increase in proteins such as SLC9A3R1, CD20 and GRB2 and a decrease in CST6, SERPINB5 and ARG1 were associated with regional LN metastasis. By contrast, increased metastatic activities in distant organ metastatic melanomas were related to higher levels of CEACAM1, MC1R, AKT1 and MMP3‐9 and decreased levels of CDKN2A, SDC1 and SDC4 proteins. Furthermore, machine learning analysis classified the lesions with up to 92% accuracy based on their metastatic status. The findings from this study provide up to date proteome‐level information about the progression of melanomas to regional LN and distant organs, leading to the identification of protein signatures with potential for clinical translation. [ABSTRACT FROM AUTHOR]

Background: Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune central nervous system (CNS) inflammatory and demyelinating disorder that can lead to serious disability and mortality. Humoral fluid biomarkers with specific, convenient, and efficient profiles that could characterize and monitor disease activity or severity are very useful. We aimed to develop a sensitive and highthroughput liquid chromatography-tandem mass spectrometry (LC-MS)/MSbased analytical method for novel biomarkers finding in NMOSD patients and verified its function tentatively. Methods: Serum samples were collected from 47 NMOSD patients, 18 patients with other neurological disorders (ONDs), and 35 healthy controls (HC). Cerebrospinal fluid (CSF) samples were collected from 18 NMOSD and 17 OND patients. Three aromatic amino acids (phenylalanine, tyrosine, and tryptophan) and nine important metabolites that included phenylacetylglutamine (PAGln), indoleacrylic acid (IA), 3-indole acetic acid (IAA), 5-hydroxyindoleacetic acid (HIAA), hippuric acid (HA), I-3-carboxylic acid (I-3-CA), kynurenine (KYN), kynurenic acid (KYNA), and quinine (QUIN) were analyzed by using the liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. The profile of IA was further analyzed, and its function was verified in an astrocyte injury model stimulated by NMO-IgG, which represents important events in NMOSD pathogenesis. Results: In the serum, tyrosine and some of the tryptophan metabolites IA and I-3-CA decreased, and HIAA increased significantly in NMOSD patients. The CSF levels of phenylalanine and tyrosine showed a significant increase exactly during the relapse stage, and IA in the CSF was also increased markedly during the relapse and remission phases. All conversion ratios had similar profiles with their level fluctuations. In addition, the serum IA levels negatively correlated with glial fibrillary acidic protein (GFAP), and neurofilament light (NfL) levels in the serum of NMOSD patients were measured by using ultra-sensitive single-molecule arrays (Simoa). IA showed an anti-inflammatory effect in an in vitro astrocyte injury model. Conclusion: Our data suggest that essential aromatic amino acid tryptophan metabolites IA in the serum or CSF may serve as a novel promising biomarker to monitor and predict the activity and severity of NMOSD disease. Supplying or enhancing IA function can promote anti-inflammatory responses and may have therapeutic benefits. [ABSTRACT FROM AUTHOR]

An immobilized metal affinity (IMAC) adsorbent was prepared for selective enrichment of adenine type CKs, via grafting polymer chain pendant with iminodiacetic acid (IDA) from polydopamine (PDA)/polyethyleneimine (PEI)-coated magnetic graphene oxide (magGO) via surface-initiated-atom transfer radical polymerization (SI-ATRP). The prepared IMAC sorbent exhibited remarkable adsorption performances and good selectivity for adenine-type CKs and was utilized as a sorbent of magnetic solid-phase extraction (MSPE) for effective enrichment of four adenine-type CKs in bean sprouts. Under the optimized extraction conditions, an analytical method for four adenine type CKs in bean sprouts was established by combining the MSPE combined with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The recoveries of the analytes were between 80.4 ± 1.9% and 114.6 ± 1.5% (n = 3). The limits of detection (LODs) range from 0.63 to 2.30 pg⋅mL−1. The relative standard deviations of intra-day and inter-day were less than 12.6%. The established method was successfully applied to the selective extraction and sensitive detection of trace adenine-type CKs in plant samples. [ABSTRACT FROM AUTHOR]

Honey is known to have antimicrobial, immunomodulatory and wound healing properties. The biological properties of honey have been attributed to phytochemicals derived from their source plants and research has focused on identifying the bioactive phytochemicals with therapeutic potential. In this study, we determined the ability of 5 honeys from Kazakhstan and manuka honey to stimulate TNF-α and TGF-β production by human keratinocytes. TNF-α and TGF-β levels increased over time in honey treated and untreated keratinocytes, whereas cells treated with sugar solutions that matched those of the honeys had reduced levels of both cytokines. This suggests that the non-sugar phytochemical components of the honeys may have prevented this decrease. Analysis by LC-MS confirmed that the honeys contained a diverse range of phytochemicals. Some phytochemicals e.g. pinobanksin and vanillin were present at different levels across the honey types, whereas other components, e.g. dicarboxylic acids and their glycosides, were abundant in all honeys. [ABSTRACT FROM AUTHOR]

Manipulating the crop load in peach trees determines carbon supply and optimum balance between fruit yield and quality potentials. The impact of carbon supply on peach fruit quality was assessed in three development stages (S2, S3, S4) on fruit of equal maturity from trees that were carbon (C) starved (unthinned) and sufficient (thinned). Previous studies determined that primary metabolites of peach fruit mesocarp are mainly linked with developmental processes, thus, the secondary metabolite profile was assessed using non-targeted liquid chromatography mass-spectrometry (LC-MS). Carbon sufficient (C-sufficient) fruit demonstrated superior quality attributes as compared to C-starved fruit. Early metabolic shifts in the secondary metabolome appear to prime quality at harvest. Enhanced C-availability facilitated the increased and consistent synthesis of flavonoids, like catechin, epicatechin and eriodyctiol, via the phenylpropanoid pathway, providing a link between the metabolome and fruit quality, and serving as signatures of C-sufficiency during peach fruit development. [Display omitted] • Optimized crop loads enhance C-supply priming peach fruit metabolism and quality. • Primary metabolism is mainly linked with peach fruit developmental processes. • Non-targeted LC-MS revealed that flavonoids up-accumulate in the C-sufficient fruit. • Phenylpropanoids are signatures of C-sufficiency during peach fruit development. • Phenylpropanoids link primary/secondary metabolism with superior fruit quality. [ABSTRACT FROM AUTHOR]

