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

  1. Metabolomics. 2022 Dec 28. 19(1): 4
      INTRODUCTION: Feature annotation is crucial in untargeted metabolomics but remains a major challenge. The large pool of metabolites collected under various instrumental conditions is underrepresented in publicly available databases. Retention time (RT) and collision cross section (CCS) measurements from liquid chromatography ion mobility high-resolution mass spectrometers can be employed in addition to MS/MS spectra to improve the confidence of metabolite annotation. Recent advancements in machine learning focus on improving the accuracy of predictions for CCS and RT values. Therefore, high-quality experimental data are crucial to be used either as training datasets or as a reference for high-confidence matching.METHODS: This manuscript provides an easy-to-use workflow for the creation of an in-house metabolite library, offers an overview of alternative solutions, and discusses the challenges and advantages of using open-source software. A total of 100 metabolite standards from various classes were analyzed and subjected to the described workflow for library generation.
    RESULTS AND DISCUSSION: The outcome was an open-access available NIST format metabolite library (.msp) with multidimensional information. The library was used to evaluate CCS prediction tools, MS/MS spectra heterogeneities (e.g., multiple adducts, in-source fragmentation, radical fragment ions using collision-induced dissociation), and the reporting of RT.
    Keywords:  Collision cross section; Ion mobility; Open-source; RMassBank; Retention time; Tandem mass spectrometry
  2. Brief Bioinform. 2022 Dec 26. pii: bbac553. [Epub ahead of print]
      BACKGROUND: Global or untargeted metabolomics is widely used to comprehensively investigate metabolic profiles under various pathophysiological conditions such as inflammations, infections, responses to exposures or interactions with microbial communities. However, biological interpretation of global metabolomics data remains a daunting task. Recent years have seen growing applications of pathway enrichment analysis based on putative annotations of liquid chromatography coupled with mass spectrometry (LC-MS) peaks for functional interpretation of LC-MS-based global metabolomics data. However, due to intricate peak-metabolite and metabolite-pathway relationships, considerable variations are observed among results obtained using different approaches. There is an urgent need to benchmark these approaches to inform the best practices.RESULTS: We have conducted a benchmark study of common peak annotation approaches and pathway enrichment methods in current metabolomics studies. Representative approaches, including three peak annotation methods and four enrichment methods, were selected and benchmarked under different scenarios. Based on the results, we have provided a set of recommendations regarding peak annotation, ranking metrics and feature selection. The overall better performance was obtained for the mummichog approach. We have observed that a ~30% annotation rate is sufficient to achieve high recall (~90% based on mummichog), and using semi-annotated data improves functional interpretation. Based on the current platforms and enrichment methods, we further propose an identifiability index to indicate the possibility of a pathway being reliably identified. Finally, we evaluated all methods using 11 COVID-19 and 8 inflammatory bowel diseases (IBD) global metabolomics datasets.
    Keywords:  LC-MS; global metabolomics; identifiability index; pathway enrichment analysis; peak annotation
  3. Forensic Toxicol. 2022 Dec 31.
      PURPOSE: The detection of hydrolysis products of Novichok agents in biological samples from victims is important for confirming exposure to these agents. However, Novichok agents are new class of nerve agent and there have been only few reports on analyses of Novichok agent degradation products. Here, we developed hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry (MS/MS) methods to detect Novichok agent degradation products in human urine with simple pretreatment and high sensitivity.METHODS: A Poroshell 120 HILIC-Z column was used to analyze six Novichok agent degradation products. For urine samples, we used a simple pretreatment method, which consisted of deproteinization with acetonitrile and microfiltration. We calculated the pKa values of the OH groups, the log P values, and the molecular weights to investigate the difference in chromatographic behaviors of the Novichok agent degradation products and the degradation products of conventional nerve agents.
    RESULTS: Six Novichok agent degradation products, including N-(bis-(diethylamino)methylidene)-methylphosphonamidic acid (MPGA), which could not be detected by our previous method, could be analyzed with sufficient peak shape and mutual separation. The detection limits of six Novichok agent degradation products were sufficiently low (1-50 ng/mL) and the calibration curves showed sufficient linearity. The physicochemical parameters of Novichok agent degradation products were different from those of conventional nerve agent degradation products, and this explains the difference in chromatographic behaviors.
    CONCLUSION: Six Novichok agent degradation products were successfully analyzed by HILIC-MS/MS. Due to the absence of a derivatization step, throughput performance was higher than our previous derivatization-liquid chromatography-MS/MS method.
