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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. In this study, a comprehensive strategy combining ultra‐performance liquid chromatography tandem quadrupole time‐of‐flight mass spectrometry qualitative analysis and high‐performance liquid chromatography diode array detector fingerprinting analysis was used for a rapid, efficient and sensitive quality control of Liuweizhiji Gegen‐Sangshen oral liquid. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: In this study the comparison of human liver microsomes in in vitro incubation as well as ZnO‐ and TiO2‐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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A nontargeted protein identification method was developed to screen for adulterations in skimmed‐milk powder (SMP). There are indications of falsified SMP content due to the addition of plant proteins. To demonstrate the reliability and accuracy of the developed comparative LC‐MS method using a quadrupole TOF MS instrument, adulterated SMP samples were prepared by the addition of protein isolates of soy and pea to skimmed‐milk before pasteurisation and evaporation. The comparative LC‐MS approach enabled unequivocal discrimination of those SMP samples containing soy and pea protein from nonadulterated SMP. To identify the source of (plant) proteins present in the adulterated SMP, data‐dependent LC‐MS/MS was used in combination with an include list of differential peptides. Numerous peptides originating from the major seed proteins of soy (glycinin, β‐conglycin) and pea (legumin, vicilin) could be identified in this way. [ABSTRACT FROM AUTHOR]

Recent work using chemical cross-linking to define interresidue distance constraints in proteins has shown that these constraints are useful for testing tertiary structural models. We applied this approach to the G-protein-coupled receptor bovine rhodopsin in its native membrane using lysine- and cysteine-targeted bifunctional cross-linking reagents. Cross-linked proteolytic peptides of rhodopsin were identified by combined liquid chromatography and FT-ICR mass spectrometry with automated data-reduction and assignment software. Tandem mass spectrometry was used to verify cross-link assignments and locate the exact sites of cross-link attachment. Cross-links were observed to form between 10 pairs of residues in dark-state rhodopsin. For each pair, cross-linkers with a range of linker lengths were tested to determine an experimental distance-of-closest-approach (DCA) between reactive side-chain atoms. In all, 28 cross-links were identified using seven different cross-linking reagents. Molecular mechanics procedures were applied to published crystal structure data to calculate energetically achievable theoretical DCAs between reactive atoms without altering the position of the protein backbone. Experimentally measured DCAs are generally in good agreement with the theoretical DCAs. However, a cross-link between C316 and K325 in the C-terminal region cannot be rationalized by DCA simulations and suggests that backbone reorientation relative to the crystal coordinates occurs on the timescale of cross-linking reactions. Biochemical and spectroscopic data from other studies have found that the C-terminal region is highly mobile in solution and not fully represented by X-ray crystallography data. Our results show that chemical cross-linking can provide reliable three-dimensional structural information and insight into local conformational dynamics in a membrane protein. [ABSTRACT FROM AUTHOR]