bims-metlip 2026-01-11 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–01–11
thirty-one papers selected by
Sofia Costa, Matterworks

An LC-MS/MS Method for the Quantitation of Metabolites M9, M12, and M20 of Bexicaserin in Human Cerebrospinal Fluid.
A simple, sensitive and rapid bioanalytical method for quantifying cannabidiol (CBD), 7-OH-CBD and 7-COOH-CBD in human plasma.
Analytical method validation with development for the detection and quantification of kratom alkaloids using LC - MS/MS.
High-throughput screening of per- and polyfluoroalkyl substances in human plasma using biocompatible solid-phase microextraction coupled with mass spectrometry via microfluidic open interface.
An Overview of Liquid Chromatography-Mass Spectrometry (LC-MS) Methods for the Quantification of Antibody-Drug Conjugates.
prepIMS: a robust data preprocessing workflow for ion mobility mass spectrometry imaging.
Establishment of a QuEChERS-FaPEx Rapid Analytical Method for N-Nitrosamines in Meat Products.
Quantitative GC-MS/MS in the clinical analysis of eicosanoids: A critical review and discussion, a 40-years historical retrospect, and possible implications for LC-MS/MS.
RP-UHPLC/MS/MS provides enhanced lipidomic profiling of human serum in pancreatic cancer.
A Sensitive Method for Quantitative Profiling of Estrogens, Progesterone, Androgens, and Corticosteroids in Human Serum by LC-MS/MS.
Sample preparation of Drosophila melanogaster larvae for MALDI imaging.
Evaluation of Three Quantification Methods for Monitoring Twelve Antiseizure Medications in Human Plasma by LC-MS/MS.
Metabolite Fraction Libraries for Quantitative NMR Metabolomics.
Development and validation of a multi-class solid-phase extraction and liquid chromatography tandem mass spectrometry method for emerging contaminants in water.
Quantitative multiclass cyanotoxin analysis by paper spray mass spectrometry.
Development and validation of a qNMR method for the simultaneous quantification of creatine and taurine in sports supplements.
Development of a chiral mass spectrometry derivatization reagent for amino acid enantiomer analysis: Metabolic investigation of DL-amino acids in the serum of nephritis patients.
Emerging analytical techniques for lipid profiling in food products: insights into processing effects and quality control.
NITCD: A streamlined metabolomics strategy based on bromine isotopes and MS-TDF software.
Mapping C═C Positions in Branched Fatty Acid Esters of Hydroxy Fatty Acids by Oxygen Attachment Dissociation Mass Spectrometry Coupled with Chemical Labeling.
1H-NMR Analysis of Wine Metabolites: Method Development and Validation.
A Comparative Review of Hydrophilic Interaction Chromatography(HILIC) with Traditional Chromatography in the Analysis of Aminoglycoside Antibiotics.
Simultaneous Determination of Lyso-Gb1 and Lyso-Gb3 in Plasma Using Salt-Assisted Liquid-Liquid Extraction Combined with LC-MS/MS.
Phase Model-Driven Deep Learning for Robust Phase Correction in High-Throughput NMR-Based Metabolomics.
In-Source Fragmentation of Pyrrolidine-Containing KRAS Scaffolds Leads to Enhanced Structure Elucidation and Tandem Mass Spectral Quality.
Evaluation of the MAGLUMI non-competitive angiotensin I chemiluminescence immunoassay for measuring renin activity in human plasma.
A GC-MS Data Analysis Platform for Untargeted Metabolomics with Enhanced Coeluting Peak Resolution.
Equine fetal-sex determination from mid-gestation to term using serum conjugated steroid profiling by liquid chromatography-tandem mass spectrometry.
Targeted Determination of Residual Sex Hormones in Cosmetics Using Magnetic Solid-Phase Extraction with Isotope-Labeled Internal Standards by UHPLC-MS/MS.
Tacrolimus Assay Comparison: Analytical and Operational Considerations for Immunoassay vs Mass Spectrometry Testing in a High Volume Clinical Laboratory.
Lipidomic Comparison of Detergent-Soluble and Detergent-Resistant Lipid Domains Isolated From Rat Cerebellum and the Effect of Hypoxia-Ischemia Using UPLC-HDMSE.

Biomed Chromatogr. 2026 Feb;40(2): e70341

An LC-MS/MS Method for the Quantitation of Metabolites M9, M12, and M20 of Bexicaserin in Human Cerebrospinal Fluid.

Raja Reddy Kallem, Rosa Chan, Maisy Yeager, Katie Neal, Katharine Fletcher, Nuggehally R Srinivas.

  The investigational molecule bexicaserin is a highly selective 5-HT2C superagonist currently in Phase 3 clinical studies for the treatment of seizures associated with developmental and epileptic encephalopathies (DEEs). To facilitate its clinical development, a simple LC-MS/MS method was developed and validated for the quantitative estimation of its metabolites, M9, M12, and M20, in human cerebrospinal fluid (CSF). The sample preparation involves sample dilution extraction of metabolites and SIL-IS internal standards from 25 μL of human CSF. The chromatographic separation of three metabolites was achieved on an Acquity HSS T3, C18 column using an 8.0-min gradient program. The intraday and interday precision and accuracy for all three metabolites were well within the range of acceptable limits. Stability studies in CSF showed that metabolites were stable in the presence of bexicaserin on the bench for 24 h, in the auto-sampler over 92 h, and extracted stability at 6°C for 48 h. Metabolites were stable in the presence of bexicaserin after three freeze-thaw cycles and long-term storage at -20°C and -80°C for 41 days. This method has since been successfully applied in a clinical study for the quantitative estimation of metabolite concentrations in CSF from a clinical study.

Keywords:  CSF; DEEs; LC–MS/MS; bexicaserin; human; metabolites; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.70341
Bioanalysis. 2026 Jan 09. 1-12

A simple, sensitive and rapid bioanalytical method for quantifying cannabidiol (CBD), 7-OH-CBD and 7-COOH-CBD in human plasma.

Nattapon Jaisupa, Michael Ashton, Sofia Birgersson.

   BACKGROUND: Reliable methods for measuring plasma concentrations of cannabidiol (CBD) and its metabolites are essential for pharmacokinetic studies. Several methods have been published, some involve complex procedures. This study aimed to develop a simple yet robust method for quantitating CBD, 7-OH-CBD and 7-COOH-CBD in human plasma.
RESEARCH DESIGN AND METHODS: Sample preparation was evaluated using various solvent systems. Analytical parameters were examined and optimized. The method was validated for sensitivity, accuracy, precision, selectivity, specificity, carryover, dilution integrity, long-term stability, freeze-thaw stability and recovery.
RESULTS: A simple protein precipitation method using acetonitrile provided optimal sample preparation. All analytes were quantified in negative ionization mode, with complete separation achieved on a C18 column within 3.5 minutes using an isocratic mobile phase. The method met all validation criteria. Analytes remained stable under long-term storage at -30°C, through freeze-thaw cycles and at refrigerated temperatures, although 7-COOH-CBD showed a slight decrease after two months at refrigerated temperatures.
CONCLUSION: This study presents a simple, rapid and robust bioanalytical method for the simultaneous quantification of CBD and its major metabolites across clinically relevant plasma concentrations. The method's reliability and efficiency make it highly suitable for routine clinical use and pharmacokinetic applications.

Keywords:  Cannabidiol; HPLC-MS/MS; bioanalytical method; plasma concentration; validation

DOI:  https://doi.org/10.1080/17576180.2026.2612921
J Pharmacol Toxicol Methods. 2026 Jan 05. pii: S1056-8719(26)00002-X. [Epub ahead of print]137 108409

Analytical method validation with development for the detection and quantification of kratom alkaloids using LC - MS/MS.

Md Harunur Rashid, Conor O'Croinin, Tyson S Le, Zeinab Abdi, Raimar Loebenberg, Neal M Davies.

