bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒11‒12
thirty-two papers selected by
Sofia Costa, Matterworks

  1. Anal Methods. 2023 Nov 06.
      Vitamin D (VD) metabolites are involved in a variety of important metabolic processes and physiological effects in organisms. Profiling of VD metabolites favors a deep understanding of the physiological role of VD. However, VD metabolites are difficult to detect due to their high chemical structural rigidity, structural similarity, and low sensitivities under liquid chromatography-tandem mass spectrometry (LC-MS). Herein, we present a chemical derivatization assisted LC-MS/MS strategy for the detection of VDs, in which 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is employed to derivatize the conjugated diene of VD metabolites and provides sensitizing reporters for MS detection. After PTAD derivatization, the sensitivities of seven VD metabolites increased by 24-276 folds, with the limits of detection ranging from 3 to 20 pg mL-1. Using this method, we achieved a sensitive and accurate quantification of 7 VD metabolites (vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3, and 1,24,25-trihydroxyvitamin D3) of the VD metabolic pathway in different trace biological samples, including human serum, mouse tissues (namely liver, kidney, lung, and spleen), and cells. We believe that the present method can provide a promising tool for an in-depth analysis of VD metabolism.
  2. Angew Chem Int Ed Engl. 2023 Nov 09. e202312275
      Spatial lipidomics based on mass spectrometry imaging (MSI) is a powerful tool for fundamental biology studies and biomarker discovery. But the structure-resolving capability of MSI is limited because of the lack of multiplexed tandem mass spectrometry (MS/MS) method, primarily due to the small sample amount available from each pixel and the poor ion usage in MS/MS analysis. Here, we report a mobility-modulated sequential dissociation (MMSD) strategy for multiplex MS/MS imaging of distinct lipids from biological tissues. With ion mobility-enabled data-independent acquisition and automated spectrum deconvolution, MS/MS spectra of a large number of lipid species from each tissue pixel are acquired, at no expense of imaging speed. MMSD imaging is highlighted by MS/MS imaging of 24 structurally distinct lipids in the mouse brain and the revealing of the correlation of a structurally distinct phosphatidylethanolamine isomer (PE 18:1_18:1) from a human hepatocellular carcinoma (HCC) tissue. Mapping of structurally distinct lipid isomers is now enabled and spatial lipidomics becomes feasible for MSI.
    Keywords:  mass spectrometry, structural analysis, lipidomics, ion mobility, molecular fragmentation
  3. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Oct 31. pii: S1570-0232(23)00316-1. [Epub ahead of print]1230 123906
      An important area within clinical research is in vivo metabolism of ketone bodies (β-hydroxybutyrate and acetoacetate) and in connection metabolites that may affect their production and/or cellular transport such as the keto-acids from the branched-chain amino acids, lactate and pyruvate. To determine in vivo metabolite turnover, availability of accurate and sensitive methods for analyzing the plasma concentrations of these metabolites and their stable isotopically labeled enrichments is mandatory. Therefore, the present study describes a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous analysis of ketone bodies, α-keto acids, lactate, pyruvate, and their tracer enrichments in humans using 2 different derivatization techniques with 4-bromo-N-methylbenzylamine and O-benzylhydroxylamine as derivatization reagents, and 1-ethyl-3-dimethylaminopropyl carbodiimide as coupling compound followed by a single LC-MS/MS run. The method was validated for matrix effects, linearity, accuracy, precision, recovery, stability, and enrichment (ratio) analysis of a stable isotopically labelled analytes (tracers) continuously infused in humans divided by the unlabeled endogenous analyte (tracee) that makes it possible to quantify the analyte in vivo synthesis and degradation rates. The applied parallel derivatization procedure yielded good sensitivity for all analytes of interest and their tracers. Despite the double derivatization method, mixing the ethyl acetate portions at the final stage made it possible to simultaneously analyze all compounds in a single LC-MS/MS run. Moreover, the liquid chromatography method was optimized to robustly quantify the keto acids derived from leucine (α-keto-isocaproic acid) and isoleucine (α-keto-β-methylvaleric acid), the compounds with similar chemical structure and identical molecular weights. The presented method is designed and validated for human plasma. However, care should be taken in blood sampling and processing procedures as well as quick freezing and storage at -80 °C due to the instability of especially acetoacetate.
    Keywords:  Acetoacetate; LC-MS/MS; Lactate; Plasma; Pyruvate; Tracer enrichment; α-keto acids; β-hydroxybutyrate
  4. Sci Rep. 2023 11 07. 13(1): 19281
      Untargeted lipidomics has been increasingly adopted for hypothesis generation in a biological context or discovery of disease biomarkers. Most of the current liquid chromatography mass spectrometry (LC-MS) based untargeted methodologies utilize a data dependent acquisition (DDA) approach in pooled samples for identification and MS-only acquisition for semi-quantification in individual samples. In this study, we present for the first time an untargeted lipidomic workflow that makes use of the newly implemented Quadrupole Resolved All-Ions (Q-RAI) acquisition function on the Agilent 6546 quadrupole time-of-flight (Q-TOF) mass spectrometer to acquire MS2 spectra in data independent acquisition (DIA) mode. This is followed by data processing and analysis on MetaboKit, a software enabling DDA-based spectral library construction and extraction of MS1 and MS2 peak areas, for reproducible identification and quantification of lipids in DIA analysis. This workflow was tested on lipid extracts from human plasma and showed quantification at MS1 and MS2 levels comparable to multiple reaction monitoring (MRM) targeted analysis of the same samples. Analysis of serum from Ceramide Synthase 2 (CerS2) null mice using the Q-RAI DIA workflow identified 88 lipid species significantly different between CerS2 null and wild type mice, including well-characterized changes previously associated with this phenotype. Our results show the Q-RAI DIA as a reliable option to perform simultaneous identification and reproducible relative quantification of lipids in exploratory biological studies.
