Search

Avocado fruit growth and development, unlike that of other fruits, is characterized by the accumulation of oil and C7 sugars (in most fruits, the carbohydrates that prevail are C6). There are five essential carbohydrates which constitute 98% of the total content of soluble sugars in this fruit; these are fructose, glucose, sucrose, d-mannoheptulose, and perseitol, which together with quinic acid and chlorogenic acid have been the analytes under study in this work. After applying an efficient extraction procedure, a novel methodology based on hydrophilic interaction liquid chromatography coupled to mass spectrometry was applied to determine the levels of these seven substances in tissues─exocarp, seed, and mesocarp─from avocado fruits of two different varieties scarcely studied, Bacon and Fuerte , at three different ripening stages. Quantitative characterization of the selected tissues was performed, and the inter-tissue distribution of metabolites was described. For both varieties, d-mannoheptulose was the major component in the mesocarp and exocarp, whereas perseitol was predominant in the seed, followed by sucrose and d-mannoheptulose. Sucrose was found to be more abundant in seed tissues, with much lower concentrations in avocado mesocarp and exocarp. Quinic acid showed a predominance in the exocarp, and chlorogenic acid was exclusively determined in exocarp samples.

Mass spectrometry is a powerful analytical technique used to identify unknown compounds, to quantify known compounds, and to elucidate the structure and chemical properties of molecules. Nevertheless, the transfer of data from one instrument to another is one of the main problems, and obtaining the same or similar information from an analogous instrument but from a different manufacturer or even with the same instrument after carrying out the analyses in different times spacing is not possible. Hence, a general methodology to provide a chromatographic signal (or chromatogram) independent of the instrument is needed. In this sense, this book chapter describes the standardization procedure of chromatographic signals obtained from mass spectrometry platforms to obtain instrument-agnostic chromatographic signals for the determination of standard retention scores. This parameter may be used for the quantification of compounds when different mass spectrometry platforms coupled to ultrahigh-performance liquid chromatography are employed., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Untargeted lipidomics using liquid chromatography high-resolution mass spectrometry (LC-HRMS) was performed using polarity switching, and in positive and negative polarity separately on the same set of serum samples, and the performances of the methods were evaluated. Polarity switching causes an increase in the cycle time of the HRMS measurements (1.18 s/cycle vs 0.27 s/cycle), resulting in fewer data points across chromatographic peaks. The coefficient of variation (CV) was on average lower for the added isotopically labelled standards in pooled samples (QC) and patient samples using separate polarities (QC = 5.6%, samples = 12.5%) compared to polarity switching (QC = 8.5%, samples = 13.4%), but the difference was not statistically significant. For the endogenous features measured in the QCs polarity switching resulted in on average significantly higher CVs (3.80 (p = 4.25e-30) and 3.3 percentage points (p = 6.84e-40), for positive and negative modes, respectively) however still acceptable for an untargeted method (mean CVs of 17.9% and 12.2% in positive and negative modes respectively). A slightly larger number of endogenous features were detected using the separate polarities, but the large majority of features (>95%) were detected with both methodologies. The overlap of features detected in both positive and negative polarities was low (4.1%) demonstrating the importance of using both polarities for untargeted lipidomics. When investigating the effects of a treatment on multiple sclerosis patients it was found that both methodologies gave highly similar biological results, further confirming the applicability of polarity switching., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Walnut kernel, a well-known TCM, is often used after being defatted in tradition. And defatted walnut powder extract (DWPE) has the actions of tonifying the liver and kidney, dissipating stagnation and removing blood stasis, which has the effect on non-alcoholic fatty liver disease (NAFLD). However, the effective components of DWPE in vivo were unclear and the multiple mechanisms of DWPE against NAFLD have not been explored., Aim of the Study: The studies were performed to screen the effective substances in vivo by identification of the metabolites of DWPE in rats and to seek the potential mechanisms of DWPE on NAFLD by construction of the network pharmacology based on metabolites and verification of the highly correlated pathway., Materials and Methods: To explore the effective substances in vivo, the metabolites of DWPE were identified in SD rats' bio-samples through UPLC-Q-Exactive Orbitrap MS. To analyze the mechanisms of DWPE on NAFLD, a Metabolite-Target-Disease network was established and the potential mechanisms were predicted. Then, highly correlated pathway was verified in animal and cells studies., Results: A total of 52 metabolites of DWPE were identified in vivo, which were derived from gallic acid, ellagic acid (EA) and glansreginin A (Gla A). The possible metabolic pathways were phase Ⅰ (hydroxylation, hydrolyzation, etc) and phase Ⅱ metabolic reactions (methylation, sulfation and glucuronidation). Furthermore, in the network pharmacology, 54 core targets were enriched into pathways in cancer, nitrogen metabolism and other 9 pathways, which were essential pathways of DWPE against NAFLD. And the mechanism of nitrogen metabolism was verified in both of animal and cells studies. The results showed that DWPE could decline the concentration of ammonia and increase the expressions of carbonic anhydrase 2 (CA2) and carbamoylphosphate synthetase (CPS1) in nitrogen metabolism., Conclusion: Taken together, the study revealed the absorption components and their metabolic pathways and demonstrated the mechanism of nitrogen metabolism of DWPE on anti-NAFLD., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Rapid and precise diagnostic methods are required to control emerging infectious diseases effectively. Human body fluids are attractive clinical samples for discovering diagnostic targets because they reflect the clinical statuses of patients and most of them can be obtained with minimally invasive sampling processes. Body fluids are good reservoirs for infectious parasites, bacteria, and viruses. Therefore, recent clinical proteomics methods have focused on body fluids when aiming to discover human- or pathogen-originated diagnostic markers. Cutting-edge liquid chromatography-mass spectrometry (LC-MS)-based proteomics has been applied in this regard; it is considered one of the most sensitive and specific proteomics approaches. Here, the clinical characteristics of each body fluid, recent tandem mass spectroscopy (MS/MS) data-acquisition methods, and applications of body fluids for proteomics regarding infectious diseases (including the coronavirus disease of 2019 [COVID-19]), are summarized and discussed.

Acidic N-linked glycan content is often associated with a protein drug's stability, efficacy and immune response. It has often been a challenge to analyze these types of glycans, including those that are differentiated by the incorporation of N-acetyl (NANA) and N-glycolyl neuraminic acid (NGNA) residues. In this study, a strategy for rapid N-glycan profiling by mixed mode chromatography is proposed as a complement to established HILIC methodologies. Hybrid silica chromatographic surfaces are used to improve recoveries during a column's initial use and to eliminate the need for column conditioning. In addition, the loss of labeled acidic glycans, especially phosphorylated glycan species, during SPE purification is addressed through the use of a citrate containing eluent. Yields for both singly and doubly phosphorylated glycan species are markedly improved. Combined with a mixed mode anion exchange reversed phase separation, these advances afford a class separation of glycans derivatized with labels designed to enhance positive ion mode MS detection. These labeled glycan species are separated according to their charge and with an added level of resolution imparted by the reversed phase retention mechanism. The separation technique itself can be accomplished with a low ionic strength gradient running from 0 to 22 mM ammonium formate such that high sensitivity detection can be achieved by both fluorescence and mass spectrometry. Using analytical scale chromatography, features in an N-glycan profile were easily interrogated to well below a 0.1% relative abundance. As such, it became possible to characterize N-glycans from recombinant beta glucuronidase and to quickly identify a number of unique phosphorylated glycan species., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels ( Mytilus galloprovincialis ), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary.

