bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒01‒22
thirty-one papers selected by
Sofia Costa

  1. Methods Mol Biol. 2023 ;2625 57-63
      Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a powerful tool for identification and classification of lipids. Ultra-high performance liquid chromatography (UHPLC) allows for robust separations of complex mixtures, while high-resolution mass spectrometry (HRMS) identifies compounds with efficiency and accuracy (Zullig T and Kofeler HC, Mass Spectrom Rev 40:162-176, 2021). The high specificity and sensitivity of mass spectrometry makes it the method of choice when analyzing lipids (Kofeler HC, J Lipid Res 62:100138, 2021). Untargeted mass spectrometry identifies all lipids within a sample and is useful for identification and further discovery. This chapter describes the use of a Q Exactive mass spectrometer to perform an untargeted LC-MS/MS lipidomics analysis.
    Keywords:  Lipidomics; Mass spectrometry; Ultra-high performance liquid chromatography
  2. Anal Chim Acta. 2023 Feb 08. pii: S0003-2670(23)00028-4. [Epub ahead of print]1241 340807
      The metabolome and lipidome are critical components in illustrating biological processes and pathological mechanisms. Generally, two or more independent methods are required to analyze the two compound panels due to their distinct chemical properties and polarity differences. Here, a novel strategy integrating stepwise solid-phase extraction (SPE) and dansyl chemical derivatization was proposed for all-in-one injection LC-MS/MS analysis of serum metabolome and lipidome. In this workflow, a stepwise elution procedure was firstly optimized to separate the metabolome and lipidome fractions using an Ostro plate. Dansyl chemical derivatization was then applied to label amine/phenol, carboxyl, and carbonyl-containing sub-metabolomes. Our results demonstrated that the dansyl labeling could significantly improve chromatographic separation, enhance the MS response, and overcome the matrix effect of co-eluting lipids. Ultimately, an all-in-one injection LC-MS/MS method measuring 256 lipids (covering 20 subclasses) and 212 metabolites (including amino acids, bile acids, fatty acids, acylcarnitines, indole derivatives, ketones and aldehydes, nucleic acid metabolism, polyamines, etc.) was established. This method was applied to investigate the metabolic changes in cisplatin-induced nephrotoxicity in rats and the results were compared with previous untargeted metabolomics. The presented strategy could predominantly improve the analytical coverage and throughput and can be of great use in discovering reliable potential biomarkers in various applications.
    Keywords:  Chemical derivatization; Dansyl labeling; Lipidome; Liquid chromatography-tandem mass spectrometry; Metabolome; Solid-phase extraction
  3. Methods Mol Biol. 2023 ;2625 115-127
      Endocannabinoids (eCBs) are a family of lipid molecules with important regulatory function in the brain and immune system. The two well-studied eCBs are arachidonic acid derivatives, N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG). Currently one of the most important methods for quantitative analysis of eCBs and related lipids from biological matrices is liquid chromatographic separation coupled with tandem mass spectroscopy (LC-MS/MS) owing to its high sensitivity and selectivity, as well as no derivatization procedures needed. Here we describe pretreatment procedures using solid-phase extraction for tissue sampling and an in vivo brain microdialysis approach prior to LC-MS/MS analysis, followed by detailed steps of LC-MS/MS analytic method to demonstrate the potential and application of this method in quantification of eCBs and congeners from various biological matrices.
    Keywords:  Brain; Endocannabinoids (eCBs); LC-MS/MS; Microdialysis; Serum; Skeletal muscle; Solid-phase extraction
  4. Methods Mol Biol. 2023 ;2625 313-321
      Advances in computational and data processing technology have enabled the development of many novel tools for analyzing metabolomic and lipidomic data. These advances involved the catalyst for the creation of publicly accessible complex web-based databases such as the Metabolomics Workbench. Open Source internet-based software packages such as MetaboAnalyst 5.0 enable researchers to perform a wide range of analyte identification and statistical analyses of their own and other researchers' data in order to identify biomarkers and classify compounds. In this paper, we set forth a protocol for obtaining experimental data of interest from a public data repository (Metabolomics Workbench), converting the data into a format suitable for submission to MetaboAnalyst 5.0, and then uploading the data to the MetaboAnalyst server for identification and statistical analysis.