Neuropathic pain is a condition affecting the quality of life of a substantial part of the population, but biomarkers and treatment options are still limited. While this type of pain is caused by nerve damage, in which lipids play key roles, lipidome alterations related to nerve injury remain poorly studied. Here, we assessed blood lipidome alterations in a common animal model, the rat sciatic nerve crush injury. We analyzed alterations in blood lipid abundances between seven rats with nerve injury (NI) and eight control (CL) rats in a time-course experiment. For these rats, abundances of 377 blood lipid species were assessed at three distinct time points: immediately after, two weeks, and five weeks post injury. Although we did not detect significant differences between NI and CL at the first two time points, 106 lipids were significantly altered in NI five weeks post injury. At this time point, we found increased levels of triglycerides (TGs) and lipids containing esterified palmitic acid (16:0) in the blood plasma of NI animals. Lipids containing arachidonic acid (20:4), by contrast, were significantly decreased after injury, aligning with the crucial role of arachidonic acid reported for NI. Taken together, these results indicate delayed systematic alterations in fatty acid metabolism after nerve injury, potentially reflecting nerve tissue restoration dynamics. [ABSTRACT FROM AUTHOR]

Hepatic AL amyloidosis patients have hepatomegaly and elevated alkaline phosphatase levels; and gastrointestinal AL amyloidosis presents with dysphagia or loss of appetite. AL amyloidosis, also known as primary systemic amyloidosis, is a rare disease with an estimated incidence of 9.7 to 14.0 cases per million person years.1 Ninety-five percent of AL amyloidosis patients are >40 years old with a median age of 64 to 70 years old; 65% to 70% of patients are male. Keywords: amyloid; light chain; liquid chromatography mass spectrometry EN amyloid light chain liquid chromatography mass spectrometry 889 892 4 09/21/22 20221001 NES 221001 INTRODUCTION The three main types of systemic amyloidosis are: primary amyloid light-chain (AL), secondary, and familial amyloidosis. [Extracted from the article]

• 21T FT-ICR MS molecular formula library annotated 53% of diatom exometabolome. • Formula library increased annotations 10x relative to MS/MS spectra matching. • FT-ICR MS assignments corroborated 94% of MS/MS spectra matching assignments. • Novel approach identified 11 protein-like compounds exuded in response to Fe stress. Increased accessibility of liquid chromatography mass spectrometry (LC-MS) metabolomics instrumentation and software have expanded their use in studies of dissolved organic matter (DOM) and exometabolites released by microbes. Current strategies to annotate metabolomes generally rely on matching tandem MS/MS spectra to databases of authentic standards. However, spectral matching approaches typically have low annotation rates for DOM. An alternative approach is to annotate molecular formula based on accurate mass and isotopic fine structure measurements that can be obtained from state-of-the-art ultrahigh resolution Fourier Transform Ion Cyclotron Resonance mass spectrometry (FT-ICR MS), but instrument accessibility for large metabolomic studies is generally limited. Here, we describe a strategy to annotate exometabolomes obtained from lower resolution LC-MS systems by matching metabolomic features to a molecular formula library generated for a representative sample analyzed by LC 21T- FT-ICR MS. The molecular formula library approach successfully annotated 53% of exometabolome features of the marine diatom Phaeodactylum tricornutum – a nearly ten-fold increase over the 6% annotation rate achieved using a conventional MS/MS approach. There was 94% agreement between assigned formula that were annotated with both approaches, and mass error analysis of the discrepancies suggested that the FT-ICR MS formula assignments were more reliable. Differences in the exometabolome of P. tricornutum grown under iron replete and iron limited conditions revealed 668 significant metabolites, including a suite of peptide-like molecules released by P. tricornutum in response to iron deficiency. These findings demonstrate the utility of FT-ICR MS formula libraries for extending the accuracy and comprehensiveness of metabolome annotations. [ABSTRACT FROM AUTHOR]

• Bidens pilosa plant contains two genes encoding acyltransferases responsible for tartaric acid esters. • The SMRT sequencing approach allowed for full length transcripts of acyl transferases. • Bidens pilosa contains heterogenous hydroxycinnamoyl-tartaric acid esters. Bidens pilosa is an edible plant with highly sought-after nutraceutical properties. The purported bioactivities of this plant can be correlated to the high number of metabolites. Amongst these metabolites, different derivatives of hydroxy‑cinnamoyl esters have been shown to exist in high proportions. However, the enzymatic machinery, thus the biosynthetic pathways responsible for the accumulation of these compounds in the plant have not yet been identified. For the first time, we report the putative identification of two genes with sequence homology to hydroxycinnamoyl-CoA: tartaric acid hydroxycinnamoyl transferase (HTT) in B. pilosa. The full-length sequence of the two isoforms of the HTT gene was achieved using single-molecule real-time (SMRT) sequencing approach. Analyses of methanolic extracts of B. pilosa through Liquid-chromatography hyphenated with mass spectrometry (LC-MS) technique revealed the existence of heterogeneous hydroxycinnamoyl-tartaric acid esters consisting of different hydroxycinnamoyl derivatives. To the best of our knowledge, this is a first report on these molecules from B. pilosa. Taken altogether, this plant utilises hydroxycinnamoyl-CoA tartaric hydroxycinnamoyl transferase (HTT) genes to diversify its metabolite composition through esterification of tartaric acid acceptor molecule by acylating it with either homogeneous or heterogenous hydroxycinnamic acids (HCA) derivatives. Therefore, B. pilosa is a source of structurally diverse isomeric compounds with purported nutraceutical values. The enzyme products of the two identified HTT genes are therefore pointed out as possible catalysts which can be further exploited by incorporating them in other economically viable plants to enhance the nutraceutical values thereof. [ABSTRACT FROM AUTHOR]

