bims-metlip 2025-08-10 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–08–10
23 papers selected by
Sofia Costa, Matterworks

mzrtsim: Raw Data Simulation for Reproducible Gas/Liquid Chromatography-Mass Spectrometry-Based Nontargeted Metabolomics Data Analysis.
Analysis of plant metabolomics data using identification-free approaches.
An Improved Metabolomics Workflow Enables Untargeted Data Acquisition and Targeted Data Analysis Using Liquid Chromatography-Mass Spectrometry.
One-Step Labeling and Purification: Accelerating FAHFA Detection in Vegetable Oils via Chemoselective Probe-Assisted LC-MS.
Leveraging open cheminformatics tools for non-targeted metabolomics analysis of C. elegans: a workflow comparison and application to strains related to xenobiotic metabolism and neurodegeneration.
Therapeutic drug monitoring of busulfan and cyclophosphamide in hematologic malignancies: An LC-MS/MS development, validation and application.
An Analytical Evaluation of Tools for Lipid Isomer Differentiation in Imaging Mass Spectrometry.
Method of Confirmation and Predictive Metabolomics: A Novel Multidimensional Analytical Framework for Characterizing New Psychoactive Substances.
A Rapid and Simple Reversed-Phase HPLC-MS/MS Determination of Baloxavir in Rat Plasma and Its Application in Pharmacokinetic Study.
Clinical validation of a liquid chromatography single quadrupole mass spectrometry (LC-MS) method using Waters Kairos™ Amino Acid Kit reagents.
A reliable UHPLC-MS/MS method for the determination of legacy and emerging bisphenol analogues in human urine.
Integrating Electrostatic Adsorption-Laser Desorption/Ionization-MS with a Derivative Full-Spectrum Internal Standard Approach for Acylcarnitine Quantification in Clinical Samples.
Development of an Efficient Method to Quantitatively Estimate Dichloroacetic Acid Genotoxic Impurity in Cetirizine Dihydrochloride by Liquid Chromatography-Mass Spectrophotometry.
Development of Enhanced Separation Techniques for Oligonucleotides Utilizing Mixed-Mode Chromatography and 2D-LC/UV/MS Analysis.
Insect Lipidomics: Advances, Applications, and Physiological Insights.
Quantification of Belzutifan in Biological Samples: LC-MS/MS Method Validation and Pharmacokinetic Study in Rats.
Leveraging chemical synthesis to discover metabolites from the gut microbiome.
Analysis of Perfluoroalkyl Substances (PFASs) in Settled Dust by Liquid Chromatography with Tandem Mass Spectrometry: Method Validation and Application to Vietnamese House and Road Dusts.
High-throughput quantitation of acetaldehyde and ethanol in mice using gas chromatography/mass spectrometry positive chemical ionization.
Batchwise data analysis with inter-batch feature alignment in large scale platelet lipidomics study using UHPLC-ESI-QTOF-MS/MS by data-independent SWATH acquisition.
Quantitative HILIC-Q-TOF-MS Analysis of Glycosaminoglycans and Non-reducing End Carbohydrate Biomarkers via Glycan Reductive Isotopic Labeling.
Unlocking spatial metabolomics with isotopically labelled internal standards.
Q2C: A software for managing mass spectrometry facilities.

Anal Chem. 2025 Aug 05.

mzrtsim: Raw Data Simulation for Reproducible Gas/Liquid Chromatography-Mass Spectrometry-Based Nontargeted Metabolomics Data Analysis.

Miao Yu, Vivek Philip.

Reproducibility of data analysis is pivotal in the context of nontargeted metabolomics based on mass spectrometry coupled with chromatography. While various algorithms have been proposed for feature or peak extraction, their validation often revolves around a limited set of known compounds or standards. While data simulation is widely used in other omics studies, simulations are focused on the feature level, neglecting uncertainties inherent in the feature or peak extraction process for metabolomics mass spectrometry data. In this technique note, we introduce an R package called "mzrtsim"' to simulate gas/liquid chromatography full scan raw data in the mzML format. Unlike simulations solely based on virtual features, our approach leverages experimental spectral data from MassBank of North America (MoNA) and the human metabolome database (HMDB). We developed algorithms to simulate chromatographic peaks, accounting for the tailing factor. The results of our study demonstrate the potential of this tool for comparing established metabolomics software (e.g., XCMS, mzMine, and OpenMS) against ground truth. We found that the investigated software introduced false positive peaks and/or loss of compounds with fewer peaks. They also showed different sensitivity to the tailing and leading peaks. This R package is free and available online (https://github.com/yufree/mzrtsim).

DOI: https://doi.org/10.1021/acs.analchem.5c01213
Appl Plant Sci. 2025 Jul-Aug;13(4):13(4): e70001

Analysis of plant metabolomics data using identification-free approaches.

Xinyu Yuan, Nathaniel S S Smith, Gaurav D Moghe.

