bims-metlip 2026-05-17 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–05–17
eighteen papers selected by
Sofia Costa, Matterworks

The Combined Quantification of the 15 Human Bile Acids and Their Precursor 7α-Hydroxy-4-Cholesten-3-One in Human Serum: LC-MS/MS Method Development and Validation.
Scanning central carbon metabolism: a HILIC-HR-TOF-MS metabolome method.
An isotopologue-mixture-based internal standard strategy for LC-MS/MS quantification in plasma.
A Rapid and Sensitive HPLC-MS/MS Method for Quantification of Propranolol in Rat Plasma and the Application to Pharmacokinetic Study.
Simultaneous UPLC-MS/MS quantification of angiotensin (I and II) and eight adrenal steroids for the assessment of plasma renin activity and secondary hypertension subtyping.
3D-printed target plates for high-throughput ambient mass spectrometry analysis of nanoliter biofluids.
In-syringe solid-phase extraction coupled with UPLC-MS/MS for simultaneous determination of reducing sugars, animo acids and Amadori compounds in tobacco.
Simultaneous quantitation of structurally analogous bile acid isomers via charge-state-engineered UPLC-MS.
An Overview of Metabolomic Approaches to Polyphenol Profiling for Nutraceutical Development.
Data-Driven Filter for Detector Oscillation Artifacts in Time-of-Flight Mass Spectrometry.
Application of Ambient Ionization Mass Spectrometry to the Analysis of Cannabis.
Forward-Backward-Flushing Valve-Assisted Selectivity Tuning (FBF-VAST) for LC × LC: Principles and Demonstration of a Modulation Mechanism.
Lipid Analysis in Live Caenorhabditis elegans Using Solution-State NMR Spectroscopy.
Structural Diversity and Analytical Characterization of Acylhomocarnitines.
Reliable Molecular Retrieval from Mass Spectra Using Conformal Prediction.
Comparative development of volatile-oriented multi-SPME and derivatisation-based GC×GC-TOFMS workflows for non-targeted faecal metabolomics.
Sensitive and selective quantification of elcatonin in equine plasma and urine using LC-FAIMS-MS/MS.
Liquid Atmospheric Pressure MALDI (LAP-MALDI) Mass Spectrometry: A Versatile Tool for the Characterization of Synthetic Polymers.

J Mass Spectrom. 2026 Jun;61(6): e70064

The Combined Quantification of the 15 Human Bile Acids and Their Precursor 7α-Hydroxy-4-Cholesten-3-One in Human Serum: LC-MS/MS Method Development and Validation.

Zuweina Al-Hadidi, Sathish Pandian, Mohammed Al-Tobi, Fahad Al-Zadjali, Amani Al-Khaifi.

  Bile acids (BAs) have been considered potential biomarkers for various metabolic diseases, which prompted the need for accurate and sensitive quantification methods. This study aimed to develop a sensitive, validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying primary, secondary, free, and conjugated BAs, along with their precursor, 7α-hydroxy-4-cholesten-3-one (C4), in human serum. The analytical approach involved a rapid protein precipitation-based extraction technique in the presence of deuterated internal standards. Mass spectrometry detection employed electrospray ionization in negative and positive ion modes for BAs and C4, respectively. BA levels obtained by LC-MS/MS were validated by comparing them to total BAs measured by a sensitive enzymatic assay. The chromatographic cycle time for the method was 21 min per injection, employing multiple reaction monitoring. All BAs were separated with negligible carryover, matrix effect, and good recovery. Good linearity was observed in the concentration range of (0.0-10.0) μM for BAs and (0.0-0.4) μM for C4. The detection limit for BAs was between 0.0002 and 0.0138 μmol/L, and the sensitivity was 0.00156-0.0125 μmol/L. Acceptable precision results were obtained on intraday and interday analyses for BAs and C4. The LC-MS/MS quantification of total BAs closely matched levels from the colorimetric assay. This study presents a straightforward LC-MS/MS method for simultaneously quantifying BAs and their precursor, C4, in human serum. The low sample volume, rapid extraction, validated results, and detailed protocol make the established method reproducible and suitable for use.

Keywords:  CYP7A1 biomarker; human bile acids; liquid chromatography–tandem mass spectrometry

DOI:  https://doi.org/10.1002/jms.70064
Metabolomics. 2026 May 13. pii: 71. [Epub ahead of print]22(3):

Scanning central carbon metabolism: a HILIC-HR-TOF-MS metabolome method.

Victoria Pozo Garcia, Valentina Ferro, Jolene Rier, Sofia Moco.