    Keywords:  Lipidomics; MetaboAnalyst; Metabolomics; Metabolomics Workbench; Statistical analysis
  5. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jan 04. pii: S1570-0232(22)00495-0. [Epub ahead of print]1216 123590
      Accelerator mass spectrometry (AMS) is the method of choice for quantitation of low amounts of 14C-labeled biomolecules. Despite exquisite sensitivity, an important limitation of AMS is its inability to provide structural information about the analyte. This limitation is not critical when the labeled compounds are well-characterized prior to AMS analysis. However, analyte identity is important in other experiments where, for example, a compound is metabolized and the structures of its metabolites are not known. We previously described a moving wire interface that enables direct AMS measurement of liquid sample in the form of discrete drops or HPLC eluent without the need for individual fraction collection, termed liquid sample-AMS (LS-AMS). We now report the coupling of LS-AMS with a molecular mass spectrometer, providing parallel accelerator and molecular mass spectrometry (PAMMS) detection of analytes separated by liquid chromatography. The repeatability of the method was examined by performing repeated injections of 14C-labeled tryptophan, and relative standard deviations of the 14C peak areas were ≤10.57% after applying a normalization factor based on a standard. Five 14C-labeled amino acids were separated and detected to provide simultaneous quantitative AMS and structural MS data, and AMS results were compared with solid sample-AMS (SS-AMS) data using Bland-Altman plots. To demonstrate the utility of the workflow, yeast cells were grown in a medium with 14C-labeled tryptophan. The cell extracts were analyzed by PAMMS, and 14C was detected in tryptophan and its metabolite kynurenine.
    Keywords:  Accelerator mass spectrometry; Carbon-14; Isotope ratio mass spectrometry; Liquid chromatography-mass spectrometry; Parallel accelerator and molecular mass spectrometry
  6. Methods Mol Biol. 2023 ;2625 149-161
      Imaging mass spectrometry (IMS) allows for spatial visualization of proteins, lipids, and metabolite distributions in a tissue. Identifying these compounds through mass spectrometry, combined with mapping the compound distribution in the sample in a targeted or untargeted approach, renders IMS a powerful tool for lipidomics. IMS analysis for lipid species such as phosphatidylcholine and phosphatidylserine allows researchers to pinpoint areas of lipid deficiencies or accumulations associated with ocular disorders such as age-related macular degeneration and diabetic retinopathy. Here, we describe an end-to-end IMS approach from sample preparation to data analysis for phosphatidylcholine and phosphatidylserine analysis.
    Keywords:  AP/MALDI; High-resolution mass spectrometry; Imaging mass spectrometry; Lipid localization; Phosphatidylcholine; Phosphatidylserine
  7. Methods Mol Biol. 2023 ;2625 299-311
      Accurate determination of prostaglandins (PGs) from biological samples is critical for understanding their biological functions and interactions during physiological and pathological processes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a highly sensitive, accurate, and high-throughput approach for simultaneous detection of ultra-trace PGs from a single biological sample. Here we describe LC-MS/MS techniques and related sample pretreatment methods including both off-line and on-line SPE for the determination of PGs in biological samples.
    Keywords:  LC–MS/MS; Prostaglandins; Restricted access media (RAM)-based on-line SPE-LC-MS/MS; Sample preparation; Solid phase extraction
  8. Rapid Commun Mass Spectrom. 2023 Jan 18. e9472
      The present work shows a comprehensive chromatographic methods and MS conditions that have been developed according to the chemical properties of each lipid subclass to detect low abundance molecular species. This manuscript shows that the developed methods can detect low and/or very low-abundant lipids like phosphatidic acid (PA) in the glycerophospholipids method, dhCer, dhSPB in the ceramides method, and LPA, LPI, LPG, SPBP in the lysolipids method. Abundant lipid subclasses in human plasma are chromatographically separated from low abundance lipids prior to detection, avoiding the need for derivatisation. Lipid subclasses from the de novo lipogenesis and sphingolipids pathways are presented in this work. Three chromatographic methods here were implemented using a tertiary pumping system to allow for the inclusion of a gradient for analyte separation using A and B pumps, while an isocratic wash elutes interfering compounds. The isocratic wash enabled elution of lipid subclasses not targeted within the method that would otherwise cause background signal in the subsequent sample injection and reduction in column lifetime. Four chromatographic methods coupled with mass spectrometry using targeted and untargeted approaches to separate high and low abundance lipid subclasses are described here. An optimised extraction method for lysolipids is also used in addition to Folch extraction in human plasma.
  9. J Chromatogr A. 2023 Jan 10. pii: S0021-9673(23)00016-X. [Epub ahead of print]1690 463788
      For the first time, a very simple and fast method combining the use of a guard column coupled to tandem mass spectrometry (guard column-MS/MS) has been proposed for the determination of plasticizer metabolites in urine. Briefly, samples (1.0 mL) were submitted to enzymatic hydrolysis for 10 min, filtered, diluted 1/10 v/v with ultrapure water and directly injected into the system. A fast run of only 2 min (3 min including the injection cycle) allowed the determination of 19 analytes. Enzymatic hydrolysis, filtering material, and guard column-MS/MS conditions were optimized. Intra-day precision at the low-level concentration (expressed as relative standard deviation, %RSD) obtained from the analysis of synthetic urine samples varied between 11 and 20%. Limits of quantification ranged from 2.8 to 60 ng/mL. Trueness values, calculated as apparent recoveries, ranged from 70 to 135%. To correct for matrix effects, analyte concentrations in real urine were quantified by the standard addition method. To confirm the results obtained by guard column-MS/MS, an ultra(high)-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was also applied (total chromatographic run time 17 min, including column re-equilibration). Concentrations measured with both methods were in good agreement. Hence, we propose the use of guard column-MS/MS to analyse a large number samples in a very short time (semi-quantification), and apply the chromatographic analysis only to those samples with levels close to/higher than the concentrations equivalent to the safe maximum daily intakes of the parent compounds (confirmation). This double strategy (semi-quantification by guard column-MS/MS and confirmation-when needed-by UHPLC-MS/MS) implies important savings in time and money.