  Plant metabolomes are structurally diverse. One of the most popular techniques for sampling this diversity is liquid chromatography-mass spectrometry (LC-MS), which typically detects thousands of peaks from single organ extracts, many representing true metabolites. These peaks are usually annotated using in-house retention time or spectral libraries, in silico fragmentation libraries, and increasingly through computational techniques such as machine learning. Despite these advances, over 85% of LC-MS peaks remain unidentified, posing a major challenge for data analysis and biological interpretation. This bottleneck limits our ability to fully understand the diversity, functions, and evolution of plant metabolites. In this review, we first summarize current approaches for metabolite identification, highlighting their challenges and limitations. We further focus on alternative strategies that bypass the need for metabolite identification, allowing researchers to interpret global metabolic patterns and pinpoint key metabolite signals. These methods include molecular networking, distance-based approaches, information theory-based metrics, and discriminant analysis. Additionally, we explore their practical applications in plant science and highlight a set of useful tools to support researchers in analyzing complex plant metabolomics data. By adopting these approaches, researchers can enhance their ability to uncover new insights into plant metabolism.

Keywords:  data analysis; evolution; liquid chromatography–mass spectrometry; machine learning; metabolic diversity; metabolomics; phytochemistry; statistical analyses

DOI:  https://doi.org/10.1002/aps3.70001
J Proteome Res. 2025 Aug 07.

An Improved Metabolomics Workflow Enables Untargeted Data Acquisition and Targeted Data Analysis Using Liquid Chromatography-Mass Spectrometry.

Yi Wu, Yang Wang.

  In this study, we present an improved metabolomics methodological framework that synergistically integrates untargeted data acquisition with targeted data analysis using chemical derivatization combined with ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF MS) analysis. Data-independent acquisition (DIA)-based mass spectrometry (MS1) data were used to conduct conventional untargeted analysis for biomarker discovery. The data-dependent acquisition (DDA) method was applied to obtain high-quality tandem mass spectrometry (MS2) information for quantitative data analysis. 1-Aminopiperidine (1AP) served as the derivatization reagent for sample preparation, which selectively reacts with carboxyl-containing compounds. Fatty acid (FA) standards were used to examine the derivatization reaction, and the results of LC-MS analysis showed that protonated FA + 1AP-H2O was the precursor. The ion at m/z 84.08, along with the product ion from a neutral loss of 45.02 Da, emerged as characteristic fragments, facilitating compound annotation and quantitative analysis. Method validation results demonstrated the proposed method with excellent repeatability, stability, and linearity. The lung tissue samples were successfully analyzed using this method, which was further employed to evaluate the therapeutic efficacy of Zhuye Shigao Decoction (ZSD) against lipopolysaccharide (LPS)-induced acute pneumonia in mice.

Keywords:  1-Aminopiperidine; liquid chromatography−mass spectrometry; metabolomics; targeted data analysis; untargeted data acquisition

DOI:  https://doi.org/10.1021/acs.jproteome.5c00427
J Agric Food Chem. 2025 Aug 06. 73(31): 19813-19822

One-Step Labeling and Purification: Accelerating FAHFA Detection in Vegetable Oils via Chemoselective Probe-Assisted LC-MS.

Xin-Ze Wu, Kai-Wen Tan, Xu Zhang, Jun-Jie Tan, Na An, Fang Cong, Wenbin Wu, Azamat Temerdashev, Quan-Fei Zhu, Hong Wang, Yu-Qi Feng.

  Fatty acid esters of hydroxy fatty acids (FAHFAs) are emerging bioactive lipids in edible oils, but their trace-level presence and matrix complexity hinder accurate analysis. Here, we designed a novel chemoselective probe piperazine-modified silica (SiO2-PP) that specifically captures FAHFAs while removing almost totally triglycerides in a one-step labeling reaction. The probe enabled single-batch sample pretreatment within 30 min, integrating capture, purification, and signal amplification. Coupled with liquid chromatography-mass spectrometry, the method achieved high sensitivity (LOD: 0.0039-0.033 ng/mL) and enabled quantification of 47 FAHFA isomers in 47 vegetable oil samples, with concentrations ranging from 0.56 to 1.76 × 104 ng/g. Five dominant isomers (e.g., 10-OAHSA) were identified, with olive and peanut oils exhibiting the highest FAHFA concentrations. This approach provides a promising platform for high-throughput functional lipid profiling, effectively overcoming the inefficiencies of traditional methods while significantly enhancing the assessment of vegetable oil quality.

Keywords:  chemoselective probe; fatty acid esters of hydroxy fatty acids; high-throughput quantification; liquid chromatography−mass spectrometry; vegetable oils

DOI:  https://doi.org/10.1021/acs.jafc.5c06401
Anal Bioanal Chem. 2025 Aug 08.

Leveraging open cheminformatics tools for non-targeted metabolomics analysis of C. elegans: a workflow comparison and application to strains related to xenobiotic metabolism and neurodegeneration.

Gianfranco Frigerio, Yunjia Lai, Emma L Schymanski, Gary W Miller.

  Caenorhabditis elegans (C. elegans) is a well-established nematode model for studying metabolism and neurodegenerative disorders, such as Alzheimer's (AD) and Parkinson's disease (PD). Non-targeted metabolomics via liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has proven useful for uncovering metabolic changes in biological systems. Here, we present workflows for C. elegans metabolomics, leveraging advanced open science tools. We compared two metabolite extraction methods: a monophasic extraction, which provided broader metabolite coverage in analyses conducted in hydrophilic interaction with positive polarity (HILIC POS), and a biphasic extraction, which yielded more features in reverse-phase C18 chromatography with negative polarity (RPLC NEG) analyses. Data were processed using patRoon, integrating IPO, XCMS, CAMERA, and MetFrag, which incorporated PubChemLite compounds and C. elegans-specific metabolites from an expanded WormJam database enhanced with PubChem and literature sources. MS-DIAL was also employed for data processing, allowing for expanded annotations with predicted spectra for the expanded WormJam metabolites calculated using CFM-ID. Significant metabolite differences were identified when comparing the Bristol (N2) wild-type strain with two knockout strains of xenobiotic-metabolizing enzymes and two transgenic strains related to neurodegenerative pathways. Pooled quality control (QC) samples for each strain ensured robust data quality and the detection of strain-related metabolites. Our study demonstrates the potential of non-targeted metabolomics for metabolite discovery employing open science tools in model organisms.

