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The potential of the green macroalga Ulva lactuca is increasingly recognized, not only for its environmental benefits, but also for its applications in various industries, including food, pharmaceuticals, and cosmetics. Given this insight, a comprehensive analysis of the chemical profile of U. lactuca from the Adriatic Sea was carried out. The hydrodistillate, rich in ( Z , Z , Z )-hexadeca-7,10,13-trienal and hexadecanoic acid, underlines its importance for health-related uses, particularly in lipid metabolism and cellular integrity. Fatty acid analysis showed a predominance of palmitic acid and a favorable n-6/n-3 polyunsaturated fatty acid ratio, suggesting that U. lactuca can make a valuable contribution to a balanced diet. In addition, essential amino acids, including leucine, valine, and isoleucine, support its use as a functional ingredient for muscle repair and metabolic health. The ethanol extract contained 56 compounds, including derivatives of fatty acids, phenolic acids, pigments, flavonoids, and steroids. Many of them, such as hexadecasphinganine, azelaic acid, 5-sulfosalicylic acid, and pheophytin a , have proven roles or potentials in promoting human health. These results confirm that U. lactuca is a rich source of bioactive compounds, emphasizing its potential in scientific research and its expanding industrial applications in health, nutrition, and cosmetics.

Magnolia bark is a traditional Chinese medicine used for hypoglycaemia. With the widespread use of Magnolia bark, its resources are facing a serious shortage. To address this issue, a strategy based on high-coverage mass spectrometry (HCMS) and multidimensional chemical-biological analysis (MCBA) was proposed for the comprehensive exploration of Magnolia officinalis which is the main source of Magnolia bark. The strategy is divided into three main steps. In the first step, the stem bark, stem xylem, root bark, root xylem, leaf and rootlet of Magnolia officinalis were comprehensively analyzed using high-coverage mass spectrometry. In the second step, multivariate statistical analysis was used to explore the heterogeneity of the six parts and detect differential chemical components. In the third step, a combination of experimental screening and molecular docking was used to explore α-glucosidase inhibitors from Magnolia officinalis. Multidimensional chemical-biological analysis (MCBA) of Magnolia officinalis was achieved by combining the last two steps. Finally, a total of 103 compounds were identified from the whole plant of Magnolia officinalis. Differential components of stem bark, stem xylem, leaf, root bark, root xylem and rootlet were systematically revealed. A pair of positional isomers, namely magnolol and honokiol, were found to be α-glucosidase inhibitors. The activity of their combination is superior to that of each single compound, indicating that magnolol and honokiol are in a synergistic relationship. This strategy contributes to comprehensive exploitation of functional plants and effective alleviation of resource shortage. This study also provides a research paradigm for other similar traditional Chinese medicinal plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Huanglian Jiedu Decoction (HJD) is a well-known Traditional Chinese Medicine formula that has been used for liver protection in thousands of years. However, the therapeutic effects and mechanisms of HJD in treating drug-induced liver injury (DILI) remain unknown. In this study, a total of 26 genes related to both HJD and DILI were identified, which are corresponding to a total of 41 potential active compounds in HJD. KEGG analysis revealed that Tryptophan metabolism pathway is particularly important. The overlapped genes from KEGG and GO analysis indicated the significance of CYP1A1, CYP1A2, and CYP1B1. Experimental results confirmed that HJD has a protective effect on DILI through Tryptophan metabolism pathway. In addition, the active ingredients Corymbosin, and Moslosooflavone were found to have relative strong intensity in UPLC-Q-TOF-MS/MS analysis, showing interactions with CYP1A1, CYP1A2, and CYP1B1 through molecule docking. These findings could provide insights into the treatment effects of HJD on DILI., (© 2024. The Author(s).)

Introduction: Liuweizhiji Gegen-Sangshen (LGS) oral liquid is a Chinese patent medicine that is widely used for the prevention and treatment of alcoholic liver disease in clinical practice. However, the chemical complexity of LGS has not yet been investigated., Objective: The aim of this study was to rapidly identify chemical constituents of LGS and establish a quality control method based on fingerprint and quantitative analysis., Methodology: A comprehensive strategy was used by combining qualitative analysis by ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and fingerprint analysis by high-performance liquid chromatography with diode array detection (HPLC-DAD)., Results: A total of 162 chemical components in LGS, including 91 flavonoids, 31 organic acids, and 20 phenolic compounds, were identified or preliminarily characterized in both positive and negative ion modes based on the UPLC-Q-TOF-MS results. Of these, 37 were confirmed with the reference standards. In fingerprint analysis, 23 peaks were chosen as common peaks and used to evaluate the similarity of different batches of LGS. Subsequently, a rapid quantification method was optimized and validated for the simultaneous determination of multiple chemical markers in LGS. The validated quantitative method was successfully used to analyze different batches of LGS samples., Conclusion: The proposed comprehensive strategy combining HPLC-DAD fingerprinting and multi-component quantification demonstrated satisfactory results with high efficiency, accuracy, and reliability. This can be used as a reference for the overall quality consistency evaluation of Chinese patent medicines., (© 2024 John Wiley & Sons Ltd.)

The main objective of this study was to investigate the metabolism of miconazole, an azole antifungal drug. Miconazole was subjected to incubation with human liver microsomes (HLM) to mimic phase I metabolism reactions for the first time. Employing a combination of an HLM assay and UHPLC-HRMS analysis enabled the identification of seven metabolites of miconazole, undescribed so far. Throughout the incubation with HLM, miconazole underwent biotransformation reactions including hydroxylation of the benzene ring and oxidation of the imidazole moiety, along with its subsequent degradation. Additionally, based on the obtained results, screen-printed electrodes (SPEs) were optimized to simulate the same biotransformation reactions, by the use of a simple, fast, and cheap electrochemical method. The potential toxicity of the identified metabolites was assessed using various in silico models.