    Keywords:  Guard column; Phthalate metabolites; Tandem mass spectrometry; Ultra(high)-performance liquid chromatography; Urine
  10. Anal Chim Acta. 2023 Feb 01. pii: S0003-2670(22)01282-X. [Epub ahead of print]1240 340711
      Over the last two decades, liquid chromatography coupled to mass-spectrometry (LC‒MS) has become the gold standard to perform qualitative and quantitative analyses of small molecules. When quantitative analysis is developed, an analyst usually refers to international guidelines for analytical method validation. In this context, the design of calibration curves plays a key role in providing accurate results. During recent years and along with instrumental advances, strategies to build calibration curves have dramatically evolved, introducing innovative approaches to improve quantitative precision and throughput. For example, when a labeled standard is available to be spiked directly into the study sample, the concentration of the unlabeled analog can be easily determined using the isotopic pattern deconvolution or the internal calibration approach, eliminating the need for multipoint calibration curves. This tutorial aims to synthetize the advances in LC‒MS quantitative analysis for small molecules in complex matrices, going from fundamental aspects in calibration to modern methodologies and applications. Different work schemes for calibration depending on the sample characteristics (analyte and matrix nature) are distinguished and discussed. Finally, this tutorial outlines the importance of having international guidelines for analytical method validation that agree with the advances in calibration strategies and analytical instrumentation.
    Keywords:  Absolute quantification; Calibration methodologies; Relative quantification; Semiquantification; Surrogate analyte; Surrogate matrix
  11. Foods. 2023 Jan 07. pii: 281. [Epub ahead of print]12(2):
      Sample preparation is usually a complex and time-consuming procedure, which can directly affect the quality of the analysis. Recent efforts have been made to establish analytical methods involving minimal sample preparation, automatized and performed online with the analytical techniques. Online Extraction coupled with Liquid Chromatography-Mass Spectrometry (OLE-LC-MS) allows a fully connected extraction, separation, and analysis system. In this work, the lecithin profile was investigated in commercial sunflower, almonds, peanuts, and pistachio seeds to demonstrate that the concept of extraction, followed by the online analysis of the extract, could be applied to analyze this class of analytes in such complex solid matrices without a prior off-line solvent extraction step. The extraction phase gradient method was optimized. Two different analytical columns were explored, one being a conventional C18 (50 × 2.1 mm, 1.7 µm SPP) and the other a novel self-packed SIGO-C18ec (100 × 0.5, 5 µm FPP), which resulted in better separation. The analysis repeatability was investigated, and suggestions to improve it were pointed out. A characteristic ion with a m/z of 184, related to lysophosphatidylcholine structure, was used to identify the lecithin compounds. The temperature effect on the chromatograms was also explored. In short, it was found that the OLE-LC-MS approach is suitable for the analysis of lecithin compounds in seeds, being a promising alternative for lipidomics approaches in the near future.
    Keywords:  OLE–LC–MS/MS; SiGO-C18ec column; lecithin; lipidomics; lipids; online sample preparation; seeds
  12. Forensic Toxicol. 2023 Jan;41(1): 71-80
      PURPOSE: The detection of hydrolysis products of nerve agents (alkyl methylphosphonic acids; RMPAs) in biological samples from victims is important to confirm exposure to nerve agents. However, analysis of RMPAs is difficult due to their high hydrophilicity. The aim of this study was to develop ion chromatography-tandem mass spectrometry (IC-MS/MS) methods using commercially available equipment and columns to analyze RMPAs in human urine and serum with high sensitivity and without using complicate techniques.METHODS: A Dionex IonPac AS11-HC anion-exchange column was used to analyze six RMPAs (MPA, EMPA, IMPA, iBuMPA, CHMPA, and PMPA). For pretreatments of biological fluids, we developed two pretreatment methods (Method 1: dilution and ultrafiltration; Method 2: removal of chloride ions with Ag cartridges).
    RESULTS: Six RMPAs including highly hydrophilic methylphosphonic acid and ethyl methylphosphonic acid could be analyzed with sufficient retention times and peak shape. The detection limits of RMPAs were improved using Dionex OnGuard II Ba/Ag/H cartridges and MetaSEP IC-Ag cartridges (urine: 0.5-5 ng/mL; serum: 1-5 ng/mL). These methods were also applied to the test samples for the Organisation for the Prohibition of Chemical Weapons Biomedical Proficiency Tests.
