bims-metlip 2025-06-22 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–06–22
twenty-six papers selected by
Sofia Costa, Matterworks

Untargeted analysis of hydrophilic metabolites using enhanced LC-MS separation with a pentafluoro phenylpropyl-functionalized column and prediction-based MS/MS spectrum annotation.
Quantification of Endogenous Steroids and Hormonal Contraceptives in Human Plasma via Surrogate Calibration and UHPLC-MS/MS.
High-resolution accurate mass- mass spectrometry based- untargeted metabolomics: Reproducibility and detection power across data-dependent acquisition, data-independent acquisition, and AcquireX.
LC-MS Orbitrap-based metabolomics using a novel hybrid zwitterionic hydrophilic interaction liquid chromatography and rigorous metabolite identification reveals doxorubicin-induced metabolic perturbations in breast cancer cells.
Simultaneous Determination of Atezolizumab and Bevacizumab by LC-MS/MS in Rat Plasma and Its Application to a Pharmacokinetic Study.
CSU-MS2: A Contrastive Learning Framework for Cross-Modal Compound Identification from MS/MS Spectra to Molecular Structures.
Untargeted and semi-targeted metabolomics approach for profiling small intestinal and fecal metabolome using high-resolution mass spectrometry.
Comprehensive lipid structure annotation via photochemical epoxidation and mass spectrometry.
An isotope dilution-liquid chromatography-tandem mass spectrometry based candidate reference measurement procedure for the simultaneous quantification of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum and plasma.
Development and Validation of an LC-MS/MS Assay for Quantitative Analysis of Nusinersen in Human CSF and Plasma.
Rapid determination of sodium pentachlorophenate in bamboo and wooden cutting boards via ultrasonic-assisted liquid-liquid extraction coupled with ultra-performance liquid chromatography-high resolution mass spectrometry.
Quantitative determination of Selinexor concentrations in plasma samples from children with non-rhabdomyosarcoma soft-tissue sarcomas: Troubleshooting plasma instability issues.
A LC-MS/MS Assay for Quantification of Amodiaquine and Desethylamodiaquine in Dried Blood Spots on Filter Paper.
Determination of testosterone by liquid Chromatography-Tandem mass spectrometry in dried plasma obtained from capillary blood using the HealthID PSD microsampling device.
Structure Elucidation for MALDI Mass Spectrometry Imaging Using Infrared Ion Spectroscopy.
Orthogonal assessment of six extraction methods for GC-MS based short-chain fatty acid quantification in fecal samples.
Efficient Stereochemical Analysis of Hydroxy Fatty Acids Using PGME and PAME Derivatization.
Quantitative Mass Spectrometry Imaging by Targeted-DESI-MSI in MRM Mode Provides Higher Sensitivity and Specificity for Fast Quantification of Chloroquine Drug in Mice Kidney.
Accurate Quantification of Multifunctional C2-3 Organosulfates in Atmospheric Aerosols Using Liquid Chromatography-Electrospray Ionization Mass Spectrometry: Overcoming Matrix Effects and Underestimation.
Broadband Collision-Induced Dissociation Mass Spectrometry Imaging.
A New Comprehensive Platform for Profile-Mode-Based Untargeted Metabolomics for Efficient Data Mining to Improve Compound Extraction and Identification.
GC-MS/MS and HR-LCMS-QTOF analysis of various extracts of Saraswata Ghrita: A comprehensive dataset on phytochemical compounds.
Data Set Analysis to Reduce Uncertainty in Formula Assignments of Ultrahigh Resolution Mass Spectra.
Efficient Denoising of Shot-Noise in Mass Spectrometry Images by PCA-Assisted Self-Supervised Deep Learning.
Characterizing the Spin System Using 3D TOCSY J-Resolved Spectroscopy to Facilitate Deep Annotation of Individual Peaks within Interleaved Multiplets in High-Resolution 1D 1H Spectra of Cellular Metabolic Mixtures.
Faecal amino acids stability: investigating optimal sampling conditions for analysis.

Metabolomics. 2025 Jun 14. 21(4): 79

Untargeted analysis of hydrophilic metabolites using enhanced LC-MS separation with a pentafluoro phenylpropyl-functionalized column and prediction-based MS/MS spectrum annotation.

Masaru Sato, Kazutaka Ikeda.

   INTRODUCTION: Wide-targeted metabolome analysis has been applied in studies on the biology of mammals, plants, and microorganisms. However, there are still issues regarding both analytical and informatics technologies for establishing an untargeted and comprehensive analysis of hydrophilic primary and secondary metabolites.
OBJECTIVES: This study aimed to develop an improved chromatographic method for analyzing hydrophilic metabolites and an annotation method for these diverse metabolites.
METHODS: We investigated the performance of a pentafluoro phenylpropyl-functionalized column (PFP column) for the comprehensive analysis of hydrophilic metabolites by liquid chromatography-mass spectrometry (LC-MS). Peaks were annotated using MS/MS spectral similarity searches of the predicted and experimental MS/MS spectra in metabolite structure databases.
RESULTS: The improved retention and peak shapes of the standard compounds were obtained using LC-MS analysis with a PFP column. The mobile phases comprised water with 0.1% formic acid and methanol with 0.1% formic acid and 10 mM ammonium formate. From the annotation results of the 48 standard compounds, the chemical structures were correctly annotated in 54% of the compounds. However, over 70% of the compounds were annotated as biologically relevant based on the natural product classification. When these methods were applied to the analysis of tomato fruits, 658 and 458 peaks were detected and annotated in the positive and negative ion analyses, respectively.
CONCLUSION: Metabolome analysis combined with LC-MS analysis and annotation can contribute to the comprehensive analysis of hydrophilic metabolites.

Keywords:  Hydrophilic metabolite; LC-MS analysis; Metabolite annotation; Untargeted metabolome analysis

DOI:  https://doi.org/10.1007/s11306-025-02272-w
Anal Chem. 2025 Jun 20.

Quantification of Endogenous Steroids and Hormonal Contraceptives in Human Plasma via Surrogate Calibration and UHPLC-MS/MS.

Min Su, Bernhard Drotleff, Tamara Janker, Zoé Bürger, Ann-Christin S Kimmig, Birgit Derntl, Michael Lämmerhofer.