Clinical metabolomics studies often have to cope with limited sample amounts, thus miniaturized liquid chromatography (LC) systems are a promising alternative. Their applicability has already been demonstrated in various fields, including a few metabolomics studies that mainly used reversed-phase chromatography. However, hydrophilic interaction chromatography (HILIC), which is widely used in metabolomics due to its particular suitability for the analysis of polar molecules, has rarely been tested for miniaturized LC-MS analysis of small molecules. In the present work, the suitability of a capillary HILIC (CapHILIC)-QTOF-MS system for non-targeted metabolomics was evaluated based on extracts of porcine formalin-fixed, paraffin-embedded (FFPE) tissue samples. The performance was assessed with respect to the number and retention time span of metabolic features as well as the analytical repeatability, the signal-to-noise ratio and the signal intensity of 16 annotated metabolites from different compound classes. The results were compared with a well established narrow-bore HILIC-QTOF-MS system. Both platforms have detected a similar number of features and performed excellent with respect to retention time stability (median RT span <0.05 min) and analytical repeatability (>75% of features with CV < 20%). The signal areas of all metabolites assessed were increased up to 18-fold by the use of CapHILIC, although the signal-to-noise ratio was only improved for 50% of the metabolites. An even better reproducibility (median CV = 5.2%) and up to 80-fold increase in signal intensity were observed after optimization of CapHILIC conditions for analysis of bile acid standard solutions. Even though the observed improvement for specific bile acids (e.g. taurocholic acid) in biological matrix needs to be evaluated, the platform comparison indicates, that the tested CapHILIC system is particularly suitable for analyses of a less broad metabolite spectrum, and specifically optimized chromatography., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction and Objective: Taking into consideration the challenges of lipid analytics, present study aims to design the best high-throughput workflow for detection and annotation of lipids., Material and Methods: Serum lipid profiling was performed on CSH-C18 and EVO-C18 columns using UHPLC Q-TOF-MS and generated lipid features were annotated based on m/z and fragment ion using different software., Result and Discussion: Better detection of features was observed in CSH-C18 than EVO-C18 with enhanced resolution except for Glycerolipids (triacylglycerols) and Sphingolipids (sphingomyelin)., Conclusion: The study revealed an optimized untargeted Lipidomics-workflow with comprehensive lipid profiling (CSH-C18 column) and confirmatory annotation (LipidBlast)., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In this study, nine forced degradation products of maraviroc were found using chemometric analysis. This antiretroviral drug was subjected to photolytic, oxidative, as well as neutral, basic and acidic hydrolysis stress conditions. Additionally, its electrochemical transformation on platinum, gold and glassy carbon screen-printed electrodes was examined. This study showed that maraviroc is especially susceptible to UVA, H 2 O 2 and electrochemical degradation, while being resistant to neutral and acidic hydrolysis. A cluster analysis showed that the electrochemical transformation, with particular reference to the platinum electrode, is able to partially simulate the forced degradation processes, especially in the context of redox reactions. These findings indicate that the electrochemical methods can be considered as quick and relatively low-cost supplements to the commonly applied forced degradation procedures.

In recent years, metabolomics has emerged as a pivotal approach for the holistic analysis of metabolites in biological systems. The rapid progress in analytical equipment, coupled to the rise of powerful data processing tools, now provides unprecedented opportunities to deepen our understanding of the relationships between biochemical processes and physiological or phenotypic conditions in living organisms. However, to obtain unbiased data coverage of hundreds or thousands of metabolites remains a challenging task. Among the panel of available analytical methods, targeted and untargeted mass spectrometry approaches are among the most commonly used. While targeted metabolomics usually relies on multiple-reaction monitoring acquisition, untargeted metabolomics use either data-independent acquisition (DIA) or data-dependent acquisition (DDA) methods. Unlike DIA, DDA offers the possibility to get real, selective MS/MS spectra and thus to improve metabolite assignment when performing untargeted metabolomics. Yet, DDA settings are more complex to establish than DIA settings, and as a result, DDA is more prone to errors in method development and application. Here, we present a tutorial which provides guidelines on how to optimize the technical parameters essential for proper DDA experiments in metabolomics applications. This tutorial is organized as a series of rules describing the impact of the different parameters on data acquisition and data quality. It is primarily intended to metabolomics users and mass spectrometrists that wish to acquire both theoretical background and practical tips for developing effective DDA methods., (© 2021 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd.)

Pigeonpea ( Cajanus cajan ) is an important crop in semi-arid regions and a significant source of dietary proteins in India. The plant is sensitive to salinity stress, which adversely affects its productivity. Based on the dosage-dependent influence of salinity stress on the growth and ion contents in the young seedlings of pigeonpea, a comparative proteome analysis of control and salt stressed (150 mM NaCl) plants was conducted using 7 days-old seedlings. Among various amino acids, serine, aspartate and asparagine were the amino acids that showed increment in the root, whereas serine, aspartate and phenylalanine showed an upward trend in shoots under salt stress. Furthermore, a label-free and gel-free comparative Q-Tof, Liquid Chromatography-Mass spectrometry (LC-MS) revealed total of 118 differentially abundant proteins in roots and shoots with and without salt stress conditions. Proteins related to DNA-binding with one finger (Dof) transcription factor family and glycine betaine (GB) biosynthesis were differentially expressed in the shoot and root of the salinity-stressed seedlings. Exogenous application of choline on GB accumulation under salt stress showed the increase of GB pathway in C. cajan . Gene expression analysis for differentially abundant proteins revealed the higher induction of ethanolamine kinase ( CcEthKin ), choline-phosphate cytidylyltransferase 1-like ( CcChoPh ), serine hydroxymethyltransferase ( CcSHMT ) and Dof protein ( CcDof29) . The results indicate the importance of, choline precursor, serine biosynthetic pathways and glycine betaine synthesis in salinity stress tolerance . The glycine betaine protects plant from cellular damages and acts as osmoticum under stress condition. Protein interaction network (PIN) analysis demonstrated that 61% of the differentially expressed proteins exhibited positive interactions and 10% of them formed the center of the PIN. Further, The PIN analysis also highlighted the potential roles of the cytochrome c oxidases in sensing and signaling cascades governing salinity stress responses in pigeonpea., Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-021-01116-w., Competing Interests: Conflict of interestThere was not any commercial and financial conflict of interest., (© Prof. H.S. Srivastava Foundation for Science and Society 2021.)

