RESUMO
In this chapter, we describe a multi-purpose, reversed-phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS) workflow for acquiring high-quality, non-targeted exposomics data utilizing data-dependent acquisition (DDA) combined with the use of toxicant inclusion lists for semi-targeted analysis. In addition, we describe expected retention times for >160 highly diverse xenobiotics in human plasma and serum samples. The method described is intended to serve as a generic LC-HRMS exposomics workflow for research and educational purposes. Moreover, it may be employed as a primer, allowing for further adaptations according to specialized research needs, e.g., by including reference and/or internal standards, by expanding to data-independent acquisition (DIA), or by modifying the list of compounds prioritized in fragmentation experiments (MS2).
Assuntos
Espectrometria de Massas , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Fluxo de Trabalho , Metabolômica/métodos , Xenobióticos/análise , Cromatografia de Fase Reversa/métodos , Espectrometria de Massas em Tandem/métodos , Exposição Ambiental/análiseRESUMO
Metabolomics can be used for a multitude of purposes, including monitoring of treatment effects and for increasing the knowledge of the pathophysiology of a wide range of diseases. Global (commonly referred to as "untargeted") metabolomics is hypothesis-generating and provides the opportunity to discover new biomarkers. Being versatile and having a high degree of selectivity and sensitivity, liquid chromatography-mass spectrometry (LC-MS) is the most common technique applied for metabolomics. We here present our global metabolomics LC-electrospray ionization-MS/MS method. The sample preparation procedures for plasma, serum, dried blood spots, urine, and cerebrospinal fluid are simple and nonspecific to reduce the risk of analyte loss. The method is based on reversed-phase chromatography using a diphenyl column. The high-resolution Q Exactive Orbitrap MS with data-dependent acquisition provides MS/MS spectra of a wide range of analytes. Our method covers a large part of the metabolome regarding hydrophobicity and compound class.
Assuntos
Metabolômica , Espectrometria de Massas em Tandem , Metabolômica/métodos , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Biomarcadores/sangue , Biomarcadores/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , Metaboloma , Teste em Amostras de Sangue Seco/métodos , Cromatografia de Fase Reversa/métodos , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Metabolomics has emerged as a pivotal field in understanding cellular function, particularly in the context of disease. In numerous diseases, including cancer, alterations in metabolism play an essential role in disease progression and drug response. Hence, unraveling the metabolic rewiring is of importance to find novel diagnostic and therapeutic strategies. Isotope tracing is a powerful technique for delving deeper into the metabolic wiring of cells. By tracking an isotopically labeled substrate through biochemical reactions in the cell, this technique provides a dynamic understanding of cellular metabolism. This chapter outlines a robust isotope tracing protocol utilizing high-resolution mass spectrometry coupled to liquid chromatography in cell culture-based models. We cover essential aspects of experimental design and analyses, providing a valuable resource for researchers aiming to employ isotopic tracing.
Assuntos
Marcação por Isótopo , Espectrometria de Massas , Metabolômica , Marcação por Isótopo/métodos , Cromatografia Líquida/métodos , Metabolômica/métodos , Espectrometria de Massas/métodos , Humanos , Animais , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Untargeted metabolomics is a powerful profiling tool for the discovery of possible biomarkers of disease onset and progression. Analytical pipelines applying liquid chromatography (LC) and mass spectrometry (MS)-based methods are widely used to survey a broad range of metabolites within various metabolic pathways, including organic acids, amino acids, nucleosides, and lipids. Accurate and complete identification of putative metabolites is an ongoing challenge in untargeted metabolomics studies. Highly sensitive instrumentation can result in the detection of adduct and fragment ions that form reproducibly and contain identifiable ions that are difficult to distinguish from metabolic pathway intermediates, which may result in false-positive identification. At concentrations as low as 10 µM, free fatty acids have been found to form homo- and heterodimers in untargeted metabolomics pipelines that resemble the lipid class fatty acid esters of hydroxy fatty acids (FAHFAs), resulting in misidentification. This chapter details a protocol for LC-MS-based untargeted metabolomics using hydrophilic interaction chromatography (HILIC) that specifically aids in distinguishing artifactual fatty acid dimers from endogenous FAHFAs.
