bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2026–02–08
37 papers selected by
Sofia Costa, Matterworks
  1. Metabolomics Analysis of Trypanosomes Using Ion Mobility-Enhanced Mass Spectrometry.
  2. [Development of an LC-MS/MS method for 32 steroid hormones and exploration of their gestational variations].
  3. A candidate reference measurement procedure for aldosterone measurement in human serum and urine on the basis of isotope dilution liquid chromatography-tandem mass spectrometry.
  4. Double derivatization-enhanced LC-MS/MS for isomer-resolved identification and quantification of fatty acids.
  5. Development and validation of a sensitive and rapid UHPLC-MS/MS method for the simultaneous quantification of CG-0255 and its active metabolite in human plasma and its application to Phase I studies.
  6. Box-Behnken optimized salting-out assisted liquid-liquid extraction coupled with LC-MS/MS for sustainable amisulpride quantification in human plasma.
  7. Simultaneous quantification of fourteen urinary tobacco biomarkers via UHPLC-Q-Orbitrap HRMS and isotope dilution.
  8. Desorption Electrospray Ionization-Mass Spectrometry Imaging (DESI-MSI) of Metabolites in Heart Samples Infected with Trypanosoma cruzi.
  9. Development and Validation of an LC-MS/MS Method for the Quantitative Estimation of Bexicaserin in Human Cerebrospinal Fluid.
  10. Overcoming challenges in LC separation of oxysterols through guided design space modelling: A comparison of three stationary phases.
  11. Validated LC-MS/MS Method for Quantification and Pharmacokinetic Analysis of Talazoparib and Enzalutamide in Rats.
  12. MS/MS Mass Spectrometry Filtering Tree for Bile Acid Regio- and Stereoisomer Annotation.
  13. Development and validation of an UPLC-MS/MS method for simultaneous determination of meropenem and its open-ring metabolite in human serum and cerebrospinal fluid with application to clinical samples.
  14. Easy synthesis and analytical application of deuterated alkanesulfonates as internal standards for determination using mass spectrometry.
  15. A Highly Sensitive and High-Throughput Quantitative HILIC-MS/MS Method for Systematic Profiling of RNA Modifications.
  16. Comparability of Liquid Chromatography Tandem Mass Spectrometry Analysis of Dissolved Organic Matter across Laboratories.
  17. Determination of hydrocortisone and cortisone in artificial saliva by spray assisted fine droplet formation liquid phase microextraction coupled to LC-MS/MS.
  18. Liquid chromatography-tandem mass spectrometry-based urinary steroid profiling applied to primary aldosteronism diagnosis.
  19. Anabasine analysis in human plasma using liquid chromatography coupled to tandem mass spectrometry to Verify tobacco use: empirical challenges and limitations.
  20. Rapid quantification of bile acids in serum by LC-MS/MS and application to serum bile acid profile in voriconazole administered patients with invasive fungal infections.
  21. Dynamic multiple reaction monitoring for high throughput detection and quantitation of polycyclic aromatic compounds.
  22. The mystery of post-mortem Gamma-hydroxybutyrate formation - method development and validation for the detection of endogenous GHB and its related compounds.
  23. Efficient and Cost-Effective Enzyme Deposition onto Tissues for Mass Spectrometry Imaging of N-Glycans Using a Mini-Humidifier.
  24. Analytical validation of an advanced U-HPLC-MS/MS method for lactose detection in food supplements and pharmaceuticals.
  25. Low-Input Proteomics Protocols for Laser-Captured FFPE Samples with Orbitrap Astral DIA-MS.
  26. Determination of antigen components in inactivated SARS-CoV-2 vaccine using ultra-high-performance liquid chromatography tandem mass spectrometry.
  27. A high-throughput detection platform for dried blood spots constructed with DESI-MS/MS.
  28. Development of a chromatographic method for the analysis of risdiplam in serum extracts.
  29. Accurate quantification of quinolones in marine sediment by UPLC-MS/MS after ultrasonic extraction and automated SPE clean-up.
  30. Analytical Method Development and Validation of Treosulfan and Its Impurities by Ultra Performance Liquid Chromatography (UPLC) and Identification of Degradation Products by LC-MS/MS.
  31. Highly sensitive separation and characterization of multiple mycotoxins by supercritical fluid chromatography combined with ion mobility quadrupole time-of-flight mass spectrometry.
  32. Micro-QuEChERS extraction method optimization for quantification of designer benzodiazepines in forensic postmortem blood samples.
  33. FPS_P/N: A two-dimensional mass spectrometry utilization program with precursor ion determination for accurately distinguishing anthocyanin from other flavonoids.