Untargeted liquid chromatographic-high-resolution mass spectrometric (LC-HRMS) metabolomics for potential exposure marker (PEM) discovery in nutrikinetic studies generates complex outputs. The correct selection of statistically significant PEMs is a crucial analytical step for understanding nutrition-health interactions. Hence, in this paper, different chemometric selection workflows for PEM discovery, using multivariate or univariate parametric or non-parametric data analyses, were comparatively tested and evaluated. The PEM selection protocols were applied to a small-sample-size untargeted LC-HRMS study of a longitudinal set of serum samples from 20 volunteers after a single intake of (poly)phenolic-rich Vaccinium myrtillus and Vaccinium corymbosum supplements. The non-parametric Games-Howell test identified a restricted group of significant features, thus minimizing the risk of false-positive retention. Among the forty-seven PEMs exhibiting a statistically significant postprandial kinetics, twelve were successfully annotated as purine pathway metabolites, benzoic and benzodiol metabolites, indole alkaloids, and organic and fatty acids, and five (i.e. octahydro-methyl-β-carboline-dicarboxylic acid, tetrahydro-methyl-β-carboline-dicarboxylic acid, citric acid, caprylic acid, and azelaic acid) were associated to Vaccinium berry consumption for the first time. The analysis of the area under the curve of the longitudinal dataset highlighted thirteen statistically significant PEMs discriminating the two interventions, including four intra-intervention relevant metabolites (i.e. abscisic acid glucuronide, catechol sulphate, methyl-catechol sulphate, and α-hydroxy-hippuric acid). Principal component analysis and sample classification through linear discriminant analysis performed on PEM maximum intensity confirmed the discriminating role of these PEMs., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Liquid chromatography (LC) - mass spectrometry quantitative analysis of substances in biological samples is usually performed in the multiple reaction monitoring (MRM) variant. In complex biological matrices, strong interferences can be observed when using the LC-MRM method. Interference levels can be significantly reduced by using LC - multiple reaction monitoring cubed (MRM 3 ). 6-sulfatoxymelatonin (6-SM) is a metabolite of melatonin, an important regulator of many biological processes. The quantitative analysis of 6-SM in urine allows monitoring of the melatonin level in the blood. The aim of the present work was to evaluate the LC-MRM 3 method for the quantitative determination of 6-SM in urine. We found that for 6-SM in aqueous solutions, under some parameters of the MRM 3 experiment, the effect of degradation of the MRM 3 signal is observed. When analyzing 6-SM in urine, this signal degradation effect was significantly reduced. We have shown that optimization of such parameters of the MRM 3 method as the linear ion trap fill time, the number of scans to sum, and the range of triple-stage scan allows obtaining the LC-MRM 3 method, which is comparable to the LC-MRM in sensitivity and significantly exceeds it in selectivity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The ever-increasing complexity of biological samples to be analysed by mass spectrometry has led to the necessity of sophisticated separation techniques, including multidimensional separation. Despite a high degree of orthogonality, the coupling of liquid chromatography (LC) and capillary zone electrophoresis (CZE) has not gained notable attention in research. Here, we present a heart-cut nanoLC-CZE-ESI-MS platform to analyse intact proteins. NanoLC and CZE-MS are coupled using a four-port valve with an internal nanoliter loop. NanoLC and CZE-MS conditions were optimised independently to find ideal conditions for the combined setup. The valve setup enables an ideal transfer efficiency between the dimensions while maintaining good separation conditions in both dimensions. Due to the higher loadability, the nanoLC-CZE-MS setup exhibits a 280-fold increased concentration sensitivity compared to CZE-MS. The platform was used to characterise intact human alpha-1-acid glycoprotein (AGP), an extremely heterogeneous N-glycosylated protein. With the nanoLC-CZE-MS approach, 368 glycoforms can be assigned at a concentration of 50 μg/mL as opposed to the assignment of only 186 glycoforms from 1 mg/mL by CZE-MS. Additionally, we demonstrate that glycosylation profiling is accessible for dried blood spot analysis (25 μg/mL AGP spiked), indicating the general applicability of our setup to biological matrices. The combination of high sensitivity and orthogonal selectivity in both dimensions makes the here-presented nanoLC-CZE-MS approach capable of detailed characterisation of intact proteins and their proteoforms from complex biological samples and in physiologically relevant concentrations., (© 2021. The Author(s).)

β-Lactam antibiotics are most commonly used in the critically ill, but their effective dosing is challenging and may result in sub-therapeutic concentrations that can lead to therapy failure and even promote antimicrobial resistance. In this study, we present the analytical tool enabling specific and sensitive determination of the sole biologically active fraction of piperacillin and imipenem in biological material from the critically ill. Thin-film microextraction sampling technique, followed by rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, was optimized and validated for the quantitative determination of antibiotics in blood and bronchoalveolar lavage (BAL) specimens collected from intensive care unit (ICU) patients suffering from ventilation-associated pneumonia (n = 18 and n = 9, respectively). The method was optimized and proved to meet the criteria of US Food and Drug Administration (FDA) guidelines for bioanalytical method validation. Highly selective, sensitive, accurate and precise analysis by means of thin-film microextraction-LC-MS/MS, which is not affected by matrix-related factors, was successfully applied in clinical settings, revealing poor penetration of piperacillin and imipenem from blood into BAL fluid (reflecting the site of bacterial infection), nonlinearity in antibiotic binding to plasma-proteins and drug-specific dependence on creatinine clearance. This work demonstrates that only a small fraction of biologically active antibiotics reach the site of infection, providing clinicians with a high-throughput analytical tool for future studies on personalized therapeutic drug monitoring when tailoring the dosing strategy to an individual patient.

We evaluated the performance of three different single-phase extraction methods to be used before untargeted lipidomics analysis by Liquid Chromatography High-Resolution Mass Spectrometry. Lipids were extracted from a pool of healthy human donors' plasma in triplicates and run in both positive and negative ESI. The most satisfactory results were attained using methanol/chloroform (2:1, v/v) mixture. Eventually, we evaluated whether a filtration of the samples could be beneficial to yield cleaner and more mass-friendly extracts. Instead of using syringes, we set up a method we called tip-tip filtration, which requires the usage of a filtrating pipette tip. This way of purification led to superior results than the solvent extraction method alone. This additional procedure not only increased reproducibility but also allowed the same lipid coverage. In addition, it permitted to spare time and money, as tip-tip filtration is not particularly expensive nor time-consuming and hopefully it may be useful to increase analytical column lifetime., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Exosomes are extracellular vesicles that play important roles in various physiological and pathological functions. Previous studies have demonstrated that exosome-derived contents are promising biomarkers to inform the pathogenesis and diagnosis of major depressive disorder and schizophrenia., Methods: We used ultraperformance liquid chromatography-tandem mass spectrometry to analyze the differentially expressed metabolites in serum exosomes of patients with bipolar disorder (BD) and evaluated the potential of exosomal metabolites as biomarkers for BD., Results: Our results showed 26 differentially expressed serum exosomal metabolites in patients with BD ( n = 32) when compared with healthy control (HC) subjects ( n = 40), and these differentially expressed metabolites were enriched in pathways related to sugar metabolism. We then utilized random forest classifier and identified 15 exosomal metabolites that can be used to classify samples from patients with BD and HC subjects with 0.838 accuracy (95% CI, 0.604-1.00) in the training set of participants. These 15 metabolites showed excellent performance in differentiating between patients with BD and HC subjects in the testing set of participants, with 0.971 accuracy (95% CI, 0.865-1.00). Importantly, the 15 exosomal metabolites also showed good to excellent performance in differentiating between BD patients and other major psychiatric diseases (major depressive disorder and schizophrenia)., Conclusion: Collectively, our findings for the first time revealed a potential role of exosomal metabolite dysregulations in the onset and/or development of BD and suggested that blood exosomal metabolites are strong candidates to inform the diagnosis of BD., Competing Interests: There is a patent pending for application related to the results in the article., (Copyright © 2022 Yang Du et al.)