    Keywords:  Biological fluids; HILIC–MS/MS; Nerve agents; Novichok agents; Warfare agents
  4. J Chromatogr A. 2022 Dec 18. pii: S0021-9673(22)00918-9. [Epub ahead of print]1688 463727
      In this study, the chromatographic behavior of mixed-mode and hydrophilic interaction liquid chromatography (HILIC) with the mixed-mode HILIC/strong anion-exchange (SAX) column HILICpak VT-50 2D and the two HILIC columns Atlantis Premier BEH Z-HILIC and Acquity Premier BEH Amide was assessed with regard to their separation capability of the metabolites from the glycolysis and pentose phosphate pathways. Chromatographic conditions were evaluated with the aim of achieving separation of the isomeric glycolytic phosphorylated carbohydrate metabolites free from isomeric interferences and thus allowing for selective targeted analysis by liquid chromatography with tandem mass spectrometry (MS/MS) using multiple reaction monitoring acquisition. The effects of pH values (8.0/9.0/10.0) of the ammonium bicarbonate buffer and gradient time were investigated during HILIC-MS/MS analysis, with the optimal conditions found at pH = 10.0. Separation of the pentose phosphate isomers (ribose 5- and 1-phosphate, xylulose 5-phosphate and ribulose 5-phosphate) was achieved on the mixed-mode HILIC/SAX (HILICpak VT-50 2D) column and HILIC BEH Amide column. Column performance was evaluated based on the direct comparison of chromatographic parameters, i.e. peak width at 50% and peak tailing factors of the individual metabolites. Parity plots were generated allowing a direct comparison between the normalized retention times and assessment of orthogonality of all 3 stationary phases evaluated. Separation of 7 biologically relevant hexose monophosphates metabolites turned out to be challenging by HILIC-MS/MS, with the BEH Amide providing the best individual results for such a separation. However, fructose 6-phosphate and glucose 1-phosphate co-eluted. Therefore, an on-line heart-cutting HILIC-Mixed Mode 2D-LC-QToF experiment was conducted, allowing the separation of this critical isomer pair. In this setup, the BEH Amide column in the 1D separated the majority of target metabolites, while a heart-cut of the peak from totally coeluted fructose 6-phosphate and glucose 1-phosphate was separated in the 2D with HILICpak VT50-2D column, thus allowing undisturbed determination of the glycolytic phosphorylated carbohydrate metabolites due to their chromatographic separation from hexose monophosphate metabolites. The assay specificity towards 7 common hexose monophosphates was characterized (glucose 1- and 6-phosphate, galactose 1- and 6-phosphate, fructose 6-phosphate, mannose 1- and 6-phosphate). The selectivity of some rare hexose monophosphates (allose 6-phosphate, tagatose 6-phosphate, sorbose 1-phosphate) was also tested.
    Keywords:  HILIC; Metabolomics; Mixed-mode chromatography; Sugar phosphates; Two-dimensional liquid chromatography
  5. Anal Chem. 2022 Dec 27.
      mRNA-based medicines are a promising modality for preventing virus-caused illnesses, including COVID-19, and treating various types of cancer and genetic diseases. To develop such medicines, methods to characterize long mRNA molecules are needed for quality control and metabolic analysis. Here, we developed an analytical platform based on isotope-dilution liquid chromatography-mass spectrometry (LC-MS) that quantitatively characterizes long, modified mRNAs by comparing them to a stable isotope-labeled reference with an identical sequence to that of the target medicine. This platform also includes database searching using the mass spectra as a query, which allowed us to confirm the primary structures of 200 to 4300 nt mRNAs including chemical modifications, with sequence coverage at 100%, to detect/identify defects in the sequences, and to define the efficiencies of the 5'-capping and integrity of the polyadenylated tail. Our findings indicated that this platform should be valuable for quantitatively characterizing mRNA vaccines and other mRNA medicines.