  34. Multi-analytical dataset on Lekhaniya Mahakashaya: HRLC-MS/MS Orbitrap profiling, HPTLC fingerprinting with marker estimation, and FTIR spectroscopy.
  35. Initial testing procedure based on microextraction followed by LC-HRMS analysis to determine 107 xenobiotics and their metabolites in serum/plasma.
  36. Tailoring of metal organic framework with electrospun polyacrylonitrile for on-chip electromembrane extraction and determination of anticancer drugs in biological samples.
  37. Green evaluation of human plasma levels of metformin, linagliptin, and empagliflozin using HPLC and HPTLC methods: a pharmacokinetic study.
  1. Methods Mol Biol. 2026 ;3013 299-313
    Metabolomics Analysis of Trypanosomes Using Ion Mobility-Enhanced Mass Spectrometry.
    Andrew MacKenzie, Fraser Massie, Tessa Moses.
      Metabolomics, the comprehensive study of low-molecular-weight metabolites, provides a direct snapshot of an organism's physiological state and complements genomics and proteomics. Ion mobility (IM) enhanced metabolomics adds crucial gas-phase separation, resolving isobaric and isomeric metabolites for deeper biological insights. Here we describe a method coupling rapid liquid chromatography to IM-enhanced mass spectrometry, which allows high-throughput metabolomics analysis. This method was successfully applied to trypanosomes to elucidate the mode of action of a small-molecule inhibitor in Trypanosoma brucei.
    Keywords:  Collision cross-section; Drift time; Global metabolomics; Liquid chromatography; Untargeted metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-5142-1_16
  2. Nan Fang Yi Ke Da Xue Xue Bao. 2026 Feb 20. pii: 1673-4254(2026)02-0301-15. [Epub ahead of print]46(2): 301-315
    [Development of an LC-MS/MS method for 32 steroid hormones and exploration of their gestational variations].
    Qiqi Zhou, Junhe Chen, Chengtao Nie, Qian Wang, Fangbo Xia, Hongwei Zhou.
       OBJECTIVES: To establish a highly sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based analytical method for detecting steroid hormones in human serum.
    METHODS: Solid-phase extraction (SPE) was used for sample pretreatment. Chromatographic conditions were optimized to achieve baseline separation, and mass spectrometry parameters were adjusted to ensure accurate detection of trace amounts of hormones. After validation of the linear range, accuracy, and precision, the method was applied in detection of serum samples from normal pregnant women for analyzing dynamic variation characteristics of 32 steroid hormones during the first, second, and third trimesters of pregnancy.
    RESULTS: The linear range of the method was 0.001-7500 ng/mL, with a limit of detection (LOD) of 0.0007 ng/mL. Both intra-day and inter-day coefficients of variation (CV) of the method were less than 10%, the spiked recovery ranged from 86.21% to 112.67%, the extraction efficiency was 85.05%-106.3%, and the matrix effects were 85.60%-113.22%. The results of detection using this method for serum steroid hormones during pregnancy revealed significant elevation of most of the hormones (such as 11-deoxycortisol, 11β-hydroxyprogesterone, dehydroepiandrosterone sulfate, and estriol) with the progression of pregnancy, indicating a close correlation with the development of fetal-placental function. Some hormones increased in the third trimester, possibly due to maternal adaptive regulation and maturation of fetal adrenal function.
    CONCLUSIONS: The detection results using the method established in this study reveal dynamic variations of steroid hormones during pregnancy, which provides clues for further physiological and pathological studies of these hormones.
    Keywords:  liquid chromatography-tandem mass spectrometry; solid-phase extraction; steroid hormones
    DOI:  https://doi.org/10.12122/j.issn.1673-4254.2026.02.08
  3. Anal Methods. 2026 Feb 06.
    A candidate reference measurement procedure for aldosterone measurement in human serum and urine on the basis of isotope dilution liquid chromatography-tandem mass spectrometry.
    Siyu Chen, Junyi Wu, Zihan Liu, Yuchen Wan, Chuanfen Xie, Yingguo Wang, Zechen Liu, Ke Chen, Jiashu Yang, Xinyi Rao, Hui Yuan.