Metabolomics deals with the large-scale analysis of metabolites, belonging to numerous compound classes and showing an extremely high chemical diversity and complexity. Lipidomics, being a subcategory of metabolomics, analyzes the cellular lipid species. Both require state-of-the-art analytical methods capable of accessing the underlying chemical complexity. One of the major techniques used for the analysis of metabolites and lipids is Liquid Chromatography-Mass Spectrometry (LC-MS), offering both different selectivities in LC separation and high sensitivity in MS detection. Chromatography can be divided into different modes, based on the properties of the employed separation system. The most popular ones are Reversed-Phase (RP) separation for non- to mid-polar molecules and Hydrophilic Interaction Liquid Chromatography (HILIC) for polar molecules. So far, no single analysis method exists that can cover the entire range of metabolites or lipids, due to the huge chemical diversity. Consequently, different separation methods have been used for different applications and research questions. In this review, we explore the current use of LC-MS in metabolomics and lipidomics. As a proxy, we examined the use of chromatographic methods in the public repositories EBI MetaboLights and NIH Metabolomics Workbench. We extracted 1484 method descriptions, collected separation metadata and generated an overview on the current use of columns, eluents, etc. Based on this overview, we reviewed current practices and identified potential future trends as well as required improvements that may allow us to increase metabolite coverage, throughput or both simultaneously., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Interleukin-33 (IL-33), a member of the IL-1 superfamily cytokines, is an endogenous danger signal and a nuclear-associated cytokine. It is one of the essential mediators of both innate and adaptive immune responses. Aberrant IL-33 signaling has been demonstrated to play a defensive role against various infectious and inflammatory diseases. Although the signaling responses mediated by IL-33 have been previously reported, the temporal signaling dynamics are yet to be explored. To this end, we applied quantitative temporal phosphoproteomics analysis to elucidate pathways and proteins induced by IL-33 in THP-1 monocytes. Employing a TMT labeling-based quantitation and titanium dioxide (TiO 2 )-based phosphopeptide enrichment strategy followed by mass spectrometry analysis, we identified and quantified 9448 unique phosphopeptides corresponding to 3392 proteins that showed differential regulation. Of these, 171 protein kinases, 60 phosphatases and 178 transcription factors were regulated at different phases of IL-33 signaling. In addition to the confirmed activation of canonical signaling modules including MAPK, NFκB, PI3K/AKT modules, pathway analysis of the time-dependent phosphorylation dynamics revealed enrichment of several cellular processes, including leukocyte adhesion, response to reactive oxygen species, cell cycle checkpoints, DNA damage and repair pathways. The detailed quantitative phosphoproteomic map of IL-33 signaling will serve as a potentially useful resource to study its function in the context of inflammatory and pathological conditions.

During biologics development, manufacturers must demonstrate clearance of host cell impurities and contaminants to ensure drug purity, manufacturing process consistency, and patient safety. Host cell proteins (HCPs) are a major class of process-related impurities and require monitoring and documentation of their presence through development and manufacturing. Even in residual amounts, they are known to affect product quality and efficacy as well as patient safety. HCP analysis using enzyme-linked immunosorbent assay (HCP-ELISA) is the standard technique, due to its simple handling, short analysis time, and high sensitivity for protein impurities. Liquid chromatography mass spectrometry (LC-MS) is an orthogonal method for HCP analysis and is increasingly included in regulatory documentation. LC-MS offers advantages where HCP-ELISA has drawbacks, e.g., the ability to identify and quantify individual HCPs. This article summarizes the available knowledge about monitoring HCPs in biologics and presents the newest trends in HCP analysis with current state-of-the-art HCP measurement tools. Through case studies, we present examples of HCP control strategies that have been used in regulatory license applications, using an MS-based coverage analysis and HCP-ELISA and LC-MS for HCP quantification. This provides novel insight into the rapid evolving strategy of HCP analysis. Improvements in technologies to evaluate HCP-ELISA suitability and the implementation of orthogonal LC-MS methods for HCP analysis are important to rationally manipulate, engineer, and select suitable cell lines and downstream processing steps to limit problematic HCPs., (© 2021. The Author(s).)

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method was developed with the aim to unambiguously identify a large number of lipid species from multiple lipid classes in human plasma. The optimized RP-UHPLC/MS method employed the C18 column with sub-2-μm particles with the total run time of 25 min. The chromatographic resolution was investigated with 42 standards from 18 lipid classes. The UHPLC system was coupled to high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) measuring full-scan and tandem mass spectra (MS/MS) in positive- and negative-ion modes with high mass accuracy. Our identification approach was based on m/z values measured with mass accuracy within 5 ppm tolerance in the full-scan mode, characteristic fragment ions in MS/MS, and regularity in chromatographic retention dependences for individual lipid species, which provides the highest level of confidence for reported identifications of lipid species including regioisomeric and other isobaric forms. The graphs of dependences of retention times on the carbon number or on the number of double bond(s) in fatty acyl chains were constructed to support the identification of lipid species in homologous lipid series. Our list of identified lipid species is also compared with previous publications investigating human blood samples by various MS-based approaches. In total, we have reported more than 500 lipid species representing 26 polar and nonpolar lipid classes detected in NIST Standard reference material 1950 human plasma., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Gambierdiscus spp. are epi-benthic dinoflagellates that have been associated with ciguatera poisoning. These microalgae can have complex secondary metabolite profiles including ciguatoxins, maitotoxins, and gambierones, with varying compositions and toxicities across species and strains. Given this chemical diversity there is a need to develop selective and sensitive methods for secondary metabolite profiling. In this study, we used a cultured Caribbean strain of Gambierdiscus silvae to develop sample preparation and analysis strategies for characterizing vic-diol containing secondary metabolites. A pooled cellular extract was first screened by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for ciguatoxin-related compounds, which resulted in the confirmation of gambierone (1) and a novel isomer of 44-methylgambierone (3). Treatment of the extract with periodate confirmed that the gambierones each contained one reactive vic-diol, which was exploited for the development of a selective extraction procedure using m-aminophenylboronic acid gel and the non-aqueous binding solvent chloroform. Using this non-traditional boronate affinity procedure, LC-HRMS also revealed the presence of additional sulfated polycyclic ethers in the gambierone-containing vic-diol fraction, while pigments and other contaminants were removed. The developed tools could be applied to screen collections of Gambierdiscus and other benthic algae to provide additional chemical characterization of gambierone-related compounds. The selective extraction procedure may also prove useful as a step in the isolation of these sulfated polyethers for structural, toxicological and biotransformation studies., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