   PURPOSE: In this investigation, a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was designed both to develop and to detect kratom alkaloids that are naturally occurring organic compounds present in the foliage and flowers of the Mitragyna speciosa tree.
METHOD: We utilize a deuterated analog of mitragynine (mitragynine-D3) as an internal standard (IS) that is spiked into and extracted from kratom extracts using liquid-liquid extraction with ethanol. Water and acetonitrile with 0.1 % formic acid constituted the mobile phase operated at a flow-rate of 0.75 mL/min through a Luna 5 μm C18(2) 100 Å, LC Column 150 × 4.6 mm analytical column and kept at 40 °C. The gradient program started at 0 min with 20 % B, then at 2 min increased to 43.7 % at 12.50 min. We used a period of 12.50 to 14.50 min, increased to 90 % B and held for 19 min, used for washing. Then, an equilibration period of the mobile phase B was maintained at 20 % for 25 min. The LC-MS/MS detection was carried out by electrospray positive ionization in the multiple-reaction monitoring (MRM) mode.
RESULT: We established an LC-MS/MS method to simultaneously quantify multiple kratom alkaloids. The optimized MRM transitions are mitragynine, speciogynine, speciociliatine, and mitracillatine (m/z 399.20 → 174.00); corynantheidine (369.20 → 144.05); paynantheine (397.20 → 174.00); 7-OH mitragynine (415.20 → 190.00); ajmalicine (353.00 → 144.05); mitraphylline (369.00 → 159.95); and the IS (402.20 → 177.10). Retention times ranged from 6.15 to 11.98 min. The assay (total run time 25 min) had excellent linearity over the concentration range of 5-100 ng/mL (R2 > 0.99), with a Lower Limit of Quantification of 5 ng/mL and Limit of Detection 1.5 ng/mL with a two μL injection. Analytes were well-resolved with the method, which exhibited acceptable intra- and inter-day accuracy and precision, along with confirmed stability under various conditions. Application of kratom extracts yielded reproducible and reliable quantification of alkaloid content.
CONCLUSION: The findings provide a standard analytical technique for testing kratom-based drug release, dissolution, comparison of different kratom products, and development of formulation. This method is also applicable for regulatory compliance and for producing validated results in fields like kratom adulteration that are unregulated and non-standardized.

Keywords:  Alkaloids; Kratom; Liquid chromatography; Mass spectrometry; Method validation

DOI:  https://doi.org/10.1016/j.vascn.2026.108409
Anal Chim Acta. 2026 Jan 22. pii: S0003-2670(25)01334-0. [Epub ahead of print]1384 344940

High-throughput screening of per- and polyfluoroalkyl substances in human plasma using biocompatible solid-phase microextraction coupled with mass spectrometry via microfluidic open interface.

Yaping Li, Emir Nazdrajić, Wei Zhou, Janusz Pawliszyn.

  Per- and polyfluoroalkyl substances (PFAS) are of increasing concern due to their environmental persistence, bioaccumulative nature, and association with adverse health outcomes. The growing need for large-scale monitoring and long-term exposure assessment studies necessitates the development of high-throughput, sustainable analytical methodologies. In this work, a solid-phase microextraction-microfluidic open interface-mass spectrometry (SPME-MOI-MS) platform was developed for the rapid screening of 18 PFAS compounds in human plasma. By bypassing the liquid chromatography separation, the method achieves high-throughput performance with an average analysis time of 3.7 min per sample. A novel SPME coating, comprising hydrophilic-lipophilic balanced mixed-mode weak anion exchange sorbent (HLB-WAX) particles embedded in a polyacrylonitrile (PAN) binder, enabled efficient extraction and effective cleanup of complex biological matrices, facilitating direct MS analysis. The method demonstrated excellent linearity (1-100 ng/mL) and low limits of detection (0.11-0.86 ng/mL) across target PFAS compounds. For practical application, PFOA and PFNA were detected in human plasma samples during these initial investigations, demonstrating the potential of the SPME-MOI-MS approach for large-scale PFAS biomonitoring and exposure assessment.

Keywords:  Human plasma; Mass spectrometry; Microfluidic open interface; Solid phase microextraction; per- and polyfluoroalkyl substances

DOI:  https://doi.org/10.1016/j.aca.2025.344940
Biomed Chromatogr. 2026 Feb;40(2): e70334

An Overview of Liquid Chromatography-Mass Spectrometry (LC-MS) Methods for the Quantification of Antibody-Drug Conjugates.

Pauline L M Buitelaar, Hilde Rosing, Jos H Beijnen, Neeltje Steeghs, Alwin D R Huitema.

  Antibody-drug conjugates (ADCs) are innovative drugs composed of cytotoxic molecules (payload) linked to antibodies, that selectively target and kill cancer cells upon internalization. In vivo, ADCs exist as intact molecules, naked antibodies, or released, unconjugated (linker-)payload. Accurate quantification of these entities is crucial for understanding ADCs pharmacokinetics. Ligand-binding assays are commonly used to measure ADC concentrations and total antibody concentrations, whereas LC-MS/MS is used to analyze the payload. Due to limitations in ligand-binding assays, this review focuses on quantitative LC-MS methods for the different ADC entities. Quantitative LC-MS assays were described for all ADC entities, available from full manuscripts and regulatory reviews of 12 ADCs evaluated by the European Medicine Agency, by January 2025. The review summarized sample pre-treatment, chromatography, mass spectrometry, validation, and stability data for each LC-MS method. Overall, critical details were often missing, particularly concerning sample pre-treatment, validation criteria, and sample stability. In conclusion, LC-MS quantification of ADC entities is feasible but current methods lack sufficient detail. Our review highlights the need for further research to develop reliable LC-MS assays for ADCs. This review may serve as a starting point and outlines key factors to consider in future LC-MS method development.

Keywords:  LC–MS; antibody‐drug conjugates; bioanalysis; literature review

DOI:  https://doi.org/10.1002/bmc.70334
Anal Chim Acta. 2026 Jan 22. pii: S0003-2670(25)01345-5. [Epub ahead of print]1384 344951

prepIMS: a robust data preprocessing workflow for ion mobility mass spectrometry imaging.

Linlin Wang, Chengyi Xie, Lei Guo, Thomas Ka Yam Lam, Chris Kong Chu Wong, Mudassir Shah, Xiangnan Xu, Jianing Wang, Jingjing Xu, Jiyang Dong, Zongwei Cai.

   BACKGROUND: Ion mobility-mass spectrometry imaging (IM-MSI) enables the spatial localization of biomolecules through two specific molecular identifiers, mass-to-charge ratio (m/z) and collision cross-section (CCS) values. IM-MSI enhances the detection of low-abundance ions and facilitates the separation of isobaric or isomeric ion species, making it particularly valuable for analyzing complex biological tissues that require higher metabolome coverage, increased analysis throughput and precise isomer separation. Preprocessing IM-MSI data is a critical step for extracting molecular features at the pixel level to reconstruct molecular images corresponding to specific m/z and CCS values, serving as a foundation for reliable downstream analysis and interpretation. However, existing preprocessing methods often struggle to effectively detect peaks from the noise-affected signals, especially for ion mobility signals with low signal-to-noise ratios.
RESULTS: We present prepIMS, a versatile and robust preprocessing workflow that incorporates a density cluster-based peak detection strategy. Experiments on the simulation dataset demonstrate that the density cluster-based peak detection in prepIMS outperforms the conventional local maxima-based strategy under noise-affected conditions, with improvements of 0.75 % in accuracy, 33.67 % in sensitivity, 17.10 % in Matthews correlation coefficient and 17.99 % in F1-score. We further validated the effectiveness of prepIMS using two whole-body mouse pup tissue datasets acquired with MALDI-TIMS, where it successfully detects and differentiates isomeric and isobaric ions within the same mass peak, underscoring its efficacy in practical applications. Furthermore, segmentation analysis of the preprocessed 4D IM-MSI data reveals that prepIMS effectively distinguishes the differences in spatial distribution from ions with subtle variations in ion mobility.
SIGNIFICANCE: prepIMS provides a powerful solution for IM-MSI data preprocessing. By effectively detecting meaningful peaks from noise-affected ion mobility signals, prepIMS facilitates the separation of isomeric and isobaric ions, and supports downstream spatial segmentation. The proposed prepIMS is expected to enhance the reliability and depth of IM-MSI data analysis, advancing data interpretation and facilitating comprehensive molecular profiling in intricate biological systems.

Keywords:  Data preprocessing; Density clustering; Ion mobility-mass spectrometry imaging; Molecular imaging; Spatial segmentation

DOI:  https://doi.org/10.1016/j.aca.2025.344951
Molecules. 2025 Dec 22. pii: 32. [Epub ahead of print]31(1):

Establishment of a QuEChERS-FaPEx Rapid Analytical Method for N-Nitrosamines in Meat Products.

