RESUMO
To improve the adsorption affinity and selectivity of fipronils (FPNs), including fipronil, its metabolites and analogs, a magnetic covalent organic framework (Fe3O4@COF-F) with copious fluorine affinity sites was innovatively designed as an adsorbent of magnetic solid-phase extraction (MSPE). The enhanced surface area, pore size, crystallinity of Fe3O4@COF-F and its exponential adsorption capacities (187.3-231.5 mg g-1) towards fipronils were investigated. Combining MSPE with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), an analytical method was established for the selective determination of fipronils in milk and milk powder samples. This method achieved high sensitivity (LODs: 0.004-0.075 ng g-1), satisfactory repeatability and accuracy with spiked recoveries ranging from 89.9% to 100.3% (RSDs≤5.1%). Overall, the constructed Fe3O4@COF-F displayed great potential for the selective enrichment of fipronils, which could be ascribed to fluorinefluorine interaction. This method proposed a feasible and promising strategy for the development of functionalized COF and broadened its application in fluorine containing hazards detection.
Assuntos
Flúor , Contaminação de Alimentos , Estruturas Metalorgânicas , Leite , Pirazóis , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Pirazóis/química , Contaminação de Alimentos/análise , Flúor/química , Leite/química , Animais , Estruturas Metalorgânicas/química , Adsorção , Cromatografia Líquida de Alta Pressão , Inseticidas/química , Inseticidas/análise , Limite de DetecçãoRESUMO
The use of direct injection ion mobility mass spectrometry (DI-IM-MS) to detect and identify betacyanin pigments in A. hortensis 'rubra' extracts was explored for the first time, with results compared to conventional LC-MS/MS analysis. The anti-inflammatory activities of leaf and seed extracts, alongside purified amaranthin and celosianin pigments, were investigated using a model of lipopolysaccharide (LPS)-activated murine macrophages. Extracts and purified pigments significantly inhibited the production of prostaglandin E2 and NO by up to 90% and 70%, respectively, and reduced the expression of Il6, Il1b, Nos2, and Cox2. Leaf and seed extracts also decreased secretion of Il6 and Il1b cytokines and reduced protein levels of Nos2 and Cox2. Furthermore, extracts and purified pigments demonstrated potent dose-dependent radical scavenging activity in a cellular antioxidant activity assay (CAA) without any cytotoxic effects. Our research highlights the promising biological potential of edible, climate-resilient A. hortensis 'rubra' as a valuable source of bioactive compounds.
Assuntos
Lipopolissacarídeos , Macrófagos , Estresse Oxidativo , Extratos Vegetais , Camundongos , Animais , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Células RAW 264.7 , Estresse Oxidativo/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Lipopolissacarídeos/farmacologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Ciclo-Oxigenase 2/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Espectrometria de Massas em TandemRESUMO
This study presents the contents of α-methylenecyclopropylglycine, a potentially toxic amino acid, in the peel, pulp and seed fractions of two well-known litchi varieties, namely Shahi and China, over a span of three harvest-seasons. For analysing α-methylenecyclopropylglycine, an LC-MS/MS-based method was validated. The method-accuracies fell within 75-110 % (RSD, <15 %) at 0.1 mg/kg (LOQ) and higher levels. A comparative evaluation of the results in peel, pulp and seed at 30 days before harvest (DBH), 15-DBH, and edible-ripe stage revealed that α-methylenecyclopropylglycine content increased as the litchi seeds grew towards maturity, regardless of the cultivar. In arils, at maturity, the concentration of α-methylenecyclopropylglycine ranged from not-detected to 11.7 µg/g dry weight. The Shahi cultivar showed slightly higher α-methylenecyclopropylglycine content in comparison to China litchi. This paper presents the first known analysis of combined seasonal data on different fruit components at various growth stages for the two chosen litchi cultivars grown in India.