  5. Biomed Chromatogr. 2023 Nov 08. e5764
      A bioanalytical method was developed and validated for determining lurasidone (LUR) in rat plasma. The analyte and internal standard were extracted from rat plasma using a liquid-liquid extraction method. The mobile phase consisted of methanol, acetonitrile and water, with an ion pairing agent, 0.1% heptafluorobutyric acid, added to minimise the matrix effect. The detection was achieved using a tandem mass spectrometer (API 2000) in positive ion multiple reaction monitoring mode. All parameters were validated, including selectivity, specificity, carry-over effect, linearity, precision, accuracy, matrix effect, sensitivity and stability. The linearity range was from 5.0 to 1200.0 ng/mL with a correlation coefficient of >0.99. The accuracy ranged from 100.00% to 110.22% across the quality control range. The mean absolute recovery from matrix samples for LUR and the internal standard was found to be 68.46% and 67.25%, respectively, and the relative recovery was found to be 73.89% and 77.44%, respectively. This method can determine LUR concentrations in rat plasma samples up to 12 h after oral administration, aiding in LUR pharmacokinetic (PK) investigations in rats. The method's reproducibility on a conventional LC-MS/MS system and a shorter run time of 3.0 min make it an appealing bioanalytical method for quantifying LUR in PK studies.
    Keywords:  liquid chromatography; lurasidone; rat plasma; tandem mass spectrometry
  6. J Agric Food Chem. 2023 Nov 10.
      Recent developments in mass spectrometry-based metabolite profiling allow unprecedented qualitative coverage of complex biological extract composition. However, the electrospray ionization used in metabolite profiling generates multiple artifactual signals for a single analyte. This leads to thousands of signals per analysis without satisfactory means of filtering those corresponding to abundant constituents. Generic approaches are therefore needed for the qualitative and quantitative annotation of a broad range of relevant constituents. For this, we used an analytical platform combining liquid chromatography-mass spectrometry (LC-MS) with Charged Aerosol Detection (CAD). We established a generic metabolite profiling for the concomitant recording of qualitative MS data and semiquantitative CAD profiles. The MS features (recorded in high-resolution tandem MS) are grouped and annotated using state-of-the-art tools. To efficiently attribute features to their corresponding extracted and integrated CAD peaks, a custom signal pretreatment and peak-shape comparison workflow is built. This strategy allows us to automatically contextualize features at both major and minor metabolome levels, together with a detailed reporting of their annotation including relevant orthogonal information (taxonomy, retention time). Signals not attributed to CAD peaks are considered minor metabolites. Results are illustrated on an ethanolic extract of Swertia chirayita (Roxb.) H. Karst., a bitter plant of industrial interest, exhibiting the typical complexity of plant extracts as a proof of concept. This generic qualitative and quantitative approach paves the way to automatically assess the composition of single natural extracts of interest or broader collections, thus facilitating new ingredient registrations or natural-extracts-based drug discovery campaigns.
    Keywords:  Charged Aerosol Detection; automated composition assessment; liquid chromatography–mass spectrometry; metabolite profiling; natural extract
  7. J Am Soc Mass Spectrom. 2023 Nov 10.
      The role of post-transcriptional modification in biological processes has been an ongoing field of study for several decades. Improvements in liquid chromatography platforms and mass spectrometry instrumentation have resulted in the enhanced identification, characterization, and quantification of modified nucleosides in biological systems. One consequence of the rapid technological improvements in the analytical acquisition of modified nucleosides has been a dearth of robust data processing workflows for analyzing more than a handful of samples at a time. To improve the utility of LC-MS/MS for batch analyses of modified nucleosides, a workflow for automated nucleoside identification has been developed. We adapted the Thermo Fisher Scientific metabolomics identification software package, Compound Discoverer, to accurately identify modified nucleosides from batch LC-MS/MS acquisitions. Three points of identification are used: accurate mass from a monoisotopic mass list, spectral matching from a spectral library, and neutral loss identification. This workflow was applied to a batch (n = 24) of urinary nucleosides, resulting in the accurate identification and relative quantification of 16 known nucleosides in less than 1 h.
  8. J Am Soc Mass Spectrom. 2023 Nov 06.
      13C-Trimethylation enhancement using diazomethane (13C-TrEnDi) is a chemical derivatization technique that uses 13C-labeled diazomethane to increase mass spectrometry (MS) signal intensities for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipid classes, both of which are of major interest in biochemistry. In silico mass spectrometry databases have become mainstays in lipidomics experiments; however, 13C-TrEnDi-modified PC and PE species have altered m/z and fragmentation patterns from their native counterparts. To build a database of 13C-TrEnDi-modified PC and PE species, a lipid extract from nutritional yeast was derivatized and fragmentation spectra of modified PC and PE species were mined using diagnostic fragmentation filtering by searching 13C-TrEnDi-modified headgroups with m/z 199 (PC) and 202 (PE). Identities of 25 PC and 10 PE species were assigned after comparing to predicted masses from the Lipid Maps Structure Database with no false positive identifications observed; neutral lipids could still be annotated after derivatization. Collision energies from 16 to 52 eV were examined, resulting in three additional class-specific fragment ions emerging, as well as a combined sn-1/sn-2 fragment ion, allowing sum-composition level annotations to be assigned. Using the Lipid Blast templates, a NIST-compatible 13C-TrEnDi database was produced based on fragmentation spectra observed at 36 eV and tested on HEK 293T cell lipid extracts, identifying 47 PC and 24 PE species, representing a 1.8-fold and 2.2-fold increase in annotations, respectively. The 13C-TrEnDi database is freely available, MS vendor-independent, and widely compatible with MS data processing pipelines, increasing the throughput and accessibility of TrEnDi for lipidomics applications.
