bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒09‒03
fifteen papers selected by
Sofia Costa, Matterworks

  1. bioRxiv. 2023 Aug 16. pii: 2023.08.16.551807. [Epub ahead of print]
      Metabolomics is an important approach for studying complex biological systems. Quantitative liquid chromatography-mass spectrometry (LC-MS)-based metabolomics is becoming a mainstream strategy but presents several technical challenges that limit its widespread use. Computing metabolite concentrations using standard curves generated from standard mixtures of known concentrations is a labor-intensive process which is often performed manually. Currently, there are few options for open-source software tools that can automatically calculate metabolite concentrations. Herein, we introduce SCALiR (Standard Curve Application for determining Linear Ranges), a new web-based software tool specifically built for this task, which allows users to automatically transform LC-MS signal data into absolute quantitative data ( ). The algorithm used in SCALiR automatically finds the equation of the line of best fit for each standard curve and uses this equation to calculate compound concentrations from their LC-MS signal. Using a standard mix containing 77 metabolites, we found excellent correlation between the concentrations calculated by SCALiR and the expected concentrations of each compound (R 2 = 0.99) and that SCALiR reproducibly calculated concentrations of mid-range standards across ten analytical batches (average coefficient of variation 0.091). SCALiR offers users several advantages, including that it (1) is open-source and vendor agnostic; (2) requires only 10 seconds of analysis time to compute concentrations of >75 compounds; (3) facilitates automation of quantitative workflows; and (4) performs deterministic evaluation of compound quantification limits. SCALiR provides the metabolomics community with a simple and rapid tool that enables rigorous and reproducible quantitative metabolomics studies.
  2. Anal Chem. 2023 Aug 31.
      Lipids are an important class of molecules involved in various biological functions but remain difficult to characterize through mass-spectrometry-based methods because of their many possible isomers. Glycolipids, specifically, play important roles in cell signaling but display an even greater level of isomeric heterogeneity as compared to other lipid classes stemming from the introduction of a carbohydrate and its corresponding linkage position and α/β anomericity at the headgroup. While liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains the gold standard technique in lipidomics, it is still unable to characterize all isomeric species, thus presenting the need for new, orthogonal, methodologies. Ion mobility spectrometry-mass spectrometry (IMS-MS) can provide an additional dimension of information that supplements LC-MS/MS workflows, but has seen little use for glycolipid analyses. Herein, we present an analytical toolbox that enables the characterization of various glycolipid isomer sets using high-resolution cyclic ion mobility separations coupled with mass spectrometry (cIMS-MS). Specifically, we utilized a combination of both permethylation and metal adduction to fully resolve isomeric sphingolipids and ceramides with our cIMS-MS platform. We also introduce a new metric that can enable comparing peak-to-peak resolution across varying cIMS-MS pathlengths. Overall, we envision that our presented methodologies are highly amenable to existing LC-MS/MS-based workflows and can also have broad utility toward other omics-based analyses.
  3. Anal Chem. 2023 Sep 01.
      Accurate quantitative analysis in liquid chromatography-mass spectrometry (LC-MS) benefits from calibration curves generated in the same matrix as the study sample. In the case of endogenous compound quantification, as no blank matrix exists, the multitargeted internal calibration (MTIC) is an attractive and straightforward approach to avoid the need for extensive matrix similarity evaluation. Its principle is to take advantage of stable isotope labeled (SIL) standards as internal calibrants to simultaneously quantify authentic analytes using a within sample calibration. An MTIC workflow was developed for the simultaneous quantification of metabolites related to chronic kidney disease (CKD) using a volumetric microsampling device to collect 20 μL of serum or plasma, followed by a single-step extraction with acetonitrile/water and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Since a single concentration of internal calibrant is necessary to calculate the study sample concentration, the instrument response function was investigated to determine the best SIL concentration. After validation, the trueness of 16 endogenous analytes in authentic human serum ranged from 72.2 to 116.0%, the repeatability from 1.9 to 11.3%, and the intermediate precision ranged overall from 2.1 to 15.4%. The proposed approach was applied to plasma samples collected from healthy control participants and two patient groups diagnosed with CKD. Results confirmed substantial concentration differences between groups for several analytes, including indoxyl sulfate and cortisone, as well as metabolite enrichment in the kynurenine and indole pathways. Multitargeted methodologies represent a major step toward rapid and straightforward LC-MS/MS absolute quantification of endogenous biomarkers, which could change the paradigm of MS use in clinical laboratories.