    CONCLUSIONS: RMPAs could be sufficiently analyzed by IC-MS/MS. In addition, the limits of detection were superior to those obtained in our previous study involving LC-MS/MS or derivatization-LC-MS/MS method. For analysis of biological samples, an appropriate pretreatment method can be chosen according to the amount of sample available for analysis and expected RMPA concentrations.
    Keywords:  Biological fluids; IC–MS/MS; Nerve agents; Warfare agents
  13. Methods Mol Biol. 2023 ;2625 291-298
      Lipids are among the major constituents of cells and play many important cellular functions. Lipid levels in the trabecular meshwork (TM) aqueous humor outflow pathway play an important role in the maintenance of aqueous humor drainage and intraocular pressure (IOP) homeostasis. Therefore, it is important to characterize the changes in the lipid contents in the aqueous humor outflow pathway tissues to better understand their functional significance in the maintenance of IOP. The multiple reaction monitoring (MRM)-based profiling aids in the analysis of the metabolome as a collection of functional groups and is utilized as an exploratory metabolomics and lipidomics approach. The MRM-based profiling utilizes tandem mass spectrometry experiments carried out on a commercial triple quadrupole mass spectrometer with three aligned quadrupole mass filters (Q1, Q2, and Q3). This screening methodology can be utilized for targeted lipidomics screening. This chapter focuses on the methodology for isolation and culturing of the TM cells, lipid extraction, and the MRM-based lipidomics approach with data analysis.
    Keywords:  Human trabecular meshwork cell culture; Human trabecular meshwork dissection; Lipid content analysis; Lipid extraction; Multiple reaction monitoring (MRM) profiling lipidomics
  14. Methods Mol Biol. 2023 ;2625 79-88
      Phospholipids are essential components of membrane lipid bilayers and serve as precursors of multiple signaling molecules, so alterations in their homeostasis are associated with the pathogenesis of numerous diseases. In this context, the application of mass spectrometry-based metabolomics has demonstrated great potential to comprehensively characterize the human phospholipidome. In this chapter, we describe an untargeted method for the determination of phospholipids and other related metabolites in a variety of biological matrices, including plasma/serum, erythrocytes, and tissues, based on the combination of high-throughput direct mass spectrometry fingerprinting and subsequent profiling by ultra-high-performance reversed-phase liquid chromatography coupled to mass spectrometry. Furthermore, we also review the characteristic fragmentation patterns of phospholipids with the aim of providing simple guidelines for their straightforward annotation.
    Keywords:  Annotation; Direct infusion; Liquid chromatography; Mass spectrometry; Metabolomics; Phospholipids
  15. J Appl Lab Med. 2023 Jan 18. pii: jfac105. [Epub ahead of print]
      BACKGROUND: Broad-spectrum drug screening is offered by many clinical laboratories to support investigation of possible drug exposures. The traditional broad-spectrum drug screen employed at our laboratory utilizes several different analytical platforms, thus requiring relatively high volumes of sample and a cumbersome workflow. Here we describe the development and validation of a consolidated broad-spectrum drug screen assay designed to qualitatively detect 127 compounds in urine (Ur) and serum/plasma (S/P) samples.METHODS: An LC-MS/MS method was developed using the Ultivo LC-MS/MS and designed to be qualitative with a 1-point calibration curve and 50% to 150% controls. Sample preparation included the addition of 122 internal standards (IS) followed by mixed-mode strong cation exchange solid-phase extraction and reverse-phase chromatographic separation on a biphenyl column.
    RESULTS: For the method described herein, ≥ 95% of analytes in urine and serum control samples had a CV of ≤20% for total imprecision. Accuracy testing included 46 external controls and demonstrated 99.9% accuracy. Method comparison studies to quantitative testing are discussed. The high level of coverage of the analytes with a stable isotope-labeled IS (SIL-IS) helped normalize for matrix effects when significant ion suppression (>25%) was present. Analyte stability in the matrix, the impact of potentially interfering compounds, and method ruggedness were demonstrated. Method limitations include limited detection of glucuronidated drugs and potential cross-contamination with samples at very high concentrations (>>100 × cutoff).
    CONCLUSIONS: The broad-spectrum drug screen method developed here qualitatively detected 127 drugs and select metabolites. This method could be used to support investigations of possible drug exposures in a clinical setting.