      Accurate quantification of aldosterone is crucial for screening and diagnosing primary aldosteronism. In clinical laboratories, chemiluminescence is commonly used for aldosterone measurement. However, significant variability exists between different measurement systems. A complete reference system is needed to achieve comparable results across laboratories. Therefore, we developed a candidate reference measurement procedure based on liquid chromatography-tandem mass spectrometry for the quantitative determination of aldosterone in serum and urine. In this study, aldosterone-d8 was used to prepare standard solutions. Serum and urine aldosterone samples were extracted via liquid-liquid extraction and solid-phase extraction, and the steps in the pre-treatment process were optimised. Chromatographic separation was performed on a ZORBAX Eclipse XDB-C18 column, and quantitative detection was carried out in negative mode via an electrospray ionization source. The performance of the method was also evaluated. Serum aldosterone exhibited linearity in the range of 4.5-3902 pg mL-1, with a limit of quantification (LOQ) of 3.1 pg mL-1. The coefficient of variation (CV) ranged from 0.5% to 1.2%. Urine aldosterone showed linearity in the range of 10-998 pg mL-1, with a LOQ of 9.9 pg mL-1. The CV ranged from 1.1% to 1.4%. The recoveries of both samples were within the range of 95-105%. This method meets the analytical requirements of a reference measurement procedure, reduces the use of hazardous chemicals during sample preparation, and may serve as a candidate reference method for the accurate quantification of aldosterone in serum and urine samples in the laboratory.
    DOI:  https://doi.org/10.1039/d5ay02023a
  4. Anal Chim Acta. 2026 Mar 08. pii: S0003-2670(26)00083-8. [Epub ahead of print]1390 345133
    Double derivatization-enhanced LC-MS/MS for isomer-resolved identification and quantification of fatty acids.
    Xingyu Shi, Xuan Liang, Shuilin Xie, Ting Zhou, Wei Xiong.
       BACKGROUND: Fatty acids (FAs) are essential metabolites involved in energy storage, signaling, and inflammation regulation, and their biological functions are closely linked to the position and geometric configuration of carbon-carbon double bonds (CC). However, the isomer-resolved characterization and highly sensitive detection of FAs in complex biological matrices remain challenging due to their co-elution, similar fragmentation, and low ionization efficiency. Therefore, there remains a critical need for an efficient approach that can simultaneously enhance chromatographic separation, enable unambiguous CC characterization, and improve detection sensitivity for FA isomers in biological samples.
    RESULTS: In this study, we developed a double derivatization strategy combining magnesium monoperoxyphthalate hexahydrate (MMPP) epoxidation with N,N-diethyl-1,2-ethanediamine (DEEA) amidation, coupled with LC-MS/MS, for isomer-resolved FA analysis. The double derivatization enhanced the chromatographic resolution of positional and cis/trans isomers, enabled reliable localization of CC positions via Δ16 Da diagnostic ion pairs, and improved detection sensitivity by 16-32 fold relative to epoxidation alone. Cis-trans configurations were further supported by parallel linear relationships between retention time and CC position under identical LC conditions, providing a standards-sparing criterion for structural assignment. Applied to mouse plasma, the strategy identified 69 FAs, including 55 unsaturated species, representing an increase of 46 over the underivatized approach. Quantitative analyses and differential analyses showed most FAs were elevated in hepatitis B virus (HBV) mice, with altered isomer ratios pointing to disrupted desaturase activity and oxidative stress.
    SIGNIFICANCE: In conclusion, this double derivatization LC-MS/MS platform provided improved structural resolution, higher detection sensitivity, and more reliable configurational assignment of FAs without requiring extensive standards. Its successful application to HBV mouse plasma demonstrated its suitability for complex biological matrices and highlighted its potential for biomarker discovery and mechanistic studies of lipid-related metabolic diseases.
    Keywords:  Biomarker discovery; Double derivatization; Fatty acid isomers; LC-MS/MS; Structural resolution
    DOI:  https://doi.org/10.1016/j.aca.2026.345133
  5. J Pharm Biomed Anal. 2026 Feb 02. pii: S0731-7085(26)00058-0. [Epub ahead of print]273 117390
    Development and validation of a sensitive and rapid UHPLC-MS/MS method for the simultaneous quantification of CG-0255 and its active metabolite in human plasma and its application to Phase I studies.
    Hanjing Chen, Jiali Li, Fei Yuan, Gongxin He, Hao Wu, Hua Yan, Hongrong Xu, Chao Liu, Lei Sheng, Xuening Li.