  4. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Aug 12. pii: S1570-0232(23)00259-3. [Epub ahead of print]1228 123849
      The field of metabolomics based on mass spectrometry has grown considerably in recent years due to the need to detect and, above all, quantify a very large number of metabolites, simultaneously. Up to now, targeted multiplexed analysis on complex samples by Liquid Chromatography coupled with tandem Mass Spectrometry (LC-MS/MS) has relied almost exclusively on compound detection based on absolute retention times, as in the Scheduled-MRM (sMRM) approach. Those methods turn out to be poorly transferable from one instrument to another and result in a time-consuming and tedious method development involving a significant number of critical parameters that need specific re-optimisation. To address this challenge, we introduce a novel acquisition mode called scout-triggered MRM (stMRM). In stMRM, a marker transition is used to trigger MS analysis for a group of dependent target analytes. These marker transitions are strategically distributed throughout the chromatographic run, and the dependent analytes are associated based on their retention times. The result is a targeted assay that remains robust even in the presence of retention time shifts. A 3 to 5-fold increase in the number of detected transitions associated to plasma metabolites was obtained when transferring from a direct application of a published sMRM to a stMRM method. This significant improvement highlights the universal applicability of the stMRM method, as it can be implemented on any LC system without the need for extensive method development. We subsequently illustrate the robustness of stMRM in modified chromatographic elution conditions. Despite a large change in metabolite's selectivity, the multiplexed assay successfully recovered 70% of the monitored transitions when consequently modifying the gradient method. These findings demonstrate the versatility and adaptability of stMRM, opening new avenues for the development of highly multiplexed LC-MS/MS methods in metabolomics. These methods are characterized by their analytical transparency and straightforward implementation using existing literature data.
    Keywords:  LC-MS/MS; Liquid chromatography; Method transfer; Multiple Reaction Monitoring; Targeted metabolomics; scout-triggered MRM
  5. Talanta. 2023 Aug 22. pii: S0039-9140(23)00825-1. [Epub ahead of print]266(Pt 2): 125074
      Central carbon and energy metabolism are the most concerned metabolic pathways in 13C-Metabolic flux analysis (13C-MFA). However, some α-keto acids, ribonucleoside triphosphate (NTPs) and deoxyribonucleoside triphosphate (dNTPs) involved in central carbon and energy metabolism pathways were unstable or reactive, leading to inaccurate metabolic flux analysis. To achieve accurate 13C-MFA of central carbon and energy metabolism, we proposed a dual strategy for the detection of 101 metabolites in glucose metabolism pathways. N-Methylphenylethylamine (MPEA) was utilized for derivatization of 4 carboxyl (α-keto acids) and 8 phosphate metabolites (NTPs and dNTPs). After derivatization, the MPEA derivatives were investigated to be stable for 4 weeks under 4 °C and detected with high intensity in ∼104 cells. On the other hand, we analyzed an additional 89 metabolites in central carbon and energy metabolic pathways were directly analyzed by liquid chromatography tandem mass spectrometry (LC-MRM-MS). The limit of detection (LODs) of our method were as low as 0.05 ng/mL and the linear range was at least two orders of magnitude with determination coefficient (R2) > 0.9701. The relative standard divisions (RSDs) of intra- and inter-day of 95% metabolites were below 20%. In addition, the isotope list of 82 detected metabolites in central carbon and energy metabolism were generated according to isotopologues and isotopomers for each metabolite resulting from 13C incorporation. Accurate assessment of mass isotopomer distributions (MIDs) of intracellular 13C-labeled metabolites was achieved in [U-13C]-glucose cultured HepG2 cells by our dual strategy. Finally, we performed MID analysis of 101 metabolites in central carbon and energy metabolism. Overall, this dual method is reproducible and robust for application on 13C-MFA and has a great potential for studying clinical isotope labeled samples.