  16. Methods Mol Biol. 2023 ;2625 183-200
      Lipids are important and abundant constituents of all biological tissues and body fluids. In particular, phospholipids (PLs) constitute a major part of the cellular membrane and play a role in signal transduction, and some selected PLs are increasingly considered as potential disease markers. Unfortunately, methods of lipid analysis are less established in comparison to techniques of protein analysis. Mass spectrometry (MS) is an increasingly used technique to analyze lipids, especially in combination with electrospray ionization MS, which is the most commonly used ionization technique in lipidomics. Matrix-assisted laser desorption/ionization coupled to time-of-flight MS (MALDI-TOF MS) has itself proven to represent a useful tool in the field of lipid analysis. 31P nuclear magnetic resonance (NMR) spectroscopy, another powerful method for PL analysis, represents a direct quantitative method and does not suffer from suppression effects.This paper gives an overview of methodological aspects of MALDI-TOF MS and 31P NMR in lipid research and summarizes the specific advantages and drawbacks of both methods. In particular, suppression effects in MS will be highlighted, and possible ways to overcome this problem, e.g., the use of different matrices and separation of the relevant lipid mixture prior to analysis, will be discussed.
    Keywords:  31P NMR spectroscopy; Lipid analysis; Lipid extracts; MALDI-TOF MS; Matrix; Phospholipids
  17. Anal Chim Acta. 2023 Feb 01. pii: S0003-2670(22)01346-0. [Epub ahead of print]1240 340775
      Development of high-throughput and rapid screening analytical method is in high demand for anti-doping and clinical point-of-care (POC) analysis. Solid-phase microextraction and mass spectrometry direct coupling (SPME-MS) has been proved as a rapid and effective way for target analysis in complex sample matrixes. An online direct coupling of in-tube SPME (IT-SPME) with MS using polymer coated open-tubular column has been developed in this work. A sharp stainless-steel needle was attached at the end of the SPME column, which enables the direct ionization of the analytes after elution from the IT-SPME column. Itaconic acid-benzene co-polymer was in-situ grown on the inner surface of the fused silica capillary and used as extraction phase. This column has low backpressure and provides both hydrophobic and weak cationic exchange interaction with the target analytes due to the chemical properties. The developed online IT-SPME-MS method showed good extraction performance towards various target analytes and good reusability at least for 60 times. As a proof-of-concept application, the above method was applied for the analysis of antiepileptic drugs (AEDs) in both plasma and urine samples with linear range (1 ng/mL-200 ng/mL), good linearity (R2 ≥ 0.99), and good reproducibility (intra-day RSDs less than 4.36%, inter-day RSDs less than 6.55%). The method exhibited high enrichment factors between 187 and 204 for the two AEDs and high sensitivity for the analysis of human plasma samples and urine samples.
    Keywords:  Antiepileptic drugs; Direct coupling; In tube solid-phase microextraction; Mass spectrometry; Open tubular column
  18. Anal Chim Acta. 2023 Feb 01. pii: S0003-2670(22)01312-5. [Epub ahead of print]1240 340741
      Supercritical Fluid Chromatography (SFC), a high-throughput separation technique, has been widely applied as a promising routine method in pharmaceutical, pesticides, and metabolome analysis in the same way as conventional liquid chromatography and gas chromatography. Unified chromatography (UC), an advanced version of SFC, which applied gradient elution with mobile phase changing continuously from supercritical to subcritical and to liquid states, can further extend the SFC applications. UC mostly applying the popular mobile phase of 95%:5%/Methanol:Water with additives allows to analyze many hydrophilic compounds. However, many of phosphorylated metabolites or multi carboxylic acids show very poor peak shapes or even can't be eluted under UC conditions, thus hampering the UC's metabolome coverage. In this study, we proposed the first proof-of-concept of UC/HILIC, a novel strategy to extend the current UC metabolome coverage by employing an aqueous gradient right after the UC gradient on a single packed column in a single measurement. The proposed method showed significant improvement regarding the chromatographic performance and metabolome coverage, while still maintaining the precision and high throughput in comparison with conventional UC methods.
    Keywords:  Highly hydrophilic metabolites; Mass spectrometry; Metabolome coverage; Supercritical fluid chromatography; UC/HILIC; Unified chromatography
  19. Methods Mol Biol. 2023 ;2625 259-267
      Analysis and quantification of ether-lipid phospholipid species-also known as plasmalogens-is a crucial step in the study of the biological functions played by these lipids. Application of analytical separation methods and high-resolution mass spectrometry has gained much attention in this regard, while resolution issues and time-consuming sequences interfered with these advances. Herein, we describe a simple and rapid method for the analysis of plasmalogen (Pl) species by HILIC-HRMS. This method is able to identify and quantify relative levels of ethanolamine-plasmalogens (PlsEtn) and choline-plasmalogens (PlsCho) in biological matrices such as whole blood, plasma, erythrocytes, and also retina. Moreover, we provide a detailed and modified lipid extraction method that is applicable to almost all biological matrices.