The effective fraction of coumarin glycosides from Hydrangea paniculata Sieb (HP) has been under development for the treatment of chronic kidney disease for years. Skimmin and apiosylskimmin are the main coumarin glycosides of HP, and the major metabolites in rats are 7-hydroxycoumarin (7-HC) and 7-hydroxycoumarin glucuronide (7-HCG). In this study, a sensitive and reliable liquid chromatography-Orbitrap mass spectrometry method was developed for the simultaneous determination of skimmin, apiosylskimmin, 7-HC and 7-HCG in rat plasma. The chromatographic separation was performed on a Zobax SB C 18 column (2.1 × 100 mm, 3.5 μm) at a flow rate of 0.3 ml/min with a gradient mobile phase of water and acetonitrile containing 0.2% formic acid. Skimmin, apiosylskimmin and 7-HCG were detected in targeted-selected-ion-monitoring mode at positive ions m/z of 325.0911, 457.1331 and 339.0703, respectively. 7-HC and the internal standard were detected in parallel-reaction-monitoring mode at m/z 163.0387 → 119.0492 and 260.1641 → 116.1071 to overcome the carryover of 7-HC. Linearity was obtained for the analytes within the ranges 20-2,000 ng/ml for skimmin, 5-500 ng/ml for apiosylskimmin and 7-HC and 100-10,000 ng/ml for 7-HCG. Validation parameters were all in line with the criteria of international guidance. The method has been applied to the pharmacokinetic study of HP in rats., (© 2021 John Wiley & Sons, Ltd.)

Aim: In response to the COVID-19 pandemic, Regeneron developed the anti-SARS-CoV-2 monoclonal antibody cocktail, REGEN-COV ® (RONAPREVE ® outside the USA). Drug concentration data was important for determination of dose, so a two-part bioanalytical strategy was implemented to ensure the therapy was rapidly available for use. Results & methodology: Initially, a liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) assay, was used to analyze early-phase study samples. Subsequently, a validated electrochemiluminescence (ECL) immunoassay was implemented for high throughput sample analysis for all samples. A comparison of drug concentration data from the methods was performed which identified strong linear correlations and for Bland-Altman, small bias. In addition, pharmacokinetic data from both methods produced similar profiles and parameters. Discussion & conclusion: This novel bioanalytical strategy successfully supported swift development of a critical targeted therapy during the COVID-19 public health emergency.

A newly introduced HIV-1 vaccination utilizes a fusion peptide (FP)-based immunogen-carrier conjugate system, where the FP is coupled to a protein carrier via a bifunctional linker. Such heterogeneous materials present a challenge for the routine product quality assessment. Peptide mapping LC-MS analysis has become an indispensable tool for assessing the site-specific conjugation ratio, estimating site occupancy, monitoring conjugation profiles, and analyzing post-translational modifications (PTMs) and disulfide bonds as well as high-order protein structures. To streamline the peptide mapping approach to match the needs of a fast-paced conjugate vaccine product characterization, a selection of signature fragment ions generated by MS E fragmentation was successfully applied to assess the product quality at the different stages of a conjugates' manufacturing process with an emphasis on monitoring the amount of a reactive linker. This technique was employed in different conjugation studies of the protein carriers, linkers, and FP compositions as well as the cross-linked species formed during stress-degradation studies. Multiple derivatives of the intermediate and final conjugated products formed during a multistaged synthesis were monitored by means of the sensitive extracted-ion chromatogram (XIC) profiling and were included in the estimation of the site-specific conjugation loads. Differentiation of the conjugates with various FP compositions was demonstrated. The conjugation site occupancy was evaluated with respect to the solvent exposure of Lys residues. The findings of these LC-MS studies greatly aided in choosing the best conjugation strategy to ensure that the final recombinant tetanus toxoid heavy chain (rTTHc) product is chemically inert and represents a safe vaccine candidate for clinical evaluation.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Ole Lagatie, Emmanuel Njumbe Ediage, Dirk Van Roosbroeck, Stijn Van Asten, Ann Verheyen, Lieve Dillen, Tom Verhaeghe, Rob Vreeken, Filip Cuyckens and Lieven J. Stuyver are current employees of Janssen Pharmaceutica NV, a Johnson & Johnson company, and may own stock or stock option in that company.

Phycotoxins occur in various marine and freshwater environments, and can accumulate in edible species such as fish, crabs, and shellfish. Human exposure to these toxins can take place, for instance, through consumption of contaminated species or supplements and through the ingestion of contaminated water. Symptoms of phycotoxin intoxication include paralysis, diarrhea, and amnesia. When the cause of an intoxication cannot directly be found, a screening method is required to identify the causative toxin. In this work, such a screening method was developed and validated for marine and freshwater phycotoxins in different matrices: fish, shellfish, water, and food supplements. Two LC methods were developed: one for hydrophilic and one for lipophilic phycotoxins. Sample extracts were measured in full scan mode with an Orbitrap high resolution mass spectrometer. Additionally, a database was created to process the data. The method was successfully validated for most matrices, and in addition, regulated lipophilic phycotoxins, domoic acid, and some paralytic shellfish poisoning toxins could be quantified in shellfish. The method showed limitations for hydrophilic phycotoxins in sea water and for lipophilic phycotoxins in food supplements. The developed method is a screening method; in order to confirm suspected compounds, comparison with a standard or an additional analysis such as NMR is required.