Tetrabromobisphenol S (TBBPS), as a substitute of tetrabromobisphenol a (TBBPA), has been used in industrial production. The environmental level of TBBPS and its influence on human body are discussed. The research progress of pretreatment methods (filtration, ultrasonic extraction, solid phase microextraction, magnetic molecularly imprinted solid phase extraction and thin layer chromatography) and detection methods (liquid chromatography, chemical sensor and liquid chromatography-mass spectrometry) of TBBPS were reviewed. [ABSTRACT FROM AUTHOR]

Olanzapine is a thienobenzodiazepine class antipsychotic that strongly antagonises the 5-HT2A serotonin receptor, but acute poisonings are reported rarely. Symptoms of an overdose include disorder of consciousness, hypersalivation, myosis, and coma. Serum concentration higher than 0.1 mg/L is toxic, while concentration above 1 mg/L can be fatal. Here we report key data about 61 patients admitted to the National Poison Control Centre in Belgrade, Serbia over olanzapine poisoning in 2017 and 2018. The ingested doses ranged from 35 to 1680 mg, and time from ingestion to determination from two to 24 hours. In 34 patients olanzapine serum concentrations were in the therapeutic range and in 27 in the toxic range. In five patients they were higher than fatal, but only one patient died. The most common symptoms of poisoning were depressed consciousness (fluctuating from somnolence to coma), tachycardia, hypersalivation, hypotension, myosis, and high creatine kinase. All patients but one recovered fully after nonspecific detoxification and symptomatic and supportive therapy. [ABSTRACT FROM AUTHOR]

Exometabolomics is an approach to assess how microorganisms alter, or react to their environments through the depletion and production of metabolites. It allows the examination of how soil microbes transform the small molecule metabolites within their environment, which can be used to study resource competition and cross-feeding. This approach is most powerful when used with defined media that enable tracking of all metabolites. However, microbial growth media have traditionally been developed for the isolation and growth of microorganisms but not metabolite utilization profiling through Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Here, we describe the construction of a defined medium, the Northen Lab Defined Medium (NLDM), that not only supports the growth of diverse soil bacteria but also is defined and therefore suited for exometabolomic experiments. Metabolites included in NLDM were selected based on their presence in R2A medium and soil, elemental stoichiometry requirements, as well as knowledge of metabolite usage by different bacteria. We found that NLDM supported the growth of 108 of the 110 phylogenetically diverse (spanning 36 different families) soil bacterial isolates tested and all of its metabolites were trackable through LC–MS/MS analysis. These results demonstrate the viability and utility of the constructed NLDM medium for growing and characterizing diverse microbial isolates and communities. [ABSTRACT FROM AUTHOR]

Several studies have shown the ability of yeast to consume peptides as a nitrogen source in single-peptide containing media. However, a suitable and cost-effective methodology to study the utilization of peptides by yeast and other microorganisms in a complex peptide mixture has yet to be put forward. This article addresses this issue by presenting a screening methodology for tracking the consumption of peptides by yeast during alcoholic fermentation. As a peptide source, the methodology makes use of an in-house prepared peptide-mapped bovine serum albumin (BSA) proteolytic digest, which was applied to a synthetic grape must. The peptide uptake was analyzed using high-throughput ultra-high-pressure liquid chromatography coupled to data-independent acquisition-based ion mobility separation-enabled high-resolution mass spectrometry (UPLC-DIA-IMS-HRMS) analysis. The relative changes of abundance of 123 di- to hexapeptides were monitored and reported during fermentations with three commercial wine strains, demonstrating different uptake kinetics for individual peptides. Using the same peptide-mapped BSA hydrolysate, the applicability of an untargeted workflow was additionally assessed for peptide profiling in unelucidated matrixes. The comparison of the results from peptide mapping and untargeted analysis experiments highlighted the ability of untargeted analysis to consistently identify small molecular weight peptides on the length and amino acid composition. The proposed method, in combination with other analytical techniques, such as gene or protein expression analysis, can be a useful tool for different metabolic studies related to the consumption of complex nitrogen sources by yeast or other microorganisms. [ABSTRACT FROM AUTHOR]

To assess the stability of Relugolix under stress conditions, identify and characterize the degradation products, it was subjected to hydrolytic and oxidative stress conditions, according to the International Council for Harmonization guideline Q1A (R2). The drug showed degradation in basic and oxidative conditions, while it was stable in acid conditions. Four degradation products were obtained, two from each basic and oxidative degradation, which was separated on a C18 column by using Auto Purification Mass Spectrometer. The four degradant products have not been reported earlier in any of the literature and are being reported here for the first time. Present work captures force degradation of Relugolix under acidic, basic, oxidative, photolytic, and thermal conditions as per International Council for Harmonization guidelines. Two degradation products were observed in basic condition marked as Degradation Product-1, Degradation Product-2 and two degradation products were formed in oxidative condition marked as Degradation Product-3, Degradation Product-4. All the four degradants are novel and are not published in any of the literature. Four degradation products were identified and characterized using various high-end analytical techniques such as Liquid Chromatography and Mass Spectrometer, Reversed Phase–High Performance Liquid Chromatography, High Resolution Mass Spectrometer and Nuclear Magnetic Resonance spectroscopy. [ABSTRACT FROM AUTHOR]

Lotus rhizome starch (LRS) is an important traditional processed product of lotus rhizome in China due to its good taste and high nutritional value. However, adulterated lotus rhizome starches are prevalent on the market. In this study, a novel method for the rapid detection of adulteration in LRS based on color reaction between ferrous sulfate (FS) and (-)-gallocatechin (GC) in LRS was studied. The polyphenols in LRS were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) as GC and (+)-catechin, and GC could show a bluish color with FS solution. LRS showed a bluish color when FS solution (0.01 mol/L) was used as the chromogenic agent, which was not observed for other ten kinds of starches tested in this work. There was an obvious color gradient when FS solution was used to react with adulterated LRS at different proportions, and the change in blue color was obvious when maize starch or cassava starch was adulterated in LRS at the ratio of 6:4. Then, a semi-quantitative method was used to make a color card according to the color gradient, which was then successfully used to detect the adulteration of four kinds of commercial LRS. The results indicate that the proposed method may facilitate the simple, effective and efficient detection of adulteration in LRS. [ABSTRACT FROM AUTHOR]