    Keywords:  (13)C-metabolic flux analysis; Carboxyl and phosphate metabolites; Central carbon and energy metabolism; Derivatization; Liquid chromatography-mass spectrometry; Mass isotopomer distributions
  6. Bio Protoc. 2023 Aug 20. 13(16): e4742
      Lipids can play diverse roles in metabolism, signaling, transport across membranes, regulating body temperature, and inflammation. Some viruses have evolved to exploit lipids in human cells to promote viral entry, fusion, replication, assembly, and energy production through fatty acid beta-oxidation. Hence, studying the virus-lipid interactions provides an opportunity to understand the biological processes involved in the viral life cycle, which can facilitate the development of antivirals. Due to the diversity and complexity of lipids, the assessment of lipid utilization in infected host cells can be challenging. However, the development of mass spectrometry, bioenergetics profiling, and bioinformatics has significantly advanced our knowledge on the study of lipidomics. Herein, we describe the detailed methods for lipid extraction, mass spectrometry, and assessment of fatty acid oxidation on cellular bioenergetics, as well as the bioinformatics approaches for detailed lipid analysis and utilization in host cells. These methods were employed for the investigation of lipid alterations in TMEM41B- and VMP1-deficient cells, where we previously found global dysregulations of the lipidome in these cells. Furthermore, we developed a web app to plot clustermaps or heatmaps for mass spectrometry data that is open source and can be hosted locally or at This protocol provides an efficient step-by-step methodology to assess lipid composition and usage in host cells.
    Keywords:  Bioenergetics; Clustergram; Lipid profiling; Lipidomics; Mass spectrometry; Seahorse assay; Virus–lipid interactions; Web tool
  7. Anal Chem. 2023 Aug 29.
      The characterization of Novichoks (NVs), a new group of nerve agents that have been implicated in two recent poisonings, has not been extensively conducted. Here, we present a novel method for analyzing NV hydrolysates using liquid chromatography-tandem mass spectrometry (LC-MS/MS) enabled by pentafluorobenzyl (PFB) derivatization followed by reaction with 1,4-diazabicyclo[2.2.2]octane (DABCO). This approach enabled efficient, simultaneous screening of six NV hydrolysates, with 1-2 orders improvement in the limit of detection in relation to that achieved through previous methods. A straightforward pretreatment using DABCO and filtration was employed for biological samples, mitigating instrument damage and allowing LC-MS/MS after a reaction with highly hydrophobic PFB bromide (PFBBr). In addition, the use of pralidoxime (PAM) significantly enhanced the detection of NV hydrolysates from NV-surrogate-spiked serum. While PAM is not a proven NV antidote, its effectiveness as an analytical reagent to aid in the detection of NV hydrolysates was demonstrated for the first time. Understanding the proposed mechanism of DABCO-mediated derivatization reagent removal in this research could broaden the range of compounds amenable to derivatization LC, thereby enhancing the capabilities of conventional derivatization techniques.
  8. Anal Bioanal Chem. 2023 Aug 29.
      Infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging (IR-MALDESI) conventionally utilizes fresh-frozen biological tissues with an ice matrix to improve the detection of analytes. Sucrose-embedding with paraformaldehyde fixation has demonstrated feasibility as an alternative matrix for analysis by IR-MALDESI by preserving tissue features and enhancing ionization of lipids. However, investigating multi-organ systems provides broader context for a biological study and can elucidate more information about a disease state as opposed to a single organ. Danio rerio, or zebrafish, are model organisms for various disease states and can be imaged as a multi-organ sample to analyze morphological and metabolomic preservation as a result of sample preparation. Herein, whole-body zebrafish were imaged to compare sucrose-embedding with paraformaldehyde fixation against conventional fresh-frozen sample preparation. Serial sections were analyzed with and without an ice matrix to evaluate if sucrose functions as an alternative energy-absorbing matrix for IR-MALDESI applications across whole-body tissues. The resulting four conditions were compared in terms of total putative lipid annotations and category diversity, coverage across the entire m/z range, and ion abundance. Ultimately, sucrose-embedded zebrafish had an increase in putative lipid annotations for the combination of putative annotations with and without the application of an ice matrix relative to fresh-frozen tissues which require the application of an ice matrix. Upon the use of an ice matrix, a greater number of high mass putative lipid annotations (e.g., glycerophospholipids, glycerolipids, and sphingolipids) were identified. Conversely, without an ice matrix, sucrose-embedded sections elucidated more putative annotations in lower molecular weight lipids, including fatty acyls and sterol lipids. Similar to the mouse brain model, sucrose-embedding increased putative lipid annotation and abundance for whole-body zebrafish.