   INTRODUCTION: Central carbon metabolism (CCM) is the primary metabolic hub of the cell, governing energy production and providing precursors essential for a myriad of biosynthetic pathways. Developing analytical tools that can identify and quantify intermediates of these metabolic reactions is crucial for studying cell metabolism in biomedical and biotechnological applications.
OBJECTIVE: This study proposes a liquid chromatography (LC)-high-resolution (HR) mass spectrometry (MS) method, covering the CCM of mammalian cell systems.
METHODS: Cells were extracted using a one-step liquid extraction, recovering the hydrophilic metabolites. A stable isotope dilution approach was employed, utilizing a U-13C-yeast internal standard (IS). A LC-HRMS metabolomics method using hydrophilic interaction liquid chromatography (HILIC) coupled to a Zeno-time-of-flight (ZenoTOF) MS was implemented for metabolite semi-quantification.
RESULTS: A total of 82 CCM metabolites is reported, of which 77 were confirmed with authentic standards, and for 63 , linearity ranges were obtained. IS normalization enhanced overall robustness, from sample preparation to metabolite semi-quantification. To study the effects on CCM by 5 chemical inhibitors (2-deoxy-D-glucose, etomoxir, UK-5099, rotenone, and 3-nitropropionic acid), our HILIC-HR-TOF-MS method was used. The approach proved efficient in capturing altered metabolite concentrations, within implicated metabolic reactions, as a consequence of inhibitor exposure.
CONCLUSION: Our HILIC-HR-TOF-MS metabolome method is efficient in mapping changes in metabolic intermediates of the CCM in mammalian cells. This approach holds potential for analysing a variety of biological samples across a range of applications, from drug development to biomedicine.

Keywords:  Central carbon metabolism; HILIC; LC–MS; Metabolomics

DOI:  https://doi.org/10.1007/s11306-026-02434-4
J Chromatogr A. 2026 May 06. pii: S0021-9673(26)00398-5. [Epub ahead of print]1781 467069

An isotopologue-mixture-based internal standard strategy for LC-MS/MS quantification in plasma.

Ping Guo, Yuan Liu, Guozhu Liu, Ming Ma, Bo Chen.

  Stable isotope-labeled internal standards (SIL-IS) are the gold standard for liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification in complex biological matrices. Their application can be limited by synthetic complexity, high cost, and the lack of commercially available labeled standards for many bioactive analytes. Here, we establish and evaluate an internal-standard strategy based on hydrogen-deuterium exchange (HDX)-derived deuterated isotopologue mixtures. Using Pd/C, Pt/C, and AlCl₃ catalytic systems, deuterated isotopologue mixtures (d₁-dₙ) of eight structurally diverse bioactive compounds were prepared by HDX in deuterated methanol and characterized by ¹H NMR and LC-MS. Isotopologues with a mass difference of Δm ≥ 3 were selected as internal-standard candidates to minimize overlap with analyte M + 1/M + 2 isotopic peaks. Isotopic-distribution screening and chromatographic evaluation showed that eligible d₃-dₙ isotopologues co-eluted with their corresponding analytes (Δt < 0.01 min), with no appreciable chromatographic isotope effect (CIE). In plasma, LC-MS/MS validation showed linearity over 1-500 ng/mL (R² ≥ 0.995), limits of detection of 0.003-0.080 ng/mL and limits of quantification of 0.01-0.25 ng/mL. Trueness ranged from 85% to 115%, while precision, expressed as relative standard deviation (RSD), was ≤8% intra-day and ≤12% inter-day. Ratio-based matrix effect (ME%) values ranged from 85% to 112%. Using clenbuterol hydrochloride as a model analyte, the isotopologue-mixture-derived internal standard showed calibration performance comparable to that of the commercial deuterated standard clenbuterol-d₉. Compared with quantification based on external calibration without internal-standard normalization, the proposed approach reduced quantitative deviation from 15 to 25% to <8%. Overall, the results support HDX-derived isotopologue mixtures as practical internal-standard sources and establish a selection-and-validation framework for LC-MS/MS quantification when commercial SIL-IS are unavailable.

Keywords:  Deuterated isotopologue mixtures; Hydrogen–deuterium exchange; Internal standards; LC–MS/MS quantification; Plasma

DOI:  https://doi.org/10.1016/j.chroma.2026.467069
Biomed Chromatogr. 2026 Jul;40(7): e70487

A Rapid and Sensitive HPLC-MS/MS Method for Quantification of Propranolol in Rat Plasma and the Application to Pharmacokinetic Study.

Li Bei, Huang Haijin, Wan Lili, Peng Guoshuang, Sun Yongbing.