A rapid, simple and generic analytical method has been developed for the analysis of veterinary drugs in pork by a quadrupole time-of-flight mass spectrometry (Q-TOF MS). This method allows for the simultaneous identification, screening and quantitation of 141 veterinary drug residues and metabolites from eighteen different classes. After extraction with acetonitrile/water and clean-up with C 18 cartridges, the samples were analyzed by HPLC-Q-TOF MS. Validation of this method consisted of confirmation of identity, selectivity, linearity, limit of detection (LOD), lowest limit of quantification (LLOQ), matrix effect, recovery, precision and applicability of the method. Identification of the analytes was based on accurate mass measurements. The characteristic fragments were obtained by collisional experiments for a more reliable identification. The procedure was then applied to real pork samples. Sulfamethazine was detected in one sample and its metabolites were successfully found in one single run. This approach proved to be satisfactory for routine analysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ciguatera poisoning (CP) is a common seafood intoxication mainly caused by the consumption of fish contaminated by ciguatoxins. Recent studies showed that Caribbean ciguatoxin-1 (C-CTX1) is the main toxin causing CP through fish caught in the Northeast Atlantic, e.g., Canary Islands (Spain) and Madeira (Portugal). The use of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) combined with neuroblastoma cell assay (N2a) allowed the initial confirmation of the presence of C-CTX1 in contaminated fish samples from the abovementioned areas, nevertheless the lack of commercially available reference materials for these particular ciguatoxin (CTX) analogues has been a major limitation to progress research. The EuroCigua project allowed the preparation of C-CTX1 laboratory reference material (LRM) from fish species ( Seriola fasciata ) from the Madeira archipelago (Portugal). This reference material was used to implement a liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) for the detection of C-CTX1, acquisition of full-scan as well as collision-induced mass spectra of this particular analogue. Fragmentation pathways were proposed based on fragments obtained. The optimized LC-HRMS method was then applied to confirm C-CTX1 in fish ( Bodianus scrofa ) caught in the Selvagens Islands (Portugal).

A procedure was established and fully validated for the screening and quantification of fourteen synthetic antidiabetic adulterants in herbal pharmaceuticals and health foods, including metformin (MF), buformin (BF), phenformin (PHF), rosiglitazone (RGZ), pioglitazone (PGZ), chlorpropamide (CPM), glipizide (GPZ), tolbutamide (TBM), gliclazide (GCZ), glibenclamide (GBM), glimepiride (GMR), repaglinide (RGN), gliquidone (GQD) and nateglinide (NGN). The samples were extracted by methanol and separated by HPLC. Retention times and ultraviolet spectra were used for the preliminary screening, and the suspected adulterants were then confirmed by liquid chromatography-quadrupole-time of flight mass spectrometry (LC-Q-TOF-MS/MS) and quantified by HPLC. The developed procedure was successfully applied to assess twenty-four herbal samples, and PHF, GCZ, GBM, MF, GPZ and BF were found in many. To the best of our knowledge, this is the first report of simultaneous screening and quantification of these fourteen synthetic antidiabetic adulterants from any matrix.

Background: Sugar phosphates are important intermediates of central carbon metabolism in biological systems, with roles in glycolysis, the pentose-phosphate pathway, tricarboxylic acid (TCA) cycle, and many other biosynthesis pathways. Understanding central carbon metabolism requires a simple, robust and comprehensive analytical method. However, sugar phosphates are notoriously difficult to analyze by traditional reversed phase liquid chromatography., Results: Here, we show a two-step derivatization of sugar phosphates by methoxylamine and propionic acid anhydride after chloroform/methanol (3:7) extraction from Populus leaf and developing wood that improves separation, identification and quantification of sugar phosphates by ultra high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS). Standard curves of authentic sugar phosphates were generated for concentrations from pg to ng/μl with a correlation coefficient R 2  > 0.99. The method showed high sensitivity and repeatability with relative standard deviation (RSD) < 20% based on repeated extraction, derivatization and detection. The analytical accuracy for Populus leaf extracts, determined by a two-level spiking approach of selected metabolites, was 79-107%., Conclusion: The results show the reliability of combined reversed phase liquid chromatography-tandem mass spectrometry for sugar phosphate analysis and demonstrate the presence of two unknown sugar phosphates in Populus extracts., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

Baby bottles made of polypropylene, Tritan® and silicone were evaluated regarding the migration of non-volatile compounds using UPLC-QTOF-MS. Twenty-seven compounds were identified. In all polypropylene samples the migration of 2.2'-(tridecylimino)bis-ethanol and derivatives thereof were detected in concentrations below the specific migration limit (1.2 mg.kg -1 ). Furthermore, clarifying agents and glycerol derivatives were detected. Tritan baby bottle showed the migration of one slip additive. On the other hand, twenty compounds were detected in silicone baby bottles. Most of them were unknown compounds derived from acrylates. Once the migrants were identified, the risk assessment was carried out using the Threshold of Toxicological Concern (TTC) approach. The risk assessment of migrants coming from silicone samples showed levels above the threshold recommended as safe for babies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

For the first time, the influence of ionising radiation on the physicochemical properties of ertapenem in solid state was studied. During our studies, we evaluated the possibility of applying radiosterilization to obtain sterile ertapenem. Spectroscopic (Fourier Transform Infrared (FT-IR)), thermal (differential scanning calorimetry (DSC), chromatography (High-Performance Liquid Chromatography (HPLC) and HPLC-MS), and X-ray powder diffraction (XRPD) studies shown that irradiation of ertapenem with the 25 kGy, the dose required to achieve sterility, does not change the physicochemical properties of the studied compound. The antimicrobial activity of ertapenem irradiated with the dose of 25 kGy was only reduced for one species. Based on the received results, we can conclude that radiostelization is a promising alternative method of obtaining sterile ertapenem. In our studies, ertapenem was also exposed to e-beam radiation with a dose of 400 kGy. It was determined that two novel degradation products that are structurally differently to degradants formed during hydrolysis and thermolysis.

Reversed-phase ultrahigh-performance-liquid chromatography-mass spectrometry (UPLC-MS) is the typical method for the lipidomic analysis of most of biological samples, which was rarely used for the comprehensive lipidomic analysis of marine shellfish. Thus, a range of columns, modifiers, and resuspension solvents were evaluated using UPLC-electrospray ionization-quadrupole time-of-flight-MS to facilitate the ionization efficiency in both the positive and negative electrospray ionization (ESI(+)/(-)) modes for abalone lipids. Optimal lipidomic coverage was acquired with 10 mM ammonium formate in both ESI(+)/(-) modes. The selected resuspension solvents also influenced ionization efficiency through the matrix effect, and resuspension in methanol enhanced the signal intensities by reducing ion suppression. Because of the higher glycerophospholipid content in shellfish, bridged ethylene hybrid C 8 columns showed clear advantages over charged surface hybrid C 18 columns. A series of glycerophospholipids, lyso-glycerophospholipids, glycerolipids, and fatty acids in different shellfish can be annotated and semiquantified in one injection by the optimized method.