      CG-0255, a thiol prodrug of clopidogrel's active metabolite H4 (CG-0236), is a novel thienopyridine P2Y12 receptor antagonist under initial clinical development for the treatment of acute coronary syndromes. Unlike clopidogrel, CG-0255 is converted to the active thiol metabolite H4 (CG-0236) in a single hydrolytic step. Compared with clopidogrel, CG-0255 exhibits more efficient and consistent H4 formation in humans, which can be quantified in plasma following either intravenous or oral administration. In this study, we developed and validated a sensitive, rapid, and robust UHPLC-MS/MS method for the simultaneous quantification of CG-0255 and its derivatized active metabolite (MP-H4, CG-0261) in human plasma. After solid-phase extraction from 94.5 μL of plasma, analytes and isotope-labeled internal standards were separated on an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) using isocratic elution with 0.1 % formic acid in water and acetonitrile (57:43, v/v) at a flow rate of 0.5 mL/min, followed by a 3.5 min column washing and re-equilibration, giving a total analytical run time of 7 min. Baseline separation of CG-0255, CG-0261, and their respective isomers was achieved. Detection was performed using positive electrospray ionization in multiple reaction monitoring mode on a Q-Trap 6500+ mass spectrometer. Calibration curves were linear over 0.05-25 ng/mL for both analytes, corresponding to 0.0353-17.65 ng/mL for H4 (CG-0236). Intra- and inter-day precision and accuracy were within ±15 % at all quality-control levels. The validated assay was successfully applied to two phase I clinical studies conducted at our center, characterizing the pharmacokinetics of CG-0255 following single-dose intravenous and multiple-dose oral administration. This UHPLC-MS/MS method provides a reliable platform for the quantitative evaluation of CG-0255 and its active metabolite in human plasma, and is well suited to support further global clinical development.
    Keywords:  Active metabolite; CG-0255; Clopidogrel prodrug; LC-MS/MS; Treated human plasma
    DOI:  https://doi.org/10.1016/j.jpba.2026.117390
  6. J Chromatogr A. 2026 Jan 21. pii: S0021-9673(26)00057-9. [Epub ahead of print]1770 466727
    Box-Behnken optimized salting-out assisted liquid-liquid extraction coupled with LC-MS/MS for sustainable amisulpride quantification in human plasma.
    Ahmed Serag, Manal E Alosaimi, Maram H Abduljabbar, Adnan Alharbi, Faisal Alsenani, Farooq M Almutairi, Muneef M Aldhafeeri, Atiah H Almalki.
      A sustainable salting-out assisted liquid-liquid extraction coupled with liquid chromatography-tandem mass spectrometry (SALLE-LC-MS/MS) method was developed and validated for amisulpride quantification in human plasma according to ICH M10 guidelines. Box-Behnken experimental design systematically optimized SALLE parameters through evaluation of sample pH (5.0-10.0), acetonitrile volume (500-1500 μL), salt concentration (2.0-6.0 mol/L), and centrifugation time (3.0-10.0 min). The developed polynomial model demonstrated strong predictive capability with subsequent numerical optimization identifying optimal conditions of pH 8.5, acetonitrile 1200 μL, ammonium acetate 4.5 mol/L, and 3-minute centrifugation. These conditions achieved 97.6% extraction recovery for amisulpride with minimal deviation from predicted values. Following optimization, chromatographic separation was achieved using a C18 column with isocratic elution (acetonitrile:water with 0.1% formic acid, 70:30 v/v) and 4-minute analysis time, with multiple reaction monitoring employing transitions m/z370.1→242.1 for amisulpride and m/z376.2→165.1 for haloperidol internal standard. Comprehensive validation subsequently demonstrated linearity across 2-1500 ng/mL with acceptable accuracy, precision, and stability under clinical storage conditions. Matrix effects ranged from 88-105% with low variability, while extraction recovery exceeded 98% across all quality control levels. Clinical application in healthy volunteers (n = 5) following 200 mg oral amisulpride administration successfully characterized key pharmacokinetic parameters including Cmax (506 ng/mL), tmax (3.8 h), and t1/2 (13.3 h) over 48 h. Finally, multi-metric sustainability assessment using CaFRI (75/100), BAGI (77.5/100), and RGB12 (83.1/100 whiteness) frameworks confirmed achievement of white analytical chemistry through balanced analytical performance, environmental friendliness, and practical implementation feasibility. The method offers significant advantages including minimal waste generation, reduced solvent consumption, and enhanced throughput while maintaining regulatory compliance for amisulpride therapeutic drug monitoring applications.
    Keywords:  Amisulpride; Box-Behnken design; Green analytical chemistry; LC-MS/MS; Salle
    DOI:  https://doi.org/10.1016/j.chroma.2026.466727
  7. Talanta. 2026 Jan 23. pii: S0039-9140(26)00103-7. [Epub ahead of print]303 129448
    Simultaneous quantification of fourteen urinary tobacco biomarkers via UHPLC-Q-Orbitrap HRMS and isotope dilution.
    Changming Ding, Yongjie Yang, Jin Yin, Guannian Liu, Yifu Lu, Xiaoming Shi.