Assuntos
Ésteres , Ácidos Graxos , Espectrometria de Massas , Metabolômica , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Ácidos Graxos/química , Cromatografia Líquida/métodos , Ésteres/análise , Ésteres/química , Ésteres/metabolismo , Metabolômica/métodos , Espectrometria de Massas/métodos , Artefatos , Dimerização , Hidroxiácidos/análise , Hidroxiácidos/metabolismo , Hidroxiácidos/química , Interações Hidrofóbicas e Hidrofílicas , Humanos , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Endocannabinoids (ECBs) are lipid-derived endogenous molecules with important physiological roles such as regulation of energy balance, immunity, or neural development. Quantitation of ECBs helps better understand their physiological role and modulation of biological processes. This chapter presents the simultaneous quantification of 14 ECBs and related molecules in the brain, liver, and muscle, as well as white and brown adipose tissue using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The dynamic range of the method has been tuned to cover the endogenous concentrations of these analytes given the fact that they are endogenously present at different orders of magnitude. Specifically, three groups are established: 0.5-5000 ng/mL for 2-oleoyl- and 2-linoleoylglycerol and arachidonic acid, 0.05-500 ng/mL for 2-arachidonoylglycerol, and 0.0005-0.5 ng/mL for anandamide, palmitoyl-, palmitoleoyl-, stearoyl-, oleoyl-, linoleoyl-, alpha-linolenoyl-, dihomo-gamma-linolenoyl-, docosahexaenoyl-, and pentadecanoylethanolamide.
Assuntos
Endocanabinoides , Espectrometria de Massas em Tandem , Endocanabinoides/análise , Endocanabinoides/metabolismo , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Animais , Encéfalo/metabolismo , Fígado/metabolismo , Fígado/química , Camundongos , Espectrometria de Massa com Cromatografia LíquidaRESUMO
The analysis of prostaglandin urinary metabolites is valuable for assessing physiological processes and identifying disease biomarkers. These metabolites, derived from the breakdown of prostaglandins, offer a noninvasive means to gauge prostaglandin production and its potential impact on various biological functions. We report an efficient LC-MS method of four commonly analyzed prostaglandin urinary metabolites including tetranor-PGEM (derived from PGE2), tetranor-PGDM, 11ß-PGF2α, and 2,3-dinor-11ß-PGF2α (derived from PGD2). Each metabolite possesses distinct characteristics and clinical applications, collectively contributing to our understanding of prostaglandin-mediated pathways.
Assuntos
Prostaglandinas , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Humanos , Cromatografia Líquida/métodos , Prostaglandinas/urina , Prostaglandinas/metabolismo , Biomarcadores/urina , Metabolômica/métodos , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Sphingolipids (SLs) are essential lipids with important functions in membrane formation and cell signaling. The presence of a long chain base (LCB) structure is common to all SLs. De novo SL synthesis is initiated by the enzyme serine-palmitoyltransferase (SPT), which forms an LCB by the conjugation from serine and fatty acyl-CoAs. SPT can metabolize a variety of acyl-CoA substrates, which form diverse LCB structures within and across species. The LCB then undergoes further metabolic modifications resulting in an extraordinarily diverse spectrum of sphingolipids formed. SL analysis, using liquid chromatography-mass spectrometry (LC-MS)-based methods, poses challenges due to the diverse range of frequently isobaric species. This complexity complicates the identification of underlying LCB structures using standard lipidomics approaches. Here, we describe a simplified method to analyze the LCB profile in cells, tissue, and blood. The procedure involves chemical hydrolysis to remove the conjugated headgroups and N-acyl chains, allowing to specifically resolve the underlying LCB structures by LC-MS. This method can also be combined with an isotope labeling approach to determine in vivo SPT activity and total SL de novo synthesis over time.