Keywords:  CYP enzyme mutant; Exposomics; FMO enzyme mutant; SV2C expression; Tau aggregation; Untargeted metabolomics

DOI:  https://doi.org/10.1007/s00216-025-06048-y
J Pharmacol Toxicol Methods. 2025 Aug 02. pii: S1056-8719(25)00809-3. [Epub ahead of print] 108389

Therapeutic drug monitoring of busulfan and cyclophosphamide in hematologic malignancies: An LC-MS/MS development, validation and application.

Jinxingyi Wang, Ruyu Tao, Hanshuai Hu, Xue Lan, Jiejie Gao, Yanping Guan, Qian Liu.

   BACKGROUND: Busulfan and cyclophosphamide (Bu-Cy) is a widely used conditioning regimen for hematopoietic stem cell transplantation (HSCT) in the treatment of hematologic malignancies. To explore the exposure-efficiency relationships of Bu-Cy regimen and evaluate the necessity of relative therapeutic drug monitoring (TDM), we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify the busulfan and cyclophosphamide in human plasma.
METHODS: A simple LC-MS/MS bioassay for simultaneous determination of busulfan and cyclophosphamide using isotope dilution internal standardization has been established. Plasma samples were subjected to protein precipitation prior to their injection into a column for subsequent separation (Thermo Accucore aQ C18, 50 × 2.1 mm, 2.6 μm). Positive electrospray ionization (ESI) mode with selected reaction monitoring (SRM) was used for analytes determination.
RESULTS: Busulfan and cyclophosphamide could be measured within 3 min, with the calibration ranges of 80-4000 ng/mL for busulfan and 25-2000 ng/mL for cyclophosphamide. The validated method showed good sensitivity and linearity. Accuracies and precisions were less than 15 %. The results of stability showed that busulfan and cyclophosphamide were stable in the plasma under room temperature for 16 h, 4 °C for 24 h, -80 °C for 2 weeks, and after three freeze-thaw processes.
CONCLUSION: The bioassay was simple, sensitive and successfully implemented in the realm of TDM for Bu-Cy, ultimately leading to improved dosing precision.

Keywords:  Busulfan; Cyclophosphamide; Hematologic malignancies; LC-MS/MS; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.vascn.2025.108389
Int J Mass Spectrom. 2024 Aug;pii: 117268. [Epub ahead of print]502

An Analytical Evaluation of Tools for Lipid Isomer Differentiation in Imaging Mass Spectrometry.

Boone M Prentice.

Imaging mass spectrometry has emerged as a powerful tool to map the spatial distributions of lipid and metabolites in biological tissues. However, these analyses are challenged by the multitude of isobaric (i.e., same nominal mass) and isomeric compounds present in most samples. Failure to adequately separate these compounds results in inaccurate or incomplete chemical identifications and produces composite images of spatial distribution arising from multiple compounds. A number of techniques have been developed to more completely resolve and identify this complex chemical milieu. These include methods that rely on condensed-phase chemical derivatization and gas-phase ion chemistry, or some combination thereof. This Young Scientist Feature focuses on summarizing the analytical figures of merit of these tools, highlighting their relative speeds, limits of detection, molecular specificities, and eases-of-use. It will also include current challenges and future perspectives for resolving structural isomers in imaging mass spectrometry experiments.

DOI: https://doi.org/10.1016/j.ijms.2024.117268
Rapid Commun Mass Spectrom. 2025 Nov 30. 39(22): e10116

Method of Confirmation and Predictive Metabolomics: A Novel Multidimensional Analytical Framework for Characterizing New Psychoactive Substances.

Anirudha Dixit, Aakanksha Soni, Astha Pandey.

   RATIONALE: The rapid emergence and structural diversity of new psychoactive substances (NPS) present significant challenges for forensic drug analysis. Existing targeted methods often fall short in accurately identifying novel compounds, increasing the risk of false positives or missed detections. This study aims to address this gap by developing a robust, non-targeted analytical approach capable of confidently screening, identifying, and confirming a broad range of NPS.
METHOD: A method was developed using Ultra-Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight Mass Spectrometry (UPLC-QTOF-MS) to analyze four selected NPS: Mephedrone (4-methylmethcathinone), Ephylone, 2C-B, and Methamphetamine. Analytical parameters such as retention time, mass accuracy (±5 ppm), diagnostic fragments, ion ratios, and isotope patterns were evaluated. The method was validated against critical criteria including precision, stability, and reproducibility. The limit of confirmation (LOC) for each analyte was established, and predictive metabolomics were performed to investigate metabolic transformations using bulk powder spiking and spiked urine matrices.
RESULTS: The UPLC-QTOF-MS method demonstrated excellent precision and reproducibility, with consistent retention times and diagnostic fragment ions within acceptable limits as per SANTE and WADA TD2023IDCR guidelines. Mass accuracy remained within ±5 ppm, confirming high analytical performance. The established LOC for all analytes was 50 ng/mL. The study also revealed significant metabolic transformations through predictive metabolomics. Compared to conventional tandem MS techniques, QTOF-MS offered enhanced non-targeted screening capabilities and the advantage of retrospective data analysis.
CONCLUSION: The validated UPLC-QTOF-MS method provides a powerful, reliable tool for the rapid detection and confirmation of emerging NPS in forensic samples. Its high mass accuracy, ability to perform non-targeted analysis, and support for retrospective interrogation make it a superior alternative to traditional mass spectrometric methods. This approach enhances forensic laboratories' ability to keep pace with evolving drug trends and strengthens the integrity of drug-related investigations.