We have investigated the metabolic profile of N-ethyl heptedrone, a new designer synthetic stimulant drug, by using data independent acquisition mass spectrometry. Phase I and phase II metabolism was studied by in vitro models, followed by liquid-chromatography coupled to mass spectrometry, to characterize and pre-select the most diagnostic markers of intake. N-ethyl heptedrone was incubated in the presence of pooled human liver microsomes. The contribution of individual enzymatic isoforms in the formation of the phase I and phase II metabolites was further investigated by using human recombinant cDNA-expressed cytochrome P450 enzymesand uridine 5'-diphospho glucuronosyltransferases. The analytical workflow consisted of liquid-liquid extraction with tert-butyl-methyl-ether at alkaline pH, performed before (to investigate the phase I metabolic profile) and after (to investigate the glucuronidation profile) enzymatic hydrolysis. The separation, identification, and determination of the compounds formed in the in vitro experiments were carried out by using liquid chromatography coupled to either high- or low-resolution mass spectrometry. Data independent acquisition method, namely sequential window acquisition of all theoretical fragment-ion spectra (SWATH®) and product ion scan were selected for high-resolution mass spectrometry, whereas multiple reaction monitoring was used for low-resolution mass spectrometry. Thirteen phase-I metabolites were isolated, formed from reactions being catalyzed mainly by CYP1A2, CYP2C9, CYP2C19 and CYP2D6 and, to a lesser degree, by CYP3A4 and CYP3A5. The phase I biotransformation pathways included hydroxylation in different positions, reduction of the ketone group, carbonylation, N-dealkylation, and combinations of the above. Most of the hydroxylated metabolites underwent conjugation reactions to form the corresponding glucurono-conjugated metabolites. Based on our in vitro observation, the metabolic products resulting from reduction of the keto group, N-dealkylation and hydroxylation of the aliphatic chain appear to be the most diagnostic target analytes to be selected as markers of exposure to N-ethyl heptedrone., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Vitamin B12 plays a key role in human biological functions and is vital in the neurological development of infants. The assessment of the vitamin B12 intake in exclusively breastfed babies depends on the reliability of its determination in milk. In this report, we present a new accurate and robust method for quantification of vitamin B12 in human milk. A highly specific sample preparation is applied, associated with chromatographic separation and detection by ICP-MS. Excellent sensitivity and accuracy are reported, with recovery values well within acceptability limits (80-120%), within- and between-day variability are lower than 10% and 15% respectively. Strong correlation with a microbiological assay was observed (r 2  = 0.9) within the validation range (40-1000 pmol/L, corresponding to 54 to 1355 ng/L). The method can be used to routinely monitor vitamin B12 in clinical or population observational studies, determine infant's intake or assess efficacy of mother's supplementation., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Monosaccharide isomers and disaccharide isomers widely exist in nature, playing a key role in a number of important biological processes. However, due to high structural similarity and high polarity, the characterization of monosaccharide isomers, disaccharide isomers, as well as the analysis of monosaccharide composition of polysaccharides by a method that does not require derivatization is an ongoing challenge. Herein, we proposed a simple method for rapid discrimination of non-derivatized neutral monosaccharide, and disaccharide isomers using hydrophilic interaction liquid chromatography coupled to quadrupole/time-of-flight mass spectrometry (HILIC-Q/TOF-MS). In this work, we optimized the experimental parameters, and detailed approaches to discriminate the precursor ions, deprotonated ions, and fragment ions are proposed, as well. To discriminate the various ions, the retention times, the relative abundance (RA) of precursor ions and fragment ions at different collision energies, the relative abundance ratio (RAR) of fragment ions to deprotonated ions or precursor ions were considered for characterization of neutral monosaccharide and disaccharide isomers. Finally, this strategy was successfully applied to analyzing the monosaccharide composition of neutral disaccharides, polysaccharides, and an aqueous extract of Moringa oleifera seeds. The experimental results revealed that the HILIC-Q/TOF-MS is an effective and convenient strategy for rapid differentiation of monosaccharide isomers and disaccharide isomers, which may serve as a general platform for the analysis of neutral polysaccharides, food, medicinal plants, and herbs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays targeting 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Whiteaker, Lundeen, Zhao, Schoenherr, Burian, Huang, Voytovich, Wang, Kennedy, Ivey, Lin, Murillo, Lorentzen, Thiagarajan, Colantonio, Caceres, Roberts, Knotts, Reading, Kaczmarczyk, Richardson, Garcia-Buntley, Bocik, Hewitt, Murray, Do, Brophy, Wilz, Yu, Ajjarapu, Boja, Hiltke, Rodriguez and Paulovich.)

Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤ 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies., Competing Interests: AH, KT, MU, JA, LE, FE No competing interests declared, DF, TM, JV, RP, SF, SH, IB, AB, LC employed by Waters Corporation, MR, RY, MP, TP, LA employed by SISCAPA Assay Technologies, (© 2021, Hober et al.)

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- 15 N 3 ) at 200 µg L -1 and detection of intrinsic 14 N-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.

Baihe Dihuang decoction is a commonly used herbal formula to treat depression and insomnia in traditional Chinese medicine. This study established a liquid chromatography-mass spectrometry method to investigate the potential active ingredients and the components absorbed in the blood and brain tissue of mice. Using a new data processing method, 94 chemical components were identified, 33 and 9 of which were absorbed in the blood and brain. More interestingly, we analyzed the substance changes during co-decoction and the characteristics of the compounds absorbed in the blood and brain. The results show that 71 newly generated chemical components were discovered from co-decoction: 38 with fragment information and five absorbed in the blood. Ultimately, the results of molecular docking show that these components have excellent performance in proteins of γ-aminobutyric acid, serotonin and melatonin receptors. The docking results of emodin with Monoamine Oxidase A and Melatonin Receptor 1A, and luteolin with Solute Carrier Family 6 Member 4, Glyoxalase I, Monoamine Oxidase B and Melatonin Receptor 1A, may explain the mechanism of action of Baihe Dihuang decoction in treating insomnia and depression. Overall, our research results may provide novel perspectives for further understanding of the effective substances in Baihe Dihuang decoction., (© 2021 Wiley-VCH GmbH.)

In this research, a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction procedure and Ultra-High Performance Liquid Chromatography-Orbitrap-Mass Spectrometry (UHPLC-Orbitrap-MS), were combined to obtain a sensitive and rapid method for the determination of multiclass pesticides in white and red wines. The optimization strategy involved the selection of buffering conditions, by applying different QuEChERS procedures and sorbents for the cleanup step in order to achieve acceptably high recoveries and low co-extractives in the final extracts. Identification was based on both accurate mass and retention time, while further confirmation was achieved by MS fragmentation. The method was evaluated in terms of linearity, recovery, precision, limit of detection (LOD) and quantification (LOQ), matrix effects (ME) and expanded uncertainty. The validated method was successfully applied to real samples (home-made and commercial) revealing the presence of two selected fungicides, in relatively low levels compared to the MRLs defined by the EU for vinification grapes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A LC-Q-Orbitrap HRMS analytical method for both qualitative screening and quantitative determination of 90 synthetic dyes including ten groups of isomers in foods has been established. An in-house synthetic dyes database and characteristic ions were also developed. Based on Q-Orbitrap HRMS, mass spectrum and fragmentation patterns of synthetic dyes were studied, which indicated that double charged ions were usually the main precursor ions. Matrix effects were successfully eliminated by the C 18 d-SPE clean-up coupled with dilute and shoot approach with methanol-water (1:4, v/v) in 100-fold. For most of the compounds, mean recoveries were satisfactory between 70% and 120% with RSD < 20% at three spiked level in the range of 0.025-1.0 mg/kg. The screening detection limits ranged from 0.025 - 1.0 mg/kg. Method validation showed that the established method was efficient, rapid and high-throughput, which has been successfully applied to the monitoring of these water-soluble synthetic dyes in foods., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Direct oral anticoagulants are an alternative to anticoagulants based on vitamin K antagonists. Monitoring of direct oral anticoagulant concentration levels is necessary in specific cases (e.g. in emergency conditions, for determination of the cause of bleeding, adverse effects, risk of drug-direct oral anticoagulants interaction); therefore, a sensitive and specific method is needed. A methanol protein precipitation method followed by liquid chromatography with high-resolution mass spectrometry was developed for simultaneous separation and determination of apixaban, betrixaban, edoxaban, dabigatran, rivaroxaban and ximelagatran. The proposed method was fully validated in terms of linearity, the limits of detection and quantification, intra- and inter-day trueness and precision, recovery, matrix effect, process efficiency and stability. The method shows a strong correlation (Pearson's correlation coefficients > 0.92) with coagulation assays of apixaban, dabigatran and rivaroxaban (dilute thrombin time for gatrans and anti Xa factor (anti-Xa) activity for xabans). In addition, the developed method was applied for the identification and determination of apixaban and dabigatran in post-mortem serum samples. The developed method is a good alternative to coagulation tests which may show various interferences., (© 2021 John Wiley & Sons, Ltd.)

