bims-metlip 2026-01-18 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–01–18
25 papers selected by
Sofia Costa, Matterworks

Development of an untargeted metabolomics analytical protocol for fecal samples by liquid chromatography-mass spectrometry.
Development and Validation of a Sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Simultaneous Quantification of Thiopurine Nucleotides in Human Red Blood Cells.
Separation of mephentermine from other structural analogues of phenethylamine and amphetamine in biological matrices using Liquid Chromatography-Tandem Mass Spectrometry.
Demonstrating Voxel-by-Voxel (V × V) Single-Point Calibration in Whole-Body Zebrafish by IR-MALDESI Quantitative MSI.
Aqueous LC-MS/MS quantification of α-/β-nicotinamide mononucleotide in dietary supplements using a pentabromophenyl column.
Validation of a Microsampling-Compatible Liquid-Liquid Extraction Method for Cannabinoid Quantitation in 50 µL of Whole Blood using LC-MS/MS.
Synthesis-driven reverse metabolomics reveals 3-hydroxy N -acyl amides as gut microbial molecules.
MALDI mass spectrometry and imaging for lipid analysis in food matrices: A review of analytical principles and perspectives.
DPM: A Deep Learning and Optimal Transport Framework for Cost-Effective Spatial Metabolomics.
Analytical Techniques for the Quantification of Pravastatin and Its Metabolites in Various Matrices: A Comprehensive Review.
TidyMass2: advancing LC-MS untargeted metabolomics through metabolite origin inference and metabolic feature-based functional module analysis.
Development of a high-throughput LLE-UPLC-QqQ-MS/MS method and comprehensive profiling of urinary steroid hormones in Chinese elderly adults.
Evolving Methodologies for the Analytical Determination of Metoclopramide.
AminoacidDB: a liquid chromatography-tandem mass spectrometry-based toolkit for the untargeted analysis of non-protein amino acids.
Beyond the native state: Revolutionizing food phenolic acids profiling through advanced chemical derivatization and sample pretreatment (2003-2025).
Development of an arrow solid-phase microextraction GC-MS/MS method for the determination of 69 odorants in water.
A highly sensitive LC-MS/MS method for quantification of AB-38b in rat plasma, liver and kidney tissues: application to a pharmacokinetic study.
Determination and Pharmacokinetic of Peiminine in Beagle Dogs by UPLC-MS/MS.
A Polymeric Zwitterionic Hydrophilic Probe for Mapping the Human Serum Endogenous Glycopeptidome.
Quantification of phthalate and DINCH metabolites in human urine and maternal breast milk: Assessing maternal body burden and infant exposure.
Ease of analysis through unification: One gas chromatographic method for the chemical profiling of essential oils.
Preparation of ultra-high efficiency sulfoalkyl betaine-type monolithic columns through amine-epoxide ring-opening reaction combined with in-situ ring-opening quaternization and their application in hydrophilic interaction chromatography.
Enhancing detection of low‑abundance metabolites in proton NMR through band‑selective suppression and presaturation.
Non-targeted metabolomic profiling of Cremastra appendiculata providing insights for phytochemical analyses.
Robust High-Performance Liquid Chromatography and Liquid Chromatography-Tandem Mass Spectrometry Methods for the Determination of Malondialdehyde in Biological Samples Using Thiobarbituric Acid Derivatization and Column Cleaning with High-Concentration Ammonium Acetate.

J Food Drug Anal. 2025 Dec 15. 33(4): 460-470

Development of an untargeted metabolomics analytical protocol for fecal samples by liquid chromatography-mass spectrometry.

Tsai-Wei Ting, Chih-Ning Cheng, Chieh-Chang Chen, Ching-Hua Kuo.

Gut microbiota has recently gained attention for its role in regulating multiple host pathways and contributing to disease developments. Fecal metabolomics using liquid chromatography-mass spectrometry (LC-MS) offers a promising approach to study gut microbial metabolites; however, it remains technically challenging due to the complex, heterogeneous nature of fecal samples and the lack of standardized protocols. This study aimed to establish a robust and reproducible untargeted fecal metabolomics workflow. We systematically evaluated sample preparation parameters-including sample amount, extraction solvent, numbers of extraction, and sample-to-solvent ratio-and assessed method reproducibility. Additionally, we compared three LC-MS data acquisition workflows using 10 samples from inflammatory bowel disease (IBD) patients and healthy controls (HC) to improve the identification of biologically relevant metabolites. In sample preparation, our results showed that 50 mg of lyophilized feces was sufficient to capture inter-individual metabolic variation. Additionally, methanol outperformed acetonitrile and showed comparable results to three binary solvent mixtures. A single extraction with methanol was sufficient, and a 1:20 (w/v) sample-to-solvent ratio maximized feature detection. Among the acquisition methods, data-dependent acquisition (DDA) with simultaneous MS1 and MS2 scans provided the highest metabolite coverage with acceptable annotation reliability. In summary, we recommend a single extraction of 50 mg lyophilized feces with 1 mL methanol and the use of DDA for sample acquisition to ensure comprehensive and reproducible untargeted analysis. This optimized protocol improves metabolite detection in human feces and offers a practical strategy to support future studies exploring gut microbial contributions to human health and disease.

DOI: https://doi.org/10.38212/2224-6614.3571
Cureus. 2025 Dec;17(12): e98953

Development and Validation of a Sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Simultaneous Quantification of Thiopurine Nucleotides in Human Red Blood Cells.

Maninder Singh, Sandeep Kaushal, Kanchan Gupta, Ajit Sood.

  Background Therapeutic drug monitoring (TDM) of thiopurine metabolites in red blood cells (RBCs) is essential to optimizing dosing regimens and minimizing adverse effects. Current analytical methods lack the desirable specificity and sensitivity needed for optimum quantification at therapeutic concentrations on a routine basis. The objectives of the current study were to validate and develop a highly repeatable and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), and 6-methylmercaptopurine (6-MMP) within human red cells. Methods Hydrophilic interaction liquid chromatography (HILIC) was employed for separation using a gradient elution system. Ammonium formate with formic acid in water (A) and acetonitrile (B) was used as the mobile phases. Multiple reaction monitoring (MRM) detection was carried out in positive mode using an electrospray ionization (ESI) source. The method was validated according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines and included testing for linearity, accuracy, recovery, precision, and specificity in the 0.5-1000 ng/mL concentration range. Results The developed method showed excellent linearity for the three analytes with a coefficient of determination (R²) of ≥0.9997. Recovery analysis showed acceptable accuracy, with the mean recoveries between 88.74% and 117.37% for all the concentration levels. The method showed high specificity without interference from endogenous matrix components. Precision study showed a relative standard deviation (RSD) of ≤15% for all the analytes. Conclusions The developed LC-MS/MS approach gives a reliable technique for the simultaneous quantification of thiopurine metabolites within erythrocytes. With its reliability and sensitivity, it makes a suitable approach for therapeutic drug monitoring at the clinical level, therefore contributing toward enhanced thiopurine therapy under the banner of personalized medicine.