Keywords:  UPLC‐QTOF; limit of confirmation; method of confirmation (MoC); new psychoactive substances (NPS)

DOI:  https://doi.org/10.1002/rcm.10116
Biomed Chromatogr. 2025 Sep;39(9): e70183

A Rapid and Simple Reversed-Phase HPLC-MS/MS Determination of Baloxavir in Rat Plasma and Its Application in Pharmacokinetic Study.

Peng Guoshuang, Wan Lili, Liu Xiang, Li Bei, Sun Yongbing.

  In this study, a novel RP-HPLC-MS/MS method was developed for the accurate quantification of baloxavir in rat plasma. The analytes were retained and separated on a C18 column, and a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source was applied for quantification. Baloxavir could be well retained in the C18 column under the appropriate chromatographic conditions. Using a simple protein precipitation method, the extraction recovery was about 100%, and the ME could be negligible. The method demonstrated excellent linearity (r = 0.9995) across the concentration range of 1.59 ~ 3180.00 ng/mL for baloxavir. The precision and accuracy were within acceptable limits. The lowest limit of quantification (LLOQ) was determined to be 1.59 ng/mL. This simple method was validated and successfully applied to support the pharmacokinetics study after baloxavir was administered to the Sprague-Dawley rats.

Keywords:  RP‐HPLC–MS/MS; baloxavir; pharmacokinetics; protein precipitation

DOI:  https://doi.org/10.1002/bmc.70183
Clin Chem Lab Med. 2025 Aug 08.

Clinical validation of a liquid chromatography single quadrupole mass spectrometry (LC-MS) method using Waters Kairos™ Amino Acid Kit reagents.

Rowan Hellier, Luke Griffiths, Chandra Sundas, Annabel Rodham, Stuart J Moat.

   OBJECTIVES: Quantitation of plasma amino acids (AA) is critical for the diagnosis and monitoring of inherited disorders of AA metabolism. AA analysis using ion-exchange chromatography (IEC) with post-column ninhydrin derivatization is time consuming, with run times of ∼2 h, limiting sample throughput. Liquid chromatography mass-spectrometry can potentially address some of the current challenges.
METHODS: Performance of components of the Waters Kairos Amino Acid Kit using liquid chromatography single quadrupole mass-spectrometry (LC-MS) following derivatization of samples with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ•Tag™ Ultra Derivatization Reagent) was evaluated. Results were compared with the Biochrom-IEC method using patient specimens (n=115), ClinChek® control and external quality assessment (EQA) material.
RESULTS: The kit reagents and our developed method had a 19-min analysis time, demonstrated acceptable inter-assay imprecision (CV<10 %) and bias vs. IEC-method (overall mean bias <2 %). Excellent correlation (concordance correlation coefficient (CCC) >0.99) with IEC was demonstrated for 10/23 analytes, good correlation (CCC >0.95) for 10/23, with the remaining three amino acids (aspartate, histidine and tryptophan) demonstrating moderate concordance (CCC ≥0.90 but <0.95). 1/23 AAs had a mean bias >10 % using EQA material. The method demonstrated a lower limit of quantitation of ≤2.5 μmol/L for all AA, making this assay suitable for CSF analysis. Calibration stability bias was <5 % over 12-weeks. Derivatized AAs were stable for ≤17 days. The analytical column supplied demonstrated good retention time stability (<0.4 %) and was capable of >2000 injections.
CONCLUSIONS: The tested methodology demonstrated good analytical performance and correlation with IEC. This approach confers practical advantages over IEC, including analytical selectivity and workflow time efficiency.

Keywords:  amino acids; derivatization; liquid chromatography; mass spectrometry

DOI:  https://doi.org/10.1515/cclm-2025-0424
J Chromatogr A. 2025 Jul 18. pii: S0021-9673(25)00579-5. [Epub ahead of print]1759 466234

A reliable UHPLC-MS/MS method for the determination of legacy and emerging bisphenol analogues in human urine.

Xiao Lin, Dejun Bao, Qi Sun, Xu Zhang, Zhuangzhuang Feng, Ying Zhu.