Quantifying endogenous and exogenous steroids at low concentrations in biological matrices remains a major analytical challenge. Immunoassay-based diagnostics are limited by cross-reactivity, particularly at low levels, prompting a shift toward (ultra)high-performance liquid chromatography-tandem mass spectrometry ((U)HPLC-MS/MS) for clinical applications. A key limitation for endogenous hormone quantification is the absence of a true blank matrix for external calibration. To address this, we developed a surrogate calibration method employing 1,2-dimethylimidazole-5-sulfonyl chloride (DMIS) derivatization for estrogens, enabling sensitive and selective quantification alongside nonderivatized steroids. Stable isotope-labeled surrogate calibrants and internal standards were used to achieve matrix-matched quantification within a clinically relevant range. Parallelism between analytes and surrogate calibrants was systematically verified in plasma across multiple calibration levels. The method was further optimized through the use of narrow-bore UHPLC columns and refined chromatographic conditions to enhance sensitivity and resolution for a broad analyte panel. Combined with efficient protein precipitation and 96-well plate-based solid-phase extraction, the developed assay achieves pg/mL-level quantification in human plasma with high precision and accuracy. This integrated approach uniquely combines surrogate calibration for endogenous steroids with external calibration for exogenous contraceptives, including sensitive DMIS-based derivatization for estrogens, enabling comprehensive hormonal profiling in a single run. Beyond its analytical scope, the method outlines a structured validation strategy, which is aligned with regulatory principles, and may therefore serve as a practical reference for future LC-MS/MS assays employing surrogate calibration.

DOI: https://doi.org/10.1021/acs.analchem.5c01912
Comput Struct Biotechnol J. 2025 ;27 2412-2423

High-resolution accurate mass- mass spectrometry based- untargeted metabolomics: Reproducibility and detection power across data-dependent acquisition, data-independent acquisition, and AcquireX.

Hanane El Boudlali, Laura Lehmicke, Uta Ceglarek.

  Untargeted metabolomics aims at the unbiased metabolic profiling and biomarker discovery but requires methods with high sensitivity and reproducibility. Here, we compare three acquisition modes-Data-Dependent Acquisition (DDA), Data-Independent Acquisition (DIA), and AcquireX -to evaluate performance and reproducibility in detecting low-abundance metabolites in a complex matrix. A system suitability test (SST) based on 14 eicosanoid standards was implemented to evaluate the suitability of our instrumental setup prior to conducting untargeted metabolomics analyses and monitor long-term system performance. Bovine liver total Lipid Extract (TLE) was spiked with decreasing levels (10-0.01 ng/mL) of the eicosanoid standard mix (StdMix) to compare the detection power of each mode. Reproducibility was evaluated over three independent measurements, spaced one week apart. Chromatographic separation was performed on a C18-Kinetex Core-Shell column and HRAM-MS/MS data were acquired using an Orbitrap Exploris 480. DIA detected and identified the highest number of metabolic features, (averaging 1036 metabolic features over three measurements), followed by DDA (18 % fewer) and AcquireX (37 % fewer). Moreover, DIA demonstrated superior reproducibility, with a coefficient of variance of 10 % across detected compounds over three measurements, compared to 17 % for DDA and 15 % for AcquireX. DIA further exhibited better compound identification consistency, with 61 % overlap between two days, compared to DDA (43 %) and AcquireX (50 %). DIA reproduced fragmentation spectra patterns with high consistency, contributing to higher reproducibility in compound identification. DIA showed the best detection power for all spiking eicosanoids at 10 and 1 ng/mL in TLE matrix. At low spiking levels, 0.1 and 0.01 ng/mL, a general cut-off was observed for the three acquisition modes. None of this assessed acquisition modes was able to detect and/or identify eicosanoids at physiologically relevant concentrations, explaining their frequent omission in routine untargeted analyses.

Keywords:  AcquireX; DDA; DIA; detection power; reproducibility

DOI:  https://doi.org/10.1016/j.csbj.2025.05.046
RSC Adv. 2025 Jun 16. 15(26): 20745-20759

LC-MS Orbitrap-based metabolomics using a novel hybrid zwitterionic hydrophilic interaction liquid chromatography and rigorous metabolite identification reveals doxorubicin-induced metabolic perturbations in breast cancer cells.

Salah Abdelrazig, Áine McCabe, Alia Yasin, Rajneil Chaudhary, Michael A Ochsenkühn, David Scicchitano, Shady A Amin.

The identification of metabolites in biological samples presents a challenge in untargeted metabolomics, mainly due to limited databases and inadequate chromatography. Current LC columns suffer from high pH instability (silica-based), low efficiencies and pressure limitations (polymer-based), or inadequate retention of polar/semi-polar metabolites (reverse-phase). In this study, a comprehensive LC-MS workflow was developed to address these limitations using a novel zwitterionic HILIC (Z-HILIC), high-resolution MS, deep-scan data-dependent acquisition (DDA), and a large chemical library comprising 990 standards. The method performance was evaluated and compared with a widely-used ZIC-pHILIC method. Z-HILIC detected 707 (71%) of the standards compared to 543 (55%) standards with the ZIC-pHILIC showing enhanced resolution, sensitivity, selectivity and retention time (RT) distribution. In triple-negative Hs578T breast cancer cell extracts spiked with the standards, Z-HILIC annotated 79.1% of the detected standards versus 66.6% with ZIC-pHILIC, demonstrating improved sensitivity, stability, and reduced matrix effects for metabolite profiling. Deep-scan DDA of the spiked cell extracts increased the number of the identified metabolites using RT, m/z and MS/MS by more than 80% compared to standard DDA. The workflow was used to investigate the metabolic signature of doxorubicin-treated Hs578T cells (n = 15). The analysis resulted in identifying 173 metabolites, of which 26 metabolites and 20 metabolic pathways were significantly altered in doxorubicin treated cells compared to controls. These pathways were associated with oxidative stress, mitochondrial dysfunction, and impaired biosynthesis, consistent with prior knowledge about the action of doxorubicin. This comprehensive workflow promises to enhance metabolite profiling across diverse metabolomics studies.

DOI: https://doi.org/10.1039/d5ra01044f
Biomed Chromatogr. 2025 Jul;39(7): e70139

Simultaneous Determination of Atezolizumab and Bevacizumab by LC-MS/MS in Rat Plasma and Its Application to a Pharmacokinetic Study.

Sudha Divya Madhuri Kallam, Mithun Rudrapal, Anoop Bodapati.

  A robust, rapid, and highly sensitive LC-MS/MS method was developed and validated for the simultaneous quantification of atezolizumab and bevacizumab in rat plasma, with potential applications in pharmacokinetic studies. The mobile phase comprised water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) in an isocratic ratio of 30:70, providing optimal separation on an Agilent Eclipse XDB column (250 mm × 4.6 mm, 5 μm). The selected peptides for atezolizumab (IYPTNGYTR) and bevacizumab (LTVLHQDWLNGK) yielded parent ions at m/z 579.78 and 686.38, and daughter ions at 746.36 and 859.44, respectively. The internal standard, abciximab, used the peptide VQWLQAGK with parent and daughter ions at m/z 471.26 and 590.32, respectively. Atezolizumab and bevacizumab were validated over linear ranges of 3.00-120 ng/mL and 1.25-50.00 ng/mL, showing excellent correlation coefficients (r2 = 0.999). Method accuracy for atezolizumab ranged from 96.76% to 99.55%, with precision (%CV) between 0.09% and 2.45% across quality controls, while bevacizumab demonstrated accuracy from 97.05% to 99.82% with precision (%CV) ranging from 0.09% to 8.82%. Matrix effects at low and high QC levels confirmed consistency, with accuracy and precision for atezolizumab and bevacizumab exceeding ICH guidelines. This optimized method offers a valuable tool for pharmacokinetic profiling of atezolizumab and bevacizumab in rat plasma, providing insights for therapeutic monitoring and drug development.