Keywords:  6-mercaptopurine; 6-thioguanine; lc-ms/ms; method validation; red blood cells; therapeutic drug monitoring; thiopurine metabolites

DOI:  https://doi.org/10.7759/cureus.98953
J Pharm Biomed Anal. 2025 Dec 31. pii: S0731-7085(25)00669-7. [Epub ahead of print]271 117328

Separation of mephentermine from other structural analogues of phenethylamine and amphetamine in biological matrices using Liquid Chromatography-Tandem Mass Spectrometry.

Tusha Tripathi, Kamna Sharma, Awanish Upadhyay, Ashok Singh, Puran Lal Sahu, Tarun Handa, Meghna Choudhary, Bikash Medhi.

  Stimulants exert euphorigenic and relaxing effects which warrant intervention of regulatory authorities in non-medical usage of these doping agents. Thus, a semi-quantitative method utilizing Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) was developed to monitor potential misuse of 10 structural analogues of amphetamine and phenylethylamine derivatives. The extraction protocol for urine was dilute and shoot (DS) with 1 mL aliquot size, while that for plasma/serum was solid-phase extraction (SPE) with a 100 µL aliquot size. Chromatographic separation was achieved over an 18-minute gradient on a Phenomenex Luna Omega UHPLC C18 column. The mobile phase consisted of water and acetonitrile, both containing formic acid as a modifier, specifically at 1 % (v/v) for urine and 0.1 % (v/v) for plasma/serum analyses. Positive electrospray ionization (ESI+) was employed for mass spectrometric detection with acquisition in multiple reaction monitoring (MRM) mode. Method validation was performed in accordance with World Anti-Doping Agency (WADA) requirements for analyzing prohibited substances with minimum reporting limits (MRLs). Limits of identification (LOI) determined for urine and plasma/serum matrices were as follows: 0.5 ng/mL and 1 ng/mL for Oxethazaine; 1 ng/mL and 2.5 ng/mL for BHPT and BHMPT; 12.5 ng/mL and 25 ng/mL for PT and 5 ng/mL and 12.5 ng/mL for MPT, NEAMP, DMAMP, 4MMAMP, NEBMPEA and NNBTPEA, respectively. An analytical method characterized by rapidity, simplicity and cost-effectiveness was implemented for the chromatographic separation of stimulant structural analogues, facilitating the determination of potential stimulant abuse within human performance sports.

Keywords:  Dilute and shoot; Doping, Stimulants; Solid-phase extraction; Structural analogues; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jpba.2025.117328
J Mass Spectrom. 2026 Feb;61(2): e70022

Demonstrating Voxel-by-Voxel (V × V) Single-Point Calibration in Whole-Body Zebrafish by IR-MALDESI Quantitative MSI.

Alena N Joignant, Stephen J Vega, David C Muddiman.

  This work describes a quantitative mass spectrometry imaging (qMSI) method comparison for the absolute quantification of arachidonic acid (AA) in whole-body zebrafish using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). Whole-body zebrafish are structurally heterogeneous samples that are complex to analyze by many qMSI methods largely due to practical sample preparation limitations. However, the multi-organ quantification is valuable in zebrafish, especially for lipid-related investigations. In the standard workflow, the ion abundance of AA was normalized to a structural analogue that was sprayed on the slide before mounting the tissue. A series of calibration spots of stable isotope label (SIL) deuterated AA were spotted onto tissue to construct a calibration curve and subsequently calculate the concentration of endogenous AA in the tissue sections. The calculated values of AA using this method provided values significantly lower than the literature. In the subsequent workflow, the structural analogue was considered in a voxel-by-voxel (V × V) calculation of the concentration of AA in the tissues resulting in an AA concentration similar to the literature within whole-body zebrafish. The V × V method proved simpler in sample preparation, and more accurately quantified AA. This implies the potential utility of single-point V × V calibration for the qMSI of highly heterogeneous tissues.

Keywords:  IR‐MALDESI; mass spectrometry imaging; quantification; voxel‐by‐voxel calibration

DOI:  https://doi.org/10.1002/jms.70022
Anal Chim Acta. 2026 Feb 08. pii: S0003-2670(25)01421-7. [Epub ahead of print]1386 345027

Aqueous LC-MS/MS quantification of α-/β-nicotinamide mononucleotide in dietary supplements using a pentabromophenyl column.

Chng Sze Hoei, Nian-Hua Wu, Chao-Ming Tsen, Ping-Wei Sun, Han-Wei Chang, Shin-Yuan Wang, Hung-Yu Lin.

  In this study, we developed an LC-MS/MS method that enables baseline separation and quantification of α- and β-nicotinamide mononucleotide (NMN) isomers under fully aqueous reversed-phase conditions. At first, three commonly used reversed-phase columns (Polar C18, F5, and PBr) were evaluated, and only the PBr phase provided sufficient stereoselectivity through enhanced π-π stacking, dipole-dipole interactions, and halogen bonding. Using 0.1 % formic acid in water as the mobile phase, the two isomers were baseline resolved within 3 min. The method was validated for linearity, sensitivity, precision, accuracy, and matrix effects. When applied to eight commercial NMN supplements, the analysis revealed substantial variation in isomer composition, including undeclared or inaccurately labeled ingredients. Overall, this work presents the first LC-MS/MS approach capable of isomer-specific NMN analysis under fully aqueous reversed-phase conditions, offering a robust and environmentally compatible platform for product authentication, quality control, and potential applications in pharmacokinetics and metabolomics.