Chun-Han Su, Peng-Wang Tan, Tsai-Hua Kao.

  This study aimed to establish a fast and efficient method for the determination of N-nitrosamines (NAs) in meat products by integrating two sample preparation techniques-QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) and FaPEx (Fast Pesticide Extraction)-with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatographic separation was performed on a Poroshell 120 Phenyl Hexyl column using a gradient elution of acetonitrile and 0.01% formic acid at a flow rate of 0.3 mL/min and a column temperature of 25 °C. Under these conditions, nine NAs and one internal standard were completely separated within 11 min with selective reaction monitoring mode (SRM) for detection. Samples were first extracted with QuEChERS powder using acetonitrile containing 0.1% formic acid, followed by purification with a FaPEx-Chl cartridge. This combined approach demonstrated superior performance compared with traditional solvent extraction or QuEChERS extraction alone. The recoveries of the developed method ranged from 76% to 111% and 52% to 103% at spiking levels of 50 ng/g and 20 ng/g, respectively. The limits of detection (LOD) and quantification (LOQ) were 0.002-0.3 ng/g and 0.006-1.00 ng/g, respectively. The inter-day and intra-day precisions (RSD%) ranged from 2.7% to 17% and 2.9% to 17%, respectively. These results indicate that the proposed method is among the most time-efficient and effective analytical approaches currently available for the determination of NAs in meat products.

Keywords:  LC-MS/MS; N-nitrosamines; QuEChERS-FaPEx; meats

DOI:  https://doi.org/10.3390/molecules31010032
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Dec 31. pii: S1570-0232(25)00465-9. [Epub ahead of print]1270 124911

Quantitative GC-MS/MS in the clinical analysis of eicosanoids: A critical review and discussion, a 40-years historical retrospect, and possible implications for LC-MS/MS.

Dimitrios Tsikas.

  Mass spectrometry was a protagonist in the discovery of prostaglandins, thromboxane, leukotrienes and other arachidonic acid-derived molecules, collectively known as the eicosanoids. Mass spectrometry has played a significant role in exploration of their metabolic pathways in humans and animals, in health and disease, and in pharmacotherapy. Clinical researchers in the United States of America and in Europe, in close cooperation with chemist analysts, were the pioneers in the application of gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) to quantitate eicosanoids and index metabolites in plasma, serum and urine samples from clinical trials by using stable-isotope labeled analogs as internal standards. In the present article, the application of the stable-isotope dilution GC-MS/MS methodology in the quantitative clinical analysis is reviewed. The focus is on prostaglandins, thromboxane, leukotrienes, and their so-called index metabolites for renal and whole-body synthesis of certain eicosanoids such as PGE2 and its major urinary metabolite (PGE-MUM), respectively. Nowadays, LC-MS/MS, which evolved later than GC-MS/MS, is increasingly used in numerous areas of research, including the eicosanoids in clinical studies. The present work critically discusses the current practice of LC-MS/MS users in the quantitative analysis of eicosanoids in biological samples. While the LC-MS/MS technology offers rapidity and high-throughput analysis, especially due to the renunciation of time-consuming analytical derivatization steps that are required in GC-MS/MS, LC-MS/MS seems to lack sufficient analytical sensitivity, i.e., lower limit of quantitation, for many eicosanoids such as thromboxane B2 and leukotriene B4. Reported data on basal concentrations of certain eicosanoids in plasma and urine samples from healthy humans as determined by LC-MS/MS are several orders of magnitude higher than originally reported by pioneering eicosanoid researchers, who developed, validated and used sophisticated, tailored GC-MS- and GC-MS/MS-based analytical methods for individual eicosanoids. Modern eicosanoids researchers would greatly benefit from the milestones and signposts set previously eicosanoids researchers from the very start. A key milestone and signpost is the concentration of primary eicosanoids and their metabolites in plasma, serum and urine samples of healthy humans. Issues for consideration in the GC-MS/MS and LC-MS/MS analysis of eicosanoids are discussed.

Keywords:  Eicosanoids; Index metabolites; Isoprostanes; Leukotrienes; Mass spectrometry; Prostaglandins; Quantitation; Thromboxane

DOI:  https://doi.org/10.1016/j.jchromb.2025.124911
Anal Chim Acta. 2026 Jan 22. pii: S0003-2670(25)01346-7. [Epub ahead of print]1384 344952

RP-UHPLC/MS/MS provides enhanced lipidomic profiling of human serum in pancreatic cancer.

Zuzana Lásko, Ondřej Peterka, Robert Jirásko, Anna Taylor, Tomáš Hájek, Beatrice Mohelníková-Duchoňová, Martin Loveček, Bohuslav Melichar, Michal Holčapek.

   BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, mainly due to the late diagnosis and the lack of reliable biomarkers. Lipidomics provides a promising approach for identifying disease-related alterations, but existing methods are often limited to lipid class profiles with insufficient molecular detail. Reversed-phase ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (RP-UHPLC/MS/MS) offers the possibility to determine lipids at the fatty acyl/alkyl level. Here, we address the need for a validated quantitative workflow that enables accurate and reproducible lipidomic profiling of human serum in the context of PDAC.
RESULTS: We developed and validated an RP-UHPLC/MS/MS method using multiple reaction monitoring, enabling the identification of 455 lipid species from 22 subclasses, with 381 species from 21 subclasses quantified. The workflow included a response factor correction for sterol esters, which markedly improved their quantification accuracy. The application to serum samples from 54 PDAC patients and 55 healthy controls yielded highly reproducible data, with clear group separation observed in both unsupervised and supervised statistical analyses. Dysregulation was most prominent in sphingolipids and phospholipids. Very long-chain saturated sphingolipids (≥C22) were downregulated, while some shorter or unsaturated chains showed mild upregulation. Phospholipid alterations were dominated by species containing polyunsaturated fatty acyls, particularly 18:2 and 20:4, with plasmalogens showing the strongest changes. These structurally resolved findings were further supported by gas chromatography - mass spectrometry analysis of fatty acid methyl esters.
SIGNIFICANCE: This validated workflow provides comprehensive quantitative coverage across 21 lipid subclasses with the structural resolution critical for biological interpretation. The detailed mapping of sphingolipid and phospholipid dysregulation in PDAC demonstrates that only the fatty acyl level annotation reveals molecular signatures that may reflect specific enzymatic activities or pathways. The method delivers a robust platform for biomarker discovery and mechanistic studies in cancer lipidomics.

Keywords:  Human serum; Lipidomics; Mass spectrometry; Pancreatic cancer; Reversed-phase liquid chromatography

DOI:  https://doi.org/10.1016/j.aca.2025.344952
J Appl Lab Med. 2026 Jan 07. pii: jfaf198. [Epub ahead of print]

A Sensitive Method for Quantitative Profiling of Estrogens, Progesterone, Androgens, and Corticosteroids in Human Serum by LC-MS/MS.

Ersilia Bifulco, Kai Triebner, Francisco Gómez Real, Bénédicte Leynaert, Steinar Hustad.

BACKGROUND: Determination of steroid hormones by mass spectrometry (MS) offers several advantages over immunoassays. Modern MS instruments have high selectivity and sensitivity and permit measurements of several compounds at picomolar concentrations in one sample volume, which is advantageous when conducting biobank studies.
METHODS: We developed a LC-MS/MS method for the determination of 17β-estradiol, 17α-ethinylestradiol, estrone, estrone 3-sulfate, estriol, progesterone, testosterone, dehydroepiandrosterone sulfate, aldosterone, cortisone, and cortisol in 300 μL human serum. Samples were subjected to robotized protein precipitation with acetonitrile and liquid-liquid extraction with ethyl acetate/heptane, and the aqueous and organic phases were used to determine conjugated and unconjugated steroids, respectively. The steroids were separated from isobars and compounds with similar mass (M + 2) on a C18 reversed phase column. Ammonium hydroxide was infused post-column to enhance ionization, and ions were measured in multiple reaction monitoring negative and positive mode.
RESULTS: The lower limits of quantification for 17β-estradiol and estrone were 3.6 (0.98 pg/mL) and 2.1 pmol/L (0.57 pg/mL), respectively. For medium concentrations of steroid hormones, total CVs were in the range 2.3% to 10.5%, and accuracies were 97% to 109%. The method was validated in terms of linearity (r2 ≥ 0.9779) and assay recovery (83%-114%), and certified control sera were analyzed. Steroid profiles were obtained in 921 women with a mean age of 56 years (range 44-66 years) from the European Community Respiratory Health Survey.
CONCLUSIONS: We developed a selective and sensitive LC-MS/MS method for 11 steroids in human serum. It is suitable for biobank studies.