Keywords:  atezolizumab; bevacizumab; bioanalysis; pharmacokinetics; rat plasma; simultaneous determination

DOI:  https://doi.org/10.1002/bmc.70139
Anal Chem. 2025 Jun 20.

CSU-MS2: A Contrastive Learning Framework for Cross-Modal Compound Identification from MS/MS Spectra to Molecular Structures.

Ting Xie, Hailiang Zhang, Qiong Yang, Jinyu Sun, Yue Wang, Jia Long, Zhimin Zhang, Hongmei Lu.

Tandem mass spectrometry (MS/MS) is a cornerstone for compound identification in complex mixtures, but conventional spectral matching approaches face critical limitations due to limited library coverage and matching algorithms. To address this, we propose CSU-MS2 (contrastively spectral-structural Unification framework for MS/MS Spectra and Molecular Structures), a novel framework that bridges MS/MS spectra and molecular structures through cross-modal contrastive learning. CSU-MS2 uniquely integrates an External Space Attention Aggregation (ESA) module to dynamically align spectral and structural features, enabling direct retrieval of molecular candidates from a unified embedding space. The framework is pretrained on large-scale in-silico MS/MS data sets generated by CFM-ID and ICEBERG, followed by fine-tuning on high-quality experimental data. Results show that CSU-MS2 achieves a Recall@1 of 75.45% when matching 1047 spectra against a reference library containing 1,001,047 compounds, significantly surpassing existing methods such as CFM-ID (68.38%), SIRIUS (64.85%), MetFrag (48.59%), and CMSSP (30.47%). Furthermore, rigorous validation on three external data sets spanning human metabolomics (MTBLS265), plant metabolites (PMhub), and the CASMI 2022 challenge demonstrates robust generalizability, with domain-specific retrieval achieving a Recall@10 of 91.67% for blood metabolites. To facilitate compound identification across various domains, we have assembled a Spectrum-searchable Structural Feature Database (SSFDB) from 23 structural databases and deployed an open-source web server supporting customizable cross-modal retrieval. All code, models, and SSFDB are publicly accessible, offering a transformative solution for high-throughput compound identification in metabolomics and beyond.

DOI: https://doi.org/10.1021/acs.analchem.5c01594
Metabolomics. 2025 Jun 19. 21(4): 84

Untargeted and semi-targeted metabolomics approach for profiling small intestinal and fecal metabolome using high-resolution mass spectrometry.

Alexandre Tronel, Morgane Roger-Margueritat, Caroline Plazy, Valérie Cunin, Ipsita Mohanty, Pieter C Dorrestein, Thomas Soranzo, Audrey Le Gouellec.

   INTRODUCTION: The gut microbiome is a complex ecosystem stratified that varies along different sections of the gut. It comprises a wide array of metabolites originating from both food, host, and microbes. Microbially-derived metabolites, such as bile acids, short-chain fatty acids, and indole derivatives, are of significant interest due to their direct interactions with host physiology and regulating function. Most current studies on the gut microbiome focus on fecal samples, which do not fully represent the upper parts of the gut due to its stratification. To collect microbiome samples from the proximal gut microbiome, endoscopic methods or new non-invasive medical devices can be used.
OBJECTIVES: To enable comprehensive profiling of the gut metabolome and analyze key metabolites, we developed a combined approach combining untargeted and semi-targeted metabolomics using a Q-Exactive Plus Orbitrap mass spectrometer.
METHODS: Initially, we selected 49 metabolites of interest for the gut metabolome based on four distinct criteria. We validated these metabolites through repeatability and linearity tests and created a compound database using the software TraceFinder (ThermoFisher Scientific). For untargeted metabolomics, we established a workflow for the annotation and discovery of molecules.
RESULTS: Finally, 37 metabolites were validated for semi-targeted metabolomics, and we conducted a proof of concept on small intestinal and fecal samples form a clinical trial (NCT05477069).
CONCLUSION: Our combined approach, facilitated by molecular networking, demonstrated the potential to discover new metabolites.

Keywords:  Combined approach; LC-MS/MS; Metabolites; Microbiome; Small intestine

DOI:  https://doi.org/10.1007/s11306-025-02288-2
Anal Bioanal Chem. 2025 Jun 20.

Comprehensive lipid structure annotation via photochemical epoxidation and mass spectrometry.

Jing Yu, Belal Alshaar, Sven Heiles.

  Cells tightly regulate lipid structures to fulfill cellular functions and to respond to external stimuli. The biochemical details of the processes that determine lipid structure alterations are often not fully understood. In this manuscript, we present a new epoxidation strategy of unsaturated lipids, which allows the annotation of lipid head group, fatty acid composition, C = C bond position, C = C bond geometry, and sn-isomerism when derivatized lipids are analyzed with corresponding separation and mass spectrometry methods. Tandem mass spectra of epoxidized deprotonated or protonated lipids provide information on head groups, fatty acids, and C = C positions, while MSⁿ of alkali metal adducts reveals sn-isomer compositions. Separation of epoxidation products via reversed-phase liquid chromatography (RPLC) not only distinguishes lipid C = C position and sn-isomers, remaining non-reacted unsaturated lipid C = C bonds photo-isomerize to reveal C = C E/Z configurations. To demonstrate the capabilities of the methodology, C = C positions of LPEs, LPCs, TGs, DGs, PCs, PSs, and PEs are annotated for bovine heart and liver extracts in RPLC-MS2 experiments, and shotgun MSn is employed to characterize 56 PC sn-isomers in HeLa and H9c2 cell lines.

Keywords:  Epoxidation; Isomers; Lipid derivatization; Mass spectrometry; Tandem mass spectrometry

DOI:  https://doi.org/10.1007/s00216-025-05953-6
Clin Chem Lab Med. 2025 Jun 20.

An isotope dilution-liquid chromatography-tandem mass spectrometry based candidate reference measurement procedure for the simultaneous quantification of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum and plasma.

Kerstin Kandler, Neeraj Singh, Friederike Bauland, Elie Fux, Andrea Geistanger, Christian Geletneky, Judith Taibon.