Keywords:  Green chromatography; LC-MS/MS; Nicotinamide mononucleotide (NMN); Pentabromophenyl column (PBr); α-/β-isomers

DOI:  https://doi.org/10.1016/j.aca.2025.345027
J Anal Toxicol. 2026 Jan 10. pii: bkag004. [Epub ahead of print]

Validation of a Microsampling-Compatible Liquid-Liquid Extraction Method for Cannabinoid Quantitation in 50 µL of Whole Blood using LC-MS/MS.

Aman A Mohammed, Mahmood Khan, Herbert Chan, Jeffrey R Brubacher.

  Recently developed dried blood analysis methods for cannabinoid quantitation utilize small blood volumes, making them microsampling-compatible, but are limited by hematocrit-related bias for dried blood spots (DBS) and higher consumable costs for volumetric absorptive microsampling (VAMS®). To address these issues, we developed a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of quantifying cannabinoids in 50 µL of liquid whole blood, providing a practical microsampling alternative to dried blood approaches. Using liquid-liquid extraction (LLE) with sodium hydroxide alkalinization and acetonitrile precipitation, followed by quantitative analysis on an Agilent 6495 liquid chromatography-triple quadrupole (LC-TQ) mass spectrometer, we achieved lower limits of quantitation (LLOQs) of 0.10 ng/mL for Δ9-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabigerol (CBG), 0.20 ng/mL for cannabidiol (CBD), 0.50 ng/mL for 11-hydroxy-THC (11-OH-THC), and 1.0 ng/mL for 11-nor-9-carboxy-THC (THC-COOH). Calibration was linear from the LLOQ to 300 ng/mL for all analytes. To our knowledge, this is one of the first validated LLE approaches for cannabinoid quantitation in less than 100 µL of liquid whole blood. Further, it achieves sub-ng/mL sensitivity, exceeding the LLOQs of most published methods which require ≥100 µL of whole blood. We anticipate particular utility for our method in obtaining evidence from suspected impaired drivers at the roadside when paired with capillary microsampling, such as via finger prick. This approach enables measurement of THC levels at the time of driving, thereby overcoming current limitations, including the decrease in THC levels that occurs with delayed blood sampling, requirement for larger sample volumes (≥100 µL), and dependence on trained phlebotomists for venipuncture.

Keywords:  cannabinoids; liquid chromatography-tandem mass spectrometry (LC-MS/MS); microsampling; whole blood; Δ9-tetrahydrocannabinol (THC)

DOI:  https://doi.org/10.1093/jat/bkag004
bioRxiv. 2026 Jan 08. pii: 2026.01.07.698252. [Epub ahead of print]

Synthesis-driven reverse metabolomics reveals 3-hydroxy N -acyl amides as gut microbial molecules.

Victoria Deleray, Vincent Charron-Lamoureux, Kyle Vittali, Helena Mannochio-Russo, Crystal X Wang, Corinn Walker, Karsten Zengler, Dionicio Siegel, Pieter C Dorrestein.

3-hydroxy N -acyl amides are bioactive lipids with reported anti-obesity and glucose-regulating effects, yet they are rarely detected in untargeted metabolomics studies because they are largely absent from existing spectral reference libraries. To address this gap, we synthesized an MS2 spectral resource comprising 436 structurally diverse 3-hydroxy N -acyl amides, spanning 3- to 18-carbon chains with a wide range of amine headgroups such as ornithine, valine, and dopamine. Using a synthesis-driven reverse metabolomics approach, we found 161,626 spectral matches across 54,744 publicly available files in untargeted metabolomics datasets revealing widespread occurrences in biological samples, including human-derived specimens. Of these molecules detected through MS2 spectral matching, 334 represent newly reported biological entities. We further confirmed their presence in human saliva, stool, and skin using retention time and ion mobility measurements. Frequent detection in microbial datasets and validation in communities of human-derived gut bacteria support microbial production. Several metabolites also showed altered abundance in individuals with diabetes mellitus, showing that this lipid class is modulated in human metabolic disease. Together, these findings establish 3-hydroxy N -acyl amides as a distinct and biologically relevant lipid class, and the accompanying MS2 spectral resource will enable their broader recognition and study in untargeted metabolomics data.

DOI: https://doi.org/10.64898/2026.01.07.698252
Anal Chim Acta. 2026 Feb 08. pii: S0003-2670(25)01328-5. [Epub ahead of print]1386 344934

MALDI mass spectrometry and imaging for lipid analysis in food matrices: A review of analytical principles and perspectives.

Ana Jano, Adrián Fuente-Ballesteros, M Teresa Martín, Ana M Ares, José Bernal.

   BACKGROUND: Lipids play essential roles in food systems, and their study through lipidomics offers insights into biochemical processes and issues such as food fraud and adulteration. Among lipid classes, glycerolipids (GLs), particularly triacylglycerols (TAGs), are the most frequently analysed in food-related studies.
RESULTS: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and MALDI mass spectrometry imaging (MSI) have emerged as powerful tools for lipidomic investigations. While traditionally applied in biological research, their use in food science is expanding to diverse matrices, including oils, dairy, cereals, algae, and meat. In terms of MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) is most commonly used in positive ion mode, whereas 9-aminoacridine (9-AA) is typically employed in negative mode. MALDI-MS has advanced lipid characterization, and MALDI-MSI now enables mapping of lipid spatial distributions, revealing processes such as lipid degradation.
SIGNIFICANCE: This review underscores the growing utility of MALDI-MS and MALDI-MSI for lipid analysis in food science. We highlight their analytical capabilities, workflows, and limitations, it demonstrates how these techniques can enhance understanding of food composition, quality, and authenticity.

Keywords:  Food; Lipids; MALDI imaging; MALDI-MSI; MALDI-TOF MS; Mass spectrometry

DOI:  https://doi.org/10.1016/j.aca.2025.344934
Anal Chem. 2026 Jan 15.

DPM: A Deep Learning and Optimal Transport Framework for Cost-Effective Spatial Metabolomics.

Bo Yao, Longfeng Yang, Chi Zhang, Yuanyuan Lv, Chenkun Yang, Li-Jun Di, Jie Luo, Shu-Hai Lin.

Mass spectrometry imaging (MSI) is a powerful technology in spatial metabolomics that enables the in situ detection and distribution analysis of metabolites in tissue sections. However, the high cost associated with high-resolution and multislice MSI acquisition remains a major limitation. Here, we introduce DeepPathMetabol (DPM), a deep learning-enhanced framework based on optimal transport theory, which accurately predicts spatial metabolite distributions in an MSI section using data from an adjacent section through an optimized mapping strategy. DPM achieves superior alignment and prediction accuracy, outperforming conventional feature similarity-based methods such as those using Euclidean or kernel-based metrics both with and without spatial distance weighting. We further demonstrated that the DPM framework can effectively enhance MSI resolution, providing a powerful tool for cost-effective and high-precision spatial metabolomics research. This approach also shows promising potential for extension to spatial transcriptomics. Collectively, our work establishes histology-facilitated MSI-to-MSI prediction as a versatile strategy for spatial biology research. DPM is open-source and available at https://github.com/LinShuhaiLAB/DeepPathMetabol.

DOI: https://doi.org/10.1021/acs.analchem.5c06903
Biomed Chromatogr. 2026 Feb;40(2): e70335

Analytical Techniques for the Quantification of Pravastatin and Its Metabolites in Various Matrices: A Comprehensive Review.

Hemn A H Barzani, Rebaz Anwar Omer, Khalamala Ibrahim Salih Barzani, Zanco Hassan Jawhar, Seerwan Hamadameen Sulaiman, Hoshyar Saadi Ali.

  Pravastatin (PRV), a highly hydrophilic statin used to treat hypercholesterolemia and prevent cardiovascular disease, presents certain analytical challenges due to its low bioavailability, tendency to lactonize, and potentially complex metabolism. This review aims to consolidate and critically evaluate the analytical procedures developed for the determination of PRV and its metabolites in pharmaceutical and biological matrices. This review aims to comprehensively summarize four decades of analytical methodologies developed for PRV and its metabolites, comparing chromatographic, spectroscopic, electrochemical, and capillary electrophoresis approaches in terms of sensitivity, selectivity, and matrix applicability. This review also evaluates essential bioanalytical nuances, including PRV's lactone-acid interconversion, matrix-induced ion suppression, pH-dependent instability, low ng-pg·mL-1 plasma levels, and metabolite-specific detection challenges alongside pharmaceutical analytical considerations. The literature demonstrates that liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography remain the most sensitive and reliable platforms for ultratrace bioanalysis, while spectrophotometric and electrochemical methods provide cost-effective alternatives for formulations and higher concentration samples, and capillary electrophoresis offers efficient separation with low solvent use. Future analytical advances should prioritize metabolite-resolved quantification, green extraction strategies, microfluidic and point-of-care designs, and AI-assisted data processing to improve sensitivity, stability assessment, and high-throughput monitoring of PRV in clinical and environmental settings.

Keywords:  HPLC; bioanalytical methods; capillary electrophoresis; electrochemical detection; matrix effects; pharmaceutical quality control; pravastatin; statins

DOI:  https://doi.org/10.1002/bmc.70335
Nat Commun. 2026 Jan 17.

TidyMass2: advancing LC-MS untargeted metabolomics through metabolite origin inference and metabolic feature-based functional module analysis.

Xiao Wang, Yijiang Liu, Chao Jiang, Zinuo Huang, Hong Yan, Sunny H Wong, Caroline H Johnson, Jingxiang Zhang, Yifei Ge, Feifan Zhang, Junli Zhang, Renfu Lai, Peng Gao, Xuebin Zhang, Xiaotao Shen.

Untargeted metabolomics provides a direct window into biochemical activities but faces critical challenges in determining metabolite origins and interpreting unannotated metabolic features. Here, we present TidyMass2, an enhanced computational framework for Liquid Chromatography-Mass Spectrometry (LC-MS) untargeted metabolomics that addresses these limitations. TidyMass2 introduces three major innovations compared to its predecessor, TidyMass: (1) a comprehensive metabolite origin inference capability that traces metabolites to human, microbial, dietary, pharmaceutical, and environmental sources through integration of 11 metabolite databases containing 532,488 metabolites with source information; (2) a metabolic feature-based functional module analysis approach that bypasses the annotation bottleneck by leveraging metabolic network topology to extract biological insights from unannotated metabolic features; and (3) a graphical interface that makes advanced metabolomics analyses accessible to researchers without programming expertise. Applied to longitudinal urine metabolomics data from human pregnancy, TidyMass2 identified diverse metabolites originating from human, microbiome, and environment, and uncovered 27 dysregulated metabolic modules. It increased the proportion of biologically interpretable metabolic features from 5.8% to 58.8%, revealing coordinated changes in steroid hormone biosynthesis, carbohydrate metabolism, and amino acid processing. By expanding biological interpretation beyond MS2 spectra-based annotated metabolites, TidyMass2 enables more comprehensive metabolic phenotyping while upholding open-source principles of reproducibility, traceability, and transparency.

DOI: https://doi.org/10.1038/s41467-026-68464-7
Anal Chim Acta. 2026 Feb 01. pii: S0003-2670(25)01388-1. [Epub ahead of print]1385 344994

Development of a high-throughput LLE-UPLC-QqQ-MS/MS method and comprehensive profiling of urinary steroid hormones in Chinese elderly adults.

Xiao Ma, Huimin Ren, Li Zhao, Xiaojie Guo, Jiran Zhang, Juan Liu, Xiaona Wang, Mengyao Wang, Yifu Lu, Shilu Tong, Hongyang Cui, Hong Chang, Yu'e Cha, Song Tang, Xiaoming Shi.

   BACKGROUND: Steroid hormones (SHs) in human urine are critical for understanding physiological processes, but their analysis is challenged by intricate urine matrices and diverse SHs physicochemical properties. Existing methods often lack sensitivity, throughput, or comprehensiveness for simultaneous qualitative and quantitative profiling. Thus, there is a need for a state-of-the-art, ultra-sensitive, high-throughput method to comprehensively and simultaneously characterize urinary SHs.
RESULTS: The improved liquid-liquid extraction (LLE) and ultra-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) method, developed by optimizing mass spectrum parameters and pretreatment conditions to address urine matrix complexity and SHs diversity, identified 133 SHs and quantified 103. The improved experimental protocol was as follows: sample volume, 1 mL; extraction solvent, methyl tert-butyl ether/ethyl acetate (MTBE/EtAc, 1/1, v/v); solvent volume, 2 mL; extraction times, 2; pH, 9; enzyme activity, 1700 U/mL; eluent A, water; eluent B, methanol. Post-extraction samples were analyzed using UPLC-QqQ-MS/MS, with quantification of method-validated SHs reliably performed through internal standards (ISs) calibration. Validated metrics included excellent linearity (R2 > 0.990, 1-500 ng/mL), the method detection/quantitation limit (MDL, 0.003-0.740 ng/mL; MQL, 0.01-2.465 ng/mL), matrix effects (ME, -0.46 %-46.56 %), recoveries (72.31 %-129.80 %), and precision/stability (RSDs <20 %). Applied to 76 elderly adults, it revealed gender-specific temporal variations, with significant 5-month declines in androgens and glucocorticoids.
SIGNIFICANCE: This method enables comprehensive, sensitive urinary SHs profiling, offering a powerful tool for in-depth research on elderly hormonal dynamics. Its ability to capture gender and temporal variations enhances understanding of age-related hormonal changes.