DOI: https://doi.org/10.1093/jalm/jfaf198
Anal Bioanal Chem. 2026 Jan 06.

Sample preparation of Drosophila melanogaster larvae for MALDI imaging.

Marius Herbst, Marie A König, Katharina Gutbrod, Johannes Volk, Peter Dörmann, Margret H Bülow, Marianne Engeser.

  Mass spectrometry-based lipidomics has emerged as a crucial field for unraveling the complexity of biological systems through comprehensive profiling of lipid species with unparalleled sensitivity and specificity. Among the available techniques, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI imaging, or MALDI-IMS) stands out as a powerful spatial lipidomics tool, enabling the visualization of distribution and localization of individual lipid classes and species directly within biological tissues. The spatial context of lipids provided by MALDI imaging is especially valuable for studying nutritional and developmental processes in heterogenic tissues. The reliability and quality of MALDI imaging data critically depend on sample preparation, which must preserve both tissue morphology and analyte integrity. Here we introduce a sample preparation protocol for Drosophila melanogaster larval tissue, addressing key limitations of standard protocols such as thin-sectioning. Owing to the small size of Drosophila tissues and the flat morphology of organs such as the fat body, our approach enables direct imaging of the inner organs of the larvae without sectioning. This not only streamlines the workflow and minimizes tissue disruption but also allows simultaneous analysis of all major internal organs in a single measurement. Our approach not only facilitates high-quality MALDI-IMS data acquisition from complex larval tissues but also enables the localization of a broad spectrum of lipid species complementary to data gained by established techniques like liquid chromatography mass spectrometry (LC-MS). These advances position MALDI imaging, when coupled with our optimized preparation workflow, as an indispensable technique for spatially resolved lipidomics studies in small model organisms such as Drosophila larvae.

Keywords:   Drosophila melanogaster ; MALDI imaging; Phospholipids; Triacylglycerides

DOI:  https://doi.org/10.1007/s00216-025-06289-x
Ther Drug Monit. 2026 Jan 07.

Evaluation of Three Quantification Methods for Monitoring Twelve Antiseizure Medications in Human Plasma by LC-MS/MS.

Donghan Wang, Shanshan Chen, Yuanyuan Zhao, Yuhang Yan, Yueyao Luan, Jiaqi Wang, Qixuan Sun, Chunhua Zhou, Jing Yu.

   BACKGROUND: The concentration of antiseizure medications in vivo has a significant influence on therapeutic and adverse effects. To use antiseizure medications safely and rationally, it is crucial to establish a method of measuring their blood concentration, provide individualized medication guidance to patients, and avoid adverse effects through therapeutic drug monitoring. Therefore, we developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the rapid detection of antiseizure medication concentrations in blood and applied it to the monitoring of clinical antiseizure medication therapy.
METHODS: A Waters HPLC-ESI TQ-D with a Waters BEH C18 column (50 × 2.1 mm, 1.7 µm) was used; the blood samples were injected after protein precipitation in 50% methanol in water, and the analysis was conducted within 3.5 minutes. The ion source for mass spectrometry was an electrospray ionization source with the multiple ion reaction monitoring mode. An isotope standard was selected as the internal standard.
RESULTS: The linearity of each epileptic drug was good in the studied concentration range (R2 > 0.9930), and the lower limit of quantification was 0.01-8.00 mcg/mL, with intraday and interday precision less than 15.00%. The recoveries of the method ranged from 91.86% to 113.97%, and the stability of the method was good and unaffected by matrix effects.
CONCLUSIONS: The established LC-MS/MS method for the determination of antiseizure medication concentration was characterized by a simple operation procedure, short analysis time, high sensitivity, and good accuracy, providing a simple and reliable assay for the monitoring of antiseizure medication therapy.

Keywords:  antiseizure medications; concentration determination; high-performance liquid chromatography–tandem mass spectrometry; therapeutic drug monitoring

DOI:  https://doi.org/10.1097/FTD.0000000000001434
bioRxiv. 2025 Dec 31. pii: 2025.12.30.696914. [Epub ahead of print]

Metabolite Fraction Libraries for Quantitative NMR Metabolomics.

Christopher Esselman, Kara Garrison, Leandro Ponce, Ricardo M Borges, Frank Delaglio, Arthur S Edison.

Nuclear Magnetic Resonance (NMR) has unique strengths in metabolomics studies, particularly in quantifying mixtures and elucidating the structures of unknown molecules. One-dimensional (1D) proton ( 1 H) NMR is the most common method; however, spectral overlap is significant, making analysis challenging. We present a new approach that utilizes chromatographically separated fractions from a pooled sample, henceforth called a metabolite fraction library (mFL). We developed an algorithm to extract highly correlated peaks from the mFL, collectively forming a metabolite basis set (mBS). The mBS can be fit to NMR profiling data, enabling comprehensive quantification. Applied to 10 mixtures of 53 metabolites, our approach accurately quantified 50, quantified an impurity and an oxidation product, and described between 91-96% of total spectral intensity. The method is demonstrated using the fungus Neurospora crassa , resulting in the identification of 45 metabolites with high confidence, 45 with medium confidence, and accounting for 94% of total spectral intensity.

DOI: https://doi.org/10.64898/2025.12.30.696914
J Chromatogr A. 2025 Dec 24. pii: S0021-9673(25)00999-9. [Epub ahead of print]1768 466655

Development and validation of a multi-class solid-phase extraction and liquid chromatography tandem mass spectrometry method for emerging contaminants in water.

Madara Weerasooriyagedara, Jordan M Partington, Wejdan Alghamdi, Mulugeta Legesse Akele, Stefano Freguia, Bradley O Clarke.

  A robust and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of 39 multi-class emerging contaminants (ECs), encompassing diverse groups including industrial chemicals (n = 6), pharmaceuticals (n = 19), personal care products (n = 4), and pesticides (n = 10). LC-MS/MS parameters were systematically optimized, including compound-specific transitions, source conditions, mobile phase composition, injection volumes, and chromatographic flow rates, to maximize signal intensity and ensure reproducible detection. Solid-phase extraction (SPE) was further optimized through evaluation of reconstitution solvent composition, evaporation settings, washing solvents, and elution strategies to improve recoveries and minimize matrix effects. The final method achieved instrument detection limits as low as 0.01 fg on-column, with method detection and quantitation limits of 0.01-1.32 ng/L and 0.03-3.96 ng/L, respectively. Calibration linearity (R² >0.99), recoveries within 70-130%, and intra-/inter-day precision (RSD ≤20%) confirmed the robustness and reproducibility of the protocol. Application to six real water matrices, bottled water, tap water, surface water, seawater, wastewater effluent, and influent, revealed the highest concentrations and widest range of analytes in influent, lower but quantifiable levels of persistent compounds in effluent, and predominantly trace-level detections or non-detects in natural and potable waters. Complementary greenness and applicability assessments using AGREE, MoGAPI, and BAGI highlighted moderate environmental sustainability and strong practical utility, reinforcing the suitability of the method for routine monitoring, source tracking, regulatory compliance, and risk assessment applications requiring ultra-trace sensitivity, high selectivity, and broad chemical coverage.

Keywords:  Emerging contaminants (ECS); Liquid chromatography mass spectrometry (LC-MS/MS); Method validation; Solid phase extraction (SPE); Water quality monitoring

DOI:  https://doi.org/10.1016/j.chroma.2025.466655
Talanta. 2026 Jan 03. pii: S0039-9140(26)00002-0. [Epub ahead of print]301 129349

Quantitative multiclass cyanotoxin analysis by paper spray mass spectrometry.

Lucas Abruzzi, Jack Gillies, Erik T Krogh, Daniel G Beach, Chris G Gill.