    Keywords:  TrEnDi; database; diazomethane; glycerophospholipids; lipidomics; lipids; mass spectrometry
  9. Rapid Commun Mass Spectrom. 2023 Dec 15. 37(23): e9643
      RATIONALE: Alcohol use disorder affects 4% to 5% of the world's population. Analysis methods are available for various biological fluids to detect this disorder. Determination of ethyl glucuronide in urine by the liquid chromatography-tandem mass spectrometry (LC/MS/MS) method is frequently used in forensic toxicology. These analyses are known to cause matrix effects.METHODS: The presented study describes the elimination of matrix effects for ethyl glucuronide. This study used two different LC/MS/MS systems containing orthogonal and z-spray ion sources. Ethyl glucuronide was analyzed in negative polarity in electrospray ionization. A different dilution method was chosen for each study. The methods were developed and validated according to the European Medicines Agency bioanalytical method validation parameters.
    RESULTS: The lower limit of quantitation of the developed methods was 0.025 μg/mL for ethyl glucuronide. The calibration curve of ethyl glucuronide was between 0.025 and 100 μg/mL with a correlation coefficient of >0.99 for the two methods.
    CONCLUSIONS: It was determined that the analyses using the z-spray ion source were more affected by the matrix effect. The two validated methods involve rapid analysis time and simple sample preparation. Also, the methods were applied to real patients' urine.
  10. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2023 Oct 20. 41(10): 854-858
      Objective: To establish a method for the rapid determination of the three metabolites of xylene, 2-methylmarmaluronic acid, 3-methylmarmaluronic acid and 4-methylmarmaluronic acid, in urine of occupationally exposed workers by ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) . Methods: In July 2022, urine samples were diluted and extracted with pH=6.86 phosphate cache solution, cleaned up by a MAX solid-phase extraction (SPE) column and separated by an Accucore Ph/Hexyl column (100 mm×2.1 mm, 2.6 μm) with a gradient of 5 mmol/L ammonium formate-0.1% formic acid aqueous solution and methanol as mobile phases. The analysis was carried out in electrospray ionization mode and full mass-data dependent secondary mass spectrometry mode, and quantified by external standard method. The characteristics of each index of this method were analyzed. Results: A good linearity was obtained in the concentration range of 1.0-200.0 μg/L for 2-methylmuramic acid, 3-methylmuramic acid and 4-methylmuramic acid with the correlation coefficients of 0.9979-0.9993. The limits of detection of the method were 0.18-0.24 μg/L. While the spiked recoveries at the three concentrations (1.0 μg/L, 100.0 μg/L, and 180.0 μg/L) were in the range of 83.0%-93.7%, with the relative standard deviations of 2.2%-7.9%. Conclusion: The UPLC-HRMS method is simple, rapid, sensitive and accurate, and is suitable for the simultaneous determination of the three metabolites of xylene in the urine of occupationally exposed workers.
    Keywords:  MAX solid-phase extraction; Metabolites of xylene; Ultra-performance liquid chromatography-high resolution mass spectrometry, UPLC-HRMS; Urine
  11. Biomed Chromatogr. 2023 Nov 08. e5769
      Sample preparation is essential for low-level compound determination. In the present work, supported liquid extraction (SLE) was used as sample preparation for the low-level determination of a new TLR7 agonist imiquimod compound, LFX453. Samples were extracted on ISOLUTE® SLE 96-well plates using tert-butyl-methyl ether followed by evaporation and dry residue reconstitution with 150 μl of a mixture of 0.1% formic acid in acetonitrile-water (50/50, v/v). Samples were eluted using a flow rate of 0.750 ml/min on a C18 column (50 × 2.1 mm, 2.7 μm) with a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Tandem mass spectrometry was used to analyze the samples in positive mode. The method run time was 6.5 min, and the low limit of quantification was 1.00 pg/ml with 0.100 ml of minipig plasma. Intra-run and inter-run precision and accuracy were within the acceptance criteria at four concentration levels over a concentration ranging from 1.00 to 200 pg/ml. There was no matrix effect and recovery, three freeze-thaw cycles and incurred samples reanalysis were validated. The method was successfully applied for measuring LFX453 in minipig plasma after application on minipig skin.
    Keywords:  LC-MS/MS method validation; LFX453; TLR7 agonist imiquimod; sample preparation; solid-liquid phase extraction
  12. J Sep Sci. 2023 Nov 09. e2300588
      Metabolomics is the study of metabolites present in a living system. It is a rapidly growing field aimed at discovering novel compounds, studying biological processes, diagnosing diseases, and ensuring quality of food products. Recently, analysis of natural samples has become important to explore novel bioactive compounds and to study how environment and genetics affect living systems. Various metabolomics techniques, databases, and data analysis tools are available for natural sample metabolomics. However, choosing the right method can be a daunting exercise because natural samples are heterogeneous and require untargeted approaches. This tutorial review aims to compile latest technologies to guide an early-career scientist on natural sample metabolomics. First, different extraction methods and their pros and cons are reviewed. Second, currently available metabolomics databases and data analysis tools are summarized. Next, recent research on metabolomics of milk, honey, and microbial samples is reviewed. Finally, after reviewing latest trends in technologies, a checklist is presented to guide an early-career researcher on how to design a metabolomics project. In conclusion, this review is a comprehensive resource for a researcher planning to conduct their first metabolomics analysis. It is also useful for experienced researchers to update themselves on latest trends in metabolomics. This article is protected by copyright. All rights reserved.