Keywords:  High-throughput simultaneous analysis; Human urine; LLE; Steroid hormones; UPLC-QqQ-MS/MS

DOI:  https://doi.org/10.1016/j.aca.2025.344994
Crit Rev Anal Chem. 2026 Jan 11. 1-25

Evolving Methodologies for the Analytical Determination of Metoclopramide.

Hemn A H Barzani, Rebaz Anwar Omer, Khalamala Ibrahim Salih Barzani, Zanco Hassan Jawhar, Seerwan Hamadameen Sulaiman.

  Metoclopramide (MCP) is a commonly prescribed prokinetic and antiemetic agent used to manage gastrointestinal motility disorders as well as nausea and vomiting. Given its extensive clinical use and the risk of dose-related adverse effects, reliable quantitative determination of MCP in pharmaceutical formulations and biological matrices is crucial for quality control, pharmacokinetic evaluation, and therapeutic monitoring. This review compiles English-language studies published over the past 25 years, retrieved from major scientific databases including Scopus, Web of Science, ScienceDirect, Google Scholar, and PubMed. It provides a critical overview of analytical techniques applied to MCP determination, focusing on chromatographic, spectrometric, and electroanalytical methods. High-performance liquid chromatography (HPLC) remains the most widely used approach because of its robustness, accuracy, and suitability for diverse sample matrices. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers superior sensitivity, achieving detection limits in the pg-ng/mL range, and is particularly valuable for advanced bioanalytical studies. Although less sensitive, HPLC-UV methods are still favored for routine pharmaceutical quality control due to their simplicity and cost-effectiveness. Spectrometric and electrochemical techniques, especially those employing nanostructured electrodes, present promising alternatives. Nonetheless, challenges such as MCP instability, matrix interferences, and cost-performance trade-offs remain, highlighting the need for robust, affordable, and portable analytical strategies.

Keywords:  Metoclopramide; analytical methods; chromatography; electrochemical analysis; spectroscopy

DOI:  https://doi.org/10.1080/10408347.2025.2612234
Analyst. 2026 Jan 16.

AminoacidDB: a liquid chromatography-tandem mass spectrometry-based toolkit for the untargeted analysis of non-protein amino acids.

Pawanjit K Sandhu, Ryland T Giebelhaus, Ryan Hayward, Tingting Zhao, Alix Tucker, Daniel Gaudet, Tao Huan, Susan J Murch.

Non-protein amino acids (npAAs) are produced by microbes, plants and humans, with previous estimates suggesting that there are ≈1000 of such metabolites. Most of the npAAs were discovered as human toxins, intermediates in metabolism and byproducts of organic and pharmaceutical synthesis. We used a text-mining approach to identify chemicals with the NHx-R-COOH moiety in PubChem and cross-checked those for classification against amino acid databases including Web of Science, LOTUS and HMDB to generate a dataset of compounds, which was cleaned and curated, resulting in a library of 332,154 amino acids. We established a standard set of 41 npAAs, selected to cover a wide array of structural and isomeric space for training the machine learning model and predicting chromatography elution using the Retip tool. Derivatization added a 6-aminoquinoline (6-AMQ) tag to the N[H] group, thus selecting amine-carrying compounds from the sample extract, which can be identified by cleaving the 6-AMQ carbonyl and producing the common product ion of 171.0555 m/z in positive ionization mode to selectively target amino acids in unknown datasets. AminoacidDB (https://www.aminoacidDB.ca) annotates amino acids by matching the features of accurate mass and retention time from untargeted mass spectrometry datasets against the aminoacidDB library. In a proof-of-concept experiment, we putatively annotated 103 amino acids and their derivatives in Arabidopsis thaliana and Cannabis sativa leaf tissues. Our original data hypothesize a wider distribution of npAAs and peptides in plants than was previously known and indicate the need for more research to understand the prevalence and metabolism of npAAs.

DOI: https://doi.org/10.1039/d5an01248a
Food Res Int. 2026 Feb 28. pii: S0963-9969(25)02493-7. [Epub ahead of print]226 118153

Beyond the native state: Revolutionizing food phenolic acids profiling through advanced chemical derivatization and sample pretreatment (2003-2025).

Xiao-Feng Huang, Ying Xue, Yue-Cai Hou, Lin-Sen Qing.

  Phenolic acids, as key bioactive constituents abundantly present in plant-based foods, play a crucial role in evaluating nutritional quality, flavor characteristics, and health-promoting effects. However, their accurate analysis is severely constrained by high polarity, low volatility, structural diversity, and matrix interference inherent to complex food matrices. Chemical derivatization has emerged as an indispensable technique to overcome these challenges. Through covalent modification of reactive functional groups, properties such as volatility, thermal stability, chromatographic performance, and detection sensitivity are improved. Over the past two decades, notable innovations have been achieved, including novel derivatization reagents for GC, advanced strategies for LC-MS, and improved sample pretreatment methodologies. This review systematically consolidates progress in derivatization-based analysis of food phenolic acids since 2003. Key chromatographic platforms are critically evaluated, with emphasis on derivatization mechanisms, such as silylation, esterification, and amidation, along with their practical implementations. Sample pretreatment techniques aimed at matrix cleanup and analyte enrichment are also comprehensively examined. Furthermore, current methodological limitations are discussed, and future directions are proposed, indicating trends toward miniaturization, automation, high-throughput capacity, metabolic research, and greener analytical principles. The overarching objective is to establish a robust scientific and technical foundation for sensitive and accurate phenolic acid analysis in food science and quality control.