  Toxic cyanobacterial blooms are increasing in frequency in many parts of the world, and the toxins they can produce threaten both recreational use and drinking water supplies. Established analytical measurement techniques suffer from either high analysis costs and longer duty cycles, or lower accuracy and precision. Additionally, the chemical diversity of the different classes of cyanotoxins makes analysis by a single technique challenging. We present the development of a rapid multiclass method for cyanotoxin analysis using paper spray mass spectrometry requiring minimal sample preparation. A preliminary paper spray mass spectrometry (PS-MS) method developed on a triple quadrupole mass spectrometer yielded sensitive detection for microcystins, nodularin, and anatoxin with limits of detection below 3 μg/L, but suffered interference between phenylalanine and anatoxin. Subsequent method development using a custom-built PS source and high-resolution MS solved the anatoxin selectivity issue and provided typical detection limits between 0.3 and 1.6 μg/L for 6 microcystins, 2 anatoxins, and nodularin-R. Quantitative results for cyanobacteria culture extracts measured by PS-MS agreed well (typically ± 20 % bias) with results obtained with a reference LC-MS method. The presented methods are promising potential strategies for rapid cyanobacterial toxin screening, and could help address the growing need for rapid and simple techniques for high throughput cyanotoxin monitoring to mitigate exposure and reduce risks.

Keywords:  Ambient ionization; Anatoxins; Culture screening; Cyanobacteria; Cyanotoxins; Harmful algal toxins; High resolution mass spectrometry; Microcystins; Nodularins; PS-MS

DOI:  https://doi.org/10.1016/j.talanta.2026.129349
Pharmazie. 2025 Dec 01. 80(11): 118-121

Development and validation of a qNMR method for the simultaneous quantification of creatine and taurine in sports supplements.

R Van Der Merwe, D P Otto, W Liebenberg, H J R Lemmer, F van der Kooy.

Creatine and taurine are frequently found together in sports supplements due to their performance-enhancing and metabolic benefits. However, discrepancies between label claims and actual content have raised concerns about product quality, regulatory compliance, and possible health impacts. Accurate quantification of these compounds is therefore essential. Liquid chromatography mass spectrometry (LC-MS/MS) is widely used for its ability to detect and quantify compounds with high sensitivity and specificity. Therefore, in a previous study, an LC-MS/MS method was developed and validated for the simultaneous quantification of creatine and taurine in sports supplements. While accurate and sensitive, its somewhat cumbersome sample preparation step makes it less suitable for a commercial setting, where typically large numbers of samples must be analysed for quality control purposes. In this study, we report the first application of quantitative nuclear magnetic resonance (qNMR) for the simultaneous quantification of creatine and taurine in sports supplements, offering a simpler alternative for quality control. A qNMR method was developed, validated, and applied to commercial sports supplements, and the results were compared to label claims. All validation parameters fell well within acceptable limits, and sample analysis revealed deviations from label claims of up to +65.97% for creatine and +141.52% for taurine. Batch-to-batch variation of the products showed better consistency with variability only as high as 8.49%. Overall, this study confirms qNMR as a reliable method demonstrating specificity, precision, accuracy, and suitability for quantitative analysis. Although high-field NMR systems remain more commonly used, the method developed here is directly transferable to modern cryogen-free benchtop NMR instruments. Benchtop NMR significantly reduces operational costs and complexity, and may arguably become a valuable, reliable, and affordable tool in quality control laboratories.

DOI: https://doi.org/10.1691/ph.2025.5078
J Chromatogr A. 2026 Jan 05. pii: S0021-9673(26)00008-7. [Epub ahead of print]1768 466677

Development of a chiral mass spectrometry derivatization reagent for amino acid enantiomer analysis: Metabolic investigation of DL-amino acids in the serum of nephritis patients.

Xinxin Kong, Zongshuo Sun, Xinyi Xu, Xi-Ling Li, Jin Ji Li, Jun Zhe Min.

  A chiral mass spectrometry derivatization reagent, (R)/(S)-(5-(2-(((1-(p-toluenesulfonylmethylamino)vinyl)oxy)carbonyl)pyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium bromide (TPP-(R)/(S)-BSA), was developed in this study. The derivatization reagent carries a permanent positive charge and incorporates an α-acyloxy acetylenamide moiety, enabling highly sensitive detection and stereochemical resolution of amino-containing enantiomers. Exposure of DL-amino acids to TPP-(R)/(S)-BSA under heating at 70 °C for 60 min resulted in formation of the corresponding diastereomers. Fine-tuning of the separation conditions enabled clear resolution of fifteen enantiomeric amino acid pairs on a BEH C18 column, with Rs values spanning 1.02-14.49. Except for Pro, Gln, and His, all DL-amino acid pairs showed Rs values above 1.5, indicating effective enantiomeric separation under standardized conditions. Based on the TPP-(S)-BSA derivatization strategy, we developed a UHPLCHRMS assay with excellent sensitivity and selectivity, enabling concurrent resolution and determination of 13 DL-amino acid enantiomeric pairs together with glycine in human serum. The method provided excellent linearity within 0.05-1000 μM (R² ≥ 0.9914), with limits of detection (LOD) between 0.2 and 4.5 fmol. For both intra-day and inter-day evaluations, the observed precision fell within the range of 0.92%-14.00%, while recovery rates averaged 97.88%-111.78%, and matrix effects were within 3.82%-14.58%. Furthermore, statistical analysis of DL-amino acid metabolic concentrations in serum from healthy volunteers and nephritis patients enabled the construction of a diagnostic model integrating differentially expressed enantiomers, providing a potential screening tool for nephritis.

Keywords:  DL-amino acids; Human serum; Nephritis; TPP-(R)/(S)-BSA; UHPLC-HRMS

DOI:  https://doi.org/10.1016/j.chroma.2026.466677
Food Chem X. 2025 Dec;32 103360

Emerging analytical techniques for lipid profiling in food products: insights into processing effects and quality control.

Deepika Kathuria, Sonal Aggarwal, Akanksha Negi, Riya Barthwal, Aroma Joshi, Narpinder Singh.

  Lipids are essential components of foods which include fatty acids, glycolipids, sphingolipids, sterols, vitamin D, isoprenoids, etc. It possesses bioactive properties that exhibit desirable impact on human health. However, understanding the complexity of food lipids, requires sophisticated analytical methods capable of capturing their full spectrum. Lipidomics (LIP), an emerging omics field derived from metabolomics focuses on the complete analysis of lipid molecules within food matrices. Mass spectrometry (MS)-based LIP, exhibit great potential for analysing food lipids. The present review discusses the application of LIP in characterizing lipid metabolites in food, including how food processing, cooking, and storage, influence lipid profiles. Advancement in MS-based LIP enabled the detection of lipid alterations, along with ensuring food quality, authenticity, and safety. Future trends suggest further advancements in MS and separation techniques and application of Large Language Models will emerge as powerful tools in supporting oil processing research and industry practices. These techniques offered innovative predictions for mining significant information from lipidomic related large volumes of scientific and technical literature that will expand the application of LIP, providing deeper insights into lipid metabolism as well as understanding lipids in relation to health and nutrition.

Keywords:  Lipids; food; food processing; mass-spectrometry; metabolomic

DOI:  https://doi.org/10.1016/j.fochx.2025.103360
Talanta. 2025 Dec 31. pii: S0039-9140(25)01833-8. [Epub ahead of print]301 129342

NITCD: A streamlined metabolomics strategy based on bromine isotopes and MS-TDF software.

Dandan Zhang, Hairong Zhang, Jiajin Yi, Di Lu, Caisheng Wu.

  In metabolomics research, derivatization methods, particularly stable isotope derivatization, are commonly employed to enhance the coverage and qualitative and quantitative accuracy of analyte compounds. In our previous work, we adopted a homolog derivatization approach combined with our in-house developed mass spectrometry triple-dimensional derivatization filter (MS-TDF) software to achieve low-cost metabolomics studies. However, differential derivatization efficiencies across homologs led to an increased false positive rate. In this study, we introduce a natural isotope triple-dimensional combinatorial derivatization (NITCD) strategy that overcomes these limitations. The approach employs 4-bromo-2-hydrazinopyridine, which provides a characteristic 1:1 isotopic doublet pattern (79Br/81Br) for intelligent metabolite identification via MS-TDF software. Combined with 2-hydrazinopyridine as a structurally matched internal standard, the system not only reduces false-positive identifications but also enables reliable relative quantification. This strategy was successfully applied to a metabolomics study on rhein treatment in inflammatory bowel disease (IBD). The experimental results demonstrate that, by using NITCD strategy, 564 target compounds can be detected in mouse plasma, with 148 in colon tissue, and 81 in spleen tissue. More importantly, these metabolites were identified, along with their dynamic changes during rhein treatment. It was found that rhein might reverse the IBD-induced alterations in arachidonic acid metabolism, tyrosine metabolism, primary bile acid biosynthesis, and tryptophan metabolism.