   OBJECTIVES: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in human serum and plasma is presented.
METHODS: Quantitative Nuclear Magnetic Resonance (qNMR) spectroscopic methodology has been utilized to assign absolute content (g/g) and International System of Units (SI)-traceability to the reference materials used as primary calibrators. This RMP was developed for the simultaneous quantification of 25OHD2 and 25OHD3 in human samples, utilizing supported liquid extraction (SLE) clean-up and a two-dimensional heart-cut ID-LC-MS/MS method to minimize matrix effects and prevent the co-elution of 3-Epi-25OHD3 and 3-Epi-25OHD2. The method underwent validation in accordance with current guidelines. Selectivity was assessed using spiked samples. To evaluate potential matrix effects, a post-column infusion experiment and a comparison of standard line slopes were performed. A 5-day validation study was conducted to determine precision, accuracy and trueness of the method. Measurement uncertainty for reference value assignment was evaluated in line with the Guide to the Expression of Uncertainty in Measurement (GUM). Equivalence to Joint Committee on Traceability in Laboratory Medicine (JCTLM) listed RMPs was demonstrated through the participation in the CDC Vitamin D Standardization-Certification Program (VDSCP) as well as the RELA scheme.
RESULTS: The RMP enabled the quantification of 25OHD2 and 25OHD3 within the range of 1.50 ng/mL-180 ng/mL (3.64-436 nmol/L for 25OHD2 and 3.74-449 nmol/L for 25OHD3), without interference from their respective epimer and no evidence of matrix effects. Intermediate precision was determined to be ≤4.0 % for 25OHD2 and ≤3.6 % for 25OHD3, while repeatability was ≤3.3 % for 25OHD2 and ≤2.9 % for 25OHD3 across all concentration levels. The relative mean bias for the secondary reference materials varied from -1.0 to 1.1 %, regardless of the analyte. For the spiked samples, the relative mean bias ranged from -4.2 to 1.0 % for 25OHD2 and from -3.9 to 0.9 % for 25OHD3, irrespective of all levels and matrices. Expanded measurement uncertainties (k=2) for target value assignment (n=6) were ≤3.9 % for 25OHD2 and ≤3.2 % for 25OHD3. Participation in the VDSCP and the RELA scheme showed a good agreement with results from the JCTLM listed RMPs and laboratories.
CONCLUSIONS: The RMP enables the accurate, precise and consistent determination of 25OHD3 and 25OHD2. The robust performance of this method supports standardization of routine assays and guarantees traceability in the measurement of individual patient samples.

Keywords:  25-hydroxyvitamin D2; 25-hydroxyvitamin D3; isotope dilution-liquid chromatography-tandem mass spectrometry; qNMR characterization; reference measurement procedure; traceability

DOI:  https://doi.org/10.1515/cclm-2024-1138
Biomed Chromatogr. 2025 Jul;39(7): e70138

Development and Validation of an LC-MS/MS Assay for Quantitative Analysis of Nusinersen in Human CSF and Plasma.

Tingting Xu, Wei Zuo, Zhuo Sun, Simin Zhang, Zhengqing Qiu, Bo Zhang, Yi Dai.

  Nusinersen is the first antisense oligonucleotide (ASO) drug approved for the treatment of spinal muscular atrophy (SMA) in China; however, its pharmacokinetics (PK) in Chinese SMA patients remains unknown. The objective of this study was to develop and validate a liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methodology for quantifying nusinersen in human plasma and cerebrospinal fluid (CSF) samples. The samples were prepared by protein precipitation, and then the gradient was eluted on a column of Acquity UPLC Xbridge C18 by using acetonitrile with 0.5% triethylamine (TEA) and 0.5% hexafluoroisopropanol (HFIP) and water with 0.5% TEA and 0.5% HFIP as the mobile phase. Detection was performed on a QTRAP6500+ tandem mass spectrometer in the negative ion multiple reaction monitoring (MRM) mode using electrospray ionization (ESI). The optimized method was successfully qualified for the nusinersen in human plasma and CSF samples over the range of 5.00 to 1000 ng/mL and 2.00 to 400 ng/mL, respectively. Importantly, our work is the first report of quantification of nusinersen in human plasma and CSF by LC-MS/MS methodology. The developed methodology is reliable and will be applied to PK study of nusinersen in Chinese SMA patients.

Keywords:  IP‐RP‐LC/MS/MS; antisense oligonucleotide; nusinersen; spinal muscular atrophy

DOI:  https://doi.org/10.1002/bmc.70138
PLoS One. 2025 ;20(6): e0326129

Rapid determination of sodium pentachlorophenate in bamboo and wooden cutting boards via ultrasonic-assisted liquid-liquid extraction coupled with ultra-performance liquid chromatography-high resolution mass spectrometry.

Ye Wang, Mingyou Hu, Fang Wang, Juan Yu, Guojian Shao.

Sodium pentachlorophenate (PCP-Na) is a toxic preservative used in wood products, posing potential health risks through food contact materials. A rapid analytical method combining ultrasonic-assisted liquid-liquid extraction with ultra-performance liquid chromatography-high resolution mass spectrometry (UA-LLE-UPLC-HRMS) was developed for the determination of PCP-Na residues in bamboo and wooden cutting boards. Sample pretreatment involved ultrasonic extraction using methanol/water (50:50 v/v, 2.0% ammonia), followed by liquid-liquid purification with n-hexane/ethyl acetate (60:40 v/v). After solvent evaporation under nitrogen, the residue was reconstituted in the initial mobile phase. Chromatographic separation was achieved on an Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm) using a gradient elution of methanol and 0.01% ammoniated aqueous solution. Detection was performed in negative electrospray ionization (ESI-) mode with targeted single ion monitoring (Targeted-SIM) scanning, utilizing pentachlorophenol-13C6 (PCP-13C6) as an isotopically labeled internal standard. The method exhibited excellent linearity across a concentration range of 1.0-500.0 μg/L (R2 ≥ 0.999), with a limit of detection (LOD) of 0.5 μg/kg and a limit of quantification (LOQ) of 1.5 μg/kg. Validation studies at three spiking levels (20.0, 200.0, and 400.0 μg/kg) demonstrated satisfactory recoveries of 97.2%-99.7% and precision with relative standard deviations (RSDs) of 0.8%-1.7% (n = 6). The total chromatographic runtime was optimized to 6 minutes. Application of this method to Seventy-five commercial cutting boards revealed PCP-Na residues in five samples, with concentrations ranging from 1.3 to 416 mg/kg. This approach features streamlined sample preparation, high sensitivity, robust accuracy, and rapid analysis, making it particularly suitable for routine monitoring of PCP-Na residues in bamboo and wooden food contact materials.

DOI: https://doi.org/10.1371/journal.pone.0326129
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jun 17. pii: S1570-0232(25)00254-5. [Epub ahead of print]1263 124700

Quantitative determination of Selinexor concentrations in plasma samples from children with non-rhabdomyosarcoma soft-tissue sarcomas: Troubleshooting plasma instability issues.

Sreenath Nair, Thandranese Owens, Abigail Stolarski, Jessica Gartrell, Christopher Tinkle, Clinton F Stewart.