Keywords:  Analytical methodology; Chemical derivatization; Food analysis; Phenolic acids; Sample pretreatment

DOI:  https://doi.org/10.1016/j.foodres.2025.118153
J Chromatogr A. 2026 Jan 10. pii: S0021-9673(26)00034-8. [Epub ahead of print]1769 466703

Development of an arrow solid-phase microextraction GC-MS/MS method for the determination of 69 odorants in water.

FeiFei Dai, Ruoyu Zhang, Dandan Nie, Shuqing Ma, Yurong Qin.

  A quantitative method for determining 69 odorants in water was developed using arrow-type headspace solid-phase microextraction (SPME) coupled with gas chromatography-tandem mass spectrometry (GC-MS/MS). Key extraction parameters, including extraction and desorption temperatures and times, NaCl addition, fiber type, and rotation speed, were systematically optimized. Under the optimized conditions, target compounds were efficiently enriched, separated on a polar chromatographic column, detected in selected reaction monitoring (SRM) mode, and quantified using external calibration. The method demonstrated high sensitivity, with limits of detection (LODs) ranging from 0.015 to 75 ng/L, and 55 compounds exhibiting LODs below 10 ng/L. All analytes showed excellent linearity (R2 > 0.995) within their respective ranges. Precision was satisfactory, with relative standard deviations (RSDs) between 1.10 % and 17.85 %, including 59 compounds with RSDs <10 %. Average recoveries at three spiking levels ranged from 80 % to 120 %. This automated and environmentally friendly method offers high sensitivity and accuracy, meeting the analytical requirements for complex odorants in water.

Keywords:  Gas chromatography-tandem mass spectrometry; Odor substances; SPME arrow; Water

DOI:  https://doi.org/10.1016/j.chroma.2026.466703
Anal Methods. 2026 Jan 12.

A highly sensitive LC-MS/MS method for quantification of AB-38b in rat plasma, liver and kidney tissues: application to a pharmacokinetic study.

Dandan Pan, Libang Chen, Haonan Luo, Haiyan Liu, Weihao Chen, Zhenzhou Jiang, Zhen Qiu, Nan Zhou, Yequan Zhou, Qiujin Xu, Tao Wang, Qian Lu, Tingting Yang.

Our research team developed AB-38b, a biphenyl diester derivative containing α,β-unsaturated carbonyl groups, inspired by investigations into the pharmacological prevention and pathophysiology of diabetic kidney disease (DKD). We have completed the synthesis and pharmacodynamic evaluation of AB-38b in preclinical DKD studies. For pharmacokinetic analysis, we established a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for quantifying AB-38b in rat plasma, as well as in liver and kidney tissues. Sample preparation involved protein precipitation using methanol, with tolvaptan employed as the internal standard (IS). Chromatographic separation was achieved on a Shim-pack VP-ODS C18 column (2.0 × 150 mm, 5 µm), with mobile phase A (5 mM ammonium acetate) and mobile phase B (methanol) under gradient elution at a flow rate of 0.2 mL min-1. AB-38b and the IS were detected and quantified using positive electrospray ionization in multiple reaction monitoring (MRM) mode at transitions of m/z 705.30 → 618.20 for AB-38b and m/z 449.30 → 252.20 for the IS. The method demonstrated excellent linearity, stability, accuracy, precision, recovery, and a non-significant matrix effect within the validated range. A one-compartment model with rapid absorption and distribution, quicker elimination, and clear tissue distribution was demonstrated by the mean blood concentration-time curve of AB-38b in rat plasma. The liver tissue contained high levels of AB-38b, with no evidence of tissue accumulation observed. These findings offer a reference for further research into the pharmacological mechanisms and potential therapeutic applications of AB-38b in metabolically related diseases, especially DKD.

DOI: https://doi.org/10.1039/d5ay01136a
Int J Anal Chem. 2026 ;2026 4144078

Determination and Pharmacokinetic of Peiminine in Beagle Dogs by UPLC-MS/MS.

Qian Cheng, Xinyu Peng, Xiaotong Li, Xueying Jia.

  This study centered on creating and validating a UPLC-MS/MS assay that is both reliable and simple, making it suitable for measuring peiminine levels in the plasma of beagle dogs. The established method was then employed with the ultimate goal of elucidating the pharmacokinetic behavior of the compound. The Acquity UPLC BEH C18 chromatographic column was used for separating peiminine and camptothecin (internal standard, ISTD). A binary mobile phase consisting of acetonitrile and 0.1% formic acid in water was used for gradient elution at 0.4 mL/min. In the multireaction monitoring mode, peiminine and a triple quadrupole mass spectrometer with an electrospray ionization source were utilized to monitor peiminine and the ISTD, and detection was performed by monitoring the following transitions: m/z 430.28 ⟶ 412.25 for peiminine and m/z 349.03 ⟶ 305.09 for the ISTD. Results indicated that the accuracy was around 100%, with both interday precision and intraday (RSD) being less than 10.37%. Additionally, a linear response for peiminine was validated over the range of 1-200 ng/mL, with the LLOQ established at 1 ng/mL. In summary, this study perfectly combined the ultrahigh chromatographic separation ability with the ultrahigh sensitivity, selectivity, and structural analysis ability of mass spectrometry, achieving rapid (2 min), accurate, and ultrasensitive (LLOQ 1 ng/mL) analysis of peiminine in samples. Using the developed method, the pharmacokinetic profile of peiminine was successfully characterized in beagle dogs following oral administration.

Keywords:  UPLC–MS/MS; beagle dog; peiminine; pharmacokinetics

DOI:  https://doi.org/10.1155/ianc/4144078
J Sep Sci. 2026 Jan;49(1): e70343

A Polymeric Zwitterionic Hydrophilic Probe for Mapping the Human Serum Endogenous Glycopeptidome.

Muhammad Salman Sajid, Shafaq Saleem, Habtom W Ressom.