Keywords:  Bromine; Inflammatory bowel disease; MS-TDF; Metabolomics; Rhein

DOI:  https://doi.org/10.1016/j.talanta.2025.129342
Anal Chem. 2026 Jan 07.

Mapping C═C Positions in Branched Fatty Acid Esters of Hydroxy Fatty Acids by Oxygen Attachment Dissociation Mass Spectrometry Coupled with Chemical Labeling.

Xin-Ze Wu, Huan-Ran Yin, Feng Ji, Na An, Xiu-Feng Fu, Xiao-Dong Li, Quan-Fei Zhu, Yu-Qi Feng.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) represent an important class of endogenous lipids with demonstrated anti-inflammatory and antidiabetic functions. However, comprehensive structural characterization, particularly the unambiguous localization of carbon-carbon double (C═C) bonds in unsaturated FAHFAs, remains analytically challenging due to structural diversity, low abundance, and the lack of reference standards. Herein, we introduce a novel mass-spectrometry-based strategy termed DISCO-MS (derivatization-initiated source and collision OAD mass spectrometry) for high-confidence FAHFA annotation. The method combines oxygen attachment dissociation (OAD) with in-source fragmentation (ISF) in a hyphenated workflow, enabling the simultaneous determination of C═C bond positions, fatty acyl chain lengths, and ester bond information in a single run. A Python-based multimass spectral generator was developed to predict and curate a comprehensive database containing 58,506 MS/MS spectra covering 19,502 putative FAHFA species. The utility of DISCO-MS was demonstrated through the analysis of six custom-synthesized unsaturated FAHFA standards and complex biological extracts, revealing a previously unresolved structural diversity. This integrated approach provides a promising platform for accelerating the discovery and functional study of FAHFAs in metabolic and nutritional research.

DOI: https://doi.org/10.1021/acs.analchem.5c05926
Molecules. 2025 Dec 24. pii: 65. [Epub ahead of print]31(1):

1H-NMR Analysis of Wine Metabolites: Method Development and Validation.

Guillaume Leleu, Rémi Butelle, Daniel Jacob, Lou-Ann Kurkiewicz, Jean-Claude Boulet, Catherine Deborde, Matthieu Dubernet, Laetitia Gaillard, Antoine Galvan, Karen Gaudin, Alexandra Gossé, Markus Herderich, Annick Moing, Sophie Rosset, Flynn Watson, Gregory Da Costa, Tristan Richard.

  Wine, as a high-value product, is vulnerable to counterfeiting. To tackle increasingly sophisticated fraud, innovative analytical approaches are required. However, they must undergo rigorous validation. Proton nuclear magnetic resonance spectroscopy (1H-NMR) is intrinsically quantitative, reproducible, and fast, making it a promising tool for official control. This study presents the development and validation of a standardised and fully automated workflow for the quantification of 20 oenologically relevant compounds, including organic acids, sugars, alcohols, esters, phenolics, and an alkaloid. The method combines optimised sample preparation, external quantification standards, spectrometer calibration, and a dedicated R package (RnmrQuant1D) for fully automated spectral processing, enabling high-throughput and operator-independent analysis. Validation was performed under intermediate precision according to OIV metrological standards, evaluating accuracy, precision, robustness, limits of quantification, and measurement uncertainty. The results demonstrated excellent linearity, trueness, and reproducibility, matching the targeted analytical performance. Measurement uncertainties were estimated both by conventional linear modelling and by a dynamic approach better suited to detection limits. The workflow is easy to implement, requires minimal sample consumption, and substantially reduces operator bias. Beyond validating a robust method, this study provides a framework for harmonised, transferable 1H-NMR workflows that could support large-scale databases, integration with chemometric models, and ultimately, 1H-NMR's recognition as a relevant method for wine authentication and quality control. This work fills a crucial gap in wine analysis by uniting practical application and rigorous methods, enabling broader adoption in control laboratories worldwide.

Keywords:  1H-NMR; metabolomics; method validation; quantification; wine

DOI:  https://doi.org/10.3390/molecules31010065
Crit Rev Anal Chem. 2026 Jan 09. 1-11

A Comparative Review of Hydrophilic Interaction Chromatography(HILIC) with Traditional Chromatography in the Analysis of Aminoglycoside Antibiotics.

Santhosh K, Sumithra M.

  Aminoglycoside antibiotics remain crucial for treating severe Gram-negative infections, yet their highly polar, polycationic nature presents ongoing analytical challenges. Traditional reversed-phase chromatography often proves inadequate, offering insufficient retention and poor peak symmetry due to limited interactions with hydrophobic stationary phases. Consequently, analytical techniques have increasingly shifted toward hydrophilic interaction liquid chromatography (HILIC), which offers improved retention, enhanced selectivity, and better compatibility with mass spectrometric detection. This review critically explores the development of chromatographic methods for aminoglycoside quantification, emphasizing methodological advancements, practical challenges, and the application of modern HILIC-MS/MS systems. Key structural elements of aminoglycosides, such as multiple amino and hydroxyl groups and the presence of 2-deoxystreptamine, significantly affect chromatographic behavior. Understanding these molecular features is essential for optimizing mobile-phase pH, buffer strength, and organic solvent composition to achieve effective separation. Recent research indicates that optimized HILIC methods can address previous issues related to ion suppression, low retention, and complex sample matrices, particularly in biological, pharmaceutical, and food safety analyses. Despite significant progress, challenges persist in method standardization, consistency across laboratories, and the development of universally applicable chromatographic conditions. This review compiles existing evidence, highlighting the strengths and limitations of current analytical methods, and suggests future directions for creating more sensitive, reproducible, and high-throughput methodologies. By integrating structural insights with modern chromatographic advancements, the review offers a comprehensive perspective aimed at enhancing aminoglycoside analysis across various research and regulatory settings.

Keywords:  Aminoglycosides; hydrophilic interaction chromatography (HILIC); normal phase chromatography (NP); reversed phase chromatography (RP-HPLC); zwitterionic columns

DOI:  https://doi.org/10.1080/10408347.2025.2610328
ACS Omega. 2025 Dec 30. 10(51): 63072-63083

Simultaneous Determination of Lyso-Gb1 and Lyso-Gb3 in Plasma Using Salt-Assisted Liquid-Liquid Extraction Combined with LC-MS/MS.

Erhan Canbay, Çetin Uzun, Yasemin Akçay, Ebru Canda, Havva Yazıcı, Sema Kalkan Uçar, Mahmut Çoker, Eser Yıldırım Sözmen.