  A novel HPLC-MS/MS method has been developed for the determination of propranolol in rat plasma in the present study. The analytes of interest were retained and separated on a C18 column, and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was applied for quantification. A simple protein precipitation method was used to pretreat the plasma; the extraction recovery (ER) was about 100%, and the matrix effect (ME) could be negligible. The method was linear over the concentration ranges of 0.625-2500.00 ng/mL for propranolol. The precision and accuracy were within acceptable limits. The lowest limit of quantification (LLOQ) was 0.625 ng/mL. This simple method was validated and successfully applied to support the pharmacokinetic study after administration of the propranolol transdermal drug delivery system to Sprague-Dawley rats.

Keywords:  HPLC‐MS/MS; pharmacokinetics; propranolol; protein precipitation

DOI:  https://doi.org/10.1002/bmc.70487
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 May 12. pii: S1570-0232(26)00203-5. [Epub ahead of print]1279 125114

Simultaneous UPLC-MS/MS quantification of angiotensin (I and II) and eight adrenal steroids for the assessment of plasma renin activity and secondary hypertension subtyping.

Yicong Yin, Ying Zhu, Jianwei Ren, Wei Luo, Xiuzhi Guo, Xiaoli Ma, Songlin Yu, Ling Qiu.

  Early screening and precise subtyping are crucial for the clinical management of secondary hypertension. However, comprehensive analytical methods for profiling multiple related biomarkers remain limited. This study aimed to develop and validate a multiplex ultra-performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of angiotensin (I and II) and eight adrenal steroids (aldosterone, 18-oxocortisol, 18-hydroxycortisol,cortisol,cortisone,corticosterone,11-deoxycortisol, and 21-deoxycortisol) in human plasma. Plasma samples were prepared utilizing solid-phase extraction and chromatographic separation was achieved on a BEH C8 column (2.1 × 50 mm, 1.7 μm). Quantitative analysis was performed using a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source. The plasma renin activity was indirectly determined by quantifying the generation of angiotensin I following a 3-hours sample incubation. The method demonstrated satisfactory linearity for all analytes (r > 0.99). The within-run and total coefficients of variation were 1.4%-8.4% and 1.6%-10.5%, respectively. Extraction recoveries ranged from 80.0% to 108.3%, and matrix effects were evaluated between 80% and 120% with negligible carryover. All validation parameters met the acceptance criteria. Ultimately, this method was successfully applied to clinical plasma samples, providing a promising analytical tool for the early screening, diagnosis, and subtyping of secondary hypertension.

Keywords:  Adrenal steroids; Renin activity; Secondary hypertension; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2026.125114
Anal Chim Acta. 2026 Jul 22. pii: S0003-2670(26)00508-8. [Epub ahead of print]1408 345558

3D-printed target plates for high-throughput ambient mass spectrometry analysis of nanoliter biofluids.

Chang Liu, Yuanyang Zhao, Junpeng Zhang, Ruixia Wang, Ligang Hu, Ming Xu.

   BACKGROUND: Ambient mass spectrometry, such as desorption electrospray ionization mass spectrometry (DESI-MS), represent a promising high-throughput platform for biomarkers analysis in biospecimens. However, its application to clinical biomarker analysis in biofluids remains limited by inherent trade-offs among sample preparation complexity, quantitative accuracy, and analytical reproducibility.
RESULTS: In this study, we report, for the first time, the fabrication of a custom-designed target plate, using three-dimensional (3D) printing technology, to enable nanodroplet array preparation for high-throughput DESI-MS analysis. A single square target plate (2 mm × 2 mm) can accommodate 188 discrete 80-nL biofluid droplets, permitting high-throughput biomarker screening in biofluids with DESI-MS. For three clinically relevant renal function biomarkers, creatinine (Cr), urea nitrogen (UN), and uric acid (UA), the limits of detection (LODs) achieved with DESI-MS range from 0.1 to 0.8 μmol/L, an approximately tenfold improvement over conventional colorimetric enzymatic assays routinely employed in clinical laboratories. More critically, DESI-MS analysis of each individual urine or serum sample requires only 6.5 s, substantially shorter than the several minutes typically needed per sample for enzymatic methods. Finally, the feasibility, reliability, and reproducibility of the developed method were demonstrated through quantifying Cr, UN, and UA in serum and urine samples from mice exposed to inorganic mercury (Hg(II)), serving as a proof-of-concept validation.
SIGNIFICANCE: We anticipate that this robust strategy for biofluid analysis will advance both biomarker discovery and mechanistic studies across clinical medicine, biology, and environmental health science.