  An efficient technique for quantitative analysis of BPA, BPF, BPS, and 11 emerging bisphenol analogues in human urine has been developed, which combined solid phase extraction (SPE) with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Stable isotope internal standards were added to 1.0 mL of urine, which was hydrolyzed by β-glucuronidase overnight, and the target analytes were enriched and purified using an HLB 96-well plate, concentrated to dryness by nitrogen blowdown and reconstituted in 0.5 mL of 20 % aqueous methanol solution. Separation of the 14 analytes was achieved using Acquity BEH C18 (100 mm × 2.1 mm, 1.7 μm) as the analytical column and gradient elution with water and methanol as the mobile phase. Qualitative and quantitative analyses were performed in multiple reaction monitoring (MRM) mode with parallel positive and negative ion detection. Fourteen analytes showed good linearity in the concentration range of 0.1-50 ng/mL with correlation coefficients (r) greater than 0.999. The method detection limits (MDLs) range from 0.01 to 0.07 ng/mL and the method quantification limits (MQLs) were between 0.02 and 0.23 ng/mL. Recoveries across three spike levels were within the range of 72 % to 127 %. Intra-day precision and inter-day precision were below 12.7 % and 15.1 %, respectively. The proposed method successfully determined bisphenol analogues in 187 urine samples from young people in Beijing, revealing high prevalence rates for BPA (98.9 %) and BPS (80.2 %), with median concentrations of 6.44 μg/L and 0.17 μg/L, respectively. No significant differences in BPA and BPS levels were observed between genders, suggesting similar exposure patterns in males and females. Notably, the detection rate of the new alternative DBSP reached 32.6 %, indicating its significant exposure in the population. This study highlights the ongoing public health concerns regarding exposure to bisphenol analogues, encompassing both traditional compounds (such as BPA and BPS) and emerging alternatives.

Keywords:  Bisphenol analogues; Human urine; Internal exposure; Solid phase extraction (SPE); UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.chroma.2025.466234
J Am Soc Mass Spectrom. 2025 Aug 06.

Integrating Electrostatic Adsorption-Laser Desorption/Ionization-MS with a Derivative Full-Spectrum Internal Standard Approach for Acylcarnitine Quantification in Clinical Samples.

Shuo Cao, Xinrong Jiang, Xiaoming Chen, Xuetong Qu, Jianmin Wu.

Acylcarnitines (ACs), essential intermediates in fatty acid β-oxidation, are increasingly recognized as biomarkers for inborn errors of metabolism (IEM), insulin resistance, heart failure, and neurodegenerative diseases. However, conventional liquid chromatography-mass spectrometry (LC-MS) and flow injection-mass spectrometry (FI-MS) methods are hindered by complex sample preparation, limited throughput, and matrix interferences. Meanwhile, matrix-assisted laser desorption/ionization-MS (MALDI-MS) suffers from low-mass background noise and poor reproducibility. Here, we present a dual-innovation mass spectrometry platform combining a substrate composed of sulfonic acid-gold nanoparticle-decorated silicon nanowires (Sulfo-Au-SiNWs) for electrostatic adsorption-laser desorption/ionization (EALDI) and a full-spectrum internal standard (FS IS) strategy based on d6-ethanol derivatization. The optimized Sulfo-Au-SiNWs offer uniform crystallinity, in situ electrostatic enrichment, efficient thermal desorption, and gold-mediated charge-hole separation, resulting in a ∼300% increase in AC signal intensity. This enabled the selective detection of over ten medium- and long-chain ACs that were previously undetectable using unmodified SiNWs. The FS IS method incorporates over 30 derivatized AC species, enabling simultaneous quantification with detection limits below 0.01 μmol/L, surpassing the sensitivity of traditional FI-MS. The platform delivers spike recovery rates of 96-112% and supports high-throughput analysis, processing each sample within seconds. Validation against FI-ESI-MS/MS in both urine and dried blood spot samples demonstrated excellent agreement (ρ > 0.95) and effectively identified IEM patient profiles via statistically significant AC elevations. With strong intra- and interbatch precision (median RSD < 12.5%) and substrate stability exceeding one month, this EALDI-MS/FS IS workflow provides a high-throughput, high-coverage, and quantitatively robust solution for clinical AC profiling and newborn screening.

DOI: https://doi.org/10.1021/jasms.5c00136
Biomed Chromatogr. 2025 Sep;39(9): e70189

Development of an Efficient Method to Quantitatively Estimate Dichloroacetic Acid Genotoxic Impurity in Cetirizine Dihydrochloride by Liquid Chromatography-Mass Spectrophotometry.

Mithun M Gharat, Pallavi T Roy, Amit N Gosar, Tabrez A Shaikh, Gurumeet C Wadhava, Nitin A Mirgane.

  An LC-MS (liquid chromatography-mass spectrometry) methodology is developed for the precise analysis of dichloroacetic acid (DCAA) genotoxic impurity with high sensitivity and selectivity in a Cetirizine dihydrochloride (CTZ) drug substance. In accordance with the "threshold of toxicological concern (TTC)," the carryover of DCAA in a CTZ is done at a limit of 25 ppm in the sample. The present methodology is validated as per "International Council for Harmonization (ICH)" guidelines, and the detection limit and the quantitation limit are 0.4 and 1.2 ppm, respectively. The linearity study was conducted, and the coefficient of regression was found to be 0.9946. The method's accuracy was confirmed by the finding the percentage of recovered spiked DCAA in the drug, which ranged from 93.47% to 99.80%. Results indicated that the methodology was reliable, precise, and reproducible. The method can be extended for the determination of DCAA genotoxic impurity in a CTZ drug substance (API) samples by LC-MS.

Keywords:  Cetirizine dihydrochloride (CTZ); dichloroacetic aicd (DCAA); genotoxic impurity; liquid chromatography–mass spectrophotometer; validation

DOI:  https://doi.org/10.1002/bmc.70189
J Sep Sci. 2025 Aug;48(8): e70238

Development of Enhanced Separation Techniques for Oligonucleotides Utilizing Mixed-Mode Chromatography and 2D-LC/UV/MS Analysis.