Assuntos
Esfingolipídeos , Cromatografia Líquida/métodos , Esfingolipídeos/metabolismo , Esfingolipídeos/análise , Esfingolipídeos/química , Lipidômica/métodos , Espectrometria de Massas/métodos , Animais , Humanos , Serina C-Palmitoiltransferase/metabolismo , Acil Coenzima A/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Sphingolipids are the most diverse class of lipids due to the numerous variations in their structural components. This diversity is also reflected in their extremely different functions. Sphingolipids are not only constituents of cell membranes but have emerged as key signaling molecules involved in a variety of cellular functions, such as cell growth and differentiation, proliferation and apoptotic cell death. Lipidomic analyses in clinical research have identified pathways and products of sphingolipid metabolism that are altered in several human pathologies. In this article, we describe how to properly design a lipidomic experiment in clinical research, how to handle plasma and serum samples for this purpose, and how to measure sphingolipids using liquid chromatography-mass spectrometry.
Assuntos
Lipidômica , Esfingolipídeos , Esfingolipídeos/metabolismo , Esfingolipídeos/sangue , Humanos , Lipidômica/métodos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodosRESUMO
Oxidative stress induces autooxidation of polyunsaturated fatty acids, producing numerous isoprostanoids and isofuranoids. These oxidized products are measurable in human plasma and urine and serve as oxidative stress biomarkers for chronic diseases. This chapter details the preparation and measurement of α-linolenic acid-derived phytoprostanes and phytofurans in human samples using liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QToF-MS/MS).
Assuntos
Ácidos Graxos Insaturados , Oxirredução , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Ácidos Graxos Insaturados/sangue , Ácidos Graxos Insaturados/urina , Cromatografia Líquida/métodos , Estresse Oxidativo , Biomarcadores/urina , Biomarcadores/sangue , Ácido alfa-Linolênico/urina , Ácido alfa-Linolênico/sangue , Ácido alfa-Linolênico/metabolismoRESUMO
Bioactive lipid mediators derived from arachidonic acid constitute an attractive pool of metabolites that reflect cellular function and signaling, as well as potential biomarkers that may respond quantitatively to disease progression or pharmacological treatment. Their quantitative measurement in biological samples is complicated by the number of isomers that share common structural features, which are not easily distinguished by immunoassays or reverse phase chromatography-tandem mass spectrometry. Here, we present a method that enables the rapid analysis of a panel of over 25 biologically important eicosanoids in a 96-well format for cell culture supernatants, plasma, and organ tissues using convergence chromatography-tandem mass spectrometry to resolve these analytes of interest.
Assuntos
Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Humanos , Eicosanoides/análise , Eicosanoides/metabolismo , Animais , Cromatografia Líquida/métodos , Lipídeos/análise , Lipídeos/química , Biomarcadores , Lipidômica/métodosRESUMO
Recent developments in LC-MS instrumentation and analytical technologies together with bioinformatics tools supporting high-throughput processing of large omics datasets significantly enhanced our capabilities and efficiency of identification and quantification of lipids in diverse biological materials. However, each biological matrix is characterized by its unique lipid composition, thus requiring optimization of analytical and bioinformatics workflows for each studied lipidome. Here, we describe an integrated workflow for deep lipidome profiling, accurate annotation, and semi-absolute quantification of complex lipidomes based on reversed phase chromatography and high resolution mass spectrometry. This chapter provides details on selection of the optimal extraction protocol, acquisition of LC-MS/MS data for accurate annotation of lipid molecular species, and design of lipidome-specific mixtures of internal standards to assist quantitative analysis of complex, native lipidomes.