  Selinexor (KPT-330), a first-in-class, CNS-penetrant oral inhibitor of Exportin-1, disrupts the nuclear export of tumor suppressor proteins, promoting their accumulation and inducing cancer cell death. In this study, a reliable and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated to quantify selinexor concentrations in human plasma. A standard solid-phase extraction method using an Oasis HLB μElution plate was utilized to isolate selinexor and its internal standard, selinexor-d3, from human plasma. The chromatographic separation was executed on a reversed-phase analytical column with a binary gradient of water and acetonitrile, both containing 0.1 % formic acid, at a flow rate of 0.5 mL/min. Mass spectrometry detection was performed in positive ion mode by tracking the mass transitions of 444.0 > 334.0 for selinexor and 447.0 > 333.9 for selinexor-d3. The developed LC-MS/MS assay for selinexor was rigorously validated over a wide range of clinically relevant concentrations (1-1000 ng/mL, r2 ≥ 0.99) in accordance with FDA bioanalytical method validation guidelines. The method exhibited inter-day accuracy, expressed as relative error (R.E.), ranging from 2.28 % to 4.38 %, with precision values not exceeding 5.92 %. Intra-day accuracy showed R.E. values between 0.24 % and 7.30 %, accompanied by precision values ≤4.81 %. Additionally, the method demonstrated high extraction recovery, ranging from 82.80 % to 87.87 %, and a negligible matrix effect. The pH adjustments applied to the plasma prior to storage and processing maintained the stability of selinexor under several experimental conditions, including multiple freeze-thaw cycles and long-term storage at -80 °C. As proof of principle, the LC-MS/MS assay was successfully applied to a phase I clinical pharmacokinetic study of selinexor in pediatric patients with non-rhabdomyosarcoma soft tissue sarcomas, yielding reliable and reproducible measurements of selinexor concentrations in plasma.

Keywords:  Bioanalysis; LC-MS/MS; Method validation; Pharmacokinetics; Selinexor (KPT-330)

DOI:  https://doi.org/10.1016/j.jchromb.2025.124700
Int J Anal Chem. 2025 ;2025 5130424

A LC-MS/MS Assay for Quantification of Amodiaquine and Desethylamodiaquine in Dried Blood Spots on Filter Paper.

Natpapat Kaewkhao, Joel Tarning, Daniel Blessborn.

  Artesunate-amodiaquine (ARS-AQ) is a first-line antimalarial treatment recommended by the World Health Organization. AQ is the long acting partner drug in this combination, and therapeutic success is correlated with the terminal exposure to AQ. Dried blood spot (DBS) sampling for AQ is a convenient and minimally invasive technique, especially suitable for clinical studies in resource limited settings and pediatric studies. Our primary aim was to develop and validate a bioanalytical method for quantification of AQ and its active metabolite in capillary blood applied onto filter paper as a DBS sample. The separation was achieved using a reverse phase column (Zorbax SB-CN 50 × 4.6 mm, I.D. 3.5 μm) and a mobile phase consisting of acetonitrile:ammonium formate 20 mM with 0.5% formic acid (15:85, v/v). A 50 μL DBS was punctured with five 3.2 mm punches from the filter paper, and the punches collected correspond to approximately 15 μL of dried blood. The blood was then extracted using a mixture of 0.5% formic acid in water:acetonitrile (50:50, v/v), along with stable isotope-labeled internal standards (AQ-D10 and desethylamodiaquine [DAQ]-D5). Mass spectrometry was used for quantification over the range of 2.03-459 ng/mL for AQ and 3.13-1570 ng/mL for DAQ. The validation of the method was carried out in compliance with regulatory requirements. The intra- and interbatch precisions were below 15% and passed all validation acceptance criteria. No carryover and no matrix effects were detected. Normalized matrix factors (analyte/internal standard) ranged from 0.96 to 1.03 for all analytes, hence no matrix effects. AQ and DAQ were stable in all conditions evaluated. Long-term stability in DBS samples was demonstrated for up to 10 years when stored at -80°C and for 15 months when stored at room temperature. The developed method was demonstrated to be reliable and accurate. This assay may be particularly useful in the context of resource limited settings and in pediatric field studies.

Keywords:  LC-MS/MS; amodiaquine; dried blood spots; malaria; validation

DOI:  https://doi.org/10.1155/ianc/5130424
Clin Chim Acta. 2025 Jun 11. pii: S0009-8981(25)00300-6. [Epub ahead of print] 120421

Determination of testosterone by liquid Chromatography-Tandem mass spectrometry in dried plasma obtained from capillary blood using the HealthID PSD microsampling device.

Amanda Pacheco Bondan, Ana Paula Grando, Eduarda Milena Reichert, Giovana Piva Peteffi, Giovanna da Silva Grassi, Maitê de Moraes Machado, Roberta Zilles Hahn, Marina Venzon Antunes, Eduardo Guimarães Camargo, Mariele Feiffer Charão, Rafael Linden.

  Monitoring testosterone (T) levels is essential in various clinical contexts, but traditional venous sampling is invasive and limits access. This study describes the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify T in dried plasma spot (DPS) samples obtained from capillary blood using the HealthID PSD microsampling device. A chloride-based correction of plasma volume in the DPS and a multiplication factor were applied to estimate venous plasma concentrations. The method showed linearity from 1.63 to 104.02 nmol/L, with accuracy ranging from 96.8 % to 105.2 % and precision between 1.90 % and 7.24 %. Matrix effects were adequately corrected by the internal standard, and extraction yield exceeded 89 %. T in DPS samples was stable for up to 10 days at room temperature and 40 °C. Clinical validation involved 104 volunteers, including cisgender men and transgender individuals on hormone therapy. A strong correlation was observed between DPS-derived and venous plasma testosterone levels (r = 0.950), and the percent total error (TE%) of 16.41 % met the desirable performance criterion derived from biological variation data. The method proved robust against hematocrit variation and sample volume differences. This is the first report on T quantification using a capillary plasma separation device, highlighting the potential of the HealthID PSD for simplified, decentralized T monitoring. The approach offers practical advantages in collection, transport, and storage, making it a promising alternative to conventional phlebotomy for clinical applications.

Keywords:  Capillary blood; Dried plasma spot; HealthID PSD; Microsampling; Testosterone

DOI:  https://doi.org/10.1016/j.cca.2025.120421
Anal Chem. 2025 Jun 16.

Structure Elucidation for MALDI Mass Spectrometry Imaging Using Infrared Ion Spectroscopy.

Jelle L Schuurman, Lara van Tetering, Kas J Houthuijs, Pieter Kooijman, Valerie Gailus-Durner, Stefanie Leuchtenberger, Helmut Fuchs, Martin Hrabe de Angelis, Udo F H Engelke, Dirk J Lefeber, Clara D M van Karnebeek, Ron A Wevers, Andrej Grgic, Benjamin Balluf, Michiel Vandenbosch, Rob Vreeken, Ron M A Heeren, Giel Berden, Jos Oomens, Jonathan Martens.