    Keywords:  Analytical tools; metabolomics; metabolomics databases; natural samples; untargeted metabolomics
  13. Biomed Chromatogr. 2023 Nov 09. e5775
      EBNA1 is an Epstein Barr virus (EBV) protein expressed in all EBV-associated cancers. EBNA1 plays a critical role in the replication and maintenance of EBV episomes in latently infected cells. VK-2019 was developed as a highly specific inhibitor of EBNA1 DNA binding activity and is currently in phase 1 development as a treatment for EBV-associated carcinomas. A sensitive and reliable method was developed to quantify VK-2019 in human plasma using liquid chromatography with tandem mass spectrometry to perform detailed pharmacokinetic studies. VK-2019 was extracted from plasma using protein precipitation with acetonitrile. Separation of VK-2019, two purported metabolites, and the internal standard, VK-2019-d6, was achieved with a Zorbax XDB C18 column using a gradient flow over 6 min. VK-2019 was detected using a SCIEX 4500 triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The assay range was 0.5-500 ng/mL and proved to be accurate and precise. Dilutions of 1:10 were accurately quantified. VK-2019 was stable in plasma at -70°C for approximately 18 months. The method was applied to assess the total plasma concentrations of VK-2019 in a patient who received a single and multiple oral daily doses of 120 mg.
    Keywords:  VK-2019 quantitative analysis; tandem mass spectrometry; validation
  14. Anal Chem. 2023 Nov 09.
      Metabolite isomers play diverse and crucial roles in various metabolic processes. However, in untargeted metabolomics analysis, it remains a great challenge to distinguish between the constitutional isomers and enantiomers of amine-containing metabolites due to their similar chemical structures and physicochemical properties. In this work, the triplex stable isotope N-phosphoryl amino acids labeling (SIPAL) is developed to identify and relatively quantify the amine-containing metabolites and their isomers by using chiral phosphorus reagents coupled with high-resolution tandem mass spectroscopy. The constitutional isomers could be effectively distinguished with stereo isomers by using the diagnosis ions in MS/MS spectra. The in-house software MS-Isomerism has been parallelly developed for high-throughput screening and quantification. The proposed strategy enables the unbiased detection and relative quantification of isomers of amine-containing metabolites. Based on the characteristic triplet peaks with SIPAL tags, a total of 854 feature peaks with 154 isomer groups are successfully recognized as amine-containing metabolites in liver cells, in which 37 amine-containing metabolites, including amino acids, polyamines, and small peptides, are found to be significantly different between liver cancer cells and normal cells. Notably, it is the first time to identify S-acetyl-glutathione as an endogenous metabolite in liver cells. The SIPAL strategy could provide spectacular insight into the chemical structures and biological functions of the fascinating amine-containing metabolite isomers. The feasibility of SIPAL in isomeric metabolomics analysis may reach a deeper understanding of the mirror-chemistry in life and further advance the discovery of novel biomarkers for disease diagnosis.
  15. J Cheminform. 2023 Nov 07. 15(1): 104
      BACKGROUND: Identifying the molecular formula and fragmentation reactions of an unknown compound from its mass spectrum is crucial in areas such as natural product chemistry and metabolomics. We propose a method for identifying the correct candidate formula of an unidentified natural product from its mass spectrum. The method involves scoring the plausibility of parent candidate formulae based on a parent subformula graph (PSG), and two possible metrics relating to the number of edges in the PSG. This method is applicable to both electron-impact mass spectrometry (EI-MS) and tandem mass spectrometry (MS/MS) data. Additionally, this work introduces the two-dimensional fragmentation plot (2DFP) for visualizing PSGs.RESULTS: Our results suggest that incorporating information regarding the edges of the PSG results in enhanced performance in correctly identifying parent formulae, in comparison to the more well-accepted "MS/MS score", on the 2016 Computational Assessment of Small Molecule Identification (CASMI 2016) data set (76.3 vs 58.9% correct formula identification) and the Research Centre for Toxic Compounds in the Environment (RECETOX) data set (66.2% vs 59.4% correct formula identification). In the extension of our method to identify the correct candidate formula from complex EI-MS data of semiochemicals, our method again performed better (correct formula appearing in the top 4 candidates in 20/23 vs 7/23 cases) than the MS/MS score, and enables the rapid identification of both the correct parent ion mass and the correct parent formula with minimal expert intervention.
    CONCLUSION: Our method reliably identifies the correct parent formula even when the mass information is ambiguous. Furthermore, should parent formula identification be successful, the majority of associated fragment formulae can also be correctly identified. Our method can also identify the parent ion and its associated fragments in EI-MS spectra where the identity of the parent ion is unclear due to low quantities and overlapping compounds. Finally, our method does not inherently require empirical fitting of parameters or statistical learning, meaning it is easy to implement and extend upon.
    SCIENTIFIC CONTRIBUTION: Developed, implemented and tested new metrics for assessing plausibility of candidate molecular formulae obtained from HR-MS data.
    Keywords:  Combinatorics; Fragmentation; Graph theory; Mass spectrometry; Metabolomics; Molecular formula; Natural products
  16. Biomed Chromatogr. 2023 Nov 09. e5772
      Triclabendazole (TCB) is widely used for prevention and treatment of parasitic infections in animals. Improper use can result in drug residues in animal tissues and cause health problems to humans through consumption. A simple and reliable analytical method for the determination of TCB and its metabolites in bovine and goat muscle using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. Analytes were extracted using acetonitrile and purified using enhanced matrix removal cartridge. Chromatographic separation was carried out on a BEH Shield RP18 column. The analytes were detected in positive-mode electrospray ionization mass spectrometry using multiple reaction monitoring. Average recoveries of 96.1%-105.6% with coefficients of variation of 1.9%-8.4% were obtained at fortification levels of 0.5, 2.5, 25, and 50 μg/kg for TCB and 5.0, 25, 250, and 500 μg/kg for its metabolites (triclabendazole sulfoxide, triclabendazole sulfone, and keto-TCB). A good linear regression was obtained with the mixed standard solutions in the range of 0.05-20 μg/L for TCB and 0.5-200 μg/L for its metabolites. The limit of quantification and limit of detection ranged from 0.05 to 0.75 μg/kg and from 0.1 to 1.5 μg/kg, respectively. Moreover, this method was successfully applied to 33 real samples.