      This study developed and validated an ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry(UHPLC-Q-Orbitrap HRMS)method for the simultaneous quantitative 14 tobacco biomarkers in human urine. This method encompasses biomarkers including nicotine metabolites, tobacco-specific nitrosamine metabolites, and minor tobacco alkaloids. Sample preparation employed a combination of enzymatic digestion and supported liquid extraction (SLE), coupled with isotopically labeled internal standards. Analyte separation was achieved rapidly using a Gemini NX-C18 column (150 mm × 2.0 mm, 3 μm), enabling detection of all target compounds within a 15-min chromatographic run time. High-resolution mass spectrometry (HRMS) operated in Full MS and parallel reaction monitoring (PRM) modes. Precise mass screening of parent ions was performed, with confirmation achieved through isotope-labeled internal standards and secondary fragment information. This method has undergone comprehensive validation, demonstrating excellent linearity for all analytes within their respective dynamic ranges. Accuracy ranged from 82.9 % to 121 %, with within-day precision between 1.0 % and 13 % and between-day precision between 1.7 % and 25 %. The limit of detection (LOD) ranged from 0.003 to 2 μg/L, while the limit of quantification (LOQ) spanned 0.01-5 μg/L. Furthermore, matrix effects, recovery rates, and stability all complied with the requirements of bioanalytical guidelines. Ultimately, this method was applied to 123 urine samples from the China National Human Biomarker Monitoring Program (CNHBM), including 30 smokers and 93 non-smokers. The results revealed significant differences in biomarker levels between groups with varying smoking statuses, demonstrating the method's potential for application in epidemiological studies and evaluation of tobacco exposure.
    Keywords:  Biomonitoring; Human urine; Nicotine; Supported liquid extraction; Tobacco exposure
    DOI:  https://doi.org/10.1016/j.talanta.2026.129448
  8. Methods Mol Biol. 2026 ;3013 245-263
    Desorption Electrospray Ionization-Mass Spectrometry Imaging (DESI-MSI) of Metabolites in Heart Samples Infected with Trypanosoma cruzi.
    Dan Chen, Guilherme M P Carrara, Laura-Isobel McCall, Zhibo Yang.
      Chagas disease is an infectious disease caused by the parasite Trypanosoma cruzi, with heart failure being a severe complication of its chronic stage. T. cruzi has three lifecycle stages: epimastigote, trypomastigote, and amastigote. Amastigotes are the intracellular form that invade mammalian host cells, replicate in their cytoplasm, and cause tissue damage. Due to the small size of T. cruzi amastigotes, fine spatial resolution would be beneficial. Current metabolomics research on Chagas disease primarily focuses on studying metabolites through widely used bulk analysis techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, conducting comprehensive molecular studies of Chagas disease is constrained by the inherent limitations, including sample loss and lack of spatial resolution, of these traditional analytical tools. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is an advanced analytical method that combines ambient sampling and ionization with mass spectrometry to produce spatially resolved chemical maps of sample surfaces. This chapter describes the general procedures of using DESI-MSI techniques to study metabolic alterations in mouse hearts infected with T. cruzi, with high spatial resolution, preserving tissue integrity and offering new insights into potential therapeutic targets.
    Keywords:  Chagas disease; Desorption electrospray ionization (DESI); Lipids; Mass spectrometry imaging (MSI); Metabolites; Trypanosoma cruzi
    DOI:  https://doi.org/10.1007/978-1-0716-5142-1_14
  9. Biomed Chromatogr. 2026 Mar;40(3): e70371
    Development and Validation of an LC-MS/MS Method for the Quantitative Estimation of Bexicaserin in Human Cerebrospinal Fluid.
    Raja Reddy Kallem, Rosa Chan, Katie Neal, Maisy Yeager, Katharine Fletcher, Nuggehally R Srinivas.
      Bexicaserin is an investigational molecule that is a highly selective superagonist of the 5-hydroxytryptamine 2C (5HT2c) serotonin receptor subtype and in Phase 3 clinical studies for the treatment of seizures associated with developmental and epileptic encephalopathies (DEEs). We have validated a simple LC-MS/MS method for the quantitative estimation of bexicaserin in human cerebrospinal fluid (CSF). The chromatographic separation of bexicaserin and the IS was achieved on an Acquity HSS T3, C18 column using a 5.5-min gradient program. The sample preparation involves the dilution extraction of bexicaserin and the isotope labeled internal standard (13CD2-bexicaserin; IS) from 25 μL of human CSF. The calibration curve was linear from a range of 0.1-100 ng/mL. The method was fully validated as per the regulatory guidelines. The intraday and interday precision for bexicaserin was in the range of 6.3%-8.7% and 6.0%-8.8%. Bexicaserin intraday and interday bias was in the range of -15.1% (LLOQ) to 12.0% and -10.0% to 1.2%. Stability studies showed that bexicaserin was stable on bench for 24 h, in autosampler for over 50 h, and extract stability at 6°C for 76 h. Bexicaserin was stable after five freeze-thaw cycles and long-term storage at -20°C for 108 days and -80°C for 102 days.