Adulteration/unintentional contamination of milk with melamine could have negative health and economic implications especially in the developing countries due to insufficient laboratory support and surveillance. This paper presents an Isotope Dilution Liquid Chromatography-Mass Spectrometry (ID LC-MS) method developed for detection of melamine in powdered milk. The rapid sample preparation involved dissolution of 1g of milk powder in 2.5% formic acid, precipitation of protein with acetonitrile, spiking of samples with melamine (triamine-15N3) at 200 µg L–1 and detection of intrinsic 14N-melamine molecular ratio to the spike. The isotope dilution calibration procedure was free from matrix effects, unlike other methods where the detector sensitivity can fluctuate up to several orders of magnitude. Limit of detection of the method was 13 µg kg–1, and the recovery of melamine at 50, 100, and 250 µg kg–1 was 78.7–126.3%. The method was used to determine melamine levels in 22 milk powder products (local and imported) available in Sri Lanka. Melamine was detected in all the samples (range = 0.33–0.96 mg kg–1). Full cream milk powders (both local and imported) contained melamine in the range of 0.39–0.84 mg kg–1, and various health and pregnancy formulas contained <0.5 mg kg–1 of melamine. Two imported infant formula samples contained the highest levels of melamine (0.96 and 0.94 mg kg–1). Although these melamine levels are below the regulatory limit in Sri Lanka (1 mg kg–1), a monitoring programme would ensure consumer safety. [ABSTRACT FROM AUTHOR]

Urine and plasma biomarker testing for lysosomal storage disorders by liquid chromatography mass spectrometry (LC-MS) currently requires multiple analytical methods to detect the abnormal accumulation of oligosaccharides, mucopolysaccharides, and glycolipids. To improve clinical testing efficiency, we developed a single LC-MS method to simultaneously identify disorders of oligosaccharide, mucopolysaccharide, and glycolipid metabolism with minimal sample preparation. We created a single chromatographic method for separating free glycans and glycolipids in their native form, using an amide column and high pH conditions. We used this glycomic profiling method both in untargeted analyses of patient and control urines using LC ion-mobility high-resolution MS (biomarker discovery), and targeted analyses of urine, serum, and dried blood spot samples by LC-MS/MS (clinical validation). Untargeted glycomic profiling revealed twenty biomarkers that could identify and subtype mucopolysaccharidoses. We incorporated these with known oligosaccharide and glycolipid biomarkers into a rapid test that identifies at least 27 lysosomal storage disorders, including oligosaccharidoses, mucopolysaccharidoses, sphingolipidoses, glycogen storage disorders, and congenital disorders of glycosylation and de-glycosylation. In a validation set containing 115 urine samples from patients with lysosomal storage disorders, all were unambiguously distinguished from normal controls, with correct disease subtyping for 88% (101/115) of cases. Glucosylsphingosine was reliably elevated in dried blood spots from Gaucher disease patients with baseline resolution from galactosylsphingosine. Glycomic profiling by liquid chromatography mass spectrometry identifies a range of lysosomal storage disorders. This test can be used in clinical evaluations to rapidly focus a diagnosis, as well as to clarify or support additional gene sequencing and enzyme studies. [ABSTRACT FROM AUTHOR]

• TOF quantified in 38 of 40 sample locations. • TOF captures up to 98 % more organic fluorine than target LC-MS/MS. • High TOF does not correlate to high quantified PFAS concentrations and vice-versa. Per- and polyfluoroalkyl substances (PFAS) are environmental contaminants of concern due to their long persistence in the environment, toxicity, and widespread presence in humans and wildlife. Knowledge regarding the extent of PFAS contamination in the environment is limited due to the need for analytical methods that can reliably quantify all PFAS , since traditional target methods using liquid chromatography (LC)-mass spectrometry (MS) fail to capture many. For a more comprehensive analysis, a total organic fluorine (TOF) method can be used as a screening tool. We combined TOF analysis with target LC-MS/MS analysis to create a statewide PFAS hotspot map for surface waters throughout South Carolina. Thirty-eight of 40 locations sampled contained detectable concentrations of organic fluorine (above 100 ng/L). Of the 33 target PFAS analyzed using LC-MS/MS, the most prevalent were perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluoroheptanoic acid (PFHpA), and perfluorohexanesulfonate (PFHxS). On average, LC-MS/MS only accounted for 2 % of the TOF measured. Locations with high TOF did not necessarily correlate to high total quantified PFAS concentrations and vice-versa, demonstrating the limitations of target PFAS analysis and indicating that LC-MS may miss highly contaminated sites. Results suggest that future surveys should utilize TOF to more comprehensively capture PFAS in water bodies. [Display omitted] [ABSTRACT FROM AUTHOR]

Curcuma mangga is a medicinal plant, and its rhizomes are often used to treat various conditions, such as fever, thorax pain, itching, stomachaches, skin diseases, gout, and asthma. Although C. mangga is commonly used, information on the relationship between its chemical constituents and the bioactivities of the rhizomes is still limited. The extraction solvents used have a strong effect on the metabolite profile and the bioactivity of the extract. A nuclear magnetic resonance (NMR)-based metabolomics approach was used to differentiate themetabolite profiles of hexane, chloroform, ethyl acetate, and methanol fractions of C. mangga rhizomes and to correlate the metabolites with a-glucosidase inhibitory activity. Primary and secondary metabolites were identified, including curcuminoids, carbohydrates, terpenoids, and amino acids. The ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis of the most active fraction (ethyl acetate) revealed the identification of additional metabolites, such as zerumin A, epigallocatechin, p-hydroxycinnamic, and copallic acids. A partial least square (PLS) biplot demonstrated that the existence of curcumin, demethoxycurcumin, curcumanggoside, calcaratarin A, labda-8(17),12-diene-15,16-dial, zerumin B, and difurocumenonol in the ethyl acetate fraction could be responsible for the a-glucosidase inhibitory activity. [ABSTRACT FROM AUTHOR]

Purpose: We aimed to investigate the association between nocturia and serum metabolites identified using metabolomics analysis. Methods: This study enrolled 66 men aged 65–80 years, recruited from the outpatient department of a university hospital. The participants were stratified as follows: Nocturia group [45 men with any total international prostate symptom score (IPSS) and an average of 3 nights ≥ 1.5 micturitions/night] and Control group (21 men with total IPSS < 8 and an average of 3 nights < 1.5 micturitions/night). The 24-h frequency–volume chart, IPSS, and Quality-of-Life questionnaire were used to evaluate micturition behavior. Serum metabolite profiles were obtained using liquid chromatography–mass spectrometry (LC–MS)-based metabolomics analysis and compared between the two groups using the unpaired t test. The relationship between serum metabolites and nocturia was determined using multivariable logistic regression analysis. Results: There were no differences in background factors between the Nocturia and Control groups. In the IPSS, mean total scores in the Nocturia and Control groups were 12.4 and 4.0, respectively. On frequency–volume chart analysis, nocturnal urine volume and micturition frequency during daytime and nighttime were significantly higher in the Nocturia group. LC–MS highlighted 13 serum metabolites as potential biomarkers of nocturia. On multivariate analysis, increased levels of palmitoylethanolamide, 4-hydroxydocosahexaenoic acid, 9-hydroxyoctadecadienoic acid, 20-hydroxydocosahexaenoic acid, 13-hydroxyoctadecadienoic acid, arachidonoylethanolamide, eicosapentaenoic acid, 12-hydroxy-eicosatetraenoic acid, and arachidonic acid were associated with nocturia. Conclusions: In aged men, the pathogenesis of nocturia involves abnormal metabolism in several signaling pathways involving omega-3 and omega-6 polyunsaturated fatty acids, as well as endocannabinoids. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Henan Agricultural Sciences is the property of Editorial Board of Journal of Henan Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Over several millennia, various native plant species in South America have been used for their healing and psychoactive properties. Chemical analysis of archaeological artifacts provides an opportunity to study the use of psychoactive plants in the past and to better understand ancient botanical knowledge systems. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze organic residues from a ritual bundle, radiocarbon dated to approximately 1,000 C.E., recovered from archaeological excavations in a rock shelter located in the Lípez Altiplano of southwestern Bolivia. The site is located at an elevation of ~3,900 m above sea level and contains evidence of intermittent human occupations during the last 4,000 years. Chemical traces of bufotenine, dimethyltryptamine, harmine, and cocaine, including its degradation product benzoylecgonine, were identified, suggesting that at least three plants containing these compounds were part of the shamanic paraphernalia dating back 1,000 years ago, the largest number of compounds recovered from a single artifact from this area of the world, to date. This is also a documented case of a ritual bundle containing both harmine and dimethyltryptamine, the two primary ingredients of ayahuasca. The presence of multiple plants that come from disparate and distant ecological areas in South America suggests that hallucinogenic plants moved across significant distances and that an intricate botanical knowledge was intrinsic to pre-Columbian ritual practices. [ABSTRACT FROM AUTHOR]

Abstract In the present work, a commercial metal-organic framework (Basolite® F300) has been applied for the first time as dispersive solid-phase extraction (dSPE) sorbent of eight phthalic acid esters (PAEs) (butyl benzyl phthalate, bis-2- n -butoxyethyl phthalate, bis-isopentyl phthalate, bis- n -pentyl phthalate, dicyclohexyl phthalate, di-(2-ethylhexyl) phthalate -DEHP-, di- n -octyl phthalate and diisononyl phthalate) and an adipate (bis (2-ethylhexyl) adipate), which is utilized as a PAE substitutive. Milli-Q, pond, tap and waste water were selected as samples. Dihexyl phthalate-3,4,5,6-d 4 was used as procedural internal standard. High-performance liquid chromatography mass spectrometry was used for the determination of the target compounds. Optimum dSPE conditions were the following: 50 mL of the water sample (pH 6.0), 120 mg of sorbent and 15 mL of ACN as elution solvent. For the validation of the method, matrix-matched calibration and trueness of the method were addressed, obtaining determination coefficients (R2) higher than 0.9905 and relative recovery values between 70 and 118 % (RSDs < 20 %). The limits of quantification of the method were in the range 22–69 ng/L. Matrix effects were also studied. Different real samples were analysed, finding DEHP in all of them at the low µg/L level. Graphical abstract fx1 Highlights • Basolite® F300 metal-organic framework was used for the dSPE of phthalates from water. • LC-IT-MS was used as separation and detection technique. • Recovery values between 70 and 118 % (RSDs < 20 %) were obtained in all cases. • The limits of quantification of the method were in the range 22–69 ng/L. • The analysis of real samples revealed the presence of DEHP at the low µg/L level. [ABSTRACT FROM AUTHOR]

NPPs (Non-phthalate plasticizers) are used as alternative plasticizers to phthalate esters, but there is limited knowledge on environmental residues, and they have not been reported in Japan. A method to analyze NPPs in seawater using solid-phase extraction was developed, and the residual burden of Diisobutyl adipate (DIBA), Acetyl tributyl citrate (ATBC), Di-(2-ethylhexyl) adipate (DEHA), Di-(2-ethylhexyl) sebacate (DEHS) and Trioctyl trimellitate (TOTM) in seawater and sediment from the Osaka Bay was measured. Using an Oasis Max column and acetone as the eluting solvent, the recovery of the target substances in seawater is >68 %. In Osaka Bay, no NPPs were detected in seawater. On the other hand, ATBC and TOTM were detected in the sediment at 36–69 ng/g and 47–131 ng/g, respectively, from about half of the 14 sites, while DEHA and DEHS were detected at 83 ng/g and 181 ng/g, respectively, from only one site. [Display omitted] • A simple SPE method for NPPs in seawater was established. • The recovery of NPPs in seawater and sediment was >63 %. • Target PPs and NPPs were DCHP, DEHP, DIBA, ATBC, DEHA, DEHS and TOTM. • ATBC, DEHA, DEHS, and TOTM were not detected in seawater but found in sediment. • This is the first study on NPPs in Japan. [ABSTRACT FROM AUTHOR]

