Search Results (171)

Background/Objectives: Sustained drug exposure is a key factor in the treatment of patients infected with human immunodeficiency virus (HIV) or hepatitis B virus (HBV) in order to achieve the intended virological response. Although influenced also by other parameters, adherence to the treatment scheme is the most important for adequate drug exposure. This can be assessed by therapeutic drug monitoring (TDM). Tenofovir (TFV) is a nucleotide analogue used in the treatment of both HIV and HBV. Although various analytical methods for the quantification of tenofovir prodrugs have been published, there is limited literature on methods for simultaneous TFV and its active metabolite, tenofovir diphosphate (TFVDP) direct determination. Methods: In this study, we describe a novel micro-liquid-chromatography-mass spectrometry (micro-LC-MS/MS) method for TDM of TFV and TFVDP in biological matrices (whole blood, plasma). The challenging separation of the high-polarity analytes was resolved on an amino stationary phase, eluted in HILIC (hydrophilic interaction liquid chromatography) mode. The sample preparation included a clean-up step with hexane for the removal of lipophilic compounds and then protein precipitation with organic solvent. Results: The achieved low limits of quantification in blood were 0.25 ng/mL for TFV, and 0.5 ng/mL for TFVDP. Linearity, accuracy (91.63–109.18%), precision (2.48–14.08), and stability were validated for whole blood matrix, meeting the guidelines performance criteria. Samples collected from treated patients were analyzed, with results being in accordance with the reported pharmacokinetics. Conclusions: The new method is adequate for analyzing samples in a clinical set-up. The measurement of both TFV and TFVDP improves clinical decision by an in-depth evaluation of long-term adherence, and together with viral load and resistance data helps guiding the treatment towards the intended virological suppression. Full article

Background: There is a continuous demand to create new, superior sensory food experiences. In the food industry, yeast-derived flavor products (YPs) are often used as ingredients in foods to create new aromas and taste qualities that are appreciated by consumers. Methods: Chicken bouillon samples containing diverse YPs were chemically and sensorially characterized using statistical multivariate analyses. The sensory evaluation was performed using quantitative descriptive analysis (QDA) by trained panelists. Thirty-four sensory attributes were scored, including odor, flavor, mouthfeel, aftertaste and afterfeel. Untargeted metabolomic profiles were obtained using stir bar sorptive extraction (SBSE) coupled to GC-MS, RPLC-MS and targeted HILIC-MS. Results: In total, 261 volatiles were detected using GC-MS, from chemical groups of predominantly aldehydes, esters, pyrazines and ketones. Random Forest (RF) modeling revealed volatiles associated with roast odor (2-ethyl-5-methyl pyrazine, 2,3,5-trimethyl-6-isopentyl pyrazine) and chicken odor (2,4-nonadienal, 2,4-decadienal, 2-acetyl furan), which could be predicted by our combined model with R² > 0.5. In total, 2305 non-volatiles were detected for RPLC-MS and 34 for targeted HILIC-MS, where fructose-isoleucine and cyclo-leucine-proline were found to correlate with roast flavor and odor. Furthermore, a list of metabolites (glutamate, monophosphates, methionyl-leucine) was linked to umami-related flavor. This study describes a straightforward data-driven approach for studying foods with added YPs to identify flavor-impacting correlations between molecular composition and sensory perception. It also highlights limitations and preconditions for good prediction models. Overall, this study emphasizes a matrix-based approach for the prediction of food taste, which can be used to analyze foods for targeted flavor design or quality control. Full article