High-resolution mass spectrometry (HRMS) in full scan mode acquires all ions present in the sample of interest offering a lot of qualitative information. This, in combination with the improved performance of the new generation HRMS systems, triggers more (bio) analysts to switch from triple quad MS systems to HRMS for quantitative analysis. Quantitative processing of HRMS data is performed based on narrow mass extraction windows rather than on nominal mass product ion chromatograms (SRM or MRM). Optimal processing of HRMS data requires different considerations and software tools and can have an impact on data processing and final results. The selection of centroid versus continuum/profile data for processing, selection of the optimal narrow mass extraction window, using theoretical versus measured accurate mass for the extraction of the ion chromatograms as well as differences in calculations and data handling residing in the different vendor software packages are tackled in the presented manuscript. These differences are illustrated on HRMS data acquired for the same plasma samples on three different platforms, i.e., a Sciex QToF, a Waters QToF, and a Thermo Orbitrap system, and processed in four different software packages, i.e., Sciex Analyst® TF, Waters Masslynx, Waters Unifi, and Thermo Xcalibur. The impact of these differences on quantitative HRMS performance was evaluated on calibration curves of eight small molecule compounds in plasma using four different ways of processing. Simple guidelines are provided for the selection of an optimal mass extraction window for continuum and centroided data. Graphical Abstract.

The influence of ionising radiation on the physicochemical properties of meropenem trihydrate in solid state was studied for doses of e-beam radiation: 25 kGy and 400 kGy. In the first part of our studies, we evaluated the possibility of applying radiosterilization to obtain sterile meropenem. No changes for meropenem irradiated with a dose of 25 kGy, the dose required to attain sterility, was confirmed in the results of spectroscopic (FT-IR), thermal (DSC, TGA) and X-ray powder diffraction (XRPD) studies. The radiation dose of 25 kGy produces no more than about 1500 ppm of radical defects. The chromatographic studies of irradiated meropenem in solutions did not show any chemical degradation. Moreover, the antimicrobial activity of meropenem irradiated with the dose of 25 kGy was unchanged. Based on the received results, we can conclude that radiostelization is a promising, alternative method for obtaining sterile meropenem. In the second part of the research, meropenem was exposed to e-beam radiation at the 400 kGy dose rate. It was confirmed, that reducing of antimicrobial activity could be connected with the degradation of β-lactam ring and changes in the trans-hydroxyethyl group. Apart from chemical changes, changes in the physical stability of irradiated meropenem (400 kGy) was also observed.

In this study the comparison of human liver microsomes in in vitro incubation as well as ZnO- and TiO 2 -assisted photocatalytic degradation of clozapine as a mimicking method of phase I metabolism transformation was performed. Based on reversed-phase UHPLC separation and high-resolution MS/MS data, eight transformation products were identified and seven of them were found to be hepatic metabolites of the parent compound. The multivariate chemometric comparison of the obtained results shows ZnO-assisted photocatalysis to be a more suitable approach to phase I metabolism simulation. The photocatalytic experiments demonstrated that the disappearance of clozapine followed pseudo-zero order kinetics., (© 2018 John Wiley & Sons, Ltd.)

Polymeric tannins from grapes have always been reported as an unresolved broad peak in HPLC chromatograms, and this has severely limited their identification to date. This study aimed to disassemble this broad peak and explore the polymeric tannin molecules inside. By applying centrifugal partition chromatography (CPC), an efficient separation approach was developed to split the broad peak of grape seed tannins into fractions. Then, the fractions were analyzed by Q-ToF (quadrupole time-of-flight mass spectrometry) to determine the corresponding structures of the tannins. The results suggest that grape seed polymeric tannins were eluted consecutively according to their degree of polymerization (DP). Condensed tannins identified in wine grape seed have a range of DP and degree of galloylation (DG) up to 20 and 11, respectively. The molecular mass of the largest molecule detected was 6067. To our knowledge, this is the first report to offer an insight into the broad peak of polymeric tannins found with HPLC and to characterize the tannins with a DP up to 20 as shown by HRMS and MS/MS data., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This paper presents a rapid, sensitive and precise method for the determination of metaldehyde in human blood, using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry and high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry. Separation was performed with a Poroshell 120 EC-C18 column; 2.7 μm atrazine‑d5 (IS) and 200 mg NaCl were added to the blood sample. Proteins in human blood were precipitated using acetonitrile; the supernatant was then analyzed with the UHPLC-Q-TOF-MS or HPLC-QqQ-MS/MS system. The results of selectivity, linearity, accuracy, precision, limits of quantification, recovery, and matrix effects were sufficient to enable the measurement of metaldehyde in human blood samples. In addition, we proposed a fragmentation pathway involving ammonium adduct fragment ions for metaldehyde., (Copyright © 2018. Published by Elsevier B.V.)

Forced degradation of toloxatone in solutions under basic, acidic, neutral, photo UV-VIS, photo UVC and oxidative stress conditions was investigated and structural elucidation of its degradation products was performed with the use of UHPLC system coupled ESI-Q-TOF mass spectrometer. Eight degradation products were found and their masses and formulas were obtained with high accuracy (0.09-3.79 ppm). The structure of unknown degradation products were elucidated from MS/MS fragmentation spectra of all analyzed compounds. Additionally, whole signals of decomposed substances were compared chemometrically. It was found that toloxatone is fragile towards basic hydrolysis, oxidative conditions and UVC irradiation. Finally, the toxicity of transformation products was computationally evaluated and compared in multivariate manner.

A procedure for the screening and quantification of 18 glucocorticoids, i.e. hydrocortisone sodium succinate (HSS), prednisolone (PDL), prednisone (PDS), hydrocortisone (HCS), methylprednisolone (MPS), betamethasone (BTM), dexamethasone (DXM), triamcinolone acetonide (TA), prednisolone acetate (PLA), hydrocortisone acetate (HA), fludrocortisone acetate (FA), prednisone acetate (PA), cortisone acetate (CA), dexamethasone acetate (DA), hydrocortisone butyrate (HB), triamcinolone acetonide acetate (TAA), fluocinonide (FN) and halcinonide (HC) from herbal pharmaceuticals and health foods was established and fully validated. The samples were extracted by methanol and separated by HPLC. The retention times and ultraviolet spectra were used for the preliminary screening, and the suspected adulterants were then confirmed by liquid chromatography-quadrupole-time of flight mass spectrometry (LC-Q-TOF-MS/MS) and quantified by HPLC. The developed procedure was successfully applied to 14 herbal samples, and 316.3 µg g -1 of DA and 13.6 µg mL -1 of BTM were found in a tablet sample and a spray sample, respectively. To our best knowledge, this is the first report of the simultaneous screening and quantification of these 18 glucocorticoids from any matrix.