• A new method for the analysis of advanced glycation end products (AGEs) is proposed. • Dansyl derivatization facilitates the separation and identification of 14 AGEs. • LODs of 1.0–43.3 ng/mL and linear ranges of two orders of magnitude are achieved. • The matrix had an insignificant effect on the recovery rate and slope changes. • The proposed method accurately determines the AGEs in foods and glycated proteins. Herein, new pre-column derivatization based on dansylation is present to resolve analytical difficulties, such as chromatographic separation difficulty, in identifying and quantifying advanced glycation end products (AGEs) owing to their high hydrophilicity, wide variety, and structural similarity. The proposed analytical method facilitated the separation of 14 AGEs, including structural isomers. Limits of detection of 1.0–43.3 ng/mL and linear ranges of the double- or triple-digit were achieved. Intra- and inter-day precisions of 1.1–3.0% and 1.3–3.1%, respectively, were achieved for standard solutions, while those for food specimens were 1.4–11.2% and 1.7–15.7%, respectively. The matrix effect was insignificant with regard to the percent recoveries and differences between slopes for both the standard solutions and food specimens. Furthermore, the quantitation results of AGEs in foods (coffee, beer, and sausage) and glycated proteins revealed the potential applicability of the developed method in various fields of food chemistry and biochemistry. [ABSTRACT FROM AUTHOR]

Using a non-invasive biofluid (saliva), we apply a powerful metabolomics workflow for unbiased biomarker discovery in Alzheimer's disease (AD). We profile and differentiate Cognitively Normal (CN), Mild Cognitive Impairment (MCI), and AD groups. The workflow involves differential chemical isotope labeling liquid chromatography mass spectrometry using dansylation derivatization for in-depth profiling of the amine/phenol submetabolome. The total sample (N = 109) was divided in to the Discovery Phase (DP) (n = 82; 35 CN, 25 MCI, 22 AD) and a provisional Validation Phase (VP) (n = 27; 10 CN, 10 MCI, 7 AD). In DP we detected 6,230 metabolites. Pairwise analyses confirmed biomarkers for AD versus CN (63), AD versus MCI (47), and MCI versus CN (2). We then determined the top discriminating biomarkers and diagnostic panels. A 3-metabolite panel distinguished AD from CN and MCI (DP and VP: Area Under the Curve [AUC] = 1.000). The MCI and CN groups were best discriminated with a 2-metabolite panel (DP: AUC = 0.779; VP: AUC = 0.889). In addition, using positively confirmed metabolites, we were able to distinguish AD from CN and MCI with good diagnostic performance (AUC > 0.8). Saliva is a promising biofluid for both unbiased and targeted AD biomarker discovery and mechanism detection. Given its wide availability and convenient accessibility, saliva is a biofluid that can promote diversification of global AD biomarker research. [ABSTRACT FROM AUTHOR]

A novel strategy for discriminating genuine and adulterated marine oils is proposed. The strategy consists of i ) determining the stereospecific distribution ( sn -1, sn -2 and sn -3) of omega 3 polyunsaturated fatty acids (ω−3 PUFA) on the backbone of triacylglycerols by using liquid chromatography tandem mass spectrometry; ii ) transforming the qualitative stereospecific information into quantitative data by means of a novel strategy; iii ) analyzing the transformed data by principal component analysis. The proposed strategy was tested on pure oils (seal, salmon, cod liver, sandeel, blue whiting, herring), a mixture of blue whiting, herring, sandeel and Norway pout and some intentionally adulterated oils. In addition, some published krill oil data were analyzed to confirm the reliability of the new approach. [ABSTRACT FROM AUTHOR]

Wastewater-based epidemiology (WBE) is used as a surveillance tool to provide data on public health, drug use, exposure to chemicals and lifestyle habits. This approach can be used to monitor the achievement of the United Nations Sustainable Development Goals, such as reducing alcohol consumption and reducing the use of tobacco. In addition, it can be applied to monitor total caffeine consumption and assess levels according to the recommended daily safety limit as suggested by the European Food Safety Authority. The purpose of this review was to summarize and discuss analytical protocols for the determination of alcohol, nicotine, and caffeine WBE biomarkers in wastewater. The WBE requirements for these biomarkers were thoroughly investigated. Furthermore, back-calculated levels of alcohol, nicotine, and caffeine in the population were reviewed. Finally, challenges were identified, and future directions were proposed to fill knowledge gaps and improve the quality of information obtained from the WBE approach. • All steps of analytical methods and WBE biomarker requirements are critically discussed. • Caffeine consumption was lower than the safe daily intake recommended by EFSA. • Studies are focused on temporal and spatial trends in developed countries. • Achievement of the UN SDG on alcohol and tobacco consumption. • Proposals are made to fill current gaps and improve the quality of data obtained by WBE. [ABSTRACT FROM AUTHOR]

Introduction: Endometriosis is an estrogen-dependent gynecological disease that causes infertility, and potential metabolomic biomarkers related to ovarian endometriosis and poor outcomes after assisted reproductive treatments are still lacking. Objectives: The present study analyzed the metabolomic profiling of follicular fluid samples from 40 patients undergoing in vitro fertilization. Methods: The follicular fluid samples were classified as controls (n = 22) and endometriosis patients (n = 18). The samples were submitted to Bligh and Dyer protocol followed by metabolomics analysis by ultra-performance liquid chromatography mass spectrometry. Clinical data was assessed by Students' T-test and metabolomics data was analyzed by multivariate statistics by MetaboAnalyst 3.0 to obtain intrinsic characteristics that allowed for groups discrimination. The Receiver Operating Characteristic curve was carried out for the proposed biomarkers, aiming to determine their specificity and sensitivity, as a set and individually. Results: From the metabolomic analysis, 20 ion masses were selected as potential biomarkers from principal component analysis, which showed that all biomarkers were more abundant in the endometriosis group when compared to controls. Tentative attribution was performed by lipid maps database, demonstrating that these potential biomarkers correspond to fatty acids, carnitines, monoacylglycerols, lysophosphatidic acids, lysophosphatidylglycerols, diacylglycerols, lysophosphatidylcholines, phosphatidylserine, lysophosphatidylinositols and Phosphatidic Acid. Conclusion: The use of mass spectrometry-based metabolomics allowed for the identification of effective biomarkers for ovarian endometriosis, which may contribute for a better comprehension of the disease and how it affects the ovary, as well as assisting in the development of accessory tools for endometriosis diagnosis and infertility management. [ABSTRACT FROM AUTHOR]

