Atriplex hortensis var. 'rubra' extracts and purified amaranthin-type pigments reduce oxidative stress and inflammatory response in LPS-stimulated RAW264.7 cells.

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.

Modular, Scalable, and Customizable LC-HRMS for Exposomics.

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).

(Pre)Clinical Metabolomics Analysis.

Metabolomics is the scientific field with the eager goal to comprehensively analyze the entirety of all small molecules of a biological system, i.e., the metabolome. Over the last few years, metabolomics has matured to become an analytical cornerstone of life science research across diverse fields, from fundamental biochemical applications to preclinical studies, including biomarker discovery and drug development. In this chapter, we provide an introduction to (pre)clinical metabolomics. We define key metabolomics aspects and provide the basis to thoroughly understand the relevance of this field in a biological and clinical context. We present and explain state-of-the-art analytical technologies devoted to metabolomic analysis as well as emerging technologies, discussing both strengths and weaknesses. Given the ever-increasing demand for handling complex datasets, the role of bioinformatics approaches in the context of metabolomic analysis is also illustrated.

Stable Isotope Tracing Experiments Using LC-MS.

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.

Quantification of Total Ketone Bodies in Biological Matrices Using Stable Isotope-Labeled Internal Standards and RP-UHPLC-MS/MS.

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.

A Method for Analysis of Oxidized Phospholipids from Biological Samples Using Mass Spectrometry.

Oxidized phospholipids (oxPLs) are generated during innate immunity and inflammation, where they play a variety of biological roles, including regulation of autoimmunity and coagulation. Some are generated by enzymatic reactions, leading to stereo- and regiospecificity, while many others can be formed through nonenzymatic oxidation and truncation and can be used as biomarkers of oxidative stress. Mass spectrometry methods have been developed over many years for oxPL analysis, which can provide robust estimations of molecular species and amounts, where standards are available. Here we present a method used for the analysis of enzymatically-generated oxPL (eoxPL), which allows quantification of mono-hydroxy oxylipin-containing species. We also show profiling of many other partially characterized structures in tissue samples and provide typical chromatograms obtained.

Reversed-Phase Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Method for High-Throughput Lipidomic Quantitation.

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.

Long Chain Base Profiling with Multiple Reaction Monitoring Mass Spectrometry.

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.

Sphingolipid Analysis in Clinical Research.

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.

Nonenzymatic Oxidized Polyunsaturated Fatty Acid Products in Human Plasma and Urine Samples by LC-QTOF-MS/MS.

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).

Quantification of Octadecanoids in Human Plasma Using Chiral Supercritical Fluid Chromatography-Tandem Mass Spectrometry.

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.

Recommendations for Accurate Lipid Annotation and Semi-absolute Quantification from LC-MS/MS Datasets.

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.

Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry in the Clinical Lipidomic Analysis.

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100 × 3 mm; 1.7 µm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4 min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8 min including column equilibration, which enables the analysis of 160 samples per day.

Rapid Analysis of Sterols in Blood-Derived Samples.

Dysregulations of cholesterol biosynthesis are known to be associated with several pathologies. Due to the rapid growth of clinical investigations in this research area, a specific, fast, and valid method for analyzing cholesterol, its precursors, and metabolites is required. Here, we describe a rapid method for sample preparation, separation, and quantification of sterols in blood-derived samples using polymeric solid phase extraction followed by gas chromatography-mass spectrometry. The validated method demonstrates a reliable quantification of cholesterol, its precursors, and metabolites.

Differential Mobility Spectrometry-Based Cardiolipin Analysis.

Cardiolipins (CL) are special lipids in many respects. First of all, CL are composed of four fatty acids linked by two phosphatidic acids, which provide CL a unique molecular structure. Secondly, in eukaryotic cells they are specific to a single organelle, mitochondria, where they are also synthetized. CL are one of the most abundant lipid classes in mitochondria, mainly localized in the inner membrane. They are key determinants of mitochondrial health and homeostasis by modulating membrane integrity and fluidity, mitochondrial shapes, and metabolic pathways. Disturbances in mitochondrial CL composition can lead to tissue malfunction and diseases. It is therefore important to develop analytical tools to study the mitochondrial lipidome, and more particularly the CL. The method described here allows the quantification of cardiolipins at the sum composition level in isolated mitochondria or in liver tissue by flow injection analysis coupled to differential mobility spectrometry (FIA-DMS), also known as DMS-based shotgun lipidomics.