In this work, metabolite profiling of seeds and antioxidant analysis of fragments of two marine seagrasses, Posidonia oceanica and Cymodocea nodosa, were carried out to identify metabolite signature involved in seed viability and to evaluate the potential of fragments as a source of bioactive compounds. Using HILIC/QTOF-MS, UHPLC-MS and spectrophotometric analysis, seed metabolites and polyphenols and antioxidant activities, such as those of radical scavenging (RSA), reduction (FRAP, CUPRAC) and complexation (CCA), of rhizome fragments were evaluated. Metabolite comparison between seeds revealed differences across development stages (germinated and non-germinated) and seed types (dormant and non-dormant), providing insights into metabolic activity potentially associated with germination processes and seed viability. Furthermore, polyphenol analysis showed the highest content of caffeic acid in mature leaves (17.00 ± 0.02 μg g⁻¹ dw for P. oceanica and 98.00 ± 0.03 μg g⁻¹ dw for C. nodosa). Total phenolic content was correlated with flavonoids and with reduction and complexation activities. The combination of radical scavenging activity and t_1/2 was higher in P. oceanica than C. nodosa and also surpassed the commercial synthetic antioxidant BHA. We conclude P. oceanica and C. nodosa exhibit distinct seed metabolite profiles related to germination and type of seeds, and that fragments are rich in antioxidants, with potential as sustainable sources of bioactive compounds. Full article

Gynecologic tumors are a leading cause of cancer-related mortality in women worldwide, with endometrial, ovarian, and cervical types being the most prevalent. Aberrant glycosylation, a key post-translational modification, plays a crucial role in tumor development, metastasis, and immune evasion. Specific glycosylation changes, such as altered sialylation and fucosylation, have been identified in gynecologic cancers and are associated with disease progression and prognosis. Understanding glycosylation alterations in gynecologic cancers holds promise for novel diagnostic and therapeutic approaches, ultimately enhancing patient outcomes. In this study, the serum N-glycome was analyzed in patients with myoma uteri, endometriosis, and cervical carcinoma by hydrophilic-interaction liquid chromatography (HILIC-UPLC) with fluorescence (FLR) and mass-spectrometric (MS) detection in order to identify their biomarker potential. Individual serum samples were deglycosylated by PNGase F digestion followed by procainamide labeling and solid-phase-extraction-based purification. All disease groups exhibited consistently higher levels of specific bi-antennary glycans (A2G2 and A2G2S1) compared to control patients. Additionally, significantly higher levels of agalactosylated and mono-sialylated glycans were found in cervical cancer, while a notable decrease in bisected N-glycans, alongside an increase in highly branched tetra-sialylated glycans, was found in endometriosis. Our study serves as proof-of-concept, demonstrating that discovering biomarkers within the serum N-glycome is a promising approach for identifying non-invasive indicators of gynecologic conditions. Full article

Background: The presence and concentration of lipids in serum of dairy cows have significant implications for both animal health and productivity and are potential biomarkers for several common diseases. However, information on serum lipid composition is rather fragmented, and lipid remodelling during the transition period is only partially understood. Methods: Using a combination of reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS), hydrophilic interaction-mass spectrometry (HILIC-MS), and lipid annotation software, we performed a comprehensive identification and quantification of serum of dairy cows in pasture-based Holstein-Friesian cows. The lipid remodelling induced by negative energy balance was investigated by comparing the levels of all identified lipids between the fresh lactation (5–14 days in milk, DIM) and full lactation (65–80 DIM) stages. Results: We identified 535 lipid molecular species belonging to 19 classes. The most abundant lipid class was cholesteryl ester (CE), followed by phosphatidylcholine (PC), sphingomyelin (SM), and free fatty acid (FFA), whereas the least abundant lipids included phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), acylcarnitine (AcylCar), ceramide (Cer), glucosylceramide (GluCer), and lactosylceramide (LacCer). Conclusions: A remarkable increase in most lipids and a dramatic decrease in FFAs, AcylCar, and DHA-containing species were observed at the full lactation compared to fresh lactation stage. Several serum lipid biomarkers for detecting negative energy balance in cows were also identified. Full article

The glycosylation process plays a crucial role in the structural integrity and biological activity of glycoproteins, where glycans are attached to a protein backbone. There are many kinds of glycans, the most common being N-glycans, which can be arranged into three classes, that is, complex, hybrid, and high mannoses, forming a structurally very diverse set of polar compounds that are difficult to detect and separate. Most commonly, N-glycans are labeled before separation by charged or fluorescence tags for better MS or fluorescence detection, respectively. This study examines the influence of ionic strength and organic modifier selection on the separation of fluorescently labeled dextran ladders in Hydrophilic Interaction Liquid Chromatography (HILIC). Using a Glycan BEH Amide column and varying the ammonium formate buffer concentration along with acetonitrile and methanol ratios, we investigated analyte retention, separation efficiency, and post-column conductivity changes. Our findings reveal that changes in the ionic strength of the mobile phase do not contribute to changes in selectivity, neither when acetonitrile nor methanol were used as organic modifiers to the mobile phase. However, the addition of methanol significantly changes the separation mechanism where two different prevailing separations mechanisms can be identified. It was assumed that the addition of methanol influences the folding pattern of dextrans around the permanent positive charge on the added tag, which influences the changes of separation selectivity. This work presents a systematic approach to altering mobile phase composition (buffer concentration, organic modifier type) to control retention and selectivity in complex glycan analysis. The discovery that methanol significantly alters separation behavior provides a potential new method for refining HILIC separations of polar compounds. Full article