    Keywords:  IR-MALDESI; Mass spectrometry imaging; PFA; Sucrose; Zebrafish
  9. Anal Chem. 2023 Aug 30.
      Epilipids, a subset of the lipidome that comprises oxidized, nitrated, and halogenated lipid species, show important biochemical activity in the regulation of redox lipid metabolism by influencing cell fate decisions, including death, health, and aging. Due to the large chemical diversity, reversed-phase liquid chromatography-high-resolution mass spectrometry (RPLC-HRMS) methods have only a limited ability to separate numerous isobaric and isomeric epilipids. Ion mobility spectrometry (IMS) is a gas-phase separation technique that can be combined with LC-HRMS to improve the overall peak capacity of the analytical platform. Here, we illustrate the advantages and discuss the current limitations of implementing IMS in LC-HRMS workflows for the analysis of oxylipins and oxidized complex lipids. Using isomeric mixtures of oxylipins, we demonstrated that while deprotonated ions of eicosanoids were poorly resolved by IMS, sodium acetate and metal adducts (e.g., Li, Na, Ag, Ba, K) of structural isomers often showed ΔCCS% above 1.4% and base peak separation with high-resolution demultiplexing (HRDm). The knowledge of the IM migration order was also used as a proof of concept to help in the annotation of oxidized complex lipids using HRDm and all-ion fragmentation spectra. Additionally, we used a mixture of deuterium-labeled lipids for a routine system suitability test with the purpose of improving harmonization and interoperability of IMS data sets in (epi)lipidomics.
  10. J Pharm Biomed Anal. 2023 Aug 20. pii: S0731-7085(23)00444-2. [Epub ahead of print]235 115675
      Cocculus hirsutus is a widely used herb in traditional systems of medicine for the treatment of various diseases. In the present study, five alkaloids (1-5), two flavonoids (6-7), one triterpenoid (8), and three steroids (9-10) were isolated from the roots of Cocculus hirsutus and further crude extract was analyzed by LC-Q-Tof-MS/MS in positive ionization mode leading to the identification of ten metabolites through comparison of exact molecular masses from their MS/MS spectra, mass fragmentation studies and with literature data. In addition, a method was developed and validated for the quantification of four bio-active compounds [Sinococuline (1), Magnoflorine (2), (E)-N-feruloyltyramine (3), and 20-Hydroxyecdysone (10)] using UPLC-QqQ-MS in multiple reaction monitoring (MRM) mode for the first time. The method has shown good linearity with correlation coefficients (r2) higher than 0.9916 for all four compounds. The intra- and inter-day precision were in the range of 0.3-6.1% and from 0.7% to 8.8%, respectively. The matrix effects of all the four analytes were found in the range of 94.7 ± 2.8-112.7 ± 3.7%. Overall, our study provides a reliable and rapid approach by hyphenated LC-MS/MS using high-resolution mass spectrometers for identification and quantification of bioactive constituents from the root extracts of Cocculus hirsutus.
    Keywords:  Chemical profiling; Cocculus hirsutus; Extraction optimization; Simultaneous quantification; UPLC-QToF-MS(e) and UPLC-QqQ-MS-MRM
  11. Bioanalysis. 2023 Aug 28.
      Background: Hybrid LC-MS assays for oligonucleotides rely on capture probes to develop assays with high sensitivity and specificity. Locked nucleic acid (LNA) probes are thermodynamically superior to existing capture probes, but are not currently used for hybrid LC-MS assays. Materials & methods: Using two lipid-conjugated double-stranded siRNA compounds as model analytes, hybrid LC-MS/MS assays using LNA probes were developed. Results: The workflows demonstrated the superiority of the LNA probes, optimized sample preparation conditions to maximize analyte recovery, evaluated the need for analyte-specific internal standards, and demonstrated that advanced mass spectrometric technology can increase assay sensitivity by up to 20-fold. Conclusion: The workflow can be used in future bioanalytical studies to develop effective hybrid LC-MS/MS methods for siRNA analytes.
    Keywords:  LC–MS/MS; capture probe; hybridization; locked nucleic acids; oligonucleotides; short interfering RNA (siRNA)
  12. J Anal Toxicol. 2023 Aug 29. pii: bkad064. [Epub ahead of print]
      Psilocin is an active substance and dephosphorylated product of psilocybin formed after ingestion of mushrooms. The low stability caused by the quick oxidation of this analyte requires sensitive methods for its determination in biological matrices. In this work, we described a method development, optimization, and validation for the quantification of psilocin in authentic oral fluid samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Liquid-liquid extraction (LLE) was performed using 100 µL of oral fluid samples collected with Quantisal™ device and t-butyl methyl ether (MTBE) as the extraction solvent. The method showed acceptable performance, with limits of detection and quantification of 0.05 ng/mL, and the calibration model was achieved between 0.05 and 10 ng/mL. Bias and imprecision results were below -14.2% and 10.7%, respectively. Ionization suppression/enhancement were lower than -30.5%, and recovery was higher than 54.5%. Dilution integrity bias was below 14.4%. No endogenous and exogenous interferences were observed analyzing oral fluid from 10 different sources and 55 pharmaceuticals and drugs of abuse, respectively. No carryover was observed at 10 ng/mL. Psilocin was stable in oral fluid at -20°C, 4°C and 24°C up to 24, 72 and 24 h, respectively, with variations lower than 17.7%. The analyte was not stable after 3 freeze/thaw cycles, with variations between -73% and -60%. This suggests the instability of psilocin in oral fluid samples, which requires timely analysis, as soon as possible after the collection. The analyte remained stable in processed samples in autosampler (at 10°C) for up to 18 h. The method was successfully applied for the quantification of five authentic samples collected from volunteers attending parties and electronic music festivals. Psilocin concentrations ranged from 0.08 to 36.4 ng/mL. This is the first work to report psilocin concentrations in authentic oral fluid samples.