Keywords:  Biofluid; Biomarker; Desorption electrospray ionization mass spectrometry; Nanodroplet; Target plate; Three-dimensional printing

DOI:  https://doi.org/10.1016/j.aca.2026.345558
J Chromatogr A. 2026 May 05. pii: S0021-9673(26)00397-3. [Epub ahead of print]1781 467068

In-syringe solid-phase extraction coupled with UPLC-MS/MS for simultaneous determination of reducing sugars, animo acids and Amadori compounds in tobacco.

Xiaoqian Wang, Yanhua Lai, Sufang Guo, Honglin Qin, Ziwang Liu, Ling Xia, Xuanjing Wang, Junzhang Wu, Gongke Li.

  Considering the critical role of Maillard reaction-related compounds in determining the sensory and chemical stability of tobacco, this study develops an in-syringe SPE coupled with UPLC-MS/MS method for simultaneous determination of 43 Maillard reaction-related compounds in tobacco, covering reducing sugars (3), amino acids (17), Amadori compounds (17), and structurally related intermediates (6). The sample pretreatment utilizes HLB-packed self-assembled columns for SPE, followed by methanol elution. The extracted solution is then analyzed using a Titank C18 column (150×2.1 mm, 1.8 μm), with acetonitrile and 0.1% formic acid aqueous solution employed as the mobile phase for gradient elution. Data were acquired in multiple reaction monitoring mode using electrospray ionization. Operation parameters were optimized systematically, including the type and amount of filler, solution pH, loading rate, elution solvent composition, and sample extraction volume. This method demonstrated excellent linearity for all 43 target compounds, with correlation coefficients exceeding 0.9980. The limit of detections was 0.10-0.50 µg/L for animo acids, 1.0×102- 2.5×102 µg/L for reducing sugars, 0.10-5.0 µg/L for related Amadori compounds. This approach was successfully applied to the analysis of tobacco samples and extracts, providing a robust tool for studying the Maillard reaction and contributing to food safety assessment and optimization of food processing techniques.

Keywords:  Amadori compounds; In-syringe solid-phase extraction; Maillard reaction; Tobacco analysis

DOI:  https://doi.org/10.1016/j.chroma.2026.467068
Analyst. 2026 May 08.

Simultaneous quantitation of structurally analogous bile acid isomers via charge-state-engineered UPLC-MS.

Desen Su, Yunyun Zheng, Xiuxian Lin, Ziqiang Chen, Minmin Huang, Qinghua Yao, Yuwu Chi.

Ursodeoxycholic, hyodeoxycholic, chenodeoxycholic, and deoxycholic acids are endogenous, dihydroxylated bile acid isomers that share a molecular mass but fulfill specific biological roles. However, their isomeric structures complicate analytical separation, while low physiological concentrations demand high detection sensitivity. Consequently, despite the use of liquid chromatography-tandem mass spectrometry for simultaneous analysis, current methodologies are often hampered by inadequate sensitivity and lengthy analysis durations. To address this analytical bottleneck, we developed an ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) method for the quantitative analysis of four bile acid isomers. This method employs a novel charge-state homogenization strategy, guided by computational prediction of ionization behavior (pH/ionic forms), to optimize chromatographic and mass spectrometric conditions. By inducing complete deprotonation and generating uniform carboxylate anions for all analytes, this approach synergistically enhances the detection sensitivity, chromatographic resolution, and analysis speed. The rigorously validated method demonstrates exceptional performance: sub-ng mL-1 sensitivity (LODs 0.10-0.22 ng mL-1), rapid analysis (<8 min), excellent linearity (R2 > 0.998 over 4 orders of magnitude), high reproducibility (intra/inter-day RSD 1.2-9.1%), and satisfactory accuracy (86.1-107.1% recovery). Its broad applicability was confirmed across diverse matrices (poultry/porcine blood, bile, concentrates), handling trace to high bile acid levels. This rapid, sensitive and highly-resolutive analytical approach addresses key challenges in bile acid isomers quantitation, offering significant advantages for pharmacokinetic research and quality control in animal health and pharmaceutical applications. Furthermore, the underlying charge-state homogenization principle provides a rational framework for optimizing chromatographic parameters (organic modifiers, stationary phases, gradients) in complex analyses.

DOI: https://doi.org/10.1039/d6an00420b
Molecules. 2026 Apr 28. pii: 1468. [Epub ahead of print]31(9):

An Overview of Metabolomic Approaches to Polyphenol Profiling for Nutraceutical Development.

Temitope Oluwaferanmi Egbeniyi, Julius Dongsogo, Titilayo Oluwayemisi Bamidele, Alberta N A Aryee.