    Keywords:  CSF; DEEs; LC–MS/MS; LP352; bexicaserin; human; pharmacokinetics
    DOI:  https://doi.org/10.1002/bmc.70371
  10. J Chromatogr A. 2026 Jan 23. pii: S0021-9673(26)00065-8. [Epub ahead of print]1770 466735
    Overcoming challenges in LC separation of oxysterols through guided design space modelling: A comparison of three stationary phases.
    Andrea Castellaneta, Ilario Losito, Claudio Crisau, Arnold Zöldhegyi, Zuzana Hrušovská, Hans-Jürgen Rieger, Imre Molnár, Tommaso R I Cataldi.
      Liquid chromatography-mass spectrometry (LC-MS) is an attractive alternative to GC-MS for oxysterol (OS) analysis, as it eliminates the need for chemical derivatization. However, in-source fragmentation during electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), usually adopted for LC-MS analysis of OS, may lead to the generation of isomeric ions from non-isomeric precursors. This prevents a complete unambiguous identification along the MS dimension and makes chromatographic separation a fundamental step. In this work, analytical design space modelling empowered by a software-based modelling tool (DryLab®) was employed to optimize the reversed-phase LC separations of ten clinically relevant OS, including isomeric ring-oxidized and side chain-oxidized sterols. Three stationary phases, octadecyl (C18), cyanopropyl (ES-CN), and pentafluorophenyl (F5), were compared in terms of separation efficacy, using HPLC columns of identical geometry (150 × 2.1 mm, 2.7 μm) packed with core-shell particles. Gradient steepness, column temperature, and composition of the organic modifier (mixture of methanol and acetonitrile) in the mobile phase were selected as critical method parameters for successive construction of the design spaces on the selected stationary phases. All the resulting optimized methods resolved positional isomers and diastereomers, excepting 25(R/S)-26-hydroxycholesterol epimers. The C18 stationary phase (at 52 °C) offered high selectivity but demanded the longest run times (ca. 80 min). ES-CN (at 50 °C) and F5 (at 25 °C) stationary phases enabled faster effective runs (< 45 min), with ES-CN also reducing retention/chemical artifacts that are presumably triggered by π-π binding interactions with the stationary phase.
    Keywords:  Analytical design space modelling; DryLab®; High-resolution mass spectrometry; Oxysterols; Reversed-phase liquid chromatography
    DOI:  https://doi.org/10.1016/j.chroma.2026.466735
  11. Biomed Chromatogr. 2026 Mar;40(3): e70381
    Validated LC-MS/MS Method for Quantification and Pharmacokinetic Analysis of Talazoparib and Enzalutamide in Rats.
    Rajesh Guntupalli, Panchumarthy Ravisankar, Sathish Kumar Konidala.
      The FDA has approved Talazoparib and Enzalutamide for metastatic castration-resistant prostate cancer (mCRPC) with homologous recombination repair (HRR) gene mutations, based on the TALAPRO-2 Phase 3 trial. This study developed and validated an LC-MS/MS method for simultaneous quantification of Talazoparib and Enzalutamide in rat plasma using Apalutamide as the internal standard. Chromatographic separation was achieved with a 70:30 (v/v) acetonitrile-0.1% formic acid system, 10 min runtime, and 1.0 mL/min flow rate. Retention times were 4.13 min (Talazoparib), 5.31 min (Enzalutamide), and 7.89 min (Apalutamide). Detection was performed in positive ionization mode using MRM transitions: m/z 381.35 → 240.56, 465.44 → 305.24, and 478.44 → 285.10, respectively. The method was linear over 0.05-2 ng/mL for Talazoparib and 4-160 ng/mL for Enzalutamide (r2 > 0.999). Accuracy and precision were within ±15%-20%, and recovery exceeded 98%. In pharmacokinetic studies on male Wistar rats, Talazoparib showed a Cmax of 0.88 ng/mL at 3 h and an AUC0-t of 20 ng·h/mL, while Enzalutamide exhibited a Cmax of 76.18 ng/mL at 1 h and an AUC0-t of 1702 ng·h/mL; both had 24 h half-lives. The validated method enables sensitive, rapid, and reliable bioanalysis for preclinical pharmacokinetic evaluation.