Jakob Haglöf, Jenny M Nilsson, Jufang Wu Ludvigsson.

  Oligonucleotides are traditionally analyzed using ion-pair reversed-phase liquid chromatography. In this project, we explored an alternative approach utilizing a reversed phase-weak anion exchange mixed-mode column, which combines separation based on both charge and hydrophobicity. Our findings indicate that the mixed-mode column provided stronger retention and superior separation compared to the C18 column, eliminating the need for ion-pairing reagents. Additionally, selectivity can be adjusted using gradient pH and buffer concentration as well as acetonitrile content. However, the use of a phosphate buffer was necessary to ensure adequate retention and separation, despite its incompatibility with MS detection. To address this issue, a 2DLC-UV-qTOF setup with multiple loops was established, where the second-dimension LC, connected to the MS detector, required only an MS-compatible mobile phase. Using a HILIC-type LUNA Omega Sugar column in the second-dimension LC, the MS intensity of the heart-cut from the first dimension was increased 100-fold, allowing for the characterization of the main peak.

Keywords:  2D liquid chromatography; mass spectrometry; mixed‐mode chromatography; oligonucleotide; reversed‐phase/weak anion‐exchange

DOI:  https://doi.org/10.1002/jssc.70238
Adv Exp Med Biol. 2025 Aug 09.

Insect Lipidomics: Advances, Applications, and Physiological Insights.

Laura Palanker Musselman, Doga Cedden, Gözde Güney, Umut Toprak.

  Lipidomics, a specialized branch of metabolomics, investigates the diversity and functionality of lipids in biological systems. Lipids serve crucial roles in energy storage, membrane composition, and environmental acclimation in insects, underpinning processes such as development and stress responses. Advances in analytical technologies, such as liquid chromatography-mass spectrometry (LC-MS), have enabled precise identification and quantification of lipid species, providing unprecedented insights into lipid metabolism and dynamics. Key lipid classes, including triacylglycerols and phospholipids, exhibit structural and functional versatility, adapting to environmental pressures through mechanisms like homeoviscous adaptation. These dynamic lipid responses are essential for maintaining cellular and cuticular integrity and functionality under stress. By exploring lipid diversity and adaptations, lipidomics offers valuable perspectives on insect physiology, survival strategies, and evolutionary ecology. This chapter summarizes methods used to study insect lipidomes and highlights comparative lipidomic studies that have advanced our understanding of insect biology.

Keywords:  Cuticular hydrocarbons; Diapause; Ecdysteroids; Insect lipidomics; Mass spectrometry

DOI:  https://doi.org/10.1007/5584_2025_878
Biomed Chromatogr. 2025 Sep;39(9): e70168

Quantification of Belzutifan in Biological Samples: LC-MS/MS Method Validation and Pharmacokinetic Study in Rats.

Kamma Harsha Sri, Panchumarthy Ravisankar, Sathish Kumar Konidala, Srinivasa Babu Puttagunta.

  Belzutifan, an inhibitor of hypoxia inducible factor-2α, is used to treat cancer associated with von Hippel Lindau disease. The quality control and pharmacokinetic study of this drug is crucial for effective chemotherapy. Since no bio-analytical method has been reported, this work aimed to develop an LC-MS/MS technique for the determination of belzutifan and its application for pharmacokinetic profiling in rat plasma. Belzutifan and apalutamide (IS) were quantified on a symmetry shield (150 × 4.6 mm, 3.5 μm) column using acetonitrile and buffer (30:70% v/v) as the mobile phase, with a run time of 7 min. The spiked samples and quality controls were extracted with an optimized protein precipitation technique. Belzutifan and IS were quantified in MRM mode. The validation of the method in compliance with the US FDA's guidelines was performed. The analyte and IS were quantified at m/z 384.3422 → 311.4205 and 478.4154 → 341.1629, respectively. The results indicate linearity between 5 and 100 ng/mL concentration with r2 = 0.9997, which proved to be accurate with % recovery between 95.0% and 97.98%, along with other essential metrics within the accepted limits. The pharmacokinetic study demonstrates that the established LC-MS/MS method accurately quantifies the drugs in rat plasma and might be useful for routine quantification of belzutifan in biological matrices.

Keywords:  LC–MS/MS; belzutifan; bio‐analytical method; pharmacokinetic study; von Hippel–Lindau disease

DOI:  https://doi.org/10.1002/bmc.70168
Ann N Y Acad Sci. 2025 Aug 04.

Leveraging chemical synthesis to discover metabolites from the gut microbiome.

Allison J Keys, Nitesh K Nandwana, Eyas Alnasser, Emily C Gentry.

  The gut microbiome has the biosynthetic potential to make a variety of secondary metabolites or natural products, which serve as molecular messages between cells and organisms. These chemical signals are capable of affecting physiology and behavior in real time, and are, therefore, bioactive and can exhibit medicinal properties including anticancer, antimicrobial, or immunomodulating activities. It is clearly important to identify signaling molecules in the human gut, but elucidating their chemical structures can be challenging since traditional isolation methods are typically not available. The discovery of microbiome-related metabolites requires multidisciplinary collaboration, where chemical synthesis often plays an essential role. This review highlights examples where synthetic chemistry was used to study novel metabolites produced by the gut microbiota. In the first part, we describe examples where organic synthesis was utilized in traditional contexts, as a last step for validating structures and sourcing material for biological testing. The final section of this review discusses next-generation applications for chemical synthesis, where integration with metabolomic or genomic analysis simultaneously uncovers both structural and biological information about small molecules from the gut.