Background: Lyso-Gb1 and Lyso-Gb3 are key biomarkers for Gaucher type 1 and Fabry diseases, offering high diagnostic accuracy and utility in disease monitoring. However, conventional sample preparation workflows for their LC-MS/MS quantification often require large volumes of organic solvents, generate hazardous waste, and involve time-consuming steps, limiting routine applicability. Objective: This study aimed to develop, validate, and apply a rapid, sensitive, and environmentally friendly LC-MS/MS method using salt-assisted liquid-liquid extraction (SALLE) for the simultaneous determination of Lyso-Gb1 and Lyso-Gb3 in human plasma. Methods: The SALLE procedure was optimized for extraction-solvent type and volume, salt type, and concentration, with heptane (450 μL) and ammonium acetate (5 M) selected as optimal conditions to achieve high recovery and minimal matrix effects. Method validation was performed according to ICH M10 guidelines, evaluating selectivity, linearity, accuracy, precision, recovery, matrix effect, carry-over, dilution integrity, stability, and reinjection reproducibility. Clinical applicability was assessed in two independent real-sample applications: (i) 31 healthy individuals and 20 Gaucher type 1 patients for Lyso-Gb1, and (ii) 29 healthy individuals and 13 Fabry disease patients for Lyso-Gb3. Result: The method demonstrated excellent linearity for Lyso-Gb1 (0.380-200 ng/mL, R 2 = 0.9890) and Lyso-Gb3 (0.756-200 ng/mL, R 2 = 0.9982), with low LLOQs (0.380 and 0.756 ng/mL, respectively), recoveries of 92.5-109.9%, and negligible matrix effects. In clinical samples, Lyso-Gb1 and Lyso-Gb3 concentrations were significantly higher in their respective patient groups compared to healthy controls (p < 0.0001). ROC analysis showed perfect discrimination for both biomarkers (AUC = 1.000), with optimal cut-offs of 5.80 ng/mL for Lyso-Gb1 and 3.334 ng/mL for Lyso-Gb3, each yielding 100% sensitivity and specificity. Conclusion: The developed SALLE-LC-MS/MS workflow is robust, high-throughput, and fully compliant with green-chemistry principles, using minimal organic solvent, reducing hazardous waste, and shortening the preparation time. Its excellent analytical performance and perfect clinical discrimination support its integration into routine diagnostic laboratories for the efficient diagnosis and monitoring of Gaucher type 1 and Fabry diseases.

DOI: https://doi.org/10.1021/acsomega.5c09235
J Phys Chem Lett. 2026 Jan 08.

Phase Model-Driven Deep Learning for Robust Phase Correction in High-Throughput NMR-Based Metabolomics.

Chuanwen Zhao, Gang Chen, Caixiang Liu, Zhao Li, Jing Zhao, Peng Sun, Hongkang Chu, Xin Zhou, Maili Liu, Peijun Song, Qingjia Bao, Chaoyang Liu.

High-throughput NMR, a key metabolomics tool, enables efficient, noninvasive profiling of large biological samples. Automatic data processing ensures scalable, consistent high-throughput NMR. A key workflow step is phase correction, critical for obtaining pure absorption-mode spectra necessary for accurate quantitative analysis. This study proposes the Phase Model-Driven Residual Attention Network (PD-RAN), a robust phase correction method that combines deep neural networks with a physically informed model. By learning low-dimensional phase features grounded in physical principles from one-dimensional NMR spectra containing thousands of data points (high-dimensional data representation), PD-RAN delivers precise and reliable phase correction. Experimental results show consistent superiority over conventional methods across diverse metabolomics samples, including brain extracts, plasma, and urine. The method demonstrates remarkable efficiency, processing 1,000 spectra in just 20 ms, rendering it highly suitable for high-throughput NMR metabolomics applications. Ablation studies further validate the effectiveness of the phase model-driven component and its robustness to noise and baseline distortions.

DOI: https://doi.org/10.1021/acs.jpclett.5c03529
J Am Soc Mass Spectrom. 2026 Jan 08.

In-Source Fragmentation of Pyrrolidine-Containing KRAS Scaffolds Leads to Enhanced Structure Elucidation and Tandem Mass Spectral Quality.

Jessica L Ochoa, Christopher M Crittenden.

The characterization of small-molecule therapeutics containing basic moieties, such as pyrrolidine groups common in KRAS G12C inhibitors, presents challenges for structure elucidation via tandem mass spectrometry (MS/MS). During fragmentation, the pyrrolidine preferentially sequesters the proton, leading to a dominant, uninformative fragment ion and a corresponding loss of structural detail. This hinders the efficient identification of related impurities and metabolites in complex mixtures. To circumvent this limitation, we developed an easily transferable analytical workflow that intentionally utilizes In-Source Fragmentation (ISF). Optimizing source parameters promotes the selective neutral loss of the pyrrolidine moiety prior to MS/MS, yielding core fragment ions (e.g., m/z 525 for GDC-6036). Tandem mass spectrometry on this ISF-generated precursor provides extensive fragmentation and structural coverage, outperforming traditional higher-energy collisional dissociation. We demonstrate the successful application of this optimized workflow to characterize GDC-6036, its synthetic intermediate, and structurally distinct KRAS inhibitors (Adagrasib and MRTX1133) across a chromatographic time scale. This approach offers a universal tool for enhancing the structure elucidation of challenging basic compounds, critical for supporting pharmaceutical process development.

DOI: https://doi.org/10.1021/jasms.5c00340
Clin Chem Lab Med. 2026 Jan 07.

Evaluation of the MAGLUMI non-competitive angiotensin I chemiluminescence immunoassay for measuring renin activity in human plasma.

Lina Wu, Baohu Zhang, Yun Zhang, Shucai Yang, Chenyan Shi, Wenfeng Wang, Zhonggang Fang, Li Zhang, Xiaosong Qin.

   OBJECTIVES: Plasma renin activity (PRA) measures the rate of renin converting angiotensinogen to angiotensin I in circulation and reflects the function of renin-angiotensin-aldosterone system (RAAS). The study aimed to demonstrate the performance of the MAGLUMI Angiotensin I assay for the measurement of PRA.
METHODS: The MAGLUMI Angiotensin I assay was evaluated for precision, linearity, limit of blank (LoB), limit of detection (LoD), limit of quantification (LoQ), interferences, cross-reactivity and reference intervals. It was compared to liquid chromatography tandem mass spectrometry (LC-MS/MS) using 54 samples, and impact of clinical factors was assessed in 181 samples.
RESULTS: The assay demonstrated repeatability of 1.5 %, 1.85%, and 1.6 % across three different pool concentrations, with corresponding within-laboratory imprecision values of 2.0 %, 2.5%, and 1.9 %. The linear range was [0.10, 24.0] ng/mL. The LoB, the LoD, and the LoQ were 0.03 ng/mL, 0.07 ng/mL, and 0.10 ng/mL, respectively. The interferences and cross-reactivity met the criterion for acceptable cross-reactivity (CR) with <±10 %. Reference interval validation shows <10 % of a total of 40 healthy individuals' plasma samples falling outside the established interval for standing and lying position. The MAGLUMI assay showed excellent agreement with LC-MS/MS for both angiotensin I (slope=1.076, intercept=0.109 ng/mL/h, r=0.987, p<0.001) and plasma renin activity (slope=1.131, intercept=-0.386 ng/mL/h, r=0.973, p<0.001).
CONCLUSIONS: The MAGLUMI Angiotensin I chemiluminescence immunoassay is a sensitive and reliable commercial kit for detecting plasma renin activity. The assay exhibited robust analytical performance and strong agreement with LC-MS/MS.

Keywords:  angiotensin I; non-competitive immunoassay; plasma renin activity; renin-angiotensin-aldosterone system

DOI:  https://doi.org/10.1515/cclm-2025-1361
Anal Chem. 2026 Jan 07.

A GC-MS Data Analysis Platform for Untargeted Metabolomics with Enhanced Coeluting Peak Resolution.

Xing-Cai Wang, Chang Yang, Hang Lv, Xue-Juan Zhang, Wan-Ting Zhou, Peng Lu, Yongjie Yu, Haiyan Fu, Yuanbin She.

Gas chromatography-mass spectrometry (GC-MS) remains challenged by the accurate resolution of coeluting peaks and the correction of retention time shift in large-scale batch analysis. Here, we introduce AntDAS-CPR, an integrated data analysis platform tailored for untargeted GC-MS-based metabolomics. The platform incorporates modules for total ion chromatogram (TIC) peak resolution, retention time shift correction, component registration, chemometric analysis, and compound identification. In this work, the TIC peak resolution module was specifically optimized through the development of a dynamic elimination multivariate curve resolution-alternating least-squares (DEMCR-ALS) algorithm, which employs multiple initialization strategies to enhance the resolution of coeluting peaks and reduce dependence on initial estimates inherent in conventional methods. The performance of AntDAS-CPR was comprehensively evaluated using standard mixtures and complex food matrix data sets. It was compared with state-of-the-art GC-MS data analysis tools, including AMDIS, ADAP-GC, MS-DIAL, and eRah. Comparative results demonstrated that AntDAS-CPR consistently outperforms existing methods in both targeted and untargeted analysis. The platform is freely accessible at http://www.pmdb.org.cn/antdascpr.