    Keywords:  LC-MS/MS; metabolites; residues; triclabendazole
  17. NMR Biomed. 2023 Nov 08. e5060
      NMR spectroscopy is a mainstay of metabolic profiling approaches to investigation of physiological and pathological processes. The one-dimensional proton pulse sequences typically used in phenotyping large numbers of samples generate spectra that are rich in information but where metabolite identification is often compromised by peak overlap. Recently developed pure shift (PS) NMR spectroscopy, where all J-coupling multiplicities are removed from the spectra, has the potential to simplify the complex proton NMR spectra that arise from biosamples and hence to aid metabolite identification. Here we have evaluated two complementary approaches to spectral simplification: the HOBS (band-selective with real-time acquisition) and the PSYCHE (broadband with pseudo-2D interferogram acquisition) pulse sequences. We compare their relative sensitivities and robustness for deconvolving both urine and serum matrices. Both methods improve resolution of resonances ranging from doublets, triplets and quartets to more complex signals such as doublets of doublets and multiplets in highly overcrowded spectral regions. HOBS is the more sensitive method and takes less time to acquire in comparison with PSYCHE, but can introduce unavoidable artefacts from metabolites with strong couplings, whereas PSYCHE is more adaptable to these types of spin system, although at the expense of sensitivity. Both methods are robust and easy to implement. We also demonstrate that strong coupling artefacts contain latent connectivity information that can be used to enhance metabolite identification. Metabolite identification is a bottleneck in metabolic profiling studies. In the case of NMR, PS experiments can be included in metabolite identification workflows, providing additional capability for biomarker discovery.
    Keywords:  band selective; broadband; metabolite identification; pure shift; strong coupling artefacts
  18. Metabolomics. 2023 Nov 08. 19(11): 93
    Metabolomics Quality Assurance and Quality Control Consortium (mQACC)
      INTRODUCTION: The Metabolomics Quality Assurance and Quality Control Consortium (mQACC) organized a workshop during the Metabolomics 2022 conference.OBJECTIVES: The goal of the workshop was to disseminate recent findings from mQACC community-engagement efforts and to solicit feedback about a living guidance document of QA/QC best practices for untargeted LC-MS metabolomics.
    METHODS: Four QC-related topics were presented.
    RESULTS: During the discussion, participants expressed the need for detailed guidance on a broad range of QA/QC-related topics accompanied by use-cases.
    CONCLUSIONS: Ongoing efforts will continue to identify, catalog, harmonize, and disseminate QA/QC best practices, including outreach activities, to establish and continually update QA/QC guidelines.
    Keywords:  Analytical batch; Blanks; Internal standards; Liquid chromatography–mass spectrometry (LC–MS); Metabolomics; Quality control (QC) samples; System suitability testing (SST)
  19. Anal Bioanal Chem. 2023 Nov 04.
      Lipids in human colostrum provide the majority of energy intake and essential fatty acids for developing infants. The fatty acid composition of human colostrum is highly variable and influenced by multiple factors. Human colostrum is a complex sample bringing challenges to fatty acid profiling. This work aimed to optimize the use of ionic liquid (IL) columns and flow-modulated comprehensive two-dimensional gas chromatography coupled to mass spectrometry (FM-GC×GC-MS) for fatty acid profiling in human colostrum. Derivatization strategies were optimized and the elution behavior of fatty acid methyl esters (FAME) on various 1D column phases (Solgel-WAX, SLB-IL60i, SLB-IL76i, and SLB-IL111i). Derivatization with sodium methoxide yielded a satisfactory recovery rate (90%) at milder conditions and reduced time. The use of IL60 as the 1D column provided superior separation, good peak shape, and better utilization of elution space. As a proof of concept, the developed method was applied to access the effects of the mode of neonatal delivery (vaginal vs. C-section) on the fatty acid profile of human colostrum samples. The integrated multidimensional gas chromatography strategy improved FAME detection and separation and can be a useful tool for accessing the effects of different factors on the fatty acid profiling of complex samples.
    Keywords:  Comprehensive two-dimensional gas chromatography; Fatty acid methyl esters; Human colostrum; Lipidomics
  20. Anal Chem. 2023 Nov 10.
      The application of NMR spectroscopy to complex mixture analysis and, in particular, to metabolomics is limited by the low sensitivity of NMR. We recently showed that dissolution dynamic nuclear polarization (d-DNP) could enhance the sensitivity of 13C NMR for complex metabolite mixtures, leading to the detection of highly sensitive 13C NMR fingerprints of complex samples such as plant extracts or urine. While such experiments provide heteronuclear spectra, which are complementary to conventional NMR, hyperpolarized 1H NMR spectra would also be highly useful, with improved limits of detection and compatibility with the existing metabolomics workflow and databases. In this technical note, we introduce an approach capable of recording both 1H and 13C hyperpolarized spectra of metabolite mixtures in a single experiment and on the same hyperpolarized sample. We investigate the analytical performance of this method in terms of sensitivity and repeatability, and then we show that it can be efficiently applied to a plant extract. Significant sensitivity enhancements in 1H NMR are reported with a repeatability suitable for metabolomics studies without compromising on the performance of hyperpolarized 13C NMR. This approach provides a way to perform both 1H and 13C hyperpolarized NMR metabolomics with unprecedented sensitivity and throughput.