Metonitazene is considered a new psychoactive substance (NPS) and emerging potent synthetic opioid, causing increased public health concern beginning in 2020. Metonitazene joins a growing list of new synthetic opioids (NSOs) contributing to deaths among people who use drugs in the United States and other parts of the world. Metonitazene (a 2-benzylbenzimidazole analogue) first appeared in mid-2020 in the recreational drug supply and subsequently began proliferating in death investigation casework towards the end of 2020. Screening and metabolite discovery were performed by liquid chromatography quadrupole time-of-flight mass spectrometry. Quantitative confirmation was performed by liquid chromatography tandem quadrupole mass spectrometry. Metonitazene was confirmed in 20 authentic forensic postmortem cases with an average concentration in blood at 6.3 ± 7.5 ng/ml (median: 3.8 ng/ml, range: 0.5-33 ng/ml, n = 18) and in urine at 15 ± 13 ng/ml (median: 11 ng/ml, range: 0.6-46 ng/ml, n = 14). Metonitazene was the only opioid identified in 30% of cases but was also found in combination with fentanyl (55%) and NPS benzodiazepines, opioids, and hallucinogens (45%). Medical examiners included metonitazene as a drug responsible for the cause of death, and the manner of death was always ruled to be an accident. The metabolism of metonitazene was found to be similar to that of isotonitazene, a closely related analogue. Toxicology laboratories and death investigators should ensure that metonitazene is included in forensic testing protocols, all while remaining vigilant for subsequent NSOs to emerge., (© 2021 John Wiley & Sons, Ltd.)

Short-chain fatty acids (SCFAs) are increasingly being monitored to elucidate the link between gut health and disease. These metabolites are routinely measured in faeces, but their determination in serum is more challenging due to their low concentrations. A method for the determination of eight SCFAs in serum is described here. High-resolution mass spectrometry and gas chromatography were used to identify the presence of isomeric interferences, which were then overcome through a combination of chromatographic separation and judicious choice of MS fragment ion. The SCFAs were derivatised to form 3-nitrophenylhydrazones before being separated on a reversed-phase column and then detected using liquid chromatography tandem mass spectrometry (LC-QQQ-MS). The LODs and LOQs of SCFAs using this method were in the range 1 to 7 ng mL -1 and 3 to 19 ng mL -1 , respectively. The recovery of the SCFAs in serum ranged from 94 to 114% over the three concentration ranges tested., (© 2021. The Author(s).)

Fentanyl and fentalogs' intake as drugs of abuse is experiencing a great increase in recent years. For this reason, there are more and more cases in which it is important to recognize and quantify these molecules and related metabolites in biological matrices. Oral fluid (OF) is often used to find out if a subject has recently used a psychoactive substance and if, therefore, the person is still under the effect of psychotropics. Given its difficulty in handling, good sample preparation and the development of instrumental methods for analysis are essential. In this work, an analytical method is proposed for the simultaneous determination of 25 analytes, including fentanyl, several derivatives and metabolites. OF was collected by means of passive drool; sample pretreatment was developed in order to be fast, simple and possibly semi-automated by exploiting microextraction on packed sorbent (MEPS). The analysis was performed by means of LC-HRMS/MS obtaining good identification and quantification of all the analytes in less than 10 min. The proposed method was fully validated according to the Scientific Working Group for Forensic Toxicology (SWGTOX) international guidelines. Good results were obtained in terms of recoveries, matrix effect and sensitivity, showing that this method could represent a useful tool in forensic toxicology. The presented method was successfully applied to the analysis of proficiency test samples.

Rationale: The World Antidoping Agency (WADA) Monitoring program concentrates analytical data from the WADA Accredited Laboratories for substances which are not prohibited but whose potential misuse must be known. The WADA List of Monitoring substances is updated annually, where substances may be removed, introduced or transferred to the Prohibited List, depending on the prevalence of their use. Retroactive processing of old sample datafiles has the potential to create information for the prevalence of use of candidate substances for the Monitoring List in previous years. MetAlign is a freeware software with functionality to reduce the size of liquid chromatography (LC)/high-resolution (HR) full-scan (FS) mass spectrometry (MS) datafiles and to perform a fast search for the presence of substances in thousands of reduced datafiles., Methods: Validation was performed to the search procedure of MetAlign applied to Anti-Doping Lab Qatar (ADLQ)-screened LC/HR-FS-MS reduced datafiles originated from antidoping samples for tramadol (TRA), ecdysterone (ECDY) and the ECDY metabolite 14-desoxyecdysterone (DESECDY) of the WADA Monitoring List. Searching parameters were related to combinations of accurate masses and retention times (RTs)., Results: MetAlign search validation criteria were based on the creation of correct identifications, false positives (FPs) and false negatives (FNs). The search for TRA in 7410 ADLQ routine LC/HR-FS-MS datafiles from the years 2017 to 2020 revealed no false identification (FPs and FNs) compared with the ADLQ WADA reports. ECDY and DESECDY were detected by MetAlign search in approximately 5% of the same cohort of antidoping samples., Conclusions: MetAlign is a powerful tool for the fast retroactive processing of old reduced datafiles collected in screening by LC/HR-FS-MS to reveal the prevalence of use of antidoping substances. The current study proposed the validation scheme of the MetAlign search procedure, to be implemented per individual substance in the WADA Monitoring program, for the elimination of FNs and FPs., (© 2021 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

The multi-attribute method (MAM) is a liquid chromatography-mass spectrometry based method that is used to directly characterize and monitor many product quality attributes and impurities on biotherapeutics, most commonly at the peptide level. It utilizes high-resolution accurate mass spectral data which are analyzed in an automated fashion. MAM is a promising approach that is intended to replace or supplement several conventional assays with a single LC-MS analysis and can be implemented in a Current Good Manufacturing Practice environment. MAM provides accurate site-specific quantitation information on targeted attributes and the nontargeted new peak detection function allows to detect new peaks as impurities, modifications, or sequence variants when comparing to a reference sample. The high resolution MAM workflow was applied here for three independent case studies. First, to monitor the behavior of monoclonal antibody product quality attributes over the course of a 12-day cell culture experiment providing an insight into the behavior and dynamics of product attributes throughout the process. Second, the workflow was applied to test the purity and identity of a product through analysis of samples spiked with host cell proteins. Third, through the comparison of a drug product and a biosimilar with known sequence variants. The three case studies presented here, clearly demonstrate the robustness and accuracy of the MAM workflow that implies suitability for deployment in the regulated environment.

Liquid chromatography-mass spectrometry (LC-MS) is one of the most widely used analytical tools. High analysis volumes and sample complexity often demand more informative LC-MS acquisition schemes to improve efficiency and throughput without compromising data quality, and such a demand has been always hindered by the prerequisite that a minimum of 13-20 MS scans (data points) across an analyte peak are required for accurate quantitation. The current study systematically re-evaluated and compared the impact of different scan numbers on quantitation analysis using both triple quadrupoles mass spectrometry (TQMS) and high-resolution mass spectrometry (HRMS). Contrary to the 13-20 minimal scan prerequisite, the data obtained from a group of eight commercial drugs in the absence and presence of biological matrices suggest that 6 scans per analyte peak are sufficient to achieve highly comparable quantitation results compared to that obtained using 10 and 20 scans, respectively. The fewer minimal scan prerequisite is presumably attributed to an improved LC system and advanced column technology, better MS detector, and more intelligent peak detection and integration algorithms leading to a more symmetric peak shape and smaller peak standard deviation. As a result, more informative acquisition schemes can be broadly set up for higher throughput and more data-rich LC-MS/MS analysis as demonstrated in a hepatocyte clearance assay in which fewer MS scans executed on HRMS led to broader metabolite coverage without compromising data quality in hepatic clearance assessment. The demonstrated acquisition scheme would substantially increase the throughput, robustness, and richness of the nonregulatory analysis, which can be broadly applied in diverse fields including pharmaceutical, environmental, forensic, toxicological, and biotechnological.