  Secondary plant metabolites such as polyphenols (flavonoids, phenolic acids, stilbenes, and lignans) are valued for their numerous benefits and commonly associated with antioxidants, anti-inflammatory, anticancer, neuroprotective, and antidiabetic effects. Comprehensive profiling facilitates their identification and quantification, with metabolomics emerging as an increasingly valuable tool. This current work provides an overview of recent application of metabolomics for investigating polyphenols with nutraceutical potential. It also highlights the influence of plant species and environmental stressors (both biotics and abiotic) inducing metabolic shifts that promote the production and accumulation of these bioactive compounds (BACs). While various analytical tools including mass spectrometry (MS) coupled with liquid chromatography (LC-MS) or gas chromatography (GC-MS), as well as nuclear magnetic resonance (NMR) spectroscopy have been utilized to identify the diverse group of polyphenol metabolites, LC-MS has been predominantly used due to its superior sensitivity and wider metabolite coverage, with flavonoids being the main compounds identified. The integration of bioinformatic tools and pathway enrichment analysis in metabolomics is providing expansive insight into the pool of polyphenols, and their bio-functional interpretation and metabolic variations beyond the narrow scope of chromatographic separation alone. This overview also identifies limitations of current methods and suggests directions for future research, aimed at facilitating the development of nutraceuticals.

Keywords:  data analysis; environmental stressors; metabolomics; nutraceuticals; phytochemicals

DOI:  https://doi.org/10.3390/molecules31091468
Anal Chem. 2026 May 12.

Data-Driven Filter for Detector Oscillation Artifacts in Time-of-Flight Mass Spectrometry.

Kas J Houthuijs, Karl J Jobst, Frederic Béen.

Time-of-flight mass spectrometry (TOF-MS) is widely used for complex mixture analysis due to its ability to detect thousands of chemical compounds in a single sample. However, high-intensity ions can saturate the detector and generate artifacts, commonly referred to as "detector ringing". These artifacts resemble true MS features and often persist after componentization, complicating data interpretation and feature prioritization. Here, we introduce a data-driven filtering approach that exploits the time-domain origin of detector ringing to remove such artifacts. Because ion flight times scale with m/z, oscillations in the detector response manifest as artifactual features with constant delays in m/z, providing a universal detection criterion independent of ion source conditions and chromatographic or ion mobility separation. The workflow was evaluated on GC-, LC-, and ion mobility-hyphenated TOF-MS datasets from multiple instrumental platforms, identifying and removing up to 5.3% of features. Comparison across instruments revealed that Δm/z spacings are not only constant but also instrument-specific and polarity-independent, suggesting potential applications in data forensics and traceability. To assess broader relevance, the filter was applied to the MassBank and NIST DART-MS Forensics fragmentation mass spectral libraries. Although the overall incidence was low relative to library size (1.7%), over 1000 mass spectra were provisionally identified as containing ringing artifacts. Clustering these spectra by their Δm/z effectively partitioned the data by TOF-MS platform, revealing distinct groupings across libraries and contributors. By specifically targeting detector-derived artifacts, this workflow reduces false positives and improves feature prioritization and compound identification, enhancing the robustness of TOF-MS-based chemical analyses.

DOI: https://doi.org/10.1021/acs.analchem.6c00762
Annu Rev Anal Chem (Palo Alto Calif). 2026 May;19(1): 25-47

Application of Ambient Ionization Mass Spectrometry to the Analysis of Cannabis.

B Garosi, R A Musah.

  To circumvent multiple challenges associated with Cannabis analysis by conventional methods such as gas chromatography (GC), liquid chromatography (LC), and hyphenated techniques such as GC and LC mass spectrometry (MS), there is increasing interest in the application of ambient ionization mass spectrometry (AIMS) for its chemical characterization, as this approach can in principle address several of the issues associated with interrogation of Cannabis-derived complex matrix samples. Among the advantages that these methods confer are rapid analysis times; limited or no need for sample pretreatment steps; detection of a range of compound classes in a single analysis, including cannabinoids, terpenes, flavonoids, and pesticides; avoidance of nuanced method development tailored to particular analyte classes; and the ability to analyze samples in their native forms. This review highlights the progress thus far in the nascent area of application of AIMS approaches to Cannabis and Cannabis-derived materials, and the further developments required in order for AIMS methods to be more widely adopted for routine analysis.

Keywords:  Cannabis; ambient ionization; cannabinoids; high-throughput analysis; mass spectrometry

DOI:  https://doi.org/10.1146/annurev-anchem-080524-100042
Anal Chem. 2026 May 11.

Forward-Backward-Flushing Valve-Assisted Selectivity Tuning (FBF-VAST) for LC × LC: Principles and Demonstration of a Modulation Mechanism.

Pattraporn Chobpradit, Kenji Hamase, Sornkanok Vimolmangkang, Thumnoon Nhujak, Chadin Kulsing.

A modulation mechanism for comprehensive two-dimensional liquid chromatography (LC × LC), termed FBF-VAST (forward-backward-flushing valve-assisted selectivity tuning), is introduced. This technique enables modulation using a simple system configuration centered around a 4-port valve. The concept lies in periodic forward-backward flushing of the first-dimension (1D) column, enabling synchronized analyte holding and release with a stepped-gradient transferred to the second dimension (2D). The modulation period (PM) is defined by the forward- and backward-flushing durations (tfwd + tbwd), with the net injection window (Δtnet = tfwd - tbwd) delivering analyte fractions into the 2D-column. These processes were simulated using advection-dispersion equations modified to include analyte retention, resulting in solvent and analyte space-time profiles, along with animations that illustrate the modulation mechanism. A unique selectivity-tuning effect was demonstrated through the 1D-column simulation and LC × LC experiments using two C18 columns operated under a unified binary gradient for red wine sample separation, yielding 27-55 separated peaks of interest. The selectivity can be effectively adjusted solely by tuning the Valve A timing (tfwd) or gradient steepness─without altering mobile phase composition or stationary phase chemistry. Repeatability studies showed %RSDs of peak areas within 5.28-11.77% (n = 6). In addition, the method enabled backflushing for online 1D-column cleaning, which may extend column life and improve robustness. This cost-effective and tunable LC × LC strategy offers enhanced control over chromatographic selectivity and long-term stability, all within a simplified instrumental design.

DOI: https://doi.org/10.1021/acs.analchem.5c08280
Bio Protoc. 2026 May 05. 16(9): e5686

Lipid Analysis in Live Caenorhabditis elegans Using Solution-State NMR Spectroscopy.

Florencia V Guastaferri, Carla B Delprato, Bruno Hernández Cravero, Gastón Prez, Diego De Mendoza, Andres Binolfi.

  Unsaturated fatty acids (UFAs) play key roles in essential cellular functions such as membrane dynamics, metabolism, and animal development. Disruptions in UFA metabolism are linked to metabolic, cardiovascular, and neurodegenerative disorders. Cellular UFAs composition and quantification are normally determined using methods such as gas chromatography and/or mass spectrometry, which require extraction procedures and prevent analysis of live specimens. Here, we describe a protocol that employs uniform 13C isotope labeling and high-resolution 2D solution-state nuclear magnetic resonance (NMR) spectroscopy to analyze lipid composition and fatty acid unsaturation directly in the model organism Caenorhabditis elegans. The approach enables in vivo assessment of lipid storage compositions with sufficient resolution and sensitivity to distinguish wild-type animals from those with altered fatty acid desaturation. Complementary analysis of total lipid extracts provides information regarding lipid molecules that are not detected in vivo, such as phospholipid molecules organized in biological membranes. Overall, this non-destructive NMR-based method offers a powerful tool for investigating lipid metabolism in C. elegans and other small model systems that can be isotopically enriched. Key features • Solution-state NMR spectroscopy is not destructive and can be used on live cells and multicellular organisms. • 13C isotopic enrichment is required for high-resolution NMR analysis of lipids in live C. elegans. • Lipid signals from live worms arise from the mobile lipid phase in lipid droplets. • NMR provides readouts of lipid compositions in live animals at a highly sensitive rate, enabling precise interpretation of the whole cell lipid metabolism.

Keywords:  13C isotope labeling; Caenorhabditis elegans; In vivo NMR spectroscopy; Lipids; Unsaturated fatty acids (UFAs)

DOI:  https://doi.org/10.21769/BioProtoc.5686
J Proteome Res. 2026 May 13.

Structural Diversity and Analytical Characterization of Acylhomocarnitines.

Jaclyn Weinberg, William J Crandall, Zachery Ryan Jarrell, Gahyun Lim, Ken Liu, Ho Young Lee, Shakshi Patel, Camilo Anthony Gacasan, Young-Mi Go, Dean P Jones.