DOI: https://doi.org/10.1021/acs.analchem.5c05471
Anim Reprod Sci. 2026 Jan 03. pii: S0378-4320(26)00001-1. [Epub ahead of print]286 108098

Equine fetal-sex determination from mid-gestation to term using serum conjugated steroid profiling by liquid chromatography-tandem mass spectrometry.

Joy Ledeck, Thomas Dubrowski, Matthieu Schoumacher, Stéphanie Peeters, Caroline Le Goff, Sophie Egyptien, Stéfan Deleuze, Etienne Cavalier, Jérôme Ponthier.

  Refined profiling of conjugated estrogens and androgens during equine pregnancy using liquid chromatography-tandem mass spectrometry (LC-MS/MS) could provide accurate fetal-sex determination. Current methods for fetal-sex prediction remain limited by timing, accuracy, and operator expertise. This study investigated sex-specific differences in maternal conjugated steroid profiles to develop a reliable, non-invasive predictive method. Samples were collected from 141 mares of various breeds starting at sixteen weeks of pregnancy. The samples were pooled according to gestational stage, divided into 28-day blocks, and analysed using a validated LC-MS/MS method. Quantified analytes included estrone, estradiol, equilin sulfates, glucuronides, and dehydroepiandrosterone sulfate. Method validation encompassed linearity, trueness, precision, accuracy, uncertainty, quantification limits, recovery, matrix effects, carryover, sensitivity, and stability. The effects of fetal sex, breed, parity, and maternal age on steroid concentrations were investigated. Data were split into a Test group for model development and a Validation group for performance assessment. Fetal sex was the principal factor influencing conjugated estrogen and dehydroepiandrosterone profiles (p = 0.01-0.0001). Female pregnancies exhibited higher classical conjugated steroid concentrations at block 5, whereas male pregnancies showed delayed elevations persisting until term. Equilin derivatives were consistently lower in males. Estrone sulfate (E1S) showed reliable univariate performance for fetal-sex prediction (area under the curve (AUC) 0.710-0.865, blocks 5-10). Multivariate models using all conjugated steroids achieved superior accuracy (AUC 0.747-0.912, blocks 5-11), providing an alternative method to univariate model and ultrasonography. The validated LC-MS/MS method improves understanding of equine feto-placental endocrinology and offers a non-invasive tool for practical fetal sexing.

Keywords:  Conjugated steroids; Fetal sex; Gestation; Liquid chromatography-tandem mass spectrometry; Mare; Serum

DOI:  https://doi.org/10.1016/j.anireprosci.2026.108098
Molecules. 2025 Dec 25. pii: 90. [Epub ahead of print]31(1):

Targeted Determination of Residual Sex Hormones in Cosmetics Using Magnetic Solid-Phase Extraction with Isotope-Labeled Internal Standards by UHPLC-MS/MS.

Yalei Dong, Shuyan Sun, Yasen Qiao, Chunhui Yu, Haiyan Wang, Lei Sun.

  As rapidly developing consumer products, cosmetics confront challenges regarding safety, especially hazardous ingredients, like sex hormones. Prolonged exposure to trace sex hormones in cosmetics can inflict immeasurable damage to human health. To accurately detect the trace amounts of sex hormones in cosmetics, a reliable method was developed and validated using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) with magnetic solid-phase extraction (MSPE) and isotope-labeled internal standards (IL-ISs). The conditions of sample pretreatment, chromatography, and mass parameters were systemically investigated. In the MSPE procedure, the commercial Fe3O4@HLB magnetic material was employed for sample pretreatment, which was beneficial for operation, as well as sample purification and analyte enrichment. The utilization of IL-ISs compensated for potential matrix effects and losses during sample preparation, thereby improving precision and accuracy. Based on the proposed MSPE technology, UHPLC-MS/MS can address the qualitative and quantitative analysis needs for target analytes in complex cosmetic matrices. At three fortification levels, recoveries were in the range of 71.7-116.2%, with a relative standard deviation (RSD) ranging from 1.6% to 8.3%. Furthermore, based on the method proposed here, a total of 116 batches of cosmetics were analyzed, and trace progestins and estrogens were discovered in 10 samples. The MSPE method, coupled with UHPLC-MS/MS using IL-ISs, was convenient, efficient, and feasible for detecting trace amounts of sex hormones in cosmetics. The method scored 0.66 (out of 1) on the AGREE metric, confirming its green profile. Based on the detected concentrations, a preliminary safety evaluation was performed to assess the potential health risks of residual progesterone in hair loss prevention cosmetics by calculating the margin of safety (MoS).

Keywords:  MSPE; UHPLC-MS/MS; cosmetics; progesterone; safety evaluation; sex hormones

DOI:  https://doi.org/10.3390/molecules31010090
J Appl Lab Med. 2026 Jan 07. pii: jfaf199. [Epub ahead of print]

Tacrolimus Assay Comparison: Analytical and Operational Considerations for Immunoassay vs Mass Spectrometry Testing in a High Volume Clinical Laboratory.

Rebekah Goldblatt, Florencia Monge, Cory Hansen, Melissa M Budelier.

BACKGROUND: Tacrolimus therapeutic drug monitoring is a routine and critical assay for managing transplant patients. Tacrolimus can be measured from whole blood samples via liquid chromatography-tandem mass spectrometry (LC-MS/MS) or immunoassay (IA). LC-MS/MS provides accurate parent drug quantification with minimal metabolite interference but requires expensive analyzers, highly trained staff, and is often a very manual process. Immunoassays offer faster, simpler workflows, but historically have been prone to significant cross-reaction with the drug's metabolites or other therapeutic drugs, leading to a falsely elevated result. Newer immunoassays show promising improvements in specificity.
METHODS: Using 73 residual patient samples, we compared our in-house LC-MS/MS tacrolimus assay to the Roche Elecsys® tacrolimus electrochemiluminescence immunoassay on the cobas® e801 analyzer. Comparative analysis was done with Deming regression and Bland-Altman comparison. Linearity, within-individual imprecision, and total imprecision post immunoassay implementation were also evaluated, along with changes and improvements to workflow and turnaround times (TAT).
RESULTS: The Roche Elecsys tacrolimus assay correlated well with our LC-MS/MS method (Deming regression equation: y = 1.004x + 0.4848). The bias was 0.52 ng/mL (an average of 8.1%) as determined by Bland-Altman comparison. When switching to the immunoassay we observed a 42%-51% improvement in TAT across the median, 75th percentile, and 90th percentile.
CONCLUSIONS: We observed excellent correlation between the Roche Elecsys tacrolimus assay on an e801 analyzer vs our in-house LC-MS/MS assay, with good precision and linearity across reportable ranges. Changing from mass spectrometry to tacrolimus immunoassay substantially improved our TAT and workflow.

DOI: https://doi.org/10.1093/jalm/jfaf199
J Mass Spectrom. 2026 Jan;61(1): e70000

Lipidomic Comparison of Detergent-Soluble and Detergent-Resistant Lipid Domains Isolated From Rat Cerebellum and the Effect of Hypoxia-Ischemia Using UPLC-HDMSE.

Samuel A Krug, Min He, Ningfeng Tang, Cynthia Bearer, Maureen A Kane.

  Hypoxic ischemic encephalopathy (HIE) due to insufficient oxygen or blood flow to the brain can result in inflammation, impaired neurodevelopment, or death. Changes to microdomain lipid composition may contribute to altered structure and function of key regulatory processes; however, knowledge regarding changes to membrane lipid composition after HIE is incomplete. Here, we describe the application of untargeted lipidomics to investigate the impact of hypoxia-ischemia on detergent-resistant membrane (DRM) and detergent-soluble membrane (DSM) domains fractionated from the cerebellum in a rat model of HIE. Lipidomics utilized ultra performance liquid chromatography coupled to data independent tandem mass spectrometry with traveling wave ion mobility. Lipid alterations specific to the DRM domains after hypoxia-ischemia included lysophospholipids, sphingomyelins, ceramides, triglycerides, and phosphatidylethanolamines, which are lipid species that have been linked to cognitive and neuronal impairment. The advances in lipidomics have enabled the tools available to study lipid composition. These data may provide insight into membrane disruption after HIE in relation to lipid composition and concentration.

Keywords:  hypoxic ischemia; lipid membrane; lipidomics; liquid chromatography–tandem mass spectrometry; membrane domains

DOI:  https://doi.org/10.1002/jms.70000