Campylobacter jejuni is the major cause of bacterial gastroenteritis in man, while it is generally regarded as a commensal of the avian gut. Consumption and handling of contaminated poultry meat products are major risk factors for human infection. The body temperature in man (37 °C) and chickens (42 °C) differ markedly, and differential gene regulation and protein expression at different temperatures may in part explain the behaviour in the two hosts. We performed proteomics analyses with C. jejuni cells grown at 37 °C and 42 °C. Time-of-flight mass spectrometry (Q-Tof) analysis was carried out after samples were digested with the Filter-Aided Sample Preparation (FASP) method and peptides were fractionated by strong anion exchanges. Differentially regulated proteins were identified by Mascot and Scaffold analyses. Triple quadrupole (QQQ) mass spectrometer analysis confirmed that a total of 33 proteins were differentially regulated between 37 °C and 42 °C. Several upregulated proteins were selected for their corresponding gene knock-out mutants to be tested for their virulence in the Galleria mellonella model. To correlate with other tissue/animal models, the GADH mutant was selected for its reduced ability to colonize chickens. At 37 °C, the mutants of outer membrane protein Omp50 and Chaperone GroEL significantly increased virulence; while at 42 °C, the mutants of YceI, Omp50, and GADH reduced virulence against Galleria mellonella compared with the wild type strains. The results of current and previous studies indicate that GADH is a virulent factor in G. mellonella and a colonization factor in chickens. The workflow of this study may prove a new way to identify stress related virulent factors. The implications of these findings are discussed for pathogenesis in the model and other hosts., Competing Interests: Conflict of Interest: All authors declare no conflicts of interest in this paper.

The ants use their venom for predation, defense, and communication. The venom of these insects is rich in peptides and proteins, and compared with other animal venoms, ant venoms remain poorly explored. The objective of this study was to evaluate the protein content of the venom in the Ponerinae ant Pachycondyla striata. Venom samples were collected by manual gland reservoir dissection, and samples were submitted to two-dimensional gel electrophoresis and separation by ion-exchange and reverse-phase high-performance liquid chromatography followed by mass spectrometry using tanden matrix-assisted laser desorption/ionization with time-of-flight (MALDI-TOF/TOF) mass spectrometry and electrospray ionization-quadrupole with time-of-flight (ESI-Q/TOF) mass spectrometry for obtaining amino acid sequence. Spectra obtained were searched against the NCBInr and SwissProt database. Additional analysis was performed using PEAKS Studio 7.0 (Sequencing de novo). The venom of P. striata has a complex mixture of proteins from which 43 were identified. Within the identified proteins are classical venom proteins (phospholipase A, hyaluronidase, and aminopeptidase N), allergenic proteins (different venom allergens), and bioactive peptides (U10-ctenitoxin Pn1a). Venom allergens are among the most expressed proteins, suggesting that P. striata venom has high allergenic potential. This study discusses the possible functions of the proteins identified in the venom of P. striata., (© 2017 Wiley Periodicals, Inc.)

Background: Toddalia asiatica root bark as an effective hemostatic natural medicine or Chinese materia medica was applied in China for long history, its complex drug action mechanisms and unclear substance basis have been constraining the development of this drug., Results: An intelligentized strategy by LC-ESI Q-TOF MS E was presented in this study for rapid identification of hemostatic chemical constituents from this natural medicine. Chromatographic separation was performed on a C18 column (150 mm × 2.1 mm, 1.8 μm), the MS E data in both negative and positive ion modes were acquired to record the high-accuracy MS and MS/MS data of all precursor ions. To reduce the false positive identifications, structural confirmation was conducted by comparison with the isolated reference standards (t R and MS, MS/MS data) or matching with natural product databases. Bioassay-guided fractionation of the extract of T. asiatica root bark was also carried out., Conclusions: As a consequence, 31 natural compounds in T. asiatica root bark got putatively characterized. There were four main coumarins, isopimpinellin (Cp.23), pimpinellin (Cp.24), coumurrayin (Cp.30) and phellopterin (Cp.34) isolated and identified from T. asiatica root bark. The present study provided candidate strategy that helps to effectively identify the primary natural compounds of TCM or other complex natural medicines, and then promote development and application of natural medicines and their medicinal resources.

Metabolite profiling of tissue samples is a promising approach for the characterization of cancer pathways and tumor classification based on metabolic features. Here, we present an analytical method for nontargeted metabolomics of kidney tissue. Capitalizing on different chemical properties of metabolites allowed us to extract a broad range of molecules covering small polar molecules and less polar lipid classes that were analyzed by LC-QTOF-MS after HILIC and RP chromatographic separation, respectively. More than 1000 features could be reproducibly extracted and analyzed (CV < 30%) in porcine and human kidney tissue, which were used as surrogate matrices for method development. To further assess assay performance, cross-validation of the nontargeted metabolomics platform to a targeted metabolomics approach was carried out. Strikingly, from 102 metabolites that could be detected on both platforms, the majority (>90%) revealed Spearman's correlation coefficients ≥0.3, indicating that quantitative results from the nontargeted assay are largely comparable to data derived from classical targeted assays. Finally, as proof of concept, the method was applied to human kidney tissue where a clear differentiation between kidney cancer and nontumorous material could be demonstrated on the basis of unsupervised statistical analysis.

Identification of soluble microbial products (SMPs) released during bacterial metabolism in mixed cultures in bioreactors is essential to understanding fundamental mechanisms of their biological production. SMPs constitute one of the main foulants (together with colloids and bacterial flocs) in membrane bioreactors widely used to treat and ultimately recycle wastewater. More importantly, the composition and origin of potentially toxic, carcinogenic, or mutagenic SMPs in renewable/reused water supplies must be determined and controlled. Certain classes of SMPs have previously been studied by GC-MS, LC-MS, and MALDI-ToF MS; however, a more comprehensive LC-MS-based method for SMP identification is currently lacking. Here we develop a UPLC-MS approach to profile and identify metabolite SMPs in the supernatant of an anaerobic batch bioreactor. The small biomolecules were extracted into two fractions based on their polarity, and separate methods were then used for the polar and nonpolar metabolites in the aqueous and lipid fractions, respectively. SMPs that increased in the supernatant after feed addition were identified primarily as phospholipids, ceramides, with cardiolipins in the highest relative abundance, and these lipids have not been previously reported in wastewater effluent.

Forced Degradation of Deflazacort drug substance in ultraviolet light condition resulted into a number of significant degradation products. Two of these degradation products were found to be unknown during the study and marked as DD-I and DD-II. Thus, the objective of this work is to investigate and identify these two novel degradation products of DFZ. The isolation method for these new degradation products were developed using a new reverse-phase high performance liquid chromatography (HPLC). DD-I and DD-II, eluting at 0.53 and 1.57 relative retention times with respect to Deflazacort (DFZ) peak respectively, were isolated from reaction mass using preparative HPLC and their structures were elucidated using high resolution MS, multidimensional NMR and FTIR spectroscopic techniques. To best of our knowledge, these two degradation products are novel impurities which are not discussed in any form of publication yet., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Coptis chinensis Franch is a traditional Chinese medical herb., Objective: In this article, ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to rapidly, qualitatively, and comprehensively identify the components in Coptis chinensis Franch., Materials and Methods: Chromatographic separation was achieved on an Agilent Zorbax RRHD Eclipse Plus C 18 column. The mobile phase consisted of 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B) with a gradient program. Qualitative analysis was performed on an Agilent 6540 quadrupole time-of-flight mass spectrometer, which was equipped with a Dual AJS ESI source operating in negative mode., Results: A total of 30 alkaloid and non-alkaloid components of Coptis chinensis Franch were identified in only 14 min., Conclusion: This study helped to provide a basis for the quality control of Coptis chinensis Franch., Summary: Qualitative analysis method of chlorogenic alkaloids and non-alkaloids in Coptis chinensis Franch is developed by Ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) method.Established UPLC-Q-TOF-MS/MS analysis method is validated with rapidness and accuracy.The developed method was successfully applied for qualitative analysis of Coptis chinensis Franch sample collected from cultivation place in China. Abbreviations used: Q-TOF-MS: quadrupole time-of-flight mass spectrometry, UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative. Q-TOF-MS: quadrupole time-of-flight mass spectrometry, UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative. UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative. pos: positive, neg: negative. neg: negative., Competing Interests: There are no conflicts of interest.

The present study demonstrates that one-step peptide backbone fragmentations can be achieved using the TEMPO [2-(2,2,6,6-tetramethyl piperidine-1-oxyl)]-assisted free radical-initiated peptide sequencing (FRIPS) mass spectrometry in a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer and a Q-Exactive Orbitrap instrument in positive ion mode, in contrast to two-step peptide fragmentation in an ion-trap mass spectrometer (reference Anal. Chem. 85, 7044-7051 (30)). In the hybrid Q-TOF and Q-Exactive instruments, higher collisional energies can be applied to the target peptides, compared with the low collisional energies applied by the ion-trap instrument. The higher energy deposition and the additional multiple collisions in the collision cell in both instruments appear to result in one-step peptide backbone dissociations in positive ion mode. This new finding clearly demonstrates that the TEMPO-assisted FRIPS approach is a very useful tool in peptide mass spectrometry research. Graphical Abstract ᅟ.

World Health Organization estimates that 34 million individuals globally are living with Human Immunodeficiency Virus (HIV). Doravirine is a non-nucleoside reverse transcriptase inhibitors (NNRTI) being evaluated by Merck for the treatment of HIV-1 infection. Drug regulation authorities require the purity of a pharmaceutical to be fully defined. This is important to ensure that the pharmacological and toxicological effects are truly those of the drug substances and not because of the impurities. Thus, understanding the drug impurity profiles is critical to the safety and potency assessment of the drug candidate for clinical trials. The impurity characterization can also provide useful information for critical assessment of pharmaceutical processes. Advances in mass spectrometry instrumentation and methods allow the identification of impurities in pharmaceuticals with a minimum of sample material and increased sensitivity. In this study, a rapid and sensitive method was developed for the structural determination of the major impurities of doravirine. The study utilizes ultra performance liquid chromatography-high-resolution-tandem mass spectrometry (UHPLC-HRMS/MS) techniques to perform structure elucidation of the unknown structures. This approach has significant impact on impurity structural elucidation, and a total of five trace-level impurities of doravirine were characterized using the developed method. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is well-known to be a powerful technique for the analysis of biological samples. By using glycerol-based liquid support matrices (LSMs) instead of conventional MALDI matrices the power of this technique can be extended further. In this study, we exploited LSMs for the identification of complex samples, that is, the Lactobacillus proteome and a bovine serum albumin (BSA) digest. Liquid and solid MALDI samples were manually and robotically prepared by coupling a nanoflow high-performance liquid chromatography (nanoHPLC) system to an automated MALDI sample spotting device. MS and MS/MS data were successfully acquired at the femtomole level using TOF/TOF as well as Q-TOF instrumentation and used for protein identification searching sequence databases. For the BSA digest analysis, liquid MALDI samples resulted in peptide mass fingerprints, which led to a higher confidence in protein identification compared with solid (crystalline) MALDI samples; however, postsource decay (PSD) MS/MS analysis of both the proteome of Lactobacillus plantarum WCFS1 cells and BSA digest showed that further optimization of the formation and detection of peptide fragment ions is still needed for liquid MALDI samples, as the MS/MS ion search score was lower than that for the solid MALDI samples, reflecting the poorer quality of the liquid MALDI-PSD spectra, which can be attributed to the differences in PSD parameters and their optimization that is currently achievable.

Background: Given the excellent performance of modern mass spectrometers, their clinical application for the analysis of macromolecules is a growing field of interest. This principle is explored by hemoglobin analysis, which is a representative example by its molecular weight and clinical relevance in e.g. screening programs for thalassemia and hemoglobin variants. Considering its abundance and cellular containment, pre-analysis is significantly reduced allowing for essential rapid acquisitions., Methods: By parallel analysis of routine diagnostics for hemoglobin variants and thalassemia, we acquired samples of adults who were consented for hemoglobinopathy screening in our clinical laboratory. The pre-analytical process comprised of red cell lysis only; without further digestion and purification steps, the samples were directly injected in an electrospray ionization quadrupole time-of-flight setup and the intact proteins were analyzed by flow injection analysis. After optimization of process parameters, the deconvoluted mass spectra revealed the presence of α- and β-globulins. The reference ranges for the average mass of both globulins and their intensity ratio (α/β-ratio) were deduced from a disease-free subgroup and patients with a hemoglobinopathy were compared., Results: The α/β-ratio is a poor marker for thalassemia patients, yet deviant α/β-ratios are found for patients with a hemoglobin variant. Mass deviations down to 1Da can be resolved; even if the patient suffers from a heterozygotic disorder, the average mass is found outside the established reference interval., Conclusions: Although subjects with mild thalassemia were not detected, all patients with a hemoglobin variant were resolved by top-down mass spectrometry using the average globulin mass and the α/β-ratio as screening parameters., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Since the concept was first introduced by Brian Chait and co-workers in 1991, mass spectrometry of proteins and protein complexes under non-denaturing conditions (native MS) has strongly developed, through parallel advances in instrumentation, sample preparation, and data analysis tools. However, the success rate of native MS analysis, particularly in heterogeneous mega-Dalton (MDa) protein complexes, still strongly depends on careful instrument modification. Here, we further explore these boundaries in native mass spectrometry, analyzing two related endogenous multipartite viruses: the Brome Mosaic Virus (BMV) and the Cowpea Chlorotic Mottle Virus (CCMV). Both CCMV and BMV are approximately 4.6 megadalton (MDa) in mass, of which approximately 1 MDA originates from the genomic content of the virion. Both viruses are produced as mixtures of three particles carrying different segments of the genome, varying by approximately 0.1 MDA in mass (~2%). This mixture of particles poses a challenging analytical problem for high-resolution native MS analysis, given the large mass scales involved. We attempt to unravel the particle heterogeneity using both Q-TOF and Orbitrap mass spectrometers extensively modified for analysis of very large assemblies. We show that manipulation of the charging behavior can provide assistance in assigning the correct charge states. Despite their challenging size and heterogeneity, we obtained native mass spectra with resolved series of charge states for both BMV and CCMV, demonstrating that native MS of endogenous multipartite virions is feasible. Graphical Abstract ᅟ.

In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted glycopeptide assignment of both N- and O-glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-glycopeptides and four out of five O-glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control.

Multiple reaction monitoring (MRM) is a universal approach for quantitative analysis because of its high specificity and sensitivity. Nevertheless, optimization of MRM parameters remains as a time and labor-intensive task particularly in multiplexed quantitative analysis of small molecules in complex mixtures. In this study, we have developed an approach named Stepped MS(All) Relied Transition (SMART) to predict the optimal MRM parameters of small molecules. SMART requires firstly a rapid and high-throughput analysis of samples using a Stepped MS(All) technique (sMS(All)) on a Q-TOF, which consists of serial MS(All) events acquired from low CE to gradually stepped-up CE values in a cycle. The optimal CE values can then be determined by comparing the extracted ion chromatograms for the ion pairs of interest among serial scans. The SMART-predicted parameters were found to agree well with the parameters optimized on a triple quadrupole from the same vendor using a mixture of standards. The parameters optimized on a triple quadrupole from a different vendor was also employed for comparison, and found to be linearly correlated with the SMART-predicted parameters, suggesting the potential applications of the SMART approach among different instrumental platforms. This approach was further validated by applying to simultaneous quantification of 31 herbal components in the plasma of rats treated with a herbal prescription. Because the sMS(All) acquisition can be accomplished in a single run for multiple components independent of standards, the SMART approach are expected to find its wide application in the multiplexed quantitative analysis of complex mixtures., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The possibility of applying radiation sterilization to cefpirome sulfate was investigated. The lack of changes in the chemical structure of cefpirome sulfate irradiated with a dose of 25 kGy, required to attain sterility, was confirmed by UV, FT-IR, Raman, DSC and chromatographic methods. Some radical defects with concentration no more than over a several dozen ppm were created by radiation. The antibacterial activity of cefpirome sulfate for two Gram-positive and three Gram-negative strains was changed. The radiation sterilised cefpirome sulfate was not in vitro cytotoxic against fibroblast cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The influence of pH on the stability of cefozopran hydrochloride (CZH) was investigated in the pH range of 0.44-13.00. Six degradation products were identified with a hybrid ESI-Q-TOF mass spectrometer. The degradation of CZH as a result of hydrolysis was a pseudo-first-order reaction. As general acid-base hydrolysis of CZH was not occurred in the solutions of hydrochloric acid, sodium hydroxide, acetate, borate and phosphate buffers, kobs = kpH because specific acid-base catalysis was observed. Specific acid-base catalysis of CZH consisted of the following reactions: hydrolysis of CZH catalyzed by hydrogen ions (kH+), hydrolysis of dications (k1H2O), monocations (k2H2O) and zwitter ions (k3H2O) and hydrolysis of zwitter ions (k1OH-) and monoanions (k2OH-) of CZH catalyzed by hydroxide ions. The total rate of the reaction was equal to the sum of partial reactions: [Formula: see text]. CZH similarly like other fourth generation cephalosporin was most stable at slightly acidic and neutral pH and less stable in alkaline pH. The cleavage of the β-lactam ring resulting from a nucleophilic attack on the carbonyl carbon in the β-lactam moiety is the preferred degradation pathway of β-lactam antibiotics in aqueous solutions.

The process of degradation was studied by using an HPLC-DAD method. Two degradation products were identified with a hybrid ESI-Q-TOF mass spectrometer. The influence of temperature and relative air humidity (RH) on the stability of cefoselis sulfate was investigated. In the solid state at increased RH the degradation of cefoselis sulfate was an autocatalytic reaction of the first order with respect to substrate concentration while in dry air was first-order reaction depending on the substrate concentration. The kinetic and thermodynamic parameters of degradation were calculated., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A new model is presented that can be used to screen for bioactivation of drugs. The evaluation of toxicity is an important step in the development of new drugs. One way to detect possible toxic metabolites is to use trapping agents such as glutathione. Often human liver microsomes are used as a metabolic model in initial studies. However, there is a need for alternatives that are easy to handle, cheap, and can produce large amounts of metabolites. In the presented study, paracetamol, mefenamic acid, and diclofenac, all known to form reactive metabolites in humans, were incubated with the fungus Cunninghamella elegans and the metabolites formed were characterized with ultra high performance liquid chromatography coupled to a quadrupole time of flight mass spectrometer. Interestingly, glutathione conjugates formed by the fungus were observed for all three drugs and their retention times and MS/MS spectra matched those obtained in a comparative experiment with human liver microsomes. These findings clearly demonstrated that the fungus is a suitable trapping model for toxic biotransformation products. Cysteine conjugates of all three test drugs were also observed with high signal intensities in the fungal incubates, giving the model a further indicator of drug bioactivation. To our knowledge, this is the first demonstration of the use of a fungal model for the formation and trapping of reactive drug metabolites. The investigated model is cheap, easy to handle, it does not involve experimental animals and it can be scaled up to produce large amounts of metabolites., (Copyright © 2014 John Wiley & Sons, Ltd.)

Glycolipids are mainly found in phototrophic organisms (like plants and cyanobacteria), in Gram-positive bacteria, and a few other bacterial phyla. Besides the function as bulk membrane lipids, they often play a role under phosphate deprivation as surrogates for phospholipids. The Gram-negative Agrobacterium tumefaciens accumulates four different glycolipids under phosphate deficiency, including digalactosyl diacylglycerol and glucosylgalactosyl diacylglycerol synthesized by a processive glycosyltransferase. The other two glycolipids have now been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as monoglucosyl diacylglycerol and glucuronosyl diacylglycerol. These two lipids are synthesized by a single promiscuous glycosyltransferase encoded by the ORF atu2297, with UDP-glucose or UDP-glucuronic acid as sugar donors. The transfer of sugars differing in their chemistry is a novel feature not observed before for lipid glycosyltransferases. Furthermore, this enzyme is the first glucuronosyl diacylglycerol synthase isolated. Deletion mutants of Agrobacterium lacking monoglucosyl diacylglycerol and glucuronosyl diacylglycerol or all glycolipids are not impaired in growth or virulence during infection of tobacco leaf discs. Our data suggest that the four glycolipids and the nonphospholipid diacylglyceryl trimethylhomoserine can mutually replace each other during phosphate deprivation. This redundancy of different nonphospholipids may represent an adaptation mechanism to enhance the competitiveness in nature.

Mass spectrometry-based metabolomics is a rapidly growing field in both research and diagnosis. Generally, the methodologies and types of instruments used for clinical and other absolute quantification experiments are different from those used for biomarkers discovery and untargeted analysis, as the former requires optimal sensitivity and dynamic range, while the latter requires high resolution and high mass accuracy. We used a Q-TOF mass spectrometer with two different types of pentafluorophenyl (PFP) stationary phases, employing both positive and negative ionization, to develop and validate a hybrid quantification and discovery platform using LC-HRMS. This dual-PFP LC-MS platform quantifies over 50 clinically relevant metabolites in serum (using both MS and MS/MS acquisitions) while simultaneously collecting high resolution and high mass accuracy full scans to monitor all other co-eluting non-targeted analytes. We demonstrate that the linearity, accuracy, and precision results for the quantification of a number of metabolites, including amino acids, organic acids, acylcarnitines and purines/pyrimidines, meets or exceeds normal bioanalytical standards over their respective physiological ranges. The chromatography resolved highly polar as well as hydrophobic analytes under reverse-phase conditions, enabling analysis of a wide range of chemicals, necessary for untargeted metabolomics experiments. Though previous LC-HRMS methods have demonstrated quantification capabilities for various drug and small molecule compounds, the present study provides an HRMS quant/qual platform tailored to metabolic disease; and covers a multitude of different metabolites including compounds normally quantified by a combination of separate instrumentation.

Soybean-based infant formulas (SBIFs) based on soybean protein isolate (90% of proteins) are an interesting alternative to cow's milk infant formulas. Different works have demonstrated the presence of bioactive peptides in different soybean-based foodstuffs. The aim of this work was the evaluation, for the first time, of antioxidant peptides in five different commercially available SBIFs. Ultrafiltration through 10 kDa molecular weight cut-off filters was the most suitable extraction method. Despite peptide concentrations ranging between 1.19 and 2.27 mg/mL, similar antioxidant capacities were detected in all SBIF extracts. Extracts were further fractionated according to their molecular weight by ultrafiltration, and fractions from 5 to 10 kDa, 3 to 5 kDa, and below 3 kDa were obtained. The most active fraction was further fractionated by off-gel isoelectrofocusing and reversed-phase chromatography. Antioxidant fractions were also submitted to simulated gastrointestinal digestion (GI) with pepsin and pancreatin to evaluate their antioxidant capacity after digestion. Peptides were identified by HPLC-ESI-Q-ToF-MS/MS. At least 120 peptides were identified in every antioxidant fraction, with 42 peptides common to all SBIFs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The process of degradation was studied by using an HPLC-DAD method. Four degradation products were identified with a hybrid ESI-Q-TOF mass spectrometer. The influence of temperature and relative air humidity (RH) on the stability of cefpirome sulfate was investigated. In the solid state the degradation of cefpirome sulfate was a first-order reaction depending on the substrate concentration. The kinetic and thermodynamic parameters of degradation were calculated., (Copyright © 2014 Elsevier B.V. All rights reserved.)

S-nitrosylation is a nitric oxide (NO)-based post-translational modification regulating protein function and signalling. We used a combination between the biotin switch method and labelling with isotope-coded affinity tag to identify endogenously S-nitrosylated peptides in Arabidopsis thaliana proteins extracted from plantlets. The relative level of S-nitrosylation in the identified peptides was compared between unstressed and cold-stress seedlings. We thereby detected 62 endogenously nitrosylated peptides out of which 20 are over-nitrosylated following cold exposure. Taken together these data provide a new repertoire of endogenously S-nitrosylated proteins in Arabidopsis with cysteine S-nitrosylation site. Furthermore they highlight the quantitative modification of the S-nitrosylation status of specific cysteine following cold stress., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

An HPLC method for the assay of the heat shock protein 90 inhibitor, PU-H71 (NSC 750424), has been developed and validated. The stress testing of PU-H71 was carried out in accordance with ICH guidelines Q1A (R2) under aqueous, acidic, alkaline, oxidative, thermolytic and photolytic conditions. The separation of PU-H71 from its impurities and degradation products was achieved within 50min on a Mac-Mod ACE 3 C18 column (150mm×4.6mm i.d., 3μm) with a gradient mobile phase comprising 20-95% acetonitrile in water, with 0.1% trifluroacetic acid in both phases. LC-quadrupole TOF/MS was used to obtain accurate mass data on various components as well as on their fragments for characterization of impurities and degradation products. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.1-0.3mg/mL, r≥0.9998), accuracy (recovery 99.7-101.1%), precision (intra-lab RSD≤1.39%, inter-lab RSD≤0.91%), sensitivity (LOD 0.08μg/mL), and ruggedness. The developed method was suitable for the assay and stability monitoring of PU-H71 drug substance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Liquid chromatography-mass spectrometry (LC-MS) is considered today as a mainstay tool for the structure characterization of minor components like impurities (IMPs) and degradation products (DPs) in drug substances and products. A multi-step systematic strategy for the purpose involves high resolution mass and multi-stage mass studies on both the drug and IMPs/DPs, followed by comparison of their fragmentation profiles. Its successful application requires consideration of many practical aspects at each step. The same are critically discussed in this review., (Copyright © 2013 Elsevier B.V. All rights reserved.)