  6. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Dec 15. pii: S1570-0232(22)00477-9. [Epub ahead of print]1215 123572
      Unsaturated fatty acids (UFAs) are essential fatty acids that execute various biological functions in the human body. Therefore, the qualitative and quantitative analysis of UFAs in biological samples can help to clarify their roles in the occurrence and development of diseases, so to reveal the mechanisms of pathogenesis and potential drug intervention strategies. Chromatography-mass spectrometry is one of the most commonly used techniques for the analysis of UFAs in biological samples. However, due to factors such as the complex structural information of UFAs (the number and specific location of CC double bonds) and the low concentration of UFAs in biological samples, it is still difficult to conduct accurate qualitative and/or quantitative studies of UFAs in complex biological samples. In recent years, the integration and application of chemical derivatization and chromatography-mass spectrometry has been widely used in the detection of UFAs. Based on this overview, we reviewed recent developments and application progress for chemical derivatization-based chromatography-mass spectrometry methods for the qualitative and/or quantitative analysis of UFAs in biological samples over the past ten years. Potential trends for the design and improvement of novel derivatization reagents were proposed.
    Keywords:  Biological samples; Chemical derivatization; Chromatography-mass spectrometry; Quantitative analysis; Unsaturated fatty acids (UFAs)
  7. J Chromatogr Sci. 2022 Dec 25. pii: bmac102. [Epub ahead of print]
      Bioanalytical method development and validation of endogenous Isotretinoin with Isotretinoin D5 as internal standard was done as per current regulatory guidelines. The method is simple, rugged and sensitive enough to estimate endogenous Isotretinoin using the chromatography-tandem mass spectrometry technique. An alternative approach has been adopted for quantitative analysis of endogenous Isotretinoin in human plasma. Isotretinoin free matrix (surrogate matrix) was prepared and further used for the development and validation of Isotretinoin. The method was validated in altered and unaltered plasma. The chromatographic optimization was done with column (ACE C18, 100 × 4.6 mm I.D. 5 μm particle size), using a mobile phase containing 1 mM ammonium acetate, pH 3.0 as a solvent A and solvent B (1 mM ammonium acetate (pH 3.0) with acetonitrile in a ratio of 10:90). A flow rate was set at 0.75 mL/min in a binary gradient mode. The analyte was recovered by liquid-liquid extraction method with diethyl ether as an extraction solvent. Multi-reaction monitoring mode in negative polarity was implemented for the quantification of endogenous Isotretinoin in plasma. The calibration curve of Isotretinoin was linear (r2 > 0.9992) over the concentration range of 0.5-1000 ng/mL. The intra-day precision was found in a range of 2.0-3.9% CV for altered samples and 0.9-3.7% CV for unaltered samples. The inter-day precision was found 2.6-6.1% CV for altered samples and 1.3-3.8% CV for unaltered samples. The average recovery of the extraction procedure was found 64.6% for altered samples and 62.2% for unaltered samples.
  8. Angew Chem Int Ed Engl. 2022 Dec 27.
      Mass spectrometry imaging (MSI) of lipids in biological tissues is useful for correlating molecular distribution with pathological results, which could provide useful information for both biological research and disease diagnosis. It is well understood that the lipidome could not be clearly deciphered without tandem mass spectrometry analysis, but this is challenging to achieve in MSI due to the limitation in sample amount at each image spot. Here we develop a multiplexed MS2 imaging (MS2I) method that can provide MS2 images for 10 lipid species or more for each sampling spot, providing spatial structural lipidomic information. Coupling with on-tissue photochemical derivatization, imaging of 20 phospholipid C=C location isomers is also realized, showing enhanced molecular images with high definition in structure for mouse brain and human liver cancer tissue sections. Spatially mapped t-distributed stochastic neighbor embedding has also been adopted to visualize the tumor margin with enhancement by structural lipidomic information.
    Keywords:  Lipidomics; MS/MS imaging; lipid biomarker; lipid isomer; mass spectrometry imaging
  9. Anal Chem. 2022 Dec 29.
      Sample preparation is the rate-limiting step in liquid chromatography-mass spectrometry (LC-MS)/MS-based clinical analysis when target analytes possess significantly different properties. Repeated solid-phase extraction (SPE) processes are typically required, resulting in low throughput and excessive consumption of labor, materials, and samples. In this study, we developed and validated a feasible and productive method to enrich target analytes with different properties during a single operation, while sufficiently removing matrix interferences to meet LC-MS/MS requirements. Gastrin determination was selected as the subject of this study. An automated magnetic-bead-assisted sequential extraction (MBASE) workflow was developed to simultaneously isolate nonsulfated gastrin-17 (G17ns), sulfated gastrin-17 (G17s), nonsulfated gastrin-34 (G34ns), and sulfated gastrin-34 (G34s) from human serum. It performs two different ion-exchange-based magnetic-bead extraction steps on one sample aliquot to produce one combined extract for LC-MS/MS analysis. When compared with the traditional SPE process, the MBASE workflow saves over 75% time and labor expenses as well as over 90% material cost, while providing even higher extraction efficiency. The MBASE LC-MS/MS method was validated as accurate and robust. Clinical sample test results demonstrated that the conventional chemiluminescence immunoassay method significantly under-estimated total gastrins in human serum, and the MBASE LC-MS/MS method could serve as an ideal tool to provide a comprehensive and accurate gastrin profile.