    Keywords:  Enzalutamide; LC‐MS/MS; Talazoparib; bioanalytical approach; pharmacokinetic analysis
    DOI:  https://doi.org/10.1002/bmc.70381
  12. Anal Chem. 2026 Feb 05.
    MS/MS Mass Spectrometry Filtering Tree for Bile Acid Regio- and Stereoisomer Annotation.
    Ipsita Mohanty, Shipei Xing, Vanessa Castillo, Julius Agongo, Abubaker Patan, Yasin El Abiead, Helena Mannochio-Russo, Wilhan D Gonçalves Nunes, Jasmine Zemlin, Itzhak Mizrahi, Dionicio Siegel, Mingxun Wang, Lee R Hagey, Pieter C Dorrestein.
      Bile acids are essential steroids with a wide range of biological roles, including the regulation of immunity, nutrient absorption, insulin signaling, appetite, and body temperature. However, due to similarities in their MS/MS spectra, spectral matching with reference MS/MS libraries generally fails to differentiate between isomers. This study introduces a proof-of-concept workflow that uses a mass spectrometry query language filtering tree to distinguish isomeric bile acids in untargeted LC-MS/MS data by leveraging intensity ratios of ions that are close to one another in the MS/MS spectrum. It can be retrospectively applied to existing LC-MS/MS data in data repositories. The filtering tree concept provides the opportunity to annotate and distinguish previously unknown bile acid isomers across LC-MS/MS data sets. To facilitate the ease of applying these filters to LC-MS/MS data sets, we developed a web-based application that simplifies the stepwise filtering tree workflow, removing the need for coding expertise. Here, we apply the multistep filtering application to a representative public data set, which revealed distinct patterns of bile acids associated with different diet types across diverse mammalian species. We further identified the previously uncharacterized bile acid deoxycholyl-N-acetyl-putrescine, which was elevated in carnivores.
    DOI:  https://doi.org/10.1021/acs.analchem.5c05677
  13. J Pharm Biomed Anal. 2026 Feb 02. pii: S0731-7085(26)00057-9. [Epub ahead of print]273 117389
    Development and validation of an UPLC-MS/MS method for simultaneous determination of meropenem and its open-ring metabolite in human serum and cerebrospinal fluid with application to clinical samples.
    XiangLong Chen, Jinhui Xu, Chengliang Wang, Lijuan Yang, Jinwei Fan, Tongtong Li, Qian Zhang, Yanxia Yu, Lian Tang, Shenjia Huang.
      Central nervous system (CNS) infections require adequate drug exposure at the site of action, yet antibiotic meropenem (MER) shows limited cerebrospinal fluid (CSF) penetration and easily undergoes non-enzymatic degradation to an inactive open-ring metabolite (ORM). In this study, we developed a simple, sensitive, and fast liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of MER and ORM in human serum and CSF. Chromatographic separation was accomplished on an Agela Venusil MP C18 column, with MER-d6 and ORM-d6 as internal standards. Methanol was found to promote methanolysis, yielding a characteristic product (m/z 416.2). Therefore, acetonitrile was selected as both the organic phase and the protein-precipitation solvent. Method validation was conducted according to the ICH M10 guideline. Follow validation, the method was successfully applied to 57 serum and 16 CSF samples. ORM concentrations in human CSF were reported for the first time. This method provides a valuable tool to support MER monitoring in patients with CNS infections.
    Keywords:  Cerebrospinal fluid; Meropenem; Open-ring metabolite; UPLC-MS/MS
    DOI:  https://doi.org/10.1016/j.jpba.2026.117389
  14. Anal Sci. 2026 Feb 02.
    Easy synthesis and analytical application of deuterated alkanesulfonates as internal standards for determination using mass spectrometry.
    Tomoki Maede, Honoka Kato, Kaname Tsutsumiuchi.
      Alkanesulfonates are widely used as anionic surfactants in detergents, requiring accurate quantitative methods for quality control. This study aimed to develop a deuterated internal standard for sodium alkanesulfonates via hydrogen/deuterium (H/D) exchange using a transition metal catalyst. Generally, sulfonate groups are known to strongly adsorb onto metal surfaces and deactivate catalysts due to their catalyst-poisoning effect. However, we found that alkanesulfonates can be deuterated with a ruthenium on carbon (Ru/C) catalyst in D2O under a hydrogen atmosphere. The D contents increased with alkyl chain length, ranging from 20 to 86%. Sodium dodecanesulfonate, which showed the highest D content, was selected as the internal standard. A model detergent sample was prepared to evaluate quantification performance. Quantitative analysis was conducted using liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) with electrospray ionization (ESI) and field desorption (FD)-TOFMS. ESI provided high sensitivity for trace analysis, while FD offered faster measurements for concentrated samples. Spike-and-recovery experiments across a concentration range (0.50-200 ppm) demonstrated that using an internal standard improved measurement accuracy. This approach offers a practical solution for quantifying sulfonate-based surfactants in complex detergent matrices.