    Keywords:  Annotation; Hydrophilic interaction liquid chromatography (HILIC); Lipid extraction; Mass spectrometry (MS); Plasmalogens (Pls)
  20. Methods Mol Biol. 2023 ;2625 65-69
      This chapter focuses on identifying gangliosides in the optic nerve of the mouse using mass spectrometry techniques. The described protocol will also permit the characterization of the sample's lipidome. Two deuterium-labeled ganglioside standards and a general lipid class standard will be utilized for extraction efficiency and quantification. Using reversed-phase high-performance liquid chromatography (HPLC) coupled to a Q Exactive mass spectrometer, the samples will be analyzed. The method will consist of both an untargeted approach and a targeted approach with a ganglioside-specific inclusion list.
    Keywords:  Gangliosides; Lipidomics; Mass spectrometry; Optic nerve
  21. Anal Chem. 2023 Jan 19.
      N-Acyl glycines (NAGlys) are an important class of metabolites in the detoxification system of the human body. They have been used in the diagnosis of several metabolic diseases. Liquid chromatography-mass spectrometry (LC-MS) is the most frequently used NAGlys detection platform. Here, we describe a simple and sensitive method of NAGlys detection by LC-MS in plasma and urine samples. This approach is based on the use of a derivatization reagent, 3-nitrophenylhydrazine. The reaction is quick in aqueous solution, and no quenching step is needed. To expand the coverage of NAGlys when standards are not available, NAGlys were first identified based on high-resolution LC-MS. Quantification was subsequently carried out on triple quadrupole LC-MS. This approach allowed a much broader measurement of NAGlys (41 NAGlys in total), especially when authentic standards are unavailable. Comprehensive analysis of NAGlys with this new method was applied in plasma and urine samples of db/db diabetic and non-diabetic db/m+ control mice. The majority of detected NAGlys were altered with high differentiation ability in plasma and urine samples from diabetic and non-diabetic mice. These identified NAGlys hold the potential to be diagnostic biomarkers for type II diabetes and diabetic complications.
  22. Methods Mol Biol. 2023 ;2625 89-102
      The emerging field of lipidomics presents the systems biology approach to identify and quantify the full lipid repertoire of cells, tissues, and organisms. The importance of the lipidome is demonstrated by a number of biological studies on dysregulation of lipid metabolism in human diseases such as cancer, diabetes, and neurodegenerative diseases. Exploring changes and regulations in the huge networks of lipids and their metabolic pathways requires a lipidomics methodology: advanced mass spectrometry that resolves the complexity of the lipidome. Here, we report a comprehensive protocol of quantitative shotgun lipidomics that enables identification and quantification of hundreds of molecular lipid species, covering a wide range of lipid classes, extracted from cultured mammalian cells.
    Keywords:  Lipid extraction; Lipidome profiling; Lipidomics; Mammalian cells; Mass spectrometry; Quantification; Shotgun lipidomics; Systems biology
  23. Nat Commun. 2023 Jan 19. 14(1): 308
      Spectral matching of MS2 fragmentation spectra has become a popular method for characterizing natural products libraries but identification remains challenging due to differences in MS2 fragmentation properties between instruments and the low coverage of current spectral reference libraries. To address this bottleneck we present Structural similarity Network Annotation Platform for Mass Spectrometry (SNAP-MS) which matches chemical similarity grouping in the Natural Products Atlas to grouping of mass spectrometry features from molecular networking. This approach assigns compound families to molecular networking subnetworks without the need for experimental or calculated reference spectra. We demonstrate SNAP-MS can accurately annotate subnetworks built from both reference spectra and an in-house microbial extract library, and correctly predict compound families from published molecular networks acquired on a range of MS instrumentation. Compound family annotations for the microbial extract library are validated by co-injection of standards or isolation and spectroscopic analysis. SNAP-MS is freely available at .
  24. Methods Mol Biol. 2023 ;2625 103-106
      Lipids serve an essential role in multiple cellular functions including signaling, metabolism, energy storage, and membrane constitution. Lipidomics, the study of lipids using analytical chemistry, allows for the study of disease states and cellular metabolism. Shotgun lipidomics is a technique that involves direct-infusion electrospray ionization (ESI) and analysis with a triple quadrupole mass spectrometer. Triple quadrupole mass spectrometry is ideally suited for lipidomics analysis because it allows for class-specific identification of lipids. Individual lipid class can be identified by the adjustment of three parameters-collision energy, ion mode, and scan type. This chapter describes the use of a triple quadrupole mass spectrometer, the TSQ Quantum Access MAX, to perform lipidomics analysis with high sensitivity, accuracy, and precision.