With increasing protein therapeutics being designed as non-mAb (non-monoclonal antibody) modalities, additional efforts and resources are required to develop and characterize such therapeutic proteins. Truncation is an emerging issue for manufacturing of non-mAb drug substances and requires sophisticated methods to investigate. In this paper, we describe two cases with complex truncation problems where traditional methods such as intact mass spectrometry led to inclusive or wrong identifications. Therefore, we developed an online top-down LC-MS (liquid chromatography-mass spectrometry) based workflow to study truncated drug substances, and we successfully identified the clipping locations. Compared to other orthogonal methods, this method provides a unique capability of solving protein clipping problems. The successful identification of truncated species and the high compatibility to routine intact MS make it a very valuable tool for resolving truncation problems during protein production in the pharmaceutical industry.

Synthetic peptides represent an important and expanding class of therapeutics. Despite having a relatively small size as compared to monoclonal antibodies and other proteins, synthetic peptides are subject to many complex structural modifications originating from the starting materials, manufacturing process, and storage conditions. Although mass spectrometry has been increasingly used to characterize impurities of synthetic peptides, systematic review of this field is scarce. In this paper, an overview of the impurities in synthetic peptide therapeutics is provided in the context of how the knowledge from detailed characterization of the impurities using liquid chromatography-mass spectrometry (LC-MS) can be used to develop the manufacturing process and control strategy for synthetic peptide therapeutics following the critical quality attribute (CQA)-driven and risk-based approach. The thresholds for identifying and controlling the impurities are discussed based on currently available regulatory guidance. Specific LC-MS techniques for identification of various types of impurities based on their structural characteristics are discussed with the focus on structural isomers and stereoisomers (i.e., peptide epimers). Absolute and relative quantitation methods for the peptide impurities are critiqued. Potential pitfalls in characterization of synthetic peptide therapeutics using LC-MS are discussed. Finally, a systematic LC-MS workflow for characterizing the impurities in synthetic peptide therapeutics is proposed, and future perspectives on applying emerging LC-MS techniques to address the remaining challenges in the development of synthetic peptide therapeutics are presented.

Bile acids (BAs) are biomolecules synthesized in the liver from cholesterol and are constituents of bile. The in-vivo BA pool includes more than 50 known diverse BAs which are unconjugated, amino acid conjugated, sulfated, and glucuronidated metabolites. Hemostasis of bile acids is known to be highly regulated and an interplay between liver metabolism, gut microbiome function, intestinal absorption, and enterohepatic recirculation. Interruption of BA homeostasis has been attributed to several metabolic diseases and drug induced liver injury (DILI), and their use as potential biomarkers is increasingly becoming important. Speciated quantitative and comprehensive profiling of BAs in various biomatrices from humans and preclinical animal species are important to understand their significance and biological function. Consequently, a versatile one single bioanalytical method for BAs is required to accommodate quantitation in a broad range of biomatrices from human and preclinical animal species. Here we report a versatile, comprehensive, and high throughput liquid chromatography-high resolution mass spectrometry (LC-HRMS) targeted metabolomics method for quantitative analysis of 50 different BAs in multiple matrices including human serum, plasma, and urine and plasma and urine of preclinical animal species (rat, rabbit, dog, and monkey). The method has been sufficiently qualified for accuracy, precision, robustness, and ruggedness and addresses the issue of nonspecific binding of bile acids to plastic for urine samples. Application of this method includes comparison for BA analysis between matched plasma and serum samples, human and animal species differences in BA pools, data analysis, and visualization of complex BA data using BA indices or ratios to understand BA biology, metabolism, and transport.

Lysophospholipids are bioactive signaling molecules derived from cell membrane glycerophospholipids or sphingolipids and are highly regulated under normal physiological conditions. Lysophosphatidic acids (LPAs) are a class of lysophospholipids that act on G-protein-coupled receptors to exert a variety of cellular functions. Dysregulation of phospholipase activity and consequently LPA synthesis in serum have been linked to inflammation, such as seen in chronic obstructive pulmonary disease (COPD). The accurate measurement of phospholipids is critical for evaluating their dysregulation in disease. In this study, we optimized experimental parameters for the sensitive measurement of LPAs. We validated the method based on matrix, linearity, accuracy, precision, and stability. An investigation into sample extraction processes emphasized that the common practice of including low concentration of hydrochloric acid in the extraction buffer causes an overestimation of lipid recovery. The liquid chromatography gradient was optimized to separate various lysophospholipid classes. After optimization, detection limits of LPA were sufficiently sensitive for subsequent analysis, ranging from 2 to 8 nM. The validated workflow was applied to a cohort of healthy donor and COPD patient sera. Eight LPA species were identified, and five unique species of LPA were quantified. Most LPA species increased significantly in COPD patients compared to healthy donors. The correlation between LPAs and other demographic parameters was further investigated in a sample set of over 200 baseline patient sera from a COPD clinical trial. For the first time, LPAs other than the two most abundant and readily detectable moieties are quantified in COPD patients using validated methods, opening the door to downstream biomarker evaluation in respiratory disease.

Chlorella vulgaris is a popular microalga used for biofuel production; nevertheless, it possesses a strong cell wall that hinders the extraction of molecules, especially lipids within the cell wall. For tackling this issue, we developed an efficient and cost-effective method for optimal lipid extraction. Microlaga cell disruption by acid hydrolysis was investigated comparing different temperatures and reaction times; after hydrolysis, lipids were extracted with n-hexane. The best recoveries were obtained at 140°C for 90 min. The microalgae were then analyzed by an untargeted approach based on liquid chromatography with high-resolution mass spectrometry, providing the tentative identification of 28 fatty acids. First, a relative quantification on the untargeted data was performed using peak area as a surrogate of analyte abundance. Then, a targeted quantitative method was validated for the tentatively identified fatty acids, in terms of recovery (78-100%), intra- and interday relative standard deviations (<10 and <9%, respectively) and linearity (R 2  > 0.98). The most abundant fatty acids were palmitic, palmitoleic, oleic, linoleic, linolenic, and stearic acids., (© 2021 The Authors. Journal of Separation Science published by Wiley-VCH GmbH.)