Assuntos
Lipidômica , Lipídeos , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Lipidômica/métodos , Lipídeos/análise , Lipídeos/química , Cromatografia Líquida/métodos , Biologia Computacional/métodos , Fluxo de Trabalho , Humanos , Cromatografia de Fase Reversa/métodos , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Polylactic acid (PLA) straws hold eco-friendly potential; however, residual diisocyanates used to enhance the mechanical strength can generate carcinogenic primary aromatic amines (PAAs), posing health risks. Herein, we present a rapid, comprehensive strategy to detecting PAAs in 18 brands of food-grade PLA straws and assessing their migration into diverse food simulants. Surface-enhanced Raman spectroscopy was conducted to rapidly screen straws for PAAs. Subsequently, qualitative determination of migrating PAAs into various food simulants (4 % acetic acid, 10 % ethanol, 50 % ethanol) occurred at 70 °C for 2 h using liquid chromatography-mass spectrometry. Three PAAs including 4,4'-methylenedianiline, 2,4'-methylenedianiline, and 2,4-diaminotoluene were detected in all straws. Specifically, 2,4-diaminotoluene in 50 % ethanol exceeded specific migration limit of 2 µg/kg, raising safety concerns. Notably, PAAs migration to 10 % and 50 % ethanol surpassed that to 4 % acetic acid within a short 2-hour period. Moreover, PLA straws underwent varying degrees of shape changes before and after migration. Straws with poly(butylene succinate) resisted deformation compared to those without, indicating enhanced heat resistance, while poly(butyleneadipate-co-terephthalate) improved hydrolysis resistance. Importantly, swelling study unveiled swelling effect wasn't the primary factor contributing to the increased PAAs migration in ethanol food simulant, as there was no significant disparity in swelling degrees across different food simulants. FT-IR and DSC analysis revealed higher PAAs content in 50 % ethanol were due to highly concentrated polar ethanol disrupting hydrogen bonds and van der Waal forces holding PLA molecules together. Overall, minimizing contact between PLA straws and alcoholic foods is crucial to avoid potential safety risks posed by PAAs.
Assuntos
Aminas , Poliésteres , Análise Espectral Raman , Poliésteres/química , Análise Espectral Raman/métodos , Cromatografia Líquida/métodos , Aminas/análise , Aminas/química , Espectrometria de Massas/métodos , Contaminação de Alimentos/análise , Embalagem de Alimentos , Espectrometria de Massa com Cromatografia LíquidaRESUMO
As an interferon-stimulating factor protein, STING plays a role in the response and downstream liaison in antiviral natural immunity. Upon viral invasion, the immediate response of STING protein leads to a series of changes in downstream proteins, which ultimately leads to an antiviral immune response in the form of proinflammatory cytokines and type I interferons, thus triggering an innate immune response, an adaptive immune response in vivo, and long-term protection of the host. In the field of antiviral natural immunity, it is particularly important to rigorously and sequentially probe the dynamic changes in the antiviral natural immunity connector protein STING caused by the entire anti-inflammatory and anti-pathway mechanism and the differences in upstream and downstream proteins. Traditionally, proteomics technology has been validated by detecting proteins in a 2D platform, for which it is difficult to sensitively identify changes in the nature and abundance of target proteins. With the development of mass spectrometry (MS) technology, MS-based proteomics has made important contributions to characterizing the dynamic changes in the natural immune proteome induced by viral infections. MS analytical techniques have several advantages, such as high throughput, rapidity, sensitivity, accuracy, and automation. The most common techniques for detecting complex proteomes are liquid chromatography (LC) and mass spectrometry (MS). LC-MS (Liquid Chromatography-Mass Spectrometry), which combines the physical separation capability of LC and the mass analysis capability of MS, is a powerful technique mainly used for analyzing the proteome of cells, tissues, and body fluids. To explore the combination of traditional proteomics techniques such as Western blotting, Co-IP (co-Immunoprecipitation), and the latest LC-MS methods to probe the anti-inflammatory pathway and the differential changes in upstream and downstream proteins induced by the antiviral natural immune junction protein STING.
Assuntos
Imunidade Inata , Proteômica , Proteômica/métodos , Cromatografia Líquida/métodos , Humanos , Western Blotting/métodos , Espectrometria de Massas/métodos , Imunoprecipitação/métodos , Animais , Proteínas de Membrana/metabolismo , Proteínas de Membrana/imunologia , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Mass spectrometers are widely used to identify protein phosphorylation sites. The process usually involves selective isolation of phosphoproteins and subsequent fragmentation to identify both the peptide sequence and phosphorylation site. Immunoprecipitation could capture and purify the protein of interest, greatly reducing sample complexity before submitting it for mass spectrometry analysis. This chapter describes a method to identify an abnormal phosphorylated site of the adaptor protein by a viral kinase through immunoprecipitation followed by LC-MS/MS.
Assuntos
Imunoprecipitação , Fosfoproteínas , Espectrometria de Massas em Tandem , Fosforilação , Espectrometria de Massas em Tandem/métodos , Imunoprecipitação/métodos , Cromatografia Líquida/métodos , Humanos , Fosfoproteínas/metabolismo , Fosfoproteínas/análise , Espectrometria de Massas/métodosRESUMO
BACKGROUND AND AIMS: Current laboratory methods for opioid detection involve an initial screening with immunoassays which offers efficient but non-specific results and a subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation which offers accurate results but requires extensive sample preparation and turnaround time. Direct Analysis in Real Time (DART) tandem mass spectrometry is evaluated as an alternative approach for accurate opioid detection with efficient sample preparation and turnaround time. MATERIALS AND METHODS: DART-MS/MS was optimized by testing the method with varying temperatures, operation modes, extraction methods, hydrolysis times, and vortex times. The method was evaluated for 12 opioids by testing the analytical measurement range, percent carryover, precision studies, stability, and method-to-method comparison with LC-MS/MS. RESULTS: DART-MS/MS shows high sensitivity and specificity for the detection of 6-acetylmorphine, codeine, hydromorphone, oxymorphone, hydrocodone, naloxone, buprenorphine, norfentanyl, and fentanyl in urine samples. However, its performance was suboptimal for norbuprenorphine, morphine and oxycodone. CONCLUSION: In this proof-of-concept study, DART-MS/MS is evaluated for its rapid quantitative definitive testing of opioids drugs in urine. Further research is needed to expand its application to other areas of drug testing.
Assuntos
Analgésicos Opioides , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Analgésicos Opioides/urina , Cromatografia Líquida/métodos , Fatores de TempoRESUMO
Bacterial keratitis (BK) is an infection that causes inflammation of the cornea and, if severe, can result in blindness. Topical fluoroquinolones combined with corticosteroids have been shown to be useful in the treatment of BK. A rapid, selective, and sensitive bioanalytical method for simultaneous quantification of Gatifloxacin (GAT) and Dexamethasone (DEX) has been developed and validated using tandem mass spectrometry (LC-MS/MS). Optimal separation was accomplished in under 5 min using an Agilent Zorbax C18 column (100 mm × 4.6 mm, 3.5 µm). The mobile phase was composed of a blend of 0.2% formic acid in triple distilled water and methanol with a flow rate of 0.65 mL/min in isocratic mode. GAT and DEX were detected in positive electrospray ionization multiple reaction monitoring mode (MRM), and the retention time was found to be at 1.64 and 2.93 min, respectively. The linearity of GAT and DEX was found to be in the range of 1.56-400 ng mL-1 with good precision and accuracy. The method was validated according to USFDA regulatory guidelines. The validated method was effectively utilized for preclinical pharmacokinetic analysis of GAT and DEX in rabbit tear fluid following the topical application of a commercial formulation.
Assuntos
Dexametasona , Gatifloxacina , Espectrometria de Massas em Tandem , Lágrimas , Animais , Coelhos , Espectrometria de Massas em Tandem/métodos , Gatifloxacina/farmacocinética , Gatifloxacina/química , Dexametasona/farmacocinética , Dexametasona/análise , Lágrimas/química , Reprodutibilidade dos Testes , Limite de Detecção , Cromatografia Líquida/métodos , Masculino , Modelos Lineares , Antibacterianos/farmacocinética , Antibacterianos/análise , Antibacterianos/sangue , Fluoroquinolonas/farmacocinética , Fluoroquinolonas/análise , Fluoroquinolonas/sangue , Soluções Oftálmicas/farmacocinética , Soluções Oftálmicas/química , Espectrometria de Massa com Cromatografia LíquidaRESUMO
RATIONALE: Anabolic steroids, also known as anabolic-androgenic steroids (AAS), encompass steroidal androgens such as testosterone, as well as synthetic counterparts with similar structures and effects. The misuse of AAS has increased over the years, leading to ethical and welfare concerns in sports. The World Anti-Doping Agency (WADA) and the International Federation for Equestrian Sports (FEI) have banned AAS in relevant sports. Methandienone is one of the most identified anabolic androgenic steroids in sports drug testing, Therefore, reliable detection methods are crucial for effective doping control and maintaining the integrity of the sports. METHODS: This study explores the use of homogenized camel liver for detecting methandienone metabolites in camels. The biotransformation pathways of methandienone in homogenized camel liver tissues are analyzed using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) to identify and characterize the phase I and phase II metabolites. Chromatographic separation was achieved using a Thermo-Hypersil C18 column. RESULTS: The study has identified 11 methandienone metabolites (M1-M11), this includes 10 phase I and one phase II metabolite. A glucuronic acid conjugate of methandienone was observed in this study, but no sulfonic acid conjugations were found. The metabolites and their possible chemical structures, along with their fragmentation patterns are confirmed using MSMS (MS2) experiments in data-independent acquisition (DIA) mode. CONCLUSIONS: These findings serve as a vital tool for the rapid detection of methandienone, combating its illicit use in camel racing. Comprehensive screenings covering both the parent drug and its metabolites are recommended to improve detection accuracy and ensure regulatory compliance in sports doping. Future research should explore methandienone's metabolite profile in administered camel samples.
Assuntos
Anabolizantes , Camelus , Dopagem Esportivo , Fígado , Detecção do Abuso de Substâncias , Animais , Dopagem Esportivo/prevenção & controle , Detecção do Abuso de Substâncias/métodos , Fígado/metabolismo , Fígado/química , Anabolizantes/análise , Anabolizantes/metabolismo , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Metandrostenolona/metabolismo , Metandrostenolona/análise , Metandrostenolona/química , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Clinical expression of coronavirus disease 2019 (COVID-19) infectionis widely variable including fatal cases and patients with mild symptoms and a rapid resolution. We studied saliva from 63 hospitalized COVID-19 patients and from 30 healthy controls by integrating large-scale proteomics, peptidomics and targeted metabolomics to assess the biochemical alterations following the infection and to obtain a set of putative biomarkers useful for noninvasive diagnosis. We used an untargeted approach by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for proteomics and peptidomics analysis and targeted LC-multiple reaction monitoring/MS for the analysis of amino acids. The levels of 77 proteins were significantly different in COVID-19 patients. Among these, seven proteins were found only in saliva from patients with COVID-19, four were up-regulated and three were down-regulated at least five-folds in saliva from COVID-19 patients in comparison to controls. The analysis of proteins revealed a complex balance between pro-inflammatory and anti-inflammatory proteins and a reduced amount of several proteins with immune activity that possibly favours the spreading of the virus. Such reduction could be related to the enhanced activity of endopeptidases induced by the infection that in turn caused an altered balance of free peptides. In fact, on a total of 28 peptides, 22 (80%) were differently expressed in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and control subjects. The multivariate analysis of such peptides permits to obtain a diagnostic algorithm that discriminate the two populations with a high diagnostic efficiency. Among amino acids, only threonine resulted significantly different between COVID-19 patients and controls, while alanine levels were significantly different between COVID-19 patients with different severity. In conclusion, the present study defined a set of molecules to be detected with a quick and easy method based on mass spectrometry tandem useful to reveal biochemical alterations involved in the pathogenesis of such a complex disease. Data are available via ProteomeXchange with identifier PXD045612.