    Keywords:  Lipidomics; Lipids; Mass spectrometry; Shotgun lipidomics; Triple quadrupole mass spectrometry
  25. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jan 13. pii: S1570-0232(23)00003-X. [Epub ahead of print]1216 123593
      Nitrosamines (NAs) are potent genotoxic agents (GAs) in several animal species, and some are classified as probable or possible human carcinogens by the International Agency for Research on Cancer (IARC). In July 2018, angiotensin II receptor blockers (ARBs) which are used to treat high blood pressure have been recalled owing to contamination with NAs. In this study, a simple and sensitive method for the determination of eleven NAs in a single analysis was developed, using atmospheric pressure chemical ionisation source coupled liquid chromatography tandem mass spectrometer (LC-APCI-MS/MS). By performing the 17 min-run in dynamic multiple reaction monitoring (dMRM) mode, eleven NAs were separated on a Poroshell HPH C18 (4.6 × 150 mm, 2.7 µm) column with gradient elution implementing mobile phase A consisting of 0.2 % formic acid in water and mobile phase B consisting of methanol. The developed analytical method was successfully applied in both active pharmaceutical ingredients (APIs) and finished products (FPs) of valsartan and irbesartan with straightforward and effective extraction procedures. Good linearity with a correlation coefficient (R2) > 0.996 was achieved over the concentration in a range of 0.5-50 ng/mL. The limits of detection (LODs) ranged in 0.001-0.008 ppm and limits of quantitation (LOQs) ranged in 0.008-0.05 ppm of the method fulfilled thresholds of US Food and Drug Administration (US-FDA) and European Medicines Agency (EMA) for testing of GAs in valsartan and irbesartan. The accuracy of the proposed method ranged from 73.1 % to 115.2 % for APIs and the relative standard deviation (RSD %) was ≤11.3 while these validation parameters were in the range of 80.2-128.5 % and ≤ 10.6 for FPs, respectively.
    Keywords:  Liquid chromatography; Mass spectrometry; N-nitrosamines; Sartans; Simultaneous determination
  26. Methods Mol Biol. 2023 ;2625 141-148
      An optimized Bligh and Dyer protocol and subsequent derivatization is described in this chapter for the extraction of free cholesterol and cholesterol esters from tissue samples. Quantification analysis of lipid species is then described utilizing gas chromatography-mass spectrometry, the ideal method for analysis of volatile organic compounds and extraction of sterols.
    Keywords:  Bligh and Dyer extraction; Cholesterol; Derivatization; Gas chromatography–mass spectrometry; Lipidomic; Optic tissue
  27. Environ Res. 2023 Jan 13. pii: S0013-9351(23)00097-X. [Epub ahead of print]221 115305
      Phenols are significant environmental endocrine disruptors that can have adverse health effects on exposed individuals. Correlating phenol exposure to potential health implications requires the development of a comprehensive and sensitive analytical method capable of analyzing multiple phenols in a single sample preparation and analytical run. Currently, no such method is available for multiple classes of phenols due to electrospray ionization (ESI) limitations in concurrent ionization and lack of sensitivity to certain phenols, particularly alkylphenols. In this study, we investigated the influence of mobile phase compositions in ESI on concurrent ionization and analytical sensitivity of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) during the analysis of multiple classes of phenols, and we propose a comprehensive and sensitive analytical method for various classes of phenols (i.e., bisphenols, parabens, benzophenones, chlorophenols, and alkylphenols). The proposed method was affected by 0.5 mM ammonium fluoride under methanol conditions. It enabled the concurrent ionization of all the phenols and significantly improved the analytical sensitivity for bisphenols and alkylphenols, which typically have poor ionization efficiency. This method, combined with a "dilute and shoot" approach, allowed us to simultaneously quantify 38 phenols with good chromatographic behavior and sensitivity. Furthermore, the method was successfully applied to the analysis of 61 urine samples collected from aquatic (swimming) and land (indoor volleyball and outdoor football) athletes.
    Keywords:  Alkylphenols; Electrospray ionization; Environmental phenols; Fluoride; Liquid chromatography; Mass spectrometry
  28. Ther Drug Monit. 2023 Jan 16.
      BACKGROUND: Bisphenol A (BPA) is a ubiquitous contaminant that has endocrine-disrupting effects. Chlorinated derivatives of BPA are formed during chlorination of drinking water and have higher endocrine-disrupting activity. Dichlorobisphenol A (Cl2BPA) is the most abundant chlorinated BPA derivative found in several human biological matrices. Recent in vitro experiments have shown that Cl2BPA is metabolized in sulpho- and glucuro-conjugated compounds. To date, no assay has been developed to quantify the sulfo- and glucuro-conjugates of 3,3'-Cl2BPA (3,3'-Cl2BPA-S and 3,3'-Cl2BPA-G, respectively).METHODS: A high-performance liquid chromatography-tandem mass spectrometry assay for the determination of 3,3'-Cl2BPA conjugated forms in plasma samples was developed and validated according to the European Medicines Agency guidelines. Quantification was performed in the multiple reaction monitoring mode for all target analytes using a SCIEX 6500 + tandem mass spectrometer with an electrospray source operating in the negative ionization mode. Chromatographic separation was achieved using a C18 column maintained at 40°C and a binary mobile phase delivered in the gradient mode at a flow rate of 0.35 mL/min. Sample was prepared via simple precipitation using acetonitrile. The assay was validated and applied to rat and human plasma samples.