In the past decade, various lipidomics methodologies have been developed using mass spectrometry based analytical technologies, enabling wide coverage lipid detection in a quantitative manner. Hence, lipidomics has become a widely-accepted approach for biomarker discovery and mechanism elucidation in both medical and biology research fields; however, there are still technical challenges. In this study, focusing on the sample preparation procedure, a single step deproteinization by a water-soluble organic solvent, such as methanol (MeOH), ethanol (EtOH), isopropanol (IPA) or acetonitrile (ACN), was evaluated and proved to be satisfactory for lipidomics analysis. Moreover, during this investigation ACN deproteinization was revealed to not be an effective method for lipid extraction because lipid decomposition was observed during the protein precipitation process through lipase activation, potentially due to the insufficient protein denaturation. Therefore, excluding ACN, protein precipitation by alcohol was evaluated as the lipid extraction reagent. Moreover, adding the MTBE-MeOH (mMM) method, one of the major liquid-liquid extraction methods for shotgun lipidomics, these four approaches were compared. Lipids were extracted from mouse plasma by these four methods and used for exhaustive lipid profiling by liquid chromatography mass spectrometry (LC/MS) analysis. Comparison of these four methods revealed that alcohol based protein precipitation was a useful sample preparation procedure for LC/MS based lipidomics analysis. Whereas MeOH extraction was appropriate for hydrophilic lipid species, IPA was effective for hydrophobic lipids such as triacylglycerols (TG). In practice, EtOH extraction is thought to be the best approach to cover wide range of lipid species using a simple preparation procedure. [ABSTRACT FROM AUTHOR]

Despite their advantages, there is concern that single-use systems used in biopharmaceutical manufacture might release potentially toxic substances during standard unit operations that negatively impact cell growth. Characterization of the extractables profile for single-use systems is necessary to know which compounds potentially become leachables under operational cell culture conditions. A key issue in the design of extractables studies is the composition of the model solvent, in particular its pH and polarity. In this study, a new approach, based on design of experiments (DoE), has been applied to determine the composition of the model solvent for extractable profiling of single-use bags (SUBs). Particular focus was placed on the determination of the degradation products of the antioxidant Irgafos 168 ® , due to evidence that some of these degradation products have cytotoxic effects on CHO cells. Results indicated that 2-propanol:water is the most appropriate solvent for the extraction of highly hydrophobic compounds with polar groups and/or acid-base properties from SUBs. The described DoE approach simplifies the number of experiments, evaluates all possible solvent water mixtures to select the best extraction solvent based on polarity, establishes the influence of each variable and provides information about variable interaction, which represents an important improvement over current best practice. The developed approach was applied to seven SUBs from different vendors and production dates facilitating the identification of potentially non-satisfactory films for cultivation of CHO cell lines under process conditions. [ABSTRACT FROM AUTHOR]

This review describes published high performance liquid chromatography/mass spectrometry (HPLC–MS) methods for the determination of anticancer drugs in human urine as non-invasive tool for monitoring of health care worker exposure to antineoplastic and cytotoxic drugs. HPLC–MS is a sensitive and specific method for analysis of anticancer drugs and their metabolites in biological fluids. In this review, a tabular summary and overview of published HPLC–MS methods are presented, as well as future trends and limitations in this area of research. [ABSTRACT FROM AUTHOR]

The performance of two derivatization and ionization techniques for the quantitative reversed phase liquid chromatography (LC)- mass spectrometry (MS) analysis of hydroxy fatty acids (OH-PUFA) in plasma was evaluated: One used AMPP ( N -(4-aminomethylphenyl)pyridinium chloride) leading to a positive charged amid-derivate which can be detected by electrospray ionization (ESI)-MS. Second yielded penta fluorobenzyl bromide (PFB) ester derivates allowing detection in electron capture atmospheric pressure chemical ionization (ecAPCI)-MS. The sensitivity of detection of a comprehensive set of hydroxy fatty acids of n6- and n3- poly unsaturated fatty acids was investigated. On the SCIEX3200 MS the applied PFB derivatization led to poor limits of detection (LOD) of 10–100 nM (0.1–1 pmol/0.03–0.3 ng on column). By contrast, AMPP derivatization led to a similar sensitivity compared to the standard ESI(-) of non derivatized analytes (LOD about 1 nM (10 fmol/3 pg on column)). For several analytes, including 9-HETE, 11-HETE and 17-HDHA the AMPP derivatization improved sensitivity enabling their detection in human plasma. However, precision was reduced by AMPP derivatization and variation in IS recovery indicated a strong matrix influence on the MS-signal. In sum, with the instrumentation used, neither of these derivatization methods improves in our hands the LC–MS based quantification of oxylipins. [ABSTRACT FROM AUTHOR]

Heparin is a structurally complex, polysaccharide anticoagulant derived from livestock, primarily porcine intestinal tissues. Low molecular weight (LMW) heparins are derived through the controlled partial depolymerization of heparin. Increased manufacturing and regulatory concerns have provided the motivation for the development of more sophisticated analytical methods for determining both their structure and pedigree. A strategy, for the comprehensive comparison of parent heparins and their LMW heparin daughters, is described that relies on the analysis of monosaccharide composition, disaccharide composition, and oligosaccharide composition. Liquid chromatography-mass spectrometry is rapid, robust, and amenable to automated processing and interpretation of both top-down and bottom-up analyses. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues and glucosamine substitution. Principal component analysis (PCA) was applied to the normalized abundance of oligosaccharides, calculated in the bottom-up analysis, to show parent and daughter correlation in oligosaccharide composition. Using these approaches, six pairs of parent heparins and their daughter generic enoxaparins from two different manufacturers were comprehensively analyzed. Enoxaparin is the most widely used LMW heparin and is prepared through controlled chemical β-eliminative cleavage of porcine intestinal heparin. Lovenox ® , the innovator version of enoxaparin marketed in the US, was analyzed as a reference for the daughter LMW heparins. The results, show similarities between LMW heparins from two different manufacturers with Lovenox ® , excellent lot-to-lot consistency of products from each manufacturer, and detects a correlation between each parent heparin and daughter LMW heparin. [ABSTRACT FROM AUTHOR]