Validated LC-MS method for the direct quantitative analysis of galantamine (acetylcholinesterase inhibitor) was developed in rat cerebrospinal fluid and brain homogenate besides rat plasma, utilizing structurally close nalbuphine as an internal standard. After a simple protein precipitation step, samples are separated on 2-μm C18 column kept at 40 °C, using isocratic flow of 80% methanol in pH 9.5 ammonium formate buffer, and retention times were about 1.8 and 2.9 min for galantamine and nalbuphine, respectively. Mass detection with electrospray ionization (ESI) and positive polarity was able to detect 0.2 ng mL -1 galantamine using single ion monitoring mode (SIM) at m/z 288 for galantamine and m/z 358 for nalbuphine. The method showed linearity within the range of 0.5 - 300 ng mL -1 . The proposed method was validated according to FDA guidelines. Trueness and precision showed acceptable values at all quality control levels, and recoveries were within 85.6 - 114.3% in all matrices at all runs and with relative standard deviations within 0.2 - 12.4%. The method was used to study in vivo brain uptake and pharmacokinetics of galantamine from brain homogenate and plasma samples following the administration of nasal galantamine-bound chitosan nanoparticles compared to oral and nasal galantamine solutions, in scopolamine-induced Alzheimer's disease rat model., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Nano-liquid chromatography (nanoLC) has proven itself as a powerful tool and its scope entails various applications in (bio)analytical fields. Operation at low (nL/min) flow rates in combination with reduced inner dimensions (ID < 100 µm), leads to significantly enhanced sensitivity when coupled with electrospray ionization-mass spectrometry (ESI-MS). Challenges that remain for the routine implementation of such miniaturized setups are related to clogging of the system and robustness in general, and thus the application of tedious sample preparation steps. To improve ruggedness, a filter placed upstream in the LC prevents particles from entering and clogging the system. This so-called online automatic filtration and filter back-flush (AFFL) system was combined with nanoLC and the direct injection principle for the sensitive confirmatory analysis of fifty different doping-relevant peptides in urine. The presented assay was fully validated for routine purposes according to selectivity and matrix interference, limit of identification (LOI), carryover, matrix effect, sample extract stability, analysis of educational external quality assessment (EQAS) samples, robustness of the online AFFL-setup and retention time stability. It was also fully compliant with the most recent minimum required performance levels (MRPL) and chromatographic/mass spectrometric identification criteria (IDCR), as imposed by the World Anti-Doping Agency (WADA). In the absence of labor-intensive sample preparation, the application of AFFL allowed for the injection of diluted urine samples without any noticeable pressure buildup in the nanoLC system. Contrary to earlier observations by our group and others, the addition of dimethylsulfoxide (DMSO) to the mobile phase did not enhance sensitivity in the presented nanoflow setup, yet was beneficial to reduce carry over. Although the robustness of the presented setup was evaluated only for the analysis of diluted urine samples, it is entirely conceivable that routine applications employing other matrices and currently running on analytical scale LC instruments could be transferred to micro/nanoLC scale systems to reach lower detection limits., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A new, fast, and automatic approach has been applied for the tentative identification of unknown substances released by food contact epoxy resin after performing a migration test with food simulant. This approach combines intelligent data acquisition with AcquireX linked to liquid chromatography-Orbitrap Tribrid high-resolution mass spectrometry using data dependent-tandem mass spectrometry with triple stage fragmentation coupled to Compound Discoverer™ software for automated data processing and compound identification. The identification of the observed features was performed using a set of identification criteria, including exact mass, isotope pattern, tandem mass spectrometry spectra match, and retention time. With these criteria, 263 substances were tentatively identified. Most of the identified compounds were additives, such as plasticisers, stabilizers, and antioxidants, used in different plastic applications. However, metabolites, biological constituents with pharmacological activity, and other substances with industrial applications were also detected. In order to perform a risk assessment of the food contact epoxy resin, threshold of toxicological concern approach was applied for the identified compounds. There was not risk associated with the migration of the identified substances., (© 2021 Wiley-VCH GmbH.)

In the present study, the anti-inflammation effect of Phellinus igniarius extract was detected on an in vitro model of RAW 264.7 cells stimulated using sodium urate. In this cell model, the content changes of inflammatory cytokines, intercellular adhesion molecule-1, and interleukin-1 beta, in cell culture supernatants were detected using an enzyme-linked immunosorbent assay. The xanthine oxidase inhibitory activity of P. igniarius extracts was determined using a microplate reader. Furthermore, in order to identify the active compounds of P. igniarius, ultrafiltration liquid chromatography mass spectrometry was utilized to screen xanthine oxidase inhibitors from the extract. Our results showed that in the presence of P. igniarius extract, the expressions of interleukin-1 beta and intercellular adhesion molecule-1 decreased (p < 0.01 and p < 0.05, respectively) compared to that in the control group. The extract effective inhibited the xanthine oxidase activity. Finally, seven compounds were screened and identified as potential xanthine oxidase inhibitors from P. igniarius. Taken together, these results demonstrate a potential anti-inflammation bioactivity of P. igniarius in vitro, providing a basis for further in vivo research for the prevention and treatment of gout., (© 2021 Wiley-VCH GmbH.)

The research was done with partial filling micellar electrokinetic chromatography, microemulsion electrokinetic chromatography, and ultra-high performance liquid chromatography. The study focuses on determination of male and female steroids from cold and hot tap water of households in Helsinki City. The district´s raw water is made run from Päijänne Lake through a water tunnel to the purification plants in Helsinki area. The effluents delivered from the plants to households as tap water were sampled and used for the study. They were concentrated with solid phase extraction to exceed the detection limits of the three methods. With partial filling method the limits were 0.50, 0.48, 0.33, and 0.50 mg/L for androsterone, testosterone, progesterone, and testosterone-glucuronide, respectively. In microemulsion method the limit values were 1.33, 1.11, and 0.40 mg/L for androsterone, testosterone, and progesterone, respectively, and 0.83, 0.45, and 0.50 mg/L for hydrocortisone, 17-α-hydroxyprogesterone, and 17-α-methyltestosterone, respectively. In the tap water samples, progesterone concentrations represented the highest values being 0.22 and 1.18 ng/L in cold and hot water, respectively. They also contained testosterone (in all samples), its glucuronide metabolite (in 25% of the samples), and androstenedione (in 75% of the samples). The ultra-high liquid chromatographic method with mass spectrometric detection was used for identification of the steroids at µg/L level., Competing Interests: Declaration of Competing Interest The authors declare that they do not have competing financial interest concerning the project. They do not have any conflicts either., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Herbs represent complex chemical systems involving various primary and secondary metabolites that are featured by large spans of acid-base property, polarity, molecular mass, and content, etc., which thus poses great challenges to characterize the metabolites contained. Here, the combination of multiple-mechanism chromatography coupled with improved data-dependent-MS 2 acquisition (DDA-MS 2 ) is presented as a strategy to support the deep metabolites characterization. Targeting Uncaria sessilifructus, a reputable medicinal herb containing alkaloids and triterpenic acids (TAs) as the main pharmacologically bioactive ingredients, a three-dimensional liquid chromatography (3D-LC) system was established by integrating ion exchange chromatography, hydrophilic interaction chromatography, and reversed-phase chromatography (IEC-HILIC-RPC). The first-dimensional chromatography, configuring a PhenoSphere SCX column eluted by methanol/20 mM ammonium acetate-0.05% formic acid in water, could well fractionate the total extract into two fractions (unretained ingredients and alkaloids). The subsequent HILIC using an XAmide column and RPC by a CSH Phenyl-Hexyl column achieved the sufficient resolution of the total TAs and total alkaloids, respectively. A polarity-switching precursor ions list-including DDA approach by Q-Orbitrap-MS enabled the high-efficiency, coverage-enhanced identification of alkaloids and TAs. This 3D-LC/Q-Orbitrap-MS system was validated as precise (RSD < 5% for intra-day/inter-day precision), Up to 308 components were separated from U. sessilifructus, and 128 thereof (including 85 alkaloids, 29 TAs, and 14 others) were identified or tentatively characterized, exhibiting superiority over the conventional one-dimensional LC/MS. Conclusively, 3D-LC/MS in an off-line mode can facilitate the flexible configuration of multiple chromatography to accomplish the fit-for-purpose characterization of the metabolites from an herbal extract or a biosample., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021. Published by Elsevier B.V.)