    Keywords:  LC-MS/MS; Psilocin; hallucinogens; oral fluid; psilocybin; stability
  13. J Anal Toxicol. 2023 Aug 26. pii: bkad063. [Epub ahead of print]
      A sensitive method for the qualitative screening of synthetic cannabinoids and opioids in whole blood was developed and validated using alkaline liquid-liquid extraction (LLE) and liquid chromatography - time-of-flight mass spectrometry (LC-QTOF-MS). Estimated limits of detection for validated compounds ranged from 0.03-0.29 µg/L (median, 0.04 µg/L) for the 27 opioids and from 0.04-0.5 µg/L (median, 0.07 µg/L) for the 23 synthetic cannabinoids. Data processing occurred in two stages; first a targeted screen was performed using an in-house database containing retention times, accurate masses and MS/MS spectra for 79 cannabinoids and 53 opioids. Suspect screening was then performed using a database downloaded from the crowd sourced NPS data website which contains mass, consensus MS/MS data and laboratory-specific predicted retention times for a far greater number of compounds. The method was applied to 61 forensic cases where synthetic cannabinoid or opioid screening was requested by the client or their use was suspected due to case information. CUMYL-PEGACLONE was detected in two cases and etodesnitazine, 5F-MDMB-PICA, 4-cyano-CUMYL-BUTINACA and carfentanil were detected in one case each. These compounds were within the targeted scope of the method but were also detected through the suspect screening workflow. The method forms a solid base for expansion as more compounds emerge onto the illicit drug market.
  14. Drug Discov Today. 2023 Aug 26. pii: S1359-6446(23)00267-2. [Epub ahead of print] 103751
      Metabolomics and lipidomics have an increasingly pivotal role in drug discovery and development. In the context of drug discovery, monitoring changes in the levels or composition of metabolites and lipids relative to genetic variations yields functional insights, bolstering human genetics and (meta)genomic methodologies. This approach also sheds light on potential novel targets for therapeutic intervention. In the context of drug development, metabolite and lipid biomarkers contribute to enhance success rates, promising a transformative impact on precision medicine. In this review, we deviate from analytical chemist-focused perspectives, offering an overview tailored to drug discovery. We provide introductory insight into state-of-the-art mass spectrometry (MS)-based metabolomics and lipidomics techniques utilized in drug discovery and development, drawing from the collective expertise of our research teams. We comprehensively outline the application of metabolomics and lipidomics in advancing drug discovery and development, spanning fundamental research, target identification, mechanisms of action, and the exploration of biomarkers.
    Keywords:  Lipids; biomarkers; clinical trials; drug discovery and development; lipidomics; mass spectrometry; metabolites; metabolomics; precision medicine; target engagement
  15. J Pharm Biomed Anal. 2023 Aug 08. pii: S0731-7085(23)00396-5. [Epub ahead of print]235 115627
      Amino acids normally have a chiral carbon and d/l-enantiomers are present. Due to the homochirality features on the present Earth, l-enantiomers are predominant in the living beings and the d-enantiomers are rare. Along with the progress and development of cutting edge analytical methods, several d-amino acids were found even in the higher animals including humans, and their biological functions and diagnostic values have also been reported. However, the amounts of these d-amino acids are much lower than the l-forms, and development/utilization of highly sensitive and selective methods are practically essential to avoid the disturbance from uncountable intrinsic substances. In the present review, multi-dimensional HPLC methods for the determination of chiral amino acids, especially two-dimensional LC-MS/MS and three-dimensional LC methods, and their applications to a variety of real-world matrices are summarized.
    Keywords:  Amino acids; Enantiomer separation; Multi-dimensional HPLC