  21. J Pharm Biomed Anal. 2023 Oct 30. pii: S0731-7085(23)00598-8. [Epub ahead of print]238 115829
      Pediatric heart failure (HF) is an important clinical disease with high hospitalization rates, morbidity, mortality and medical costs. Sacubitril/valsartan (also known as Entresto), was approved for the treatment of adult HF and is recently used in pediatrics. However, clinical therapy on children is more challenging than adults, and the pharmacokinetics of Entresto in children are still largely unknown and urgently needed. Herein, we aim to develop a simple and sensitive analytic method to monitor Entresto in pediatric patients, which is of great importance for individualized safe medication in children. Specifically, a liquid chromatography tandem mass spectrometry method for simultaneously quantification of valsartan, sacubitril and its bio-active metabolite sacubitrilat in human plasma has been developed and validated in pediatric patients. Plasma samples were pretreated with acetonitrile for protein precipitation. Elution was performed on X Select HSS T3 column (2.1 × 100 mm, 5 µm; Waters) column using an isocratic mobile phase process consisting of 0.1% formic acid aqueous solution and 0.1% formic acid acetonit rile with a total run time of 3.0 min. Valsartan-d3, sacubitrilin-d4 and sacubitrilat-d4 were used as the corresponding deuterium internal standards. According to the Bioanalytical Method Validation Guidance for Industry, the method was validated in the range of 0.5-5000 ng/mL. Intra- and inter-day accuracy of sacubitril,valsartan and sacubitrilat ranged from 93%- 108%, 98%- 109%, 91%- 102%, respectively, with relative standard deviation of precision ranging from 2.0% to 5.1%, 2.4%- 7.5%, 1,3%- 7.4%. The proposed method demonstrated good accuracy, precision and linearity. The matrix factors normalized by internal standard meet the acceptance criteria. The method was fully validated and applied in 39 children. Trough concentration of the three substances to be measured were: valsartan (11.3-938.0 ng/mL), sacubitril (0.5-395.5 ng/mL) and sacubitrilat (522.1-4890.0 ng/mL). Overall, this is the first study to simultaneously determined the plasma valsartan, sacubitril and sacubitrilat concentrations in children, which is believed to facilitate the clinical management of pediatric HF.
    Keywords:  LC-MS/MS; Pediatric patients; Sacubitril; Therapeutic drug monitoring; Valsartan
  22. Anal Chem. 2023 Nov 08.
      Ambient liquid extraction techniques enable direct mass spectrometry imaging (MSI) under ambient conditions with minimal sample preparation. However, currently an integrated probe for ambient liquid extraction-based MSI with high spatial resolution, high sensitivity, and stability is still lacking. In this work, we developed a new integrated probe made of pulled coaxial capillaries, named pulled flowprobe, and compared it with the previously reported single-probe. Mass transfer kinetics in probes was first investigated. The extraction kinetic curves during probe sampling indicate a narrower and higher peak shape for the pulled flowprobe than single-probe. Computational fluid dynamics analysis reveals that in the pulled flowprobe flow velocities are lower in liquid microjunction and higher in the transferring channels, resulting in higher extraction efficiencies and reduced band diffusion compared with single-probe and other probes with a similar flow route. Results of ambient liquid extraction-based MSI of lipids in rat cerebrum show that signals of low-abundance lipids were 2-5 times higher via a pulled flowprobe than via a single-probe, and 26 more lipid species were detected on brain tissue via a pulled flowprobe than via a single-probe. The stability of MSI with the pulled flowprobe was found to be higher than that with single-probe (averaged relative standard deviation = 18% vs 80%) by imaging a lab-made uniform ink coating. Moreover, in the pulled flowprobe, no retraction of the inner capillary from outer capillary is optimal for both sensitivity and stability. The spatial resolution of the pulled flowprobe (30-40 μm) was measured to be higher than that of a comparable size single-probe by calculation with the "80-20" rule. Finally, the new pulled flowprobe was applied to high-resolution MSI of lipids in the hippocampus, and localization of several lipids to the specific cell layers in the hippocampus region was observed. Thus, this work provides an alternative easily fabricated sampling probe with enhanced sensitivity, stability, and spatial resolution, promoting the use of ambient liquid extraction-based MSI in biological and clinical research.
  23. J Environ Sci (China). 2024 Feb;pii: S1001-0742(22)00438-7. [Epub ahead of print]136 237-247
      A method of quantification of perfluorinated compounds (PFCs) from atmospheric particulate matter (APM) is described. A single step pretreatment method, selective pressurized liquid extraction (SPLE), was developed to reduce the high matrix background and avoid contamination from commonly used multiple sample pretreatment steps. An effective sorbent was selected to purify the PFCs during SPLE, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), for quantification of PFCs. Conditions affecting the SPLE efficiency, including temperature, static extraction time, and number of extraction cycles used, were studied. The optimum conditions were found to be 120°C, 10 min, and 3 cycles, respectively. LC-MS/MS method was developed to obtain the optimal sensitivity specific to PFCs. The method detection limits (MDLs) were 0.006 to 0.48 ng/g for the PFCs studied and the linear response range was from 0.1 to 100 ng/g. To ensure accurate values were obtained, each step of the experiment was evaluated and controlled to prevent contamination. The optimized method was tested by performing spiking experiments in natural particulate matter matrices and good rates of recovery and reproducibility were obtained for all target compounds. Finally, the method was successfully used to measure 16 PFCs in the APM samples collected in Beijing over five years from 2015 to 2019. It is observed that some PFCs follow the trend of total PFC changes, and can be attributed to the environment influencing events and policy enforcement, while others don't seem to change as much with time of the year or from year to year.