  Endogenous glycopeptides in human serum are valuable candidates for disease biomarker discovery, but their low abundance and structural complexity pose significant analytical challenges. In this study, we developed and optimized a robust workflow for enriching and in-depth characterizing the endogenous glycopeptidome using a novel hydrophilic interaction liquid chromatography (HILIC) sorbent. The HILIC-based strategy effectively enriched glycopeptides from mono-glycosylated IgG and avidin digests, as well as multi-glycosylated horseradish peroxidase (HRP), demonstrating high selectivity, femtomolar-level sensitivity (down to 1 fmol), reproducibility (RSD > 1), and reusability for up to four cycles. Comprehensive analysis by mass spectrometry (MS) identified 334 endogenous intact N-linked glycopeptides, including 318 N-glycosites from 289 glycoproteins. In addition, 242 endogenous intact O-linked glycopeptides from 38 glycoproteins are identified. These represent one of the most extensive endogenous glycopeptide datasets to date. N-glycans were primarily complex biantennary types, while Core 1 structures dominated O-glycans. Motif analysis revealed a strong preference for canonical N-glycosylation motifs (NXT/NXS) and a nearly equal distribution of O-glycosylation on serine and threonine residues. Gene Ontology analysis indicated functional enrichment in membrane-associated, catalytic, and immune-related processes. This optimized HILIC-MS platform enables sensitive and reproducible glycoproteomic profiling, providing a valuable tool for biomarker discovery.

Keywords:  endogenous glycopeptidome; enrichment; human serum; mass spectrometry; polymeric zwitterionic hydrophilic probe

DOI:  https://doi.org/10.1002/jssc.70343
Ecotoxicol Environ Saf. 2026 Jan 10. pii: S0147-6513(26)00027-8. [Epub ahead of print]309 119698

Quantification of phthalate and DINCH metabolites in human urine and maternal breast milk: Assessing maternal body burden and infant exposure.

Agneta A Runkel, Žan Rekar, Neja Kosirnik, Darja Mazej, Milena Horvat, Janja Snoj Tratnik, Tina Kosjek.

  Based on toxicological evidence, human exposure to phthalates (PHs) and diisononylcyclohexane-1,2-dicarboxylate (DINCH) may contribute to adverse health effects, especially during vulnerable developmental stages. To support the exposure assessment for this group of endocrine disruptors, we developed and validated a method for the analysis of 14 PH and 3 DINCH metabolites in human urine and maternal milk, applied the method in a pilot study, and identified crucial obstacles in the path of establishing maternal milk as a routine matrix in human biomonitoring. Urine and milk samples were extracted with solid-phase extraction (SPE) and QuEChERS salts, respectively, and analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method accuracy was confirmed for urine samples via a certified standard reference material and the G-EQUAS intercomparison programme. We identified a need for sampling protocols, reference materials, and external method verification schemes in order to establish maternal milk as a routine matrix. Finally, the method was tested for its applicability in a pilot biomonitoring study on 30 paired urine and milk samples from lactating mothers, with medians ranging from <LLOQ - 15 µg/L in urine and <LLOQ - 16 µg/L in maternal milk and generally higher detection rates in urine. Furthermore, the results indicate extensive monoester formation under cooled storage conditions, resulting in potentially high infantile exposure to phthalate monoesters for which, to date, no guidance values exist despite their demonstrated toxicity.

Keywords:  DINCH; Human biomonitoring; LC-MS/MS; Maternal milk; Phthalate metabolites; Urine

DOI:  https://doi.org/10.1016/j.ecoenv.2026.119698
J Pharm Biomed Anal. 2026 Jan 08. pii: S0731-7085(26)00018-X. [Epub ahead of print]272 117350

Ease of analysis through unification: One gas chromatographic method for the chemical profiling of essential oils.

Dominik Kresnik, Bryan Bajor, Christian Steuer.

  The rising interest in essential oils (EOs) for their therapeutic, antibacterial, and antifungal properties has led to an increasing demand for high-quality products in both medicinal and industrial sectors. To meet these stringent quality requirements, robust, precise, and efficient analytical methods are essential. Gas chromatography (GC) remains the gold standard for EO analysis due to its sensitivity and resolution. Although numerous methods are available -primarily targeting similar analytes in varying combinations standardization remains a challenge, with protocols differing across ISO guidelines and international pharmacopoeias. In this study, Design of Experiments (DoE) was employed to optimize and harmonize existing GC methods, focusing on sample preparation and chromatographic parameters. A polar GC column (60 m length, 0.25 mm inner diameter, 0.25 µm film thickness) was selected for its ability to effectively separate 87 terpenes, sesquiterpenes, and related compounds commonly found in EOs. The optimized temperature gradient enabled complete separation within a 75-minute runtime, outperforming or matching existing methods in terms of resolution and reproducibility. Streamlined sample preparation protocols led to reduced solvent consumption and minimized sample requirements across all tested EOs. As a proof of concept, the final method was applied to 12 different essential oils, demonstrating comparable analytical performance and confirming its broad applicability and efficiency.

Keywords:  Design of Experiments; Essential oils; Gas chromatography; Method development; Sample preparation; Terpenes

DOI:  https://doi.org/10.1016/j.jpba.2026.117350
Anal Chim Acta. 2026 Feb 01. pii: S0003-2670(25)01406-0. [Epub ahead of print]1385 345012

Preparation of ultra-high efficiency sulfoalkyl betaine-type monolithic columns through amine-epoxide ring-opening reaction combined with in-situ ring-opening quaternization and their application in hydrophilic interaction chromatography.

Ye Chen, Zihui Yang, Xiyue Xiong, Feng Chang, Yingzhuang Chen, Ming Ma, Bo Chen.

   BACKGROUND: The separation of highly polar and ionic compounds remains a significant challenge in hydrophilic interaction chromatography (HILIC). While zwitterionic monolithic columns are promising, their preparation often involves complex, multi-step synthesis procedures or relies on monomers that are difficult to obtain, limiting their wider application and reproducibility. This study aimed to develop a novel, simplified, and highly efficient method for synthesizing a sulfoalkyl betaine-type zwitterionic monolithic column for high-performance separation of diverse polar analytes.
RESULTS: A novel sulfoalkyl betaine-type monolithic column was successfully synthesized via a simplified two-step, single-vessel method combining amine-epoxide ring-opening polymerization with in-situ ring-opening quaternization. Characterization confirmed the material possesses a homogenous, bicontinuous porous structure and zwitterionic surface properties. The column demonstrated exceptional separation performance for a wide range of polar analytes, including nucleosides, phenols, aromatic acids, amides, and bases, as well as complex BSA tryptic digests, operating under a mixed-mode retention mechanism. Critically, a "peak compression" phenomenon was observed, leading to ultra-high column efficiencies for specific analytes such as thymidine, pyrocatechol, guanine, and notably 2,5-dihydroxybenzoic acid, which achieved an efficiency of 519,350 plates/m. Systematic investigation revealed that aprotic solvents promote this phenomenon, while protic solvents inhibit it, and its effective implementation depends on the synergy of stationary phase characteristics, mobile phase composition, and appropriate additives.
SIGNIFICANCE: This work introduces a facile and robust strategy for fabricating high-performance zwitterionic monolithic columns, simplifying a previously complex process. The major contribution is the first systematic elucidation of the peak compression phenomenon in HILIC mode, identifying the key parameters that control it. This finding provides a new avenue for achieving ultra-high separation efficiency and detection sensitivity, holding significant potential for advancing the analysis of complex biological samples, biomarker discovery, and other challenging applications in hydrophilic interaction chromatography.