    RESULTS: Linearity was demonstrated over the range of 0.006-25 ng/mL for 3,3'-Cl2BPA-G and 0.391-100 ng/mL for 3,3'-Cl2BPA-S. Intraday and interday bias values were in the 95%-109% range, and the imprecision <9%. Internal standard corrected matrix effects were also investigated. This method enabled quantification of the conjugated forms of 3,3'-Cl2BPA in plasma samples.
    CONCLUSIONS: This is the first report on the development and validation of an analytical method for the quantification of 3,3'-Cl2BPA-G and 3,3'-Cl2BPA-S in the plasma matrix. This study is also the first report on the in vivo occurrence of these metabolites.
  29. Methods Mol Biol. 2023 ;2625 1-6
      Mitochondria participate in many important metabolic processes in the body. The lipid profile of mitochondria is especially important in membrane regulation and pathway signaling. The isolation and study of these lipids can provide unparalleled information about the mechanisms behind these cellular processes. In this chapter, we describe a protocol to isolate mitochondrial lipids from homogenized murine optic nerves. The lipid extraction was performed using butanol-methanol (BUME) and subsequently analyzed using liquid chromatography-mass spectrometry. Further analysis of the raw data was conducted using LipidSearch™ and MetaboAnalyst 4.0.
    Keywords:  Lipidomics; Liquid chromatography; Mitochondrial lipids; Neurodegeneration; mass spectrometry
  30. Methods Mol Biol. 2023 ;2625 241-257
      Lipid extraction is an important component of many ecological and ecotoxicological measurements. For instance, percent lipid is often used as a measure of body condition, under the assumption that those individuals with higher lipid reserves are healthier. Likewise, lipids are depleted in 13C compared with protein, and it is consequently a routine to remove lipids prior to measuring carbon isotopes in ecological studies so that variation in lipid content does not obscure variation in diet. We provide detailed methods for two different protocols for lipid extraction: Soxhlet apparatus and manual distillation. We also provide methods for polar and non-polar solvents. Neutral (non-polar) solvents remove some lipids but few non-lipid compounds whereas polar solvents remove not only most lipids but also many non-lipid compounds. We discuss each of the methods and provide guidelines for best practices. We recommend that for stable isotope analysis, researchers test for a relationship between the change in the carbon stable isotope ratio and the amount of lipid extracted to see if the degree of extraction has an impact on isotope ratios. Stable isotope analysis is widely used by ecologists, and we provide a detailed methodology that minimizes known biases.
    Keywords:  Diet reconstruction; Ecophysiology; Ecotoxicology; Lipid extraction; Neutral lipids; Polar lipids; Soxhlet apparatus; Stable isotope analysis
  31. J Pharm Biomed Anal. 2023 Jan 13. pii: S0731-7085(23)00021-3. [Epub ahead of print]226 115252
      The recent discovery of N-acyl taurines (NATs) as a class of endogenous bioactive lipids and the perspective of their possible pharmacological applications stimulated the development of mass spectrometry-based methods for their quantitative measurements in biological tissues and fluids. We report here for the first time a procedure validated both in liver surrogate matrix and neat solvent (MeOH) based on UPLC-ESI-QqQ analysis for the identification and quantification of NATs in biological tissue extracts. The LC-MS method was based on five representative lipid analogues, including saturated, monounsaturated and polyunsaturated species, namely N-palmitoyl taurine (C16:0 NAT), N-oleoyl taurine (C18:1 NAT), N-arachidonoyl taurine (C20:4 NAT), N-docosanoyl taurine (C22:0 NAT) and N-nervonoyl taurine (C24:1 NAT), and evaluated for specificity, linearity, matrix effect, recovery, repeatability and intermediate precision and accuracy. The method validated in MeOH by internal standard approach (d4-C20:4 NAT) showed excellent linearity in the range 1-300 ng/ml with R always ≥ 0.9996 for all NATs; intra-day and inter-day precision and accuracy were always within the acceptable range. Specificity was assessed on NAT standards in MeOH, applying the confirmation ratio of two diagnostic MRM ion transitions for product ions at m/z 80 and m/z 107 to true samples in the adopted BEH C18 UPLC conditions. Limit of detection (LOD) and limit of quantification (LOQ) were 0.3-0.4 and 1 ng/ml, respectively, for all compounds. The method was successfully applied to assess the levels of NATs in the mouse liver and, for the first time, in varying sections of the intestine (duodenum, jejunum, ileum and colon). NAT levels increased from duodenum to colon, evidencing a remarkable prevalence in the large intestine of C22:0 NAT, typically occurring mainly in the central nervous system. These findings prompt further studies to disclose the biological function of the various members of this class in different peripheral tissues.
    Keywords:  Endocannabinoid-like mediators; Mass spectrometry; Method validation; NAT; Quantitative analysis; UPLC