  Homocarnitine is a five-carbon analog of carnitine produced in mammals through hydroxylation of the microbiome-derived metabolite δ-valerobetaine. Here, we describe liquid chromatography-mass spectrometry methods for the measurement of fatty acyl-homocarnitines, a previously uncharacterized family of mammalian metabolites. These acyl-homocarnitines are homologs of acyl-carnitines, in which the fatty acid is extended by one carbon. We show that short-chain fatty acyl-CoAs are converted to corresponding acyl-homocarnitines by carnitine acetyltransferase and that these enzyme-generated standards exhibit retention times and ion dissociation patterns identical to acyl-homocarnitines produced by mammalian cells. In vitro 13C3-homocarnitine isotope tracer studies showed that mammalian cells produce short-, medium-, and long-chain acyl-homocarnitines. Ion dissociation analyses established diagnostic product ions to distinguish acyl-homocarnitines from isomeric acyl-carnitines. Sample preparation and chromatographic methods are provided to separate and analyze isomers in extracts of mouse tissues. These findings expand knowledge of carnitine analogs and establish analytical strategies to differentiate acyl-homocarnitines from isomeric acyl-carnitines.

Keywords:  HILIC; acylcarnitine; carnitine; chromatography; energy-resolved; fatty acid metabolism; homocarnitine; isomer; mass spectrometry; valerobetaine

DOI:  https://doi.org/10.1021/acs.jproteome.5c01255
J Chem Inf Model. 2026 May 11.

Reliable Molecular Retrieval from Mass Spectra Using Conformal Prediction.

Morteza Rakhshaninejad, Gaetan De Waele, Mira Jürgens, Willem Waegeman.

A key task in the computational analysis of liquid chromatography-tandem mass spectrometry (LC-MS/MS) data is identifying the molecular structure underlying a measured spectrum. A common approach ranks candidate molecules retrieved from chemical databases using predicted fingerprint similarities, yet standard metrics such as top-k accuracy summarize performance only at the data set level and provide no spectrum-specific reliability statement. In this work, we apply conformal prediction to candidate-based molecular retrieval to construct spectrum-specific prediction sets that contain the true molecule with a user-specified probability. We evaluate marginal and conditional conformal prediction across three experimental scenarios representing in-distribution, partially shifted, and fully out-of-distribution settings on the MassSpecGym benchmark. When calibration and test data are aligned, conformal prediction attains the target coverage with small candidate sets for most spectra. Under distribution shift, prediction sets become larger as rankings grow more ambiguous, although candidates can still be reduced when calibration remains representative. Conditional conformal prediction improves subgroup reliability across spectra of different difficulty, with the best gains obtained using confidence-based grouping. Overall, conformal prediction turns candidate rankings into reliable, spectrum-specific candidate sets with an explicit reliability-efficiency trade-off.

DOI: https://doi.org/10.1021/acs.jcim.6c00727
Talanta. 2026 May 06. pii: S0039-9140(26)00610-7. [Epub ahead of print]308 129954

Comparative development of volatile-oriented multi-SPME and derivatisation-based GC×GC-TOFMS workflows for non-targeted faecal metabolomics.

Thibaut Dejong, Kinjal Bhatt, Anaïs Rodrigues, Thibault Massenet, Siebe Lievens, Jean-François Focant, Pierre-Hugues Stefanuto.

  Gas chromatography-mass spectrometry (GC-MS) remains a key technique in metabolomics, yet most workflows rely on chemical derivatisation to enable the analysis of non-volatile metabolites. Although derivatisation broadens metabolite coverage, it increases sample preparation time and may introduce additional analytical variabilities. In contrast, solid-phase microextraction (SPME) enables rapid, solvent-free sampling of volatile and semi-volatile compounds, representing an attractive alternative for non-targeted studies. However, methodological developments integrating SPME with comprehensive two-dimensional gas chromatography (GC × GC-MS) remain limited. In this study, a simultaneous multi-SPME GC × GC-TOFMS workflow was developed for the non-targeted screening of faecal samples. Three identical fibres were used simultaneously to generate technical replicates from a single biological sample resulting strong performances in terms of relative standard deviation (10%). Dedicated fibre storage containers and optimised storage conditions were also developed to preserve analyte stability between sampling and injection. In parallel, commonly used GC × GC column configurations were evaluated. The optimised workflow was applied to stool reference materials generated within an interlaboratory metabolomics study coordinated by the National Institute of Standards and Technology (NIST), investigating the effects of diet (vegan vs. omnivore) and sample preservation (aqueous vs. lyophilised) to develop the more adequate stool reference material. SPME results were compared with derivatisation-based metabolomic and lipidomic workflows. Multivariate analysis revealed clear discrimination between diets and storage conditions, while data-fusion analysis highlighted the complementary nature of volatile, metabolomic, and lipidomic profiles. This work provides practical guidance for developing robust GC × GC-MS workflows for complex biological matrices, highlighting the need of multi-extraction approaches for comprehensive analytical coverage.