  10. ACS Omega. 2022 Dec 20. 7(50): 47372-47377
      The reliably accurate and precise quantification of biomarkers is a priceless objective in the drug development and diagnostic arenas. To employ a technique that brings such reliability and furthermore involves a simpler, faster, and inexpensive regime would only underline the potential importance of the concept and technique. To the existing established approaches for biomarker quantification in bioanalytical LC-MS, surrogate matrix (SUR-M) and surrogate analyte (SUR-A), in this Letter we present an approach that fulfills the aforementioned advantages. The concept builds on the historic method of standard addition (SA), in which one source of biological matrix is spiked with analyte to form a calibration curve. With the SA curve back-calculated, the heart of this procedure is the subsequent adjustment of the intercept to zero, the origin, and using only the slope of the curve for interpolation giving calculated sample concentrations. In SA, the concentration axis intercept indicates the endogenous analyte concentration, and our zeroing of this is equivalent to removing the endogenous level. This key shift of the calculated line to the origin unveils our novel origin-adjusted (OA) approach. It enables use akin to a regular xenobiotic method, with no need to ultimately account for the endogenous analyte level in the control matrix used for calibrants. We present a comparison of OA against the control approach of SUR-M in a representative application for kynurenine and tryptophan in human plasma by LC-MS. A numerical performance analysis performed is demonstrative of equivalence between the two approaches for both analytes.
  11. J Pharm Biomed Anal. 2022 Dec 17. pii: S0731-7085(22)00624-0. [Epub ahead of print]225 115203
      Hyperlipidemia is a disease characterized by abnormal blood lipid levels and is the leading risk factor for cardiovascular disease. 3',4'-Dimethoxy flavonol-3-O-glucoside (abbreviated DF3G) is a new lipid-lowering drug created as a flavonoid structural analog. The principal metabolite of DF3G in human plasma is the aglycone glucuronide conjugate M2. The purpose of this study is to use liquid chromatography-tandem mass spectrometry to develop and validate a quantitative analysis method for DF3G and its metabolite M2 in human plasma, and to use the method to investigate the pharmacokinetics of DF3G and M2 in a clinical trial. This method employed DF3G-d6 as the internal standard, and plasma samples were processed by protein precipitation. Isocratic separation could accurately differentiate DF3G, M2, and DF3G-d6 from endogenous components in the matrix or other components in the samples, and endogenous components in the matrix had little impact on ionization efficiency. Positive electrospray ionization with multiple reaction monitoring (MRM) transitions of m/z 461.2 → 299.0 for DF3G, m/z 475.1 → 299.1 for M2 and m/z 467.1 → 305.1 for DF3G-d6 was used for quantification. The DF3G and M2 linear range for plasma were in the range of 4.00/4.00 ng/mL to 4000/4000 ng/mL. Both the analytes and the internal standard were stable regardless of whether they were in solution or plasma samples. The accuracy of the average concentration of the quality control samples was within 15% of the theoretical value, and the RSD was less than 15%. The method is rapid, accurate, straightforward, and precise. It is appropriate for the determination of DF3G and M2 concentrations in human plasma and has been successfully applied to determine the pharmacokinetic analysis in phase I clinical trials.