Background: The metabolome of COVID-19 patients has been studied sparsely, with most research focusing on a limited number of plasma metabolites or small cohorts. This is the first study to test saliva metabolites in COVID-19 patients in a comprehensive way, revealing patterns significantly linked to disease and severity, highlighting saliva’s potential as a non-invasive tool for pathogenesis or diagnostic studies. Methods: We included 30 asymptomatic subjects with no prior COVID-19 infection or vaccination, 102 patients with mild SARS-CoV-2 infection, and 61 hospitalized patients with confirmed SARS-CoV-2 status. Saliva samples were analyzed using hydrophilic interaction liquid chromatography–mass spectrometry (HILIC-MS/MS) in positive and negative ionization modes. Results: Significant differences in metabolites were identified in COVID-19 patients, with distinct patterns associated with disease severity. Dipeptides such as Val-Glu and Met-Gln were highly elevated in moderate cases, suggesting specific protease activity related to SARS-CoV-2. Acetylated amino acids like N-acetylserine and N-acetylhistidine increased in severe cases. Bacterial metabolites, including muramic acid and indole-3-carboxaldehyde, were higher in mild–moderate cases, indicating that oral microbiota differs according to disease severity. In severe cases, polyamines and organ-damage-related metabolites, such as N-acetylspermine and 3-methylcytidine, were significantly increased. Interestingly, most metabolites that were reduced in moderate cases were elevated in severe cases. Conclusions: Saliva metabolomics offers insightful information that is potentially useful in studying COVID-19 severity and for diagnosis. Full article

Semicarbazide (SEM), a metabolite of nitrofurazone (NFZ), is widely used to detect the illegal application of NFZ in crustaceans. The conventional detection method involves chemical derivatization combined with reversed-phase liquid chromatography–tandem mass spectrometry (RPLC-MS/MS), which is both complex and time-consuming. To address this limitation, a more efficient approach was developed for SEM detection. This study introduces a modified QuEChERS pretreatment method coupled with hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) for detecting SEM in crustaceans. The proposed method is simple, fast, and highly accurate, making it universally applicable for SEM detection in crustaceans. Additionally, the method was applied to investigate NFZ metabolism in Macrobrachium rosenbergii with a kinetic model. The findings suggested a plausible mechanism for the absorption of NFZ and its subsequent transfer from meat to the shell. In conclusion, this study provides a simple and rapid technique for SEM detection in crustaceans with immense application value. Full article

Plasma contains metabolites with diverse physicochemical properties, ranging from highly polar to highly apolar, and concentrations spanning at least nine orders of magnitude. Plasma metabolome analysis is valuable for monitoring health and evaluating medical interventions but is challenging due to the metabolome’s diversity and complexity. This study aims to develop and validate targeted LC-MS/MS methods for quantifying 235 mammalian metabolites from 17 compound classes in porcine plasma without prior derivatization. Utilizing reversed-phase and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry, each analyte is identified and quantified using two selected reaction monitoring (SRM) transitions. Fast polarity switching and scheduled SRM enhance the metabolome coverage and throughput, enabling the analysis of one sample in about 40 min. A simple “dilute and shoot” sample preparation protocol was employed, with samples injected at two dilution levels to align metabolite concentrations within calibration curve ranges. Validation in porcine plasma included assessments of carryover, linearity, detection and quantification limits, repeatability and recovery. The method was further applied to plasma samples from various animal species, demonstrating its applicability to human and animal studies. This study establishes two robust LC-MS/MS methods for comprehensive porcine plasma metabolome quantification, advancing large-scale targeted metabolomics in biomedical research. Full article