    Keywords:  Atmospheric particulate matter; Liquid chromatography-mass spectrometry; Perfluorinated compounds; Pollution control; Selective pressurized liquid extraction
  24. Anal Chem. 2023 Nov 10.
      Cholesterol is a critical molecule whose dysregulation in certain brain regions is related to multiple neurological disorders. It is of pathological importance to map the distribution of cholesterol in brain. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been widely used in the molecular imaging of metabolites at a high spatial resolution. However, it is challenging to analyze cholesterol by MALDI-MS due to its difficulty in ionization. Herein, we present for the first time a type of reactive matrix for MALDI-MS of cholesterol. Methylpyridinium carboxaldehydes react with cholesterol and other hydroxyl-containing sterols, which greatly enhanced both desorption and ionization and improved the limits of detection to the low μg/mL range. Compared with previous methods, our reactive matrix requires only one step of chemical derivatization and avoids time-consuming enzymatic reaction, which simplified the sample pretreatment. The reactive matrix was successfully used in mapping the distribution of cholesterol in brain tissue sections using MALDI-MS imaging. In summary, this work has provided a sensitive and simple method for the MALDI-MS analysis of cholesterol, has proposed a novel solution to visualize the distribution of sterol metabolites, and has great potential for applications in neurological and pathological studies.
  25. SLAS Technol. 2023 Nov 02. pii: S2472-6303(23)00066-3. [Epub ahead of print]
      This study aimed to develop a streamlined method for evaluating the dilution ratio of drug dose-response plates created by automated liquid handlers in the early stages of drug discovery. The quantitative techniques commonly used for this purpose have restrictions due to their limited linear dynamic range and inaccuracies in assessing serial dilution performance. To address this challenge, we describe a method based on acoustic ejection mass spectrometry (AEMS). The method involves using standard compounds and an internal standard to evaluate each dilution point in quality control (QC) plates. The samples are transferred to a chromatography-free tandem mass spectrometry system through an acoustic source, enabling the analysis of one sample per three seconds from a microtiter plate. This approach provides precise, accurate, label-free, and rapid data acquisition to support high-throughput screening efforts.
    Keywords:  Acoustic ejection mass spectrometry; Assay development; Direct dilution; High-throughput quality control method; Serial dilution
  26. Mass Spectrom (Tokyo). 2023 ;12(1): A0133
      Carotenoids are tetraterpene pigments that are present in photosynthetic bacteria, some species of archaea and fungi, algae, plants, and animals. Carotenoids are essential pigments in photosynthetic organs along with chlorophylls. Carotenoids also act as photo-protectors, antioxidants, color attractants, and precursors of plant hormones in plants. Carotenoids in animals play important roles, such as precursors of vitamin A, photo-protectors, antioxidants, enhancers of immunity, and contributors to reproduction. More than 850 kinds of carotenoids are present in nature. The structures are similar and all of them are labile. Analysis of natural carotenoids requires the establishment of reliable methods for analyzing them. Liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry/mass spectrometry (MS/MS) coupled with photodiode array detector (DAD) is an important tool for analysis of natural carotenoids. Electrospray ionization and atmospheric pressure chemical ionization are commonly used for ionization of LC-MS of carotenoids. MS and MS/MS provide not only molecular weight information but also some structural information on carotenoids. Ultraviolet-visible spectra from DAD provide information on chromophore systems, which cannot be provided by MS spectral data. In the present review, I report the structural diversity and function of natural carotenoids, and also describe the techniques for analysis of natural carotenoids using the LC-DAD-MS and MS/MS system.
    Keywords:  LC-MS; MS/MS; analysis; carotenoids; photodiode array detector
  27. Anal Chem. 2023 Nov 09.
      Carboxylic acids are central metabolites in bioenergetics, signal transduction, and post-translation protein regulation. However, the quantitative analysis of carboxylic acids as an indispensable part of metabolomics is prohibitively challenging, particularly in trace amounts of biosamples. Here we report a diazo-carboxyl/hydroxylamine-ketone double click derivatization method for the sensitive analysis of hydrophilic, low-molecular-weight carboxylic acids. In general, our method renders a 5- to 2000-fold higher response in mass spectrometry along with improved chromatographic separation. With this method, we presented the near-single-cell analysis of carboxylic acid metabolites in 10 mouse egg cells before and after fertilization. Malate, fumarate, and β-hydroxybutyrate were found to decrease after fertilization. We also monitored the isotope labeling kinetics of carboxylic acids inside adherent cells cultured in 96-well plates during drug treatment. Finally, we applied this method to plasma or serum samples (5 μL) collected from mice and humans under pathological and physiological conditions. The double click derivatization method paves a way toward single-cell metabolomics and bedside diagnostics.
  28. Anal Methods. 2023 Nov 08.
      Hygrine and cuscohygrine, two coca leaf alkaloids, have been previously proposed as markers to differentiate legal and illegal cocaine consumption. This is a very common problem in some countries of South America, where the consumption of coca leaves has a long tradition. Analytical methods focusing on the assessment of coca leaf alkaloids, such as cuscohygrine, hygrine, tropacocaine and t-cinnamoylcocaine, in oral fluid are virtually non-existent in forensic toxicology laboratories worldwide due to their lack of application. However, the problem of differentiating legal and illegal cocaine use in criminal justice, DUID (drug-impaired driving) and WDT (workplace drug testing) programs is growing. Therefore, researchers are obliged to develop methods to measure coca leaf alkaloids (cuscohygrine, hygrine and t-cinnamoylcocaine) in biological matrices for further validation for routine analyses in forensic toxicology laboratories. This work aims to optimize a previously published separation method by protein precipitation in oral fluid by using solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) operating in multiple reaction monitoring (MRM) mode. The use of SPE allowed the matrix effect and the background to be reduced in the chromatograms due to the obtained cleaner extracts. Consequently, improved detection and quantification limits were reached. Findings showed that the detection windows for coca leaf alkaloids were longer than three hours in real oral fluid samples from volunteers who drank a cup of coca tea. These detection windows are quite higher than those previously obtained when using the method based on separation by protein precipitation.