Assuntos
Biomarcadores , COVID-19 , Metabolômica , Proteômica , SARS-CoV-2 , Saliva , Espectrometria de Massas em Tandem , Humanos , COVID-19/virologia , COVID-19/metabolismo , Saliva/química , Saliva/virologia , Espectrometria de Massas em Tandem/métodos , Masculino , Feminino , Proteômica/métodos , Pessoa de Meia-Idade , Metabolômica/métodos , Biomarcadores/análise , Biomarcadores/metabolismo , Adulto , Idoso , Cromatografia Líquida/métodos , Estudos de Casos e Controles , Proteoma/análise , Proteoma/metabolismoRESUMO
BACKGROUND: Several oxylipins including hydroxy- and epoxy-polyunsaturated fatty acids act as lipid mediators. In biological samples they can be present as non-esterified form, but the major part occurs esterified in phospholipids (PL) or other lipids. Esterified oxylipins are quantified indirectly after alkaline hydrolysis as non-esterified oxylipins. However, in this indirect analysis the information in which lipid class oxylipins are bound is lost. In this work, an untargeted liquid chromatography high-resolution mass spectrometry (LC-HRMS) method for the direct analysis of PL bearing oxylipins was developed. RESULTS: Optimized reversed-phase LC separation achieved a sufficient separation of isobaric and isomeric PL from different lipid classes bearing oxylipin positional isomers. Individual PL species bearing oxylipins were identified based on retention time, precursor ion and characteristic product ions. The bound oxylipin could be characterized based on product ions resulting from the α-cleavage occurring at the hydroxy/epoxy group. PL sn-1/sn-2 isomers were identified based on the neutral loss of the fatty acyl in the sn-2 position. A total of 422 individual oxPL species from 7 different lipid classes i.e., PI, PS, PC, PE, PC-P, PC-O, and PE-P were detected in human serum and cells. This method enabled to determine in which PL class supplemented oxylipins are incorporated in HEK293 cells: 20:4;15OH, 20:4;14Ep, and 20:5;14Ep were mostly bound to PI. 20:4;8Ep and 20:5;8Ep were esterified to PC and PE while other oxylipins were mainly found in PC. SIGNIFICANCE: The developed LC-HRMS method enables the comprehensive detection as well as the semi-quantification of isobaric and isomeric PL species bearing oxylipins. With this method, we show that the position of the oxidation has a great impact and directs the incorporation of oxylipins into the different PL classes in human cells.
Assuntos
Espectrometria de Massas , Oxilipinas , Fosfolipídeos , Oxilipinas/análise , Oxilipinas/química , Humanos , Fosfolipídeos/análise , Fosfolipídeos/química , Espectrometria de Massas/métodos , Cromatografia Líquida/métodos , IsomerismoRESUMO
Donepezil (DPZ), a piperidine-based reversible cholinesterase inhibitor, finds extensive use in treating Alzheimer's disease (AD). Originally designed as an oral formulation, DPZ encounters drawbacks such as a brief duration of action and reduced treatment effectiveness in elderly patients with memory impairment or difficulty swallowing medications. To address these issues and improve patient compliance, researchers are actively exploring alternative DPZ formulations. Consequently, reliable methods are necessary to quantitate DPZ in biological samples for in vivo assessment. Therefore, we propose an efficient, sensitive, wide-dynamic, and cost-effective method for quantitating DPZ in rat plasma. Our method employs liquid-liquid extraction (LLE) followed by liquid chromatography and tandem mass spectrometry, enabling in vivo evaluation of novel DPZ formulations. Notably, our method requires only 20 µL of rat plasma and employs icopezil as the internal standard-a cost-effective compound with chemical similarity to DPZ. We meticulously optimized LLE conditions, taking into account factor interactions through design of experiments (DOE). Our rapid and straightforward extraction and purification involved using 500 µL of pure methyl tert-butyl ether to extract DPZ from the sample within five minutes. The dynamic range of the method extends from 0.5 ng/mL to 1,000 ng/mL, demonstrating excellent sensitivity and suitability for pharmacokinetic studies across diverse DPZ formulations. Following the FDA guidelines, we rigorously validated the developed method, evaluating selectivity, linearity (with a coefficient of determination ≥0.9999), accuracy (ranging from 96.0% to 109.6%), precision (≤13.9%), matrix effect (92.2% to 103.8%), recovery (98.5% to 106.8%), the lower limit of quantitation (0.5 ng/mL), and stability. Finally, we effectively employed the validated method for the long-term pharmacokinetic assessment of a DPZ formulation. We expect that this approach will make a substantial contribution to the advancement of new DPZ formulations, ultimately benefiting individuals afflicted by AD.