Keywords:  Amine-epoxide ring-opening; Hydrophilic interaction chromatography; Peak compression phenomenon; Polar analytes; Ring-opening quaternization; Sulfoalkyl betaine-type monolithic column

DOI:  https://doi.org/10.1016/j.aca.2025.345012
Nat Prod Bioprospect. 2026 Jan 11. 16(1): 16

Enhancing detection of low‑abundance metabolites in proton NMR through band‑selective suppression and presaturation.

Upendra Singh, Renad Z Al Ahmadi, Ruba Al-Nemi, Manel Dhahri, Mohammed S Alarawi, Abdul Aziz, Faisal Abdulaziz Bushulaybi, Tamer Abdalla Mashtoly, Abdul-Hamid Emwas, Lukasz Jaremko, Mariusz Jaremko.

  Metabolomics provides powerful means to analyze metabolite profiles in biological samples, enabling insights into biochemical changes under genetic, environmental, or pathological conditions. Nuclear Magnetic Resonance (NMR) spectroscopy is central to metabolomics, but its utility is often constrained by the strong and overlapping resonances of abundant components, such as sugars in plant‑ and food‑derived materials, which obscure signals of lower‑abundance metabolites. Here, we introduce a modified NMR acquisition method that increases sensitivity and specificity by selectively suppressing dominant signals, while enhancing weaker metabolite signals across the spectrum. The method integrates water presaturation with excitation sculpting (ES), yielding a robust 1D presat‑1H‑ES pulse sequence. Validation on a range of sugar-rich samples demonstrated 2-fourfold signal enhancement for low‑abundance metabolites compared with conventional 1H‑ES. Multivariate analyses show the method improves reproducibility and discrimination, enabling detection and comparison of low‑abundance metabolites not accessible with conventional approaches'. Beyond sugar‑rich systems, the method is broadly applicable to other spectral regions where dominant metabolite classes obscure lower‑concentration compounds, including primary metabolites and structurally diverse natural products. Overall, the 1D presat‑1H‑ES significantly enhances resolution and sensitivity of NMR‑based metabolomics, shortens analysis time, and supports more precise profiling for both fundamental studies and translational applications in metabolomics and natural‑products discovery.

Keywords:  Band-selective inversion; Metabolomics; NMR spectroscopy; Signal-enhancement; Sugars

DOI:  https://doi.org/10.1007/s13659-025-00570-3
PeerJ. 2026 ;14 e20592

Non-targeted metabolomic profiling of Cremastra appendiculata providing insights for phytochemical analyses.

Rui Guan, Yuxin Shan, Hamizah Shahirah Hamezah, Somnuk Bunsupa, Hong To Quyen Duong, Yadong Zhou, Rongchun Han, Xiaohui Tong.

   Background: Cremastra appendiculata (D. Don) Makino, known as "Shan Cigu" in China, is a valuable medicinal plant historically employed for its antibacterial and anti-inflammatory properties. However, its comprehensive metabolome remains underexplored, hindering the establishment of standardized quality control.
Methods: In this study, a non-targeted metabolomics approach based on the Thermo Fisher Orbitrap Exploris 120 LC-MS (Liquid Chromatography-Mass Spectrometry) platform was employed to systematically profile the metabolites of C. appendiculata.
Results: A total of 174 compounds were annotated through a dual-validation workflow integrating Compound Discoverer 3.3 and manual tandem mass spectrometry spectral verification. Orthogonal partial least squares discriminant analysis prioritized 30 candidate quality markers, of which, six were further validated through network pharmacology-based bioactivity screening. Hierarchical clustering analysis revealed distinct metabolic patterns across the different tissues (roots, pseudobulbs, and leaves), establishing a tissue-specific chemical atlas. The integration of chemometric, network pharmacological, and chemotaxonomic analyses resulted in a robust, molecularly guided quality control framework, providing novel insights for phytochemical research and medical applications of C. appendiculata.

Keywords:  Cremastra appendiculata; Quality markers; Tissue-specific profiling; Untargeted metabolomics

DOI:  https://doi.org/10.7717/peerj.20592
J Agric Food Chem. 2026 Jan 12.

Robust High-Performance Liquid Chromatography and Liquid Chromatography-Tandem Mass Spectrometry Methods for the Determination of Malondialdehyde in Biological Samples Using Thiobarbituric Acid Derivatization and Column Cleaning with High-Concentration Ammonium Acetate.

Miyu Ujihara, Tomoko Asai, Kenji Sato.

  Malondialdehyde (MDA) is a widely used biomarker of lipid peroxidation and is commonly quantified as a red chromophore formed by the reaction with thiobarbituric acid (TBA). However, we found that previously unrecognized byproducts generated during the TBA reaction gradually accumulated on high-performance liquid chromatography (HPLC) columns, leading to poor elution and unstable retention of the TBA derivative of MDA. To address this issue, we developed a highly sensitive and reproducible HPLC-based method that incorporates a column-cleaning step by the injection of 1 M ammonium acetate during column washing, which effectively removes the retained byproducts and restores consistent chromatographic performance. As a result, MDA could be reliably quantified at concentrations as low as 1 μM by HPLC and 10 nM by liquid chromatography-tandem mass spectrometry, respectively. The method was successfully applied to detect elevated MDA levels in the blood and kidneys of mice after oral administration of fructose at a rate of 2 g per kg body weight.

Keywords:  LC−MS/MS; TBARS; fructose; malondialdehyde; oxidative stress

DOI:  https://doi.org/10.1021/acs.jafc.5c10557