Assuntos
Frutas , Litchi , Sementes , Espectrometria de Massas em Tandem , Litchi/química , Litchi/crescimento & desenvolvimento , Litchi/metabolismo , Frutas/química , Frutas/crescimento & desenvolvimento , China , Sementes/química , Sementes/crescimento & desenvolvimento , Glicina/análogos & derivados , Glicina/análise , Cromatografia Líquida de Alta Pressão , Ciclopropanos/análiseRESUMO
Fortification of human milk (HM) is often necessary to meet the nutritional requirements of preterm infants. The present experiment aimed to establish whether the supplementation of HM with either an experimental donkey milk-derived fortifier containing whole donkey milk proteins, or with a commercial bovine milk-derived fortifier containing hydrolyzed bovine whey proteins, affects peptide release differently during digestion. The experiment was conducted using an in vitro dynamic system designed to simulate the preterm infant's digestion followed by digesta analysis by means of LC-MS-MS. The different fortifiers did not appear to influence the cumulative intensity of HM peptides. Fortification had a differential impact on the release of either donkey or bovine bioactive peptides. Donkey milk peptides showed antioxidant/ACE inhibitory activities, while bovine peptides showed opioid, dipeptil- and propyl endo- peptidase inhibitory and antimicrobial activity. A slight delay in peptide release from human lactoferrin and α-lactalbumin was observed when HM was supplemented with donkey milk-derived fortifier.
Assuntos
Digestão , Equidae , Proteínas do Leite , Leite Humano , Peptídeos , Humanos , Animais , Leite Humano/química , Leite Humano/metabolismo , Proteínas do Leite/química , Proteínas do Leite/metabolismo , Proteínas do Leite/análise , Bovinos , Peptídeos/química , Peptídeos/metabolismo , Alimentos Fortificados/análise , Espectrometria de Massas em Tandem , Modelos Biológicos , Proteínas do Soro do Leite/química , Proteínas do Soro do Leite/metabolismoRESUMO
A modified QuEChERS method was developed to determine multi-class pesticide and veterinary residues in aquatic products. Chitosan microspheres were conveniently synthesized and utilized as the cleanup adsorbent in the QuEChERS procedure, showcasing rapid filtration one-step pretreatment ability for the determination of drug multi-residues in aquatic products. Compared to conventional synthetic sorbents, chitosan microspheres not only have good purification performance, but also have renewable and degradable properties. This novel sorbent worked well in the simultaneous determination of 95 pesticides and veterinary drug residues in aquatic products after being combined with an improved one-step vortex oscillating cleanup method. We achieved recoveries ranging from 64.0% to 115.9% for target drugs in shrimp and fish matrix. The limits of detection and quantification were 0.5-1.0 and 1.0-2.0 µg kg-1, respectively. Notably, hydrocortisone was detected with considerable frequency and concentration in the tested samples, underscoring the necessity for stringent monitoring of this compound in aquatic products.
Assuntos
Quitosana , Peixes , Microesferas , Espectrometria de Massas em Tandem , Drogas Veterinárias , Animais , Quitosana/química , Cromatografia Líquida de Alta Pressão , Drogas Veterinárias/análise , Drogas Veterinárias/isolamento & purificação , Contaminação de Alimentos/análise , Resíduos de Drogas/análise , Resíduos de Drogas/isolamento & purificação , Resíduos de Drogas/química , Praguicidas/isolamento & purificação , Praguicidas/análise , Praguicidas/química , Resíduos de Praguicidas/isolamento & purificação , Resíduos de Praguicidas/análise , Resíduos de Praguicidas/química , Adsorção , Extração em Fase Sólida/métodos , Extração em Fase Sólida/instrumentação , Poluentes Químicos da Água/isolamento & purificação , Poluentes Químicos da Água/química , Poluentes Químicos da Água/análise , Alimentos Marinhos/análise , Frutos do Mar/análise , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Aromatic amino acid oxidation products (AAAOPs) are newly discovered risk substances of thermal processes. Due to its significant polarity and trace level in food matrices, there are no efficient pre-treatment methods available to enrich AAAOPs. Herein, we proposed a magnetic cationic covalent organic framework (Fe3O4@EB-iCOF) as an adsorbent for dispersive magnetic solid-phase extraction (DMSPE). Benefiting from the unique charged characteristics of Fe3O4@EB-iCOF, AAAOPs can be enriched through electrostatic interaction and π-π interactions. Under the optimal DMSPE conditions, the combined HPLC-MS/MS method demonstrated good linearity (R2 ≥ 0.990) and a low detection limit (0.11-7.5 µg·kg-1) for AAAOPs. In addition, the method was applied to real sample and obtained satisfactory recoveries (86.8 % â¼ 109.9 %). Especially, we applied this method to the detection of AAAOPs in meat samples and conducted a preliminarily study on its formation rules, which provides a reliable basis for assessing potential dietary risks.
Assuntos
Aminoácidos Aromáticos , Oxirredução , Extração em Fase Sólida , Extração em Fase Sólida/métodos , Aminoácidos Aromáticos/química , Aminoácidos Aromáticos/análise , Aminoácidos Aromáticos/isolamento & purificação , Espectrometria de Massas em Tandem , Estruturas Metalorgânicas/química , Temperatura Alta , Contaminação de Alimentos/análise , Cromatografia Líquida de Alta Pressão , Animais , Adsorção , Carne/análise , Alimento ProcessadoRESUMO
Dried citrus peel (DCP), also called "Chen Pi", has edible and medicinal value. However, the specific differences among various sources remain unknown. Herein, we collected six DCP species, namely, one Citrus reticulata 'Chachi' (CZG) and five Citrus reticulata Blanco (CRB). Targeted high-performance liquid chromatography and untargeted ultra-high-performance liquid chromatography-tandem mass spectrometry were employed to comprehensively compare the phenolic compounds and metabolites in DCP. Interestingly, 13 different phenolic compounds were noted in DCP. The total phenolic compound content in all CRB samples (58.86-127.65 mg/g) was higher than that of CZG (39.47 mg/g). Untargeted metabolomic revealed 1495 compounds, with 115 differentially expressed metabolites for CRBs and CZG, particularly flavonoids (38), terpenoids (15), and phenolic acids and derivatives (9). Lastly, antioxidant assays revealed that all CRB samples exhibited higher antioxidant activities compared with CZG. Therefore, our study results provide a theoretical basis for the high-value utilization of citrus peels and their metabolites.
Assuntos
Antioxidantes , Citrus , Frutas , Metabolômica , Extratos Vegetais , Espectrometria de Massas em Tandem , Citrus/química , Citrus/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Antioxidantes/análise , Cromatografia Líquida de Alta Pressão , Frutas/química , Frutas/metabolismo , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Fenóis/metabolismo , Fenóis/química , Fenóis/análise , Flavonoides/metabolismo , Flavonoides/química , Flavonoides/análiseRESUMO
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
Acetoacetate (AcAc) and D-beta-hydroxybutyrate (D-ßOHB), the two major ketone bodies found in circulation, are linked to multiple physiological and pathophysiological states. Therefore, analytical methodologies surrounding the quantification of total ketone body (TKB) concentrations in biological matrices are paramount. Traditional methods to quantify TKBs relied on indirect spectrophotometric assays with narrow dynamic ranges, which have been significantly improved upon by modern mass spectrometry (MS)-based approaches. However, the lack of stable isotope-labeled internal standards (ISs) for AcAc and the need to distinguish D-ßOHB from its closely related structural and enantiomeric isomers pose significant obstacles. Here, we provide a protocol to synthesize and quantify a [13C] stable isotope-labeled IS for AcAc, which, in conjunction with a commercially available [2H] stable isotope-labeled IS for ßOHB, allows TKBs to be measured across multiple biological matrices. This rapid (7 min) analysis employs reverse phase ultra-high performance liquid chromatography (RP-UHPLC) coupled to tandem MS (MS/MS) to distinguish ßOHB from three structural isomers using parallel reaction monitoring (PRM), providing excellent specificity and selectivity. Finally, a method is provided that distinguishes D-ßOHB from L-ßOHB using a simple one-step derivatization to produce the corresponding diastereomers, which can be chromatographically resolved using the same rapid RP-UHPLC separation with new PRM transitions. In summary, this method provides a rigorous analytical pipeline for the analysis of TKBs in biological matrices via leveraging two authentic stable isotope-labeled ISs and RP-UHPLC-MS/MS.
Assuntos
Isótopos de Carbono , Marcação por Isótopo , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Marcação por Isótopo/métodos , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Isótopos de Carbono/química , Corpos Cetônicos/química , Acetoacetatos/química , Cromatografia de Fase Reversa/métodos , Padrões de Referência , Ácido 3-Hidroxibutírico/química , Ácido 3-Hidroxibutírico/análise , AnimaisRESUMO
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
Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 µm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.
Assuntos
Cromatografia de Fase Reversa , Lipidômica , Lipídeos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Humanos , Lipidômica/métodos , Lipídeos/análise , Espectrometria de Massas/métodos , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas em Tandem/métodos , Software , 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
Short- and medium-chain fatty acids (SMCFA) are monocarboxylic acids with a carbon chain length of 1-12 carbon atoms. They are mainly produced in humans by the gut microbiota, play crucial metabolic roles, are vital for intestinal health, and have multifaceted impact on immune and neurological functions. Accurate detection and quantification of SMCFA in different human biofluids is achieved using 3-nitro phenylhydrazine (3-NPH) derivatization of the free fatty acids followed by reverse phase liquid chromatography (RPLC) separation and detection by tandem mass spectrometry (MS/MS). Here, we describe the simultaneous measurement of 14 SMCFA and lactate in detail. All 3-NPH-SMCFA-hydrazones are separated in less than 5 min with an 8-min total run time (injection-to-injection). Linear dynamic range over 0.1-500 µM is achieved for most SCFAs, while it is 0.05-100 µM for MCFAs. Validation of the procedure depicts good linearity (R2 > 0.98) and repeatability (CV ≤ 20%). The lower limit of detection (LLOD) is 10-30 nM. The lower limit of quantification (LLOQ) is 50-100 nM for most analytes, while it is 0.5 µM for acetate. In conclusion, the method offers several benefits compared to alternative methods regarding throughput, selectivity, sensitivity, and robustness.
Assuntos
Cromatografia de Fase Reversa , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Humanos , Cromatografia de Fase Reversa/métodos , Ácidos Graxos Voláteis/análise , Ensaios de Triagem em Larga Escala/métodos , Limite de Detecção , Ácidos Graxos/análise , Ácidos Graxos/química , Reprodutibilidade dos TestesRESUMO
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
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
Octadecanoids are a subset of oxylipins derived from 18-carbon fatty acids. These compounds have historically been understudied but have more recently attracted attention to their purported biological activity. One obstacle to the study of octadecanoids has been a lack of specific analytical methods for their measurement. A particular limitation has been the need for chiral-based methods that enable separation and quantification of individual stereoisomers. The use of chirality provides an additional dimension for distinguishing analytes produced enzymatically from those formed through autoxidation. In this chapter, we describe a comprehensive method using chiral supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) for the quantification of octadecanoids in human plasma. This method stands as an effective approach for quantifying octadecanoids and is applicable to diverse research applications including clinical research.
Assuntos
Cromatografia com Fluido Supercrítico , Espectrometria de Massas em Tandem , Cromatografia com Fluido Supercrítico/métodos , Humanos , Espectrometria de Massas em Tandem/métodos , Estereoisomerismo , Oxilipinas/sangue , Oxilipinas/químicaRESUMO
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.