Keywords:  gut microbiome; mass spectrometry; metabolite discovery; metabolomics; organic synthesis; reverse metabolomics; synthetic chemistry

DOI:  https://doi.org/10.1111/nyas.70004
Bull Environ Contam Toxicol. 2025 Aug 08. 115(2): 28

Analysis of Perfluoroalkyl Substances (PFASs) in Settled Dust by Liquid Chromatography with Tandem Mass Spectrometry: Method Validation and Application to Vietnamese House and Road Dusts.

Anh Quoc Hoang, Mai Anh Luu, The Hieu Nguyen, Duc Hieu Nguyen, Thi Huyen Trang Luu, Thi Phuong Mai Nguyen, Thi Hong Thuy Do, Hoang Anh Dinh, Linh Trang Nguyen, Thi Lieu Tran, Thi Quynh Hoa Nguyen, Thi Anh Huong Nguyen.

  Information about perfluoroalkyl substances (PFASs) like perfluoroalkyl carboxylic acids (PFCAs) and sulfonates (PFSs) in settled dust from emerging and developing countries is still limited, partly due to the lack of efficient analytical methods. In this study, a reliable, simple, and cost-effective analytical procedure was developed and validated to determine 12 PFCAs and 4 PFSs in dust samples. The samples were ultrasonicated with methanol, followed by a dispersive sorbent clean-up step with graphitized carbon and quantification by liquid chromatography with tandem mass spectrometry (LC-MS/MS) method. The analytical method exhibited adequate recovery (80-120% for native standards and 50-130% for labeled standards), precision (relative standard deviations < 25%), and detection limits (0.010-0.10 ng/g). The validated method was applied to analyze PFAS concentrations in settled dusts collected from Hanoi, Vietnam. PFASs were frequently detected in the Vietnamese dust samples at relatively low concentrations (median 4.15, max 27.4 ng/g).

Keywords:  Dispersive sorbent clean-up; LC–MS/MS; PFASs; Settled dust; Ultrasonic extraction

DOI:  https://doi.org/10.1007/s00128-025-04100-2
Alcohol Clin Exp Res (Hoboken). 2025 Aug 04.

High-throughput quantitation of acetaldehyde and ethanol in mice using gas chromatography/mass spectrometry positive chemical ionization.

Yu-Hong Lin, Cheng Chen, Shoupeng Wei, Guillot Adrien, Bryan Mackowiak, Hongna Pan, Yaojie Fu, Luca Maccioni, Tianyi Ren, Li Zhang, Joseph Hibbeln, Robert Pawlosky, Bin Gao.

   BACKGROUND: Acetaldehyde, an immediate ethanol metabolite, mediates many ethanol-induced behavioral effects and is both psychoactive and toxic to animals and humans. Monitoring the kinetics of acetaldehyde using rodent models of alcohol misuse is essential for understanding and managing ethanol-associated diseases. However, quantitation of acetaldehyde in biological specimens after alcohol consumption has been challenging due to its high volatility, relatively low concentrations, and strong reactivity toward biochemical molecules. It was necessary to develop and establish an accurate and high-throughput method to quantitate acetaldehyde and ethanol.
METHODS: Gas chromatography/mass spectrometry in positive chemical ionization mode coupled with a 111-vial headspace autosampler was employed to quantitate acetaldehyde and ethanol using 2H4-acetaldehyde and 2H5-ethanol as internal standards. A multidimensional approach was used to develop the method, including sample collection and processing, instrumental data analysis, optimization, and validation. Blood and tissues collected from genetically modified mouse models and their wild-type counterparts were studied.
RESULTS: The method was validated and applied to quantitate acetaldehyde and ethanol in blood and tissues from multiple mouse studies on ethanol metabolism. Acetaldehyde and ethanol were well-resolved from chromatographic interferences with linear ranges of 6.25-800 μM for acetaldehyde and 1.25-160 mM for ethanol. Both regression coefficients for calibration curves were >0.999. The within- and between-run precisions for ethanol in plasma, whole blood, and serum were all <5.0%, and for acetaldehyde in plasma and serum were <9.0%, while in whole blood it was 19.2%. Sample throughput was on the order of 60 samples per 15 h daily, with a maximum of 111 per batch.
CONCLUSIONS: Despite some limitations, this validated method proved to be specific, accurate, and reproducible for high-throughput quantitation of acetaldehyde and ethanol in rodent plasma, whole blood, serum, and visceral organs.

Keywords:  direct PCA procedure; plasma; selected ion monitoring; serum; whole blood

DOI:  https://doi.org/10.1111/acer.70126
J Pharm Biomed Anal. 2025 Aug 05. pii: S0731-7085(25)00429-7. [Epub ahead of print]266 117088

Batchwise data analysis with inter-batch feature alignment in large scale platelet lipidomics study using UHPLC-ESI-QTOF-MS/MS by data-independent SWATH acquisition.

Kristina Dittrich, Xiaoqing Fu, Adrian Brun, Madhumita Chatterjee, Meinrad Gawaz, Michael Lämmerhofer.