Sludge is one of the most problematic residues generated during wastewater treatment. Herein, we validate a single-step, sensitive procedure for the determination of a selection of 46 basic micro-pollutants, either used as pharmaceuticals or pesticides, in sludge from municipal sewage treatment plants (STPs), using liquid chromatography tandem mass spectrometry as determination technique. The proposed method permitted to achieve accurate recoveries (values from 70% to 120%, for samples spiked at different concentration levels) using solvent-based calibration standards. This feature, combined with limits of quantification lower than 5 ng g−1 (dry weight), allowed the rapid and sensitive quantification of target compounds in freeze-dried sludge samples. Out of 46 investigated pollutants, 33 species showed detection frequencies above 85% in a group of 48 sludge samples, obtained from 45 STPs located in the Northwest of Spain. The assessment of eco-toxicological risks associated to sludge disposal as fertilizer in agriculture and/or forestry, considering average concentrations found in sludge samples, highlighted eight pollutants (sertraline, venlafaxine, N-desethyl amiodarone, amiodarone, norsertraline, trazodone, amitriptyline and ketoconazole) representing an environmental hazard based on ratios between predicted soil levels and non-effect concentrations estimated using the equilibrium partition method. [Display omitted] • Single-step, effective extraction of basic micro-pollutants from sludge. • Accurate recoveries using solvent-based calibration standards. • Detection frequencies above 85% for 33 compounds in sludge from 45 STPs. • Eight species highlighted as hazardous based on their RQs for sludge-amended soil. [ABSTRACT FROM AUTHOR]

Charge variants, especially acidic charge variants, in recombinant monoclonal antibodies are critical quality attributes, which can affect antibodies' properties in vitro and in vivo. Meanwhile, charge variants are cumulative effects of various post-translational modifications and chemical degradations on antibody. In this work, to investigate the effect of lowering culture pH in the stationary phase on acidic charge variant contents in fed-batch cultures and its mechanism, cell culture experiments in 2-L bioreactors were firstly performed to explore the changes in the charge distribution under the pH downshift condition using weak cation exchange chromatography. It is found that acidic charge variant contents were significantly decreased by pH downshift. Then, to reveal the mechanism by which the content of acidic charge variants is reduced under pH downshift condition, the variation of post-translational modifications and chemical degradations under the pH downshift condition was explored. Meanwhile, the structure of the acidic charge variants was characterized. Several analysis experiments including size exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate under non-reducing conditions, tryptic peptide map, and reduced antibody mass were applied in this study. The results show that the mechanism by which the content of acidic charge variants is reduced is that the contents of disulfide bond reduction, galactosylation, and asparagine deamination of the HC-N388 in the Fc domain were reduced by pH downshift. [ABSTRACT FROM AUTHOR]

Background: Metabolomics is increasingly recognized as an invaluable tool in the biological, medical and environmental sciences yet lags behind the methodological maturity of other omics fields. To achieve its full potential, including the integration of multiple omics modalities, the accessibility, standardization and reproducibility of computational metabolomics tools must be improved significantly. Results: Here we present our end-to-end mass spectrometry metabolomics workflow in the widely used platform, Galaxy. Named Galaxy-M, our workflow has been developed for both direct infusion mass spectrometry (DIMS) and liquid chromatography mass spectrometry (LC-MS) metabolomics. The range of tools presented spans from processing of raw data, e.g. peak picking and alignment, through data cleansing, e.g. missing value imputation, to preparation for statistical analysis, e.g. normalization and scaling, and principal components analysis (PCA) with associated statistical evaluation. We demonstrate the ease of using these Galaxy workflows via the analysis of DIMS and LC-MS datasets, and provide PCA scores and associated statistics to help other users to ensure that they can accurately repeat the processing and analysis of these two datasets. Galaxy and data are all provided pre-installed in a virtual machine (VM) that can be downloaded from the GigaDB repository. Additionally, source code, executables and installation instructions are available from GitHub. Conclusions: The Galaxy platform has enabled us to produce an easily accessible and reproducible computational metabolomics workflow. More tools could be added by the community to expand its functionality. We recommend that Galaxy-M workflow files are included within the supplementary information of publications, enabling metabolomics studies to achieve greater reproducibility. [ABSTRACT FROM AUTHOR]

Synthetic cathinones continue to present a formidable challenge to forensic toxicology laboratories despite the fact that they are often encountered in impaired driving and death investigations. Due to limitations in immunoassay-based screening technologies, many forensic toxicology laboratories must rely on more labor intensive chromatographic-based screening approaches in order to detect these drugs in biological evidence. Solid phase extraction (SPE) and liquid chromatography-quadrupole/time of flight (LC-Q/TOF) mass spectrometry were used to identify twenty-two synthetic cathinones in urine and blood. Target drugs included methcathinone, ethcathinone, pentedrone, buphedrone, 3-fluoromethcathinone (3-FMC), 4-fluoromethcathinone (4-FMC), 4-methylethcathinone (4-MEC), 4-ethylmethcathinone (4-EMC), mephedrone, methedrone, 3,4-dimethylmethcathinone (3,4-DMMC), ethylone, butylone, pentylone, eutylone, methylone, methylenedioxypyrovalerone (MDPV), 4-methylpyrrolidinobutiophenone (MPBP), 3,4-methylenedioxypyrrolidinobutiophenone (MDPBP), α-pyrrolidinopentiphenone (α-PVP), pyrovalerone, and naphyrone. A total of nine deuterated internal standards were employed. Using traditional reversed phase chromatography all positional isomers, including 3-FMC and 4-FMC, were separated in 12 min. The procedure was validated in accordance with the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation. Extraction efficiencies were 84–104% and 81–93% in urine and blood, respectively. Limits of quantitation in both matrices were 0.25–5 ng/mL. Precision, bias and matrix effect were all within acceptable thresholds and the assay was free from more than fifty interferences. The validated method was used to identify cathinones in authentic urine case samples (n = 20) and these results highlight important considerations for cathinone stability and the subsequent interpretation of results. [ABSTRACT FROM AUTHOR]