Keywords:  Data fusion; GC×GC-TOFMS; Microbiome; Multi-SPME; Non-target screening

DOI:  https://doi.org/10.1016/j.talanta.2026.129954
J Pharm Biomed Anal. 2026 May 07. pii: S0731-7085(26)00219-0. [Epub ahead of print]279 117551

Sensitive and selective quantification of elcatonin in equine plasma and urine using LC-FAIMS-MS/MS.

Kohei Ohnuma, Minori Fukazawa, Taiga Uchida, Tomohiro Kato, Michiko Sugai-Bannai, Mariko Shibuya, Masayuki Yamada, Misato Hirano-Kodaira.

  The use of drugs for enhancing bone strength is prohibited under international regulations governing horse racing and equestrian sports. Elcatonin, a synthetic peptide with analgesic and bone-strengthening properties, is routinely monitored for compliance. However, its cyclic structure and high molecular weight hinder reliable detection. In this study, we developed and validated a sensitive liquid chromatography-high-field asymmetric waveform ion mobility spectrometry-tandem mass spectrometry method for quantifying elcatonin in equine plasma and urine. Sample preparation was optimised via weak cation exchange solid-phase extraction. A pilot pharmacokinetic and pharmacodynamic study was conducted to confirm the practical applicability of our method by validating linearity, reproducibility, sensitivity, selectivity, recovery, and analyte stability, including enzymatic/proteolytic stability. The method achieved a detection limit of 10 pg/mL for urine and plasma (R2 > 0.9995 in both cases), meeting the validation criteria for quantitative analysis. Following administration, elcatonin was detected in plasma (for up to 6 h) but not in urine. Pharmacodynamic analysis revealed that elcatonin administration caused a transient small decrease in the plasma calcium levels without any other observable clinical effects, such as hypocalcaemia. The established method overcomes the challenges of detecting and quantifying cyclic peptides and has the potential to improve the detectability of elcatonin and related molecules relevant to doping control and animal healthcare.

Keywords:  Cyclic peptides; Elcatonin; Equine antidoping; High-field asymmetric waveform ion mobility spectrometry; Liquid chromatography–tandem mass spectrometry; Pharmacokinetics; Solid-phase extraction

DOI:  https://doi.org/10.1016/j.jpba.2026.117551
Rapid Commun Mass Spectrom. 2026 Aug 30. 40(16): e70102

Liquid Atmospheric Pressure MALDI (LAP-MALDI) Mass Spectrometry: A Versatile Tool for the Characterization of Synthetic Polymers.

Agata E Kowalczyk, Jeffery Brown, Michael Morris, Rainer Cramer.

RATIONALE: Liquid atmospheric pressure matrix-assisted laser desorption/ionization (LAP-MALDI) mass spectrometry (MS) has previously been applied to the analysis of biopolymers such as peptides, proteins, and DNA, producing ESI-like multiply charged analyte ions. In this study, LAP-MALDI MS has been investigated for the analysis of synthetic polymers for the first time.
METHODS: A LAP-MALDI source was interfaced with a Q-TOF mass spectrometer. The study investigated synthetic polymer standards, including polyethylene glycol (PEG), polypropylene glycol (PPG), polyacrylic acid (PAA), and polystyrene (PS) of different sizes in positive and negative ionization modes with and without ion mobility spectrometry (IMS). The acquired data were compared to ESI MS data using the same polymer standards and instrument but with a standard ESI source.
RESULTS: LAP-MALDI MS enables the detection of singly and predominantly multiply charged ions with high ion signal stability (< 10% RSD), though with lower charge states than ESI MS. Its flexible sample preparation and ion source setup allow for the controlled manipulation of charge states. Common salts facilitate ionization by metal cation adduct formation and improve detection of PEG, PPG, and PS. PAA was efficiently detected in negative ion mode by deprotonation, and PS required a silver salt for ionization. IMS provides an additional dimension in separation and manipulating charge state distributions, and its data obtained for PEG corroborate previously published ESI IMS results.
CONCLUSIONS: LAP-MALDI MS provides a rapid, less labor-intensive, and reagent-efficient way for the analysis of synthetic polymers and produces stable ion signals in both positive and negative ion modes. The source enables the detection of singly and multiply charged ions, with the flexibility to manipulate charge states over a wide range. LAP-MALDI MS tolerates diverse solvent compositions and offers a versatile platform for high-throughput polymer structural analysis, including MS/MS analysis that can replace or complement standard polymer analysis tools.

DOI: https://doi.org/10.1002/rcm.70102