  29. Anal Chem. 2023 Nov 08.
      Compound annotation using spectral-matching algorithms is vital for (MS/MS)-based metabolomics research, but is hindered by the lack of high-quality reference MS/MS library spectra. Finding and removing errors from libraries, including noise ions, is mostly done manually. This process is both error-prone and time-consuming. To address these challenges, we have developed an automated library curation pipeline, LibGen, to universally build novel spectral libraries. This pipeline corrects mass errors, denoises spectra by subformula assignments, and performs quality control of the reference spectra by calculating explained intensity and spectral entropy. We employed LibGen to generate three high-quality libraries with chemical standards of 2241 natural products. To this end, we used an IQ-X orbital ion trap mass spectrometer to generate 1947 classic high-energy collision dissociation spectra (HCD) as well as 1093 ultraviolet-photodissociation (UVPD) mass spectra. The third library was generated by an electron-activated collision dissociation (EAD) 7600 ZenoTOF mass spectrometer yielding 3244 MS/MS spectra. The natural compounds covered 140 chemical classes from prenol lipids to benzypyrans with >97% of the compounds showing <0.2 Tanimoto-similarity, demonstrating a very high structural variance. Mass spectra showed much higher information content for both UVPD- and EAD-mass spectra compared to classic HCD spectra when using spectral entropy calculations. We validated the denoising algorithm by acquiring MS/MS spectra at high concentration and at 13-fold diluted chemical standards. At low concentrations, a higher proportion of spectra showed apparent fragment ions that could not be explained by subformula losses of the parent molecule. When more than 10% of the total intensity of MS/MS fragments was regarded as noise ions, spectra were considered as low quality and were not included in the libraries. As the overall process is fully automated, LibGen can be utilized by all researchers who create or curate mass spectral libraries. The libraries we created here are publicly available at
  30. J Am Soc Mass Spectrom. 2023 Nov 06.
      Chromatographic separations at subzero temperature significantly improve the precision of back-exchange-corrected hydrogen-deuterium exchange mass spectrometry (HDX-MS) determinations. Our previously reported dual-enzyme HDX-MS analysis instrument used reversed phase liquid chromatography (RPLC) at -30 °C, but high backpressures limited flow rates and required materials and equipment rated for very high pressures. Here, we report the design and performance of a dual-enzyme HDX-MS analysis instrument comprising a RPLC trap column and a hydrophilic interaction liquid chromatography (HILIC) analytical column in a two-dimensional RPLC-HILIC configuration at subzero temperature. During operation at -30 °C, the HILIC column manifests greatly reduced backpressure, which enables faster analytical flow rates and the use of materials rated for lower maximum pressures. The average peptide eluted from a HILIC column during a 40 min gradient at -30 °C contained ≈13% more deuterium than peptides eluted from a tandem RPLC-RPLC apparatus using a conventional 8 min gradient at 0 °C. A subset of peptides eluted from the HILIC apparatus contained ≈24% more deuterium.
  31. Anal Bioanal Chem. 2023 Nov 09.
      Chemicals infiltrate our daily experiences through multiple exposure pathways. Human biomonitoring (HBM) is routinely used to comprehensively understand these chemical interactions. Historically, HBM depended on targeted screening methods limited to a relatively small set of chemicals with triple quadrupole instruments typically. However, recent advances in high-resolution mass spectrometry (HRMS) have facilitated the use of broad-scope target, suspect, and non-target strategies, enhancing chemical exposome characterization within acceptable detection limits. Despite these advancements, establishing robust and efficient sample treatment protocols is still essential for trustworthy broad-range chemical analysis. This study sought to validate a methodology leveraging HRMS-based strategies for accurate profiling of exogenous chemicals and related metabolites in urine samples. We evaluated five extraction protocols, each encompassing various chemical classes, such as pharmaceuticals, plastic additives, personal care products, and pesticides, in terms of their extraction recoveries, linearity, matrix effect, sensitivity, and reproducibility. The most effective protocol was extensively validated and subsequently applied to 10 real human urine samples using wide-scope target analysis encompassing over 2000 chemicals. We successfully identified and semi-quantified a total of 36 chemicals using an ionization efficiency-based model, affirming the methodology's robust performance. Notably, our results dismissed the need for a deconjugation step, a typically labor-intensive and time-consuming process.
    Keywords:  Deconjugation; Glucuronides; High-resolution mass spectrometry (HRMS); Human biomonitoring (HBM); Method validation; Non-target
  32. Plant J. 2023 Nov 08.
      Anionic phospholipids (PS, PA, PI, PIPs) are low-abundant phospholipids with impactful functions in cell signaling, membrane trafficking and cell differentiation processes. They can be quickly metabolized and can transiently accumulate at defined spots within the cell or an organ to respond to physiological or environmental stimuli. As even a small change in their composition profile will produce a significant effect on biological processes, it is crucial to develop a sensitive and optimized analytical method to accurately detect and quantify them. While thin-layer chromatography (TLC) separation coupled with gas chromatography (GC) detection methods already exist, they do not allow for precise, sensitive, and accurate quantification of all anionic phospholipid species. Here we developed a method based on high-performance liquid chromatography (HPLC) combined with two-dimensional mass spectrometry (MS2 ) by MRM mode to detect and quantify all molecular species and classes of anionic phospholipids in one shot. This method is based on a derivatization step by methylation that greatly enhances the ionization, the separation of each peak, the peak resolution as well as the limit of detection and quantification for each individual molecular species, and more particularly for PA and PS. Our method universally works in various plant samples. Remarkably, we identified that PS is enriched with very long chain fatty acids in the roots but not in aerial organs of Arabidopsis thaliana. Our work thus paves the way for new studies on how the composition of anionic lipids is finely tuned during plant development and environmental responses.
    Keywords:  anionic phospholipids; methylation; phosphoinositides; plant lipids; tandem mass spectrometry; technical advance