Infrared ion spectroscopy (IRIS) is a tandem mass spectrometry (MS) technique that generates structurally diagnostic vibrational spectra for mass-selected ions trapped in a mass spectrometer. Until now, IRIS applications for biological samples have primarily focused on solution-based analyses, such as body fluids (e.g., plasma and urine) and tissue homogenates, using electrospray ionization (ESI) coupled with liquid chromatography-mass spectrometry (LC-MS). In this study, we have combined matrix-assisted laser desorption/ionization (MALDI) with IRIS for the direct analysis of small molecules from biological tissues on a Fourier-transform ion cyclotron resonance mass spectrometer. We applied this technique alongside MALDI mass spectrometry imaging to analyse brain tissue from two knockout mouse models of l-lysine catabolism disorders: pyridoxine-dependent epilepsy (ALDH7A1) and glutaric aciduria type 1 (GCDH). The MALDI-IRIS platform, now available for users at HFML-FELIX, represents a significant advance in the direct structural characterization of metabolites in complex biological tissues and opens new possibilities for structure elucidation in the field of MALDI mass spectrometry imaging.

DOI: https://doi.org/10.1021/acs.analchem.5c00948
J Chromatogr A. 2025 Jun 09. pii: S0021-9673(25)00478-9. [Epub ahead of print]1757 466132

Orthogonal assessment of six extraction methods for GC-MS based short-chain fatty acid quantification in fecal samples.

Huanhuan Jiang, Qibin Li, Yang Yang, Jingcong Dai, Li Wang, Richard D Cannon, Xiaojing Dong, Ting-Li Han, Lina Hu.

  Short-chain fatty acids (SCFAs) are prominent regulators of host physiology and are closely related to the pathophysiology of many metabolic diseases. SCFAs are predominately generated by microbial fermentation of dietary fiber in the gut. Therefore, efficient, reproducible, and affordable analytical methods are needed to identify and quantify SCFAs in a complex fecal sample. In this study, five different SCFAs (acetic acid, butyric acid, propionic acid, valeric acid, and isobutyric acid) were compared using six different extraction methods (HCl-DE, NaOH-Hexane, H3PO4Butanol, H3PO4-EA, NaHCO3-MTBE, and SPME) based on gas chromatography-mass spectrometry (GC-MS) analysis. The specificity, linearity, recovery, precision, and sensitivity of different analytical approaches were systematically assessed using SCFA chemical standards and further validated with human fecal samples, ensuring cross-species applicability. Our results revealed that no single method performed optimally for all SCFAs, but rather, distinct methods exhibited compound-specific advantages. Moreover, this study provides a practical guideline for balancing recovery, reproducibility, and extraction efficiency for method selection based on research purposes. The H3PO4-Butanol method displayed superior recovery accuracy for valeric acid (100.16 ± 0.81 % and 101.82 ± 4.83 % recovery rate for low and median spiked concentrations, respectively). It also exhibited minimum intra-day repeatability (0.92 % to 5.67 % RSD values) and good linearity (R2=0.94-0.99) across chemical mixtures. However, it showed the best extracted efficiency for butyric acid and best sensitivity for isobutyruc acid in fecal samples. Moreover, the H3PO4-EA method showed the highest recovery accuracy for isobutyric acid (103.21 ± 11.07 % to 104.79 ± 3.59 % recovery rate for all three different spiked concentrations) and the smallest inter-day repeatability (3.37 % to 7.25 % RSD values). The SPME method exhibited superior recovery accuracy for acetic acid (81.16 ± 21.17 % to 94.08 ± 10.92 % recovery rate for all three different spiked concentrations) and extracted the highest levels of SCFAs for acetic acid, propionic acid, and isobutyric acid. Finally, the application of these methods in human clinical samples revealed significant alterations of SCFA levels in patients with missed abortion, supporting their potential for translational studies. Overall, our results indicated that H3PO4-Butanol and SPME are the recommended methods for studying SCFAs in fecal samples.

Keywords:  GC-MS; Liquid-liquid extraction; SCFAs; SPME; Stool

DOI:  https://doi.org/10.1016/j.chroma.2025.466132
Anal Chem. 2025 Jun 19.

Efficient Stereochemical Analysis of Hydroxy Fatty Acids Using PGME and PAME Derivatization.

Soohyun Um, Jaeyoun Lee, Seung Hyun Kim.

We present a time-efficient and cost-effective approach for the stereochemical analysis of α- and β-hydroxy fatty acids (α-HFAs and β-HFAs) using chiral derivatizing agents, phenylglycine methyl ester (PGME) and phenylalanine methyl ester (PAME). Conventional methods for stereochemical analysis, such as X-ray crystallography and Mosher's ester derivatization, require pure samples and are laborious. In contrast, PGME and PAME derivatization enables direct LC-MS analysis of crude extracts without compound isolation or extensive sample preparation. Additionally, this method eliminates the need for NMR measurement and crystallization after the reaction, thereby reducing the overall analysis time. The approach was successfully applied to newly discovered lipopeptides, demonstrating its efficiency, reproducibility, and potential for widespread use in the stereochemical characterization of bioactive natural products.

DOI: https://doi.org/10.1021/acs.analchem.5c01447
J Mass Spectrom. 2025 Jul;60(7): e5148

Quantitative Mass Spectrometry Imaging by Targeted-DESI-MSI in MRM Mode Provides Higher Sensitivity and Specificity for Fast Quantification of Chloroquine Drug in Mice Kidney.

Md Muedur Rahman, Mst Sayela Afroz, Md Al Mamun, Ariful Islam, Takumi Sakamoto, Shuhei Aramaki, Tomohito Sato, Thanai Paxton, Yutaka Takahashi, Tomoaki Kahyo, Mitsutoshi Setou.

  Quantitative mass spectrometry imaging (QMSI) is being applied for spatial quantification of drugs and metabolites using mass spectrometry imaging (MSI) tools. DESI-MSI is ideally suited to QMSI as a soft and ambient ionization technique. However, some challenging issues of QMSI include extraction efficiency, matrix effect, sensitivity, and specificity, which need to be addressed. Here, we applied targeted DESI-MSI in multiple reaction monitoring (MRM) mode for QMSI of chloroquine, an antimalarial drug, as a model in mice kidneys and carefully addressed those challenging issues. A triple quadrupole mass spectrometer coupled with a DESI source was used to quantify the chloroquine (transition m/z 320.2 → 247.1) drug. A deuterated internal standard chloroquine-d5 (transition m/z 325.2 → 147.1), was used to normalize the data from pixel to pixel. A mimetic in-tissue model was employed to constract a calibration curve demonstrating good linearity (y = 0.0041x, R2 = 0.9953) and precision (RSD < 15%). The calibration curve was validated by back-calculation, with results falling within acceptable ranges (accuracy error< ±15%). Finally, the dosed (30 mg/kg) chloroquine was quantified in three spatial regions (cortex, medulla, and pelvis) in the mice kidneys. The highest concentrations of chloroquine in the pelvis (399.85 and 436.28 ng/mg of kidney tissue at 30 and 60 min intervals) region is consistent with the previous report that a portion of the drug is eliminated from the kidney as unchanged forms. This study provides valuable insights into using DESI-MSI in MRM mode for the QMSI of drugs in biological tissues and will have implications for future research on pharmacology and toxicology.

Keywords:  DESI‐MSI; chloroquine; kidney; multiple reaction monitoring; quantitative mass spectrometry imaging

DOI:  https://doi.org/10.1002/jms.5148
Environ Sci Technol. 2025 Jun 17.

Accurate Quantification of Multifunctional C2-3 Organosulfates in Atmospheric Aerosols Using Liquid Chromatography-Electrospray Ionization Mass Spectrometry: Overcoming Matrix Effects and Underestimation.

Shumin Liang, Yuchen Wang, Hanzhe Chen, Wan Chan, Jian Zhen Yu.

  Reversed-phase liquid chromatography (RPLC) coupled with electrospray ionization-mass spectrometry (ESI-MS) is widely used to analyze polar organic compounds in atmospheric particulate matter (PM). However, its efficacy for small, polar multifunctional C2-C3 organosulfates (C2-3OSs)─conceivably key products of isoprene oxidation─is questionable. Notable matrix effects are anticipated to arise from poor retention and coelution with abundant salts in PM samples. Here, we systematically evaluated RPLC versus hydrophilic interaction liquid chromatography (HILIC) coupled with ESI-Orbitrap MS in quantifying PM-bound C2-3OSs. We synthesized three C2-3OSs, including glycolic acid sulfate, hydroxyacetone sulfate, and lactic acid sulfate. The availability of authentic standards enabled the first quantitative assessment of measurement bias for C2-3OSs using the RPLC-ESI-Orbitrap MS method, revealing an underestimation of these compounds by 1-2 orders of magnitude. The measurement bias primarily stemmed from the matrix effects arising from the coexisting bisulfate in ambient PM. In contrast, HILIC notably outperformed RPLC in retentive capacities and peak resolving abilities, effectively avoiding matrix suppression effects. Additionally, the HILIC-ESI-MS method uncovered six previously unreported C2-3OSs, expanding our knowledge of atmospheric OSs. This work enhances our capability of accurate quantification of aerosol components, thus helping to reduce constraints on studies of aerosols and their impacts.

Keywords:  C2−3 organosulfates; hydrophilic interaction liquid chromatography; matrix effects; quantitative analysis; reversed-phase liquid chromatography; secondary organic aerosol

DOI:  https://doi.org/10.1021/acs.est.5c01846
J Am Soc Mass Spectrom. 2025 Jun 19.

Broadband Collision-Induced Dissociation Mass Spectrometry Imaging.

Sumi Krupa, Wiktoria Szuberla, Joanna Nizioł, Anna Ossolińska, Krzysztof Ossoliński, Tomasz Ruman.

  This study demonstrates the first application of broadband collision-induced dissociation (bbCID) to mass spectrometry imaging (MSI) for the identification of untargeted metabolites in human tissues. The methodology integrates bbCID with laser ablation-remote atmospheric pressure photoionization/chemical ionization (LARAPPI/CI), enabling the simultaneous acquisition of precursor and fragment ion distributions during MSI for many compounds simultaneously. In this approach, an infrared (IR) laser is used to ablate biological material, which is then ionized in the gas phase by a combination of photoionization and chemical ionization at atmospheric pressure. The method was validated using reference compounds, including thymidine and commonly used synthetic dyes, to assess ionization efficiency, fragmentation behavior, and spatial colocalization of precursor and fragment ions. Subsequently, bbCID-MSI was applied to clinical tissue samples of human bladder and kidney cancer. For the bladder cancer tissue, higher intensities of heptadecanoic acid, docosahexaenoic acid (FA(22:6)), docosapentaenoic acid (FA(22:5)), and FA(16:0) were observed in tumor regions, whereas proline was more abundant in adjacent nontumorous area. In renal cell carcinoma, cancerous regions exhibited elevated levels of polyunsaturated fatty acids such as arachidonic acid (FA(20:4)) and adrenic acid (FA(22:4)), while creatine and serine were enriched in healthy tissue zones. These findings highlight the utility of bbCID-MSI for spatially resolved metabolite analysis and its potential to reveal biologically relevant metabolic alterations associated with cancer.

Keywords:  mass spectrometry imaging; metabolomics; molecular imaging; tandem mass spectrometry; tissue imaging

DOI:  https://doi.org/10.1021/jasms.5c00045
Anal Chem. 2025 Jun 16.

A New Comprehensive Platform for Profile-Mode-Based Untargeted Metabolomics for Efficient Data Mining to Improve Compound Extraction and Identification.

Xing-Cai Wang, Meng Zhai, Shu-Fang Li, Hang Lv, Hui Ma, Chang Yang, Qing-Xia Zheng, Ping-Ping Liu, Peng Lu, Yong-Jie Yu, Hai-Yan Fu, Yuanbin She.

The profile mode of ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) is commonly utilized in metabolomics for its ability to comprehensively retain compound information in mass spectra. However, current data-analysis methods have not been optimized for the entire profile-mode-based untargeted metabolomics. To address this issue, we developed a set of novel algorithms, including centroiding transformation, extracted ion chromatogram construction, and feature extraction. We integrated them into a new automatic data analysis platform, AntDAS-Profiler. The performance of these newly developed algorithms was demonstrated by distinguishing chrysanthemums from various production origins. Additionally, AntDAS-Profiler was comprehensively compared with several state-of-the-art tools such as MS-DIAL, XCMS, and MZmine. Results suggested that AntDAS-Profiler can provide researchers with a comprehensive solution for UHPLC-HRMS profile-mode-based metabolomics. AntDAS-Profiler can be accessed at http://www.pmdb.org.cn/antdasprofiler.

DOI: https://doi.org/10.1021/acs.analchem.4c05768
Data Brief. 2025 Aug;61 111675

GC-MS/MS and HR-LCMS-QTOF analysis of various extracts of Saraswata Ghrita: A comprehensive dataset on phytochemical compounds.

Robin Badal, Shivani Ranjan, Sudhanshu Kumar Jha, Lalan Kumar, Ashok Kumar Patel, Pramod Yadav, Pradeep Kumar Prajapati.