Kefir, a fermented milk product produced using kefir grains, is a symbiotic consortium of bacteria and yeasts responsible for driving the fermentation process. In this study, an in-depth analysis of kefir’s lipid profile was conducted, with a focus on its phospholipid (PL) content, employing liquid chromatography with high-resolution mass spectrometry (LC-HRMS). Nearly 300 distinct polar lipids were identified through hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization (ESI) and Fourier-transform orbital-trap MS and linear ion-trap tandem MS/MS. The identified lipids included phosphatidylcholines (PCs), lyso-phosphatidylcholines (LPCs), phosphatidylethanolamines (PEs) and lyso-phosphatidylethanolamines (LPEs), phosphatidylserines (PSs), phosphatidylglycerols (PGs), and phosphatidylinositols (PIs). The presence of lysyl-phosphatidylglycerols (LyPGs) was identified as a key finding, marking a lipid class characteristic of Gram-positive bacterial membranes. This discovery highlights the role of viable bacteria in kefir and underscores its probiotic potential. The structural details of minor glycolipids (GLs) and glycosphingolipids (GSLs) were further elucidated, enriching the understanding of kefir’s lipid complexity. Fatty acyl (FA) composition was characterized using reversed-phase LC coupled with tandem MS. A mild epoxidation reaction with meta-chloroperoxybenzoic acid (m-CPBA) was performed to pinpoint double-bond positions in FAs. The dominant fatty acids were identified as C18:3, C18:2, C18:1, C18:0 (stearic acid), C16:0 (palmitic acid), and significant levels of C14:0 (myristic acid). Additionally, two isomers of FA 18:1 were distinguished: ∆9-cis (oleic acid) and ∆11-trans (vaccenic acid). These isomers were identified using diagnostic ion pairs, retention times, and accurate m/z values. This study provides an unprecedented level of detail on the lipid profile of kefir, shedding light on its complex composition and potential nutritional benefits. Full article

The signature of human serum IgG glycosylation is critical in the defense against pathogens. Alterations of IgG N-glycome were associated with COVID-19 (Coronavirus disease 2019) severity, although knowledge on the response to vaccination is limited. IgG N-glycome was analyzed in this study in post-COVID-19 and post-vaccination patients to reveal potential glycosylation-based alterations using hydrophilic interaction liquid chromatography (HILIC-UPLC) with fluorescence (FLR) and mass-spectrometric (MS) detection. IgG antibodies were purified from serum samples through protein G affinity chromatography followed by PNGase F digestion-based deglycosylation. The released glycans were fluorescently derivatized by procainamide labeling and purified via solid-phase extraction. Higher levels of sialylation and afucosylation were identified in post-COVID-19 patients, which was further expanded by vaccination, but only in those who were previously SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) infected. Full article

Analyzing and detecting endogenous amino acids in blood is of crucial importance for the diagnosis of medical conditions and scientific research. Considering the lack of UV chromophores in most of these analytes and the presence of several interfering substances in plasma, the quantification of quite a few amino acids and related compounds presents certain technical challenges. As a blank plasma matrix lacking these endogenous substances does not exist, the surrogate matrix method is used, as well as isotopic internal standards for calibration, to ensure the accuracy and reliability of the study. Method validation was conducted for 48 target analytes, giving the following results: linearity (R² at least 0.99), limit of quantification (from 0.65 to 173.44 μM), precision (intra-day and inter-day RSD for LQC ranged from 3.2% to 14.2%, for MQC from 2.0% to 13.6%, and for HQC from 1.6% to 11.3%), accuracy, recovery, and stability of the method (all complied with the guidelines). To further investigate the applicability of this method to large-scale sample analysis, the method was successfully applied to the analysis of amino acids in plasma samples collected from 20 control individuals, demonstrating its wide application scope for clinical diagnosis and metabolic research. Full article