  Untargeted lipidomics by ultra-high-performance liquid chromatography (UHPLC) hyphenated with tandem mass spectrometry using data-independent acquisition (DIA) is a technique with increasing popularity for generating new hypotheses in support of clinical research. Its strength is its data comprehensiveness on both MS and MS/MS level. However, especially when applying SWATH acquisition for large-scale analysis, e.g. clinical studies with over 1000 s to 10,000 s of samples, simultaneous processing of acquired data in multiple batches over longer period of time may be challenging due to retention time and mass shifts as well as huge bulk of data, particularly when computer power is limited. This problem can be alleviated by a batchwise data processing strategy by inter-batch feature alignment of separately processed sample batches. After batchwise automated data processing in MS-DIAL, feature lists can be combined by aligning identical features from different batches attributed to similarity in precursor m/z and retention time, with the intention to generate a representative reference peak list for targeted data extraction. The workflow was established with detected features from three batches of platelet lipid extracts of coronary artery disease (CAD) patients (n = 120) and then applied on a clinical cohort with 1057 CAD patients measured in 22 batches. As a result, the lipidome coverage was significantly increased when several batches were used to create the target feature list compared to a single batch and the increase of annotated features levelled off with 7-8 batches. Further, the lipid identification was improved in terms of number of structurally annotated features.

Keywords:  Coronary artery disease; Data processing; Lipidomics; Mass spectrometry; Platelet

DOI:  https://doi.org/10.1016/j.jpba.2025.117088
Anal Chem. 2025 Aug 06.

Quantitative HILIC-Q-TOF-MS Analysis of Glycosaminoglycans and Non-reducing End Carbohydrate Biomarkers via Glycan Reductive Isotopic Labeling.

Amrita Basu, Stephanie Archer-Hartmann, Pradeep Chopra, Mehrnoush Taherzadeh Ghahfarrokhi, Xiaolin Dong, Neil G Patel, Yiwen Zhang, Biswa Choudhury, Kosuke Funato, Dhananjay Yellajoshyula, Geert-Jan Boons, Parastoo Azadi, Ryan J Weiss.

Glycosaminoglycans (GAGs) are linear, heterogeneous polysaccharides expressed on all animal cells. Sulfated GAGs, including heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS), are involved in numerous physiological and pathological processes; therefore, precise and robust analytical methods for their characterization are essential to correlate structure with function. In this study, we developed a method utilizing hydrophilic interaction liquid chromatography coupled with time-of-flight mass spectrometry (HILIC-Q-TOF-MS) and glycan reductive isotopic reducing end labeling (GRIL) for the quantitative compositional analysis of HS and CS/DS polysaccharides. Lyase-generated disaccharides and commercial standards were chemically tagged on the reducing end with aniline stable isotopes, thus enabling the absolute quantification of HS and CS/DS disaccharides in complex biological samples. In addition, we adapted this workflow, in conjunction with new synthetic carbohydrate standards, for the quantification of disease-specific non-reducing end (NRE) carbohydrate biomarkers that accumulate in patients with mucopolysaccharidoses (MPS), a subclass of lysosomal storage disorders. As a proof of concept, we applied this method to measure NRE biomarkers in patient-derived MPS IIIA and MPS IIID fibroblasts, as well as in cortex tissue from a murine model of MPS VII. Overall, this method demonstrates improved sensitivity compared to previous GRIL-LC/MS techniques and, importantly, avoids the use of ion-pairing reagents, which are undesirable in certain mass spectrometry instrumentation and contexts. By combining the benefits of HILIC separation with isotopic labeling, our approach offers a robust and accessible tool for the analysis of GAGs, paving the way for advancements in understanding GAG structure and function.

DOI: https://doi.org/10.1021/acs.analchem.5c02338
Nat Metab. 2025 Aug 05.

Unlocking spatial metabolomics with isotopically labelled internal standards.

DOI: https://doi.org/10.1038/s42255-025-01341-7
J Proteomics. 2025 Jul 31. pii: S1874-3919(25)00138-1. [Epub ahead of print]321 105511

Q2C: A software for managing mass spectrometry facilities.

Diogo B Lima, Max Ruwolt, Marlon D M Santos, Ke Pu, Fan Liu, Paulo C Carvalho.

  We present Q2C, an open-source software designed to streamline mass spectrometer queue management and assess performance based on quality control metrics. Q2C provides a fast and user-friendly interface to visualize projects queues, manage analysis schedules and keep track of samples that were already processed. Our software includes analytical tools to ensure equipment calibration and provides comprehensive log documentation for machine maintenance, enhancing operational efficiency and reliability. Additionally, Q2C integrates with Google™ Cloud, allowing users to access and manage the software from different locations while keeping all data synchronized and seamlessly integrated across the system. For multi-user environments, Q2C implements a write-locking mechanism that checks for concurrent operations before saving data. When conflicts are detected, subsequent write requests are automatically queued to prevent data corruption, while the interface continuously refreshes to display the most current information from the cloud storage. Finally, Q2C, a demonstration video, and a user tutorial are freely available for academic use at https://github.com/diogobor/Q2C. Data are available from the ProteomeXchange consortium (identifier PXD055186). SIGNIFICANCE: Q2C addresses a critical gap in mass spectrometry facility management by unifying sample queue management with instrument performance monitoring. It ensures optimal instrument utilization, reduces turnaround times, and enhances data quality by dynamically prioritizing and routing samples based on analysis type and urgency. Unlike existing tools, Q2C integrates queue control and QC in a single platform, maximizing operational efficiency and reliability.

Keywords:  Management queue; Mass spectrometry; Proteomics; Quality control

DOI:  https://doi.org/10.1016/j.jprot.2025.105511