  This dataset provides a comprehensive phytochemical profile of Saraswata Ghrita, a classical Ayurvedic formulation traditionally used for cognitive enhancement. To capture its diverse bioactive constituents, three different extracts-methanol, hexane, and hexane-ethanol-were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS/MS) and High-Resolution Liquid Chromatography-Mass Spectrometry-Quadrupole Time-of-Flight (HR-LCMS/MS-QTOF). The dataset includes volatile and semi-volatile compounds identified through GC-MS/MS, while HR-LCMS/MS-QTOF facilitates the characterization of non-volatile and polar metabolites. Advanced chromatographic and spectrometric techniques were employed, integrating mass spectrometric detection, retention time analysis, and cheminformatics-based compound classification. Spectral data were processed using multiple databases, ensuring accurate compound annotation. Additionally, key parameters such as molecular weight, chemical structure, base peak intensity, and ion fragmentation patterns were recorded to aid in structural elucidation. This dataset is structured for comparative metabolomics, quality control, and pharmacological exploration, offering a valuable reference for researchers investigating the phytochemical complexity of Ayurvedic formulations. The compiled raw data, including chromatograms, peak intensities, spectral fingerprints, and molecular fragmentations, are publicly available for further computational modeling and validation. The dataset can also support future drug discovery efforts, network pharmacology studies, and Ayurvedic formulation standardization, ensuring reproducibility and facilitating integrative research on traditional medicine.

Keywords:  Ayurveda formulation; Bioactive compounds; Gas chromatography; Liquid chromatography; Mass spectrometry; Metabolite profiling; Phytochemical analysis

DOI:  https://doi.org/10.1016/j.dib.2025.111675
Anal Chem. 2025 Jun 16.

Data Set Analysis to Reduce Uncertainty in Formula Assignments of Ultrahigh Resolution Mass Spectra.

Christian Dewey, Yuri Corilo, William Kew, Rene M Boiteau.

Environmental samples contain a vast array of organic compounds with diverse elemental compositions and heteroatom content. Molecular formula assignments of ultrahigh resolution mass spectra (HRMS) hold promise for elucidating the molecular composition of these compounds. However, the need to account for an assortment of heteroatoms increases the uncertainty associated with individual assignments - and ultimately the ecological, biological, and biogeochemical insights gleaned from the assignments. To address this challenge, we introduce a formula assignment strategy that leverages HRMS data sets to improve assignment confidence, filter false assignments, and mitigate bias in assignment routines. The strategy, implemented using CoreMS, first identifies the highest confidence assignment for a recurring ion in a data set by assessing the mass accuracy and isotopologue similarity of all assignments to the ion across the data set. The second component of the strategy examines the consistency of mass errors for an assigned ion throughout a data set and flags formulas with statistically unlikely deviations in mass error. We illustrate the application and utility of the strategy by comparing its results against documented misassignment patterns within a set of oceanographic samples that were measured with 21 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Because the efficacy of our strategy improves with data set size, it is particularly useful for enhancing assignment confidence in large HRMS data sets common in studies of environmental systems.

DOI: https://doi.org/10.1021/acs.analchem.4c06826
Anal Chem. 2025 Jun 17.

Efficient Denoising of Shot-Noise in Mass Spectrometry Images by PCA-Assisted Self-Supervised Deep Learning.

Claire Seydoux, Matthew Bryan, Jean-Paul Barnes, Pierre-Henri Jouneau.

Mass spectrometry imaging (MSI) often suffers from inherent noise due to signal distribution across numerous pixels and low ion counts, leading to shot noise. This can compromise the accurate interpretation, especially for trace molecules. Recent advances in self-supervised deep learning denoising have demonstrated significant potential for enhancing data quality. In this Letter, we propose an optimized approach for using the Noise2Void (N2 V) algorithm for MSI denoising by applying a principal component analysis (PCA) preprocessing step. By rotating the data along its principal components prior to denoising, our method, Principal Component-Assisted Noise2Void (PCA-n2v), outperforms direct N2 V implementations and other state-of-the-art denoising techniques. The limitations of PCA-n2v are also evaluated using a synthetic MSI data set, revealing that bleedthrough artifacts may arise in images with extremely low signal-to-noise ratios. To facilitate adoption, an easy-to-use PCA-n2v implementation is provided via a GitHub repository. Overall, PCA-n2v advances MSI data processing, enabling higher-throughput and higher-resolution experiments with improved fidelity.

DOI: https://doi.org/10.1021/acs.analchem.5c00825
Anal Chem. 2025 Jun 16.

Characterizing the Spin System Using 3D TOCSY J-Resolved Spectroscopy to Facilitate Deep Annotation of Individual Peaks within Interleaved Multiplets in High-Resolution 1D 1H Spectra of Cellular Metabolic Mixtures.

Caihong Bai, Xiu Gu, Yu Yang, Xiaomei Yu, Kaifeng Hu.

One-dimensional (1D) 1H nuclear magnetic resonance (NMR) is often employed in metabolomics studies. In this study, 3D TOCSY J-resolved spectroscopy has been proposed to facilitate the characterization of a spin system with multiple chemical shifts, multiplicities, and J coupling constants. The combined information on the whole spin system allows for unambiguous annotation of individual peaks, including those within interleaved or closely spaced multiplets in a high-resolution 1D 1H NMR spectrum using the complete set of chemical shifts, multiplicities, and J couplings from an NMR database as a distinct identifier for the metabolite. The method was demonstrated on a simple model mixture comprising five human metabolite standards and a complex cellular metabolic mixture. It facilitated deep annotation of 242 characteristic peaks in the high-resolution 1D 1H NMR spectrum and enabled unambiguous identification of 51 metabolites within the complex cellular metabolic mixture.

DOI: https://doi.org/10.1021/acs.analchem.4c06938
Metabolomics. 2025 Jun 14. 21(4): 81

Faecal amino acids stability: investigating optimal sampling conditions for analysis.

Roza C M Opperman, Eva Vermeer, Sofie Bosch, Tim G J de Meij, Nanne K H de Boer, Eduard A Struys.

   INTRODUCTION: Faecal amino acids are promising non-invasive diagnostic biomarkers, but stability remains unclear.
OBJECTIVES: This study examined whether storage conditions, sampling site within the sample, freeze-thaw cycles, and the OMNImet®·GUT device affect faecal amino acid concentrations.
METHODS: Faecal samples from three donors underwent various pre-analytical conditions. Amino acids were analysed through targeted liquid chromatography-tandem mass spectrometry.
RESULTS: Most amino acids remained stable across sampling sites and freeze-thaw cycles. Storage at -20 °C preserved integrity, whereas 4 °C and 20 °C storage led to variations. The OMNImet®·GUT device stabilised some amino acids but showed inconsistencies.
CONCLUSION: Pre-analytical conditions influence faecal amino acid concentrations. Standardisation is essential for biomarker reliability.

Keywords:  Faecal amino acids; LC-MS/MS; Pre-analytical conditions; Sample stability; Targeted metabolomics

DOI:  https://doi.org/10.1007/s11306-025-02279-3