Colorectal cancer (CRC) development is a gradual process in which progressive histological alterations of the intestinal mucosa damage occur over years. This process can be influenced by modifiable external factors such as lifestyle and diet. Most CRC cases (>80%) originate from conventional adenomas through the adenomatous pathway and usually harbour dysplastic cells, whereas the serrated pathway is less frequent (<20% cases) and comprises hyperplastic polyps and other polyps containing dysplastic cells. The aim of the present work was to shed light on alterations of the faecal metabolome associated with hyperplastic polyps and conventional adenomas. Metabolites were analysed by Reversed-Phase High-Performance Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry (RP/HPLC-Q/TOF-MS/MS) and Hydrophilic Interaction Liquid Chromatography–Quadrupole-Time of Flight Mass Spectrometry (HILIC-Q/TOF-MS/MS) and the results were integrated. Comparisons were performed between controls without mucosal lesions and the polyps’ group, hyperplastic polyps versus conventional adenomas, and hyperplastic polyps or conventional adenomas versus controls. Alterations of metabolites in specific biochemical modules differentiated hyperplastic polyps and conventional adenomas. The metabolome of the hyperplastic polyps was characterized by an enrichment in glycerophospholipids and an altered metabolism of the degradation pathways of xanthines/purines and pyrimidines, whereas the enrichment in some phenolic compounds and disaccharides, all of them from exogenous origin, was the main differential faecal signature of conventional adenomas. Further research could help to elucidate the contribution of diet and the intestinal microbiota to these metabolomics alterations. Full article

Peripheral blood mononuclear cells (PBMCs), including lymphocytes, are important components of the human immune system. These cells contain a diverse array of lipids, primarily glycerophospholipids (GPs) and sphingolipids (SPs), which play essential roles in cellular structure, signaling, and programmed cell death. This study presents a detailed analysis of GP and SP profiles in human PBMC samples using tandem mass spectrometry (MS/MS). Hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization (ESI) coupled with linear ion-trap MS/MS were employed to investigate the diagnostic fragmentation patterns that aided in determining regiochemistry in complex lipid extracts. Specifically, the study explored the fragmentation patterns of various lipid species, including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), their plasmalogen and lyso forms, phosphatidylserines (PSs), phosphatidylinositols (PIs), phosphatidylglycerols (PGs), sphingomyelins (SMs), and dihexosylceramides (Hex₂Cer). Our comprehensive analysis led to the characterization of over 200 distinct lipid species, significantly expanding our understanding of PBMC lipidome complexity. A freely available spreadsheet tool for simulating MS/MS spectra of GPs is provided, enhancing the accessibility and reproducibility of this research. This study advances our knowledge of PBMC lipidomes and establishes a robust analytical framework for future investigations in lipidomics. Full article

Background: Nicotinamide adenine dinucleotide (NAD⁺), its precursors, and its derivatives (collectively NADome) play a crucial role in cellular processes and maintain redox homeostasis. Understanding the dynamics of these metabolic pools and redox reactions can provide valuable insights into metabolic functions, especially cellular regulation and stress response mechanisms. The accurate quantification of these metabolites is challenging due to the interconversion between the redox forms. Methods: Our laboratory previously developed a zwitterionic hydrophilic interaction liquid chromatography (zic-HILIC)–tandem mass spectrometry method for the quantification of five essential pyridine nucleotides, including NAD⁺ derivatives and it’s reduced forms, with ¹³C isotope dilution and matrix-matched calibration. In this study, we have improved the performance of the chromatographic method and expanded its scope to twelve analytes for a comprehensive view of NAD⁺ biosynthesis and utilization. The analytical method was validated and applied to investigate Escherichia coli BL21 under varying oxygen supplies including aerobic, microaerobic, and anaerobic conditions. Conclusions: The intracellular absolute metabolite concentrations ranged over four orders of magnitude with NAD⁺ as the highest abundant, while its precursors were much less abundant. The composition of the NADome at oxygen-limited conditions aligned more with that in the anaerobic conditions rather than in the aerobic phase. Overall, the NADome was quite homeostatic and E. coli rapidly, but in a minor way, adapted the metabolic activity to the challenging shift in the growth conditions and achieved redox balance. Our findings demonstrate that the zic-HILIC-MS/MS method is sensitive, accurate, robust, and high-throughput, providing valuable insights into NAD⁺ metabolism and the potential significance of these metabolites in various biological contexts. Full article