Extracting Knowledge from MS Clinical Metabolomic Data: Processing and Analysis Strategies.

Assessing potential alterations of metabolic pathways using large-scale approaches plays today a central role in clinical research. Because several thousands of mass features can be measured for each sample with separation techniques hyphenated to mass spectrometry (MS) detection, adapted strategies have to be implemented to detect altered pathways and help to elucidate the mechanisms of pathologies. These procedures include peak detection, sample alignment, normalization, statistical analysis, and metabolite annotation. Interestingly, considerable advances have been made over the last years in terms of analytics, bioinformatics, and chemometrics to help massive and complex metabolomic data to be more adequately handled with automated processing and data analysis workflows. Recent developments and remaining challenges related to MS signal processing, metabolite annotation, and biomarker discovery based on statistical models are illustrated in this chapter in light of their application to clinical research.

Application of a Computational Metabolomics Workflow for the Diagnosis of Inborn Errors of Metabolism in a Laboratory Setting.

Inborn errors of metabolism constitute a set of hereditary diseases that impose severe medical and physical challenges in the affected individual, in particular, for the pediatric patient population. Timely diagnosis is crucial for these patients, as any delay could result in irreversible health damage, underscoring the importance of early initiation of personalized treatment. Current routine diagnostic screening for inborn errors of metabolism relies on various targeted analyses of established biomarkers. However, this approach is time-consuming, focuses on a limited number of tests (based on clinical information) with a relatively small number of biomarkers, and does not facilitate the identification of new markers. In contrast, untargeted metabolomics-based screening offers a more efficient diagnostic solution, by assessing thousands of metabolites across multiple metabolic pathways in a single test. This not only saves time but also conserves resources for clinicians, the diagnostic laboratory, and for patients.This chapter describes the computational workflow of our "Next Generation Metabolic Screening" approach, which is a metabolomics-based method that is currently applied at the Translational Metabolic Laboratory of the Radboud University Medical Center (the Netherlands) for the diagnosis of inborn errors of metabolism.

In Situ Isotope Tracing at Single-Cell Resolution Using Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) allows for label-free spatial molecular interrogation of tissues. With advances in the field over recent years, the spatial resolution at which MSI data can be recorded has reached the single-cell level. This makes MSI complementary to other single-cell omics technologies. As metabolism is a highly dynamic process, capturing the metabolic turnover adds a valuable layer of information. Here, we describe how to set up in situ stable isotope tracing followed by MSI-enabled spatial metabolomics to perform dynamic metabolomics at the single-cell level.

Examination of primary aromatic amines content in polylactic acid straws and migration into food simulants using SERS with LC-MS.

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.

A dopant-assisted iodide-adduct chemical ionization time-of-flight mass spectrometer based on VUV lamp photoionization for atmospheric low-molecular-weight organic acids analysis.

Formic and acetic acids are the most abundant gaseous organic acids and play the key role in the atmospheric chemistry. In iodine-adduct chemical ionization mass spectrometry (CIMS), the low utilization efficiency of methyl iodide and humidity interference are two major issues of the vacuum ultraviolet (VUV) lamp initiated CIMS for on-line gaseous formic and acetic acids analysis. In this work, we present a new CIMS based on VUV lamp, and the ion-molecular reactor is separated into photoionization and chemical ionization zones by a reducer electrode. Acetone was added to the photoionization zone, and the VUV photoionization acetone provided low-energy electrons for methyl iodide to generate I-, and the addition of acetone reduced the amount of methyl iodide by 2/3. In the chemical ionization zone, a headspace vial containing ultrapure water was added for humidity calibration, and the vial changes the sensitivity as a function of humidity from ambiguity to well linear correlation (R2 > 0.95). With humidity calibration, the CIMS can quantitatively measure formic and acetic acids in the humidity range of 0%-88% RH. In this mode, limits of detection of 10 and 50 pptv are obtained for formic and acetic acids, respectively. And the relative standard deviation (RSD) of quantitation stability for 6 days were less than 10.5%. This CIMS was successfully used to determine the formic and acetic acids in the underground parking and ambient environment of the Shandong University campus (Qingdao, China). In addition, we developed a simple model based formic acid concentration to assess vehicular emissions.