    Keywords:  3′, 4′-Dimethoxy flavonol-3-O-glucoside; Aglycone glucuronide; Human pharmacokinetic study; LC-MS/MS
  12. J Breath Res. 2022 Dec 29. 17(1):
      Applications for direct breath analysis by mass spectrometry (MS) are rapidly expanding. One of the more recent mass spectrometry-based approaches is secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS). Despite increasing usage, the SESI methodology still lacks standardization procedures for quality control and absolute quantification. In this study, we designed and evaluated a custom-built standard delivery system tailored for direct breath analysis. The system enables the simultaneous introduction of multiple gas-phase standard compounds into ambient MS setups in the lower parts-per-million (ppm) to parts-per-billion (ppb) range. To best mimic exhaled breath, the gas flow can be heated (37 °C-40 °C) and humidified (up to 98% relative humidity). Inter-laboratory comparison of the system included various SESI-HRMS setups, i.e. an Orbitrap and a quadrupole time-of-flight mass spectrometer (QTOF), and using both single- as well as multi-component standards. This revealed highly stable and reproducible performances with between-run variation <19% and within-run variation <20%. Independent calibration runs demonstrated high accuracy (96%-111%) and precision (>95%) for the single-compound standard acetone, while compound-specific performances were obtained for the multi-component standard. Similarly, the sensitivity varied for different compounds within the multi-component standard across all SESI-Orbitrap and -QTOF setups, yielding limits of detections from 3.1 ppb (forp-xylene) to 0.05 ppb (for 1,8-cineol). Routinely applying the standard system throughout several weeks, allowed us to monitor instrument stability and to identify technical outliers in exhaled breath measurements. Such routine deployment of standards would significantly improve data quality and comparability, which is especially important in longitudinal and multi-center studies. Furthermore, performance validation of the system demonstrated its suitability for reliable absolute quantification while it illustrated compound-dependent behavior for SESI.
    Keywords:  SESI-HRMS; direct breath analysis; multi-component; reference material; standardization
  13. Addict Biol. 2023 Jan;28(1): e13255
      Methamphetamine (METH) is a commonly abused addictive psychostimulant, and METH-induced neurotoxic and behavioural deficits are in a sex-specific manner. However, there is lack of biomarkers to evaluate METH addiction in clinical practice, especially for gender differences. We utilized ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to detect the serum metabolomics in METH addicts and controls, specially exploring the sex-specific metabolic alterations by METH abuse. We found that many differently expressed metabolites in METH addicts related to metabolisms of amino acid, energy, vitamin and neurological disorders. Further, METH abuse caused different patterns of metabolomics in a sex-specific manner. As to amino acid metabolism, L-phenylalanine, L-tryptophan and L-histidine in serum of male addicts and betaine in serum of female addicts were significantly changed by METH use. In addition, it seemed that purine and pyrimidine-related metabolites (e.g., xanthosine and adenosine 5'-monophosphate) in male and the metabolites of hormone (e.g., cortisol) and folate biosynthesis (e.g., 7,8-dihydrobiopterin and 4-hydroxybenzoic acid) in female were more sensitive to METH addiction. Our findings revealed that L-glutamic acid, L-aspartic acid, alpha-ketoglutarate acid and citric acid may be potential biomarkers for monitoring METH addiction in clinic. Considering sex-specific toxicity by METH, the metabolites of purine and pyrimidine metabolism in male and those of stress-related hormones in female may be used to facilitate the accurate diagnosis and treatment for METH addicts of different genders.
    Keywords:  UHPLC-MS; human serum metabolomics; methamphetamine; sex-specific metabolites
  14. Anal Chem. 2022 Dec 28.
      Surface-assisted laser desorption/ionization (SALDI) acts as a soft desorption/ionization technique, which has been widely recognized in small-molecule analysis owing to eliminating the requirement of the organic matrix. Herein, titania nanosheets (TiO2 NSs) were applied as novel substrates for simultaneous analysis and imaging of low-mass molecules and lipid species. A wide variety of representative analytes containing amino acids, bases, drugs, peptides, endogenous small molecules, and saccharide-spiked urine were examined by the TiO2 NS-assisted LDI mass spectrometry (MS). Compared with conventional organic matrices and substrates [Ag nanoparticles (NPs), Au NPs, carbon nanotubes, carbon NPs, CeO2 microparticles, and P25 TiO2], the TiO2 NS-assisted LDI MS method shows higher sensitivity and less spectral interference. Repeatability was evaluated with batch-to-batch relative standard deviations for 5-hydroxytryptophan, glucose-spiked urine, and glucose with addition of internal standard, which were 17.4, 14.9, and 2.8%, respectively. The TiO2 NS-assisted LDI MS method also allows the determination of blood glucose levels in mouse serum with a linear range of 0.5-10 mM. Owing to the nanoscale size and uniform deposition of the TiO2 NS matrix, spatial distributions of 16 endogenous small molecules and 16 lipid species from the horizontal section of the mouse brain tissue can be visualized at a 50 μm spatial resolution. These successful applications confirm that the TiO2-assisted LDI MS method has promising prospects in the field of life science.