    Keywords:  Alkanesulfonate; Deuteration; FD-MS; H/D exchange reaction; Internal standard; LC-MS
    DOI:  https://doi.org/10.1007/s44211-026-00873-6
  15. Anal Chem. 2026 Feb 02.
    A Highly Sensitive and High-Throughput Quantitative HILIC-MS/MS Method for Systematic Profiling of RNA Modifications.
    Cheng Guo, Xiujuan Hong, Yiqiu Hu, Tao Pan, Yunxiang Zhou, Haifen Lei, Yuanjiang Pan.
      Understanding the functions and regulatory mechanisms of the epitranscriptome entails robust and accurate analytical methods to identify and quantify post-transcriptional modifications in RNA. However, there are still various challenges in analyzing multiple modified nucleosides in RNA. Herein, we established a highly sensitive and high-throughput hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method, in conjunction with a stable isotope-dilution technique, for accurate quantification of 35 nucleosides. By the use of malic acid as a mobile phase additive, the HILIC-based separation of nucleosides was improved and the MS signal response of nucleosides was enhanced by 2.5- to 20.0-fold. Notably, seven groups of isomeric nucleosides with identical multiple-reaction monitoring ion transitions and six groups of nucleosides with identical or similar molecular weights that were indistinguishable by MS were well resolved by optimal HILIC separation. Thirty-five nucleosides were analyzed simultaneously within 12.5 min, and the limits of detection of these nucleosides ranged from 15.0 amol to 43.5 fmol. With this method, we conducted a comprehensive analysis and evaluation of the alteration in the RNA modification profile in breast cancer and assessed the RNA modification patterns across different breast cancer subtypes. The developed HILIC-MS/MS method has excellent capabilities for sensitive and high-throughput detection of multiple modified nucleosides, thereby providing a valuable analytical tool for deciphering the epitranscriptomic landscape and screening nucleosides as biomarkers in future clinical research.
    DOI:  https://doi.org/10.1021/acs.analchem.5c06973
  16. Environ Sci Technol. 2026 Feb 06.
    Comparability of Liquid Chromatography Tandem Mass Spectrometry Analysis of Dissolved Organic Matter across Laboratories.
    Jarmo-Charles Kalinski, Bruno Ruiz Brandão da Costa, Tilman Schramm, Lance R Buckett, Laura T Carlson, Nicole R Coffey, Tito Damiani, Elias Dechent, Yasin El Abiead, Steffen Heuckeroth, Elaine K Jennings, Jan Kaesler, Naomi L Stock, Alice M Orme, Ralph R Torres, Sara Trojahn, Helen L Whelton, Yingfei Yan, Allegra T Aron, Rene M Boiteau, Ian D Bull, Pieter C Dorrestein, Duc Huy Dang, Richard P Evershed, Marta Gledhill, Gerd Gleixner, Andreas F Haas, Martin Hansen, Tilmann Harder, Ellen C Hopmans, Anitra E Ingalls, Uwe Karst, William Kew, Melissa Kido Soule, Boris P Koch, Elizabeth B Kujawinski, Oliver J Lechtenfeld, Krista Longnecker, Tomáš Pluskal, Georg Pohnert, Zachary C Redman, Albert Rivas-Ubach, Philippe Schmitt-Kopplin, Gabriel Singer, Jan Tebben, Patrick L Tomco, Nicholas D Ward, Lihini I Aluwihare, Carsten Simon, Jeffrey Hawkes, Daniel Petras.
      Non-targeted liquid chromatography tandem high-resolution mass spectrometry (LC-MS/MS) is increasingly applied for the structure-resolved chemical analysis of dissolved organic matter (DOM). With new developments in MS instrumentation and analysis software, the approach has gained substantial momentum over the past decade. However, achieving high-quality analytical data that is reproducible and comparable across laboratories can be a bottleneck in non-targeted metabolomics and organic matter chemical analysis, especially for data reuse in repository-scale analyses. Understanding the capabilities as well as challenges of comparing LC-MS/MS data from different laboratories is necessary for inferring global trends from public data sets. To illuminate instