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.

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.

Taking into consideration the challenges of lipid analytics, present study aims to design the best high-throughput workflow for detection and annotation of lipids.
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.
Better detection of features was observed in CSH-C18 than EVO-C18 with enhanced resolution except for Glycerolipids (triacylglycerols) and Sphingolipids (sphingomyelin).
The study revealed an optimized untargeted Lipidomics-workflow with comprehensive lipid profiling (CSH-C18 column) and confirmatory annotation (LipidBlast).

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, H2O2 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.

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.
BACKGROUND: The online version contains supplementary material available at 10.1007/s12298-021-01116-w.

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.

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 C18 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.

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).

UNASSIGNED: 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.
UNASSIGNED: 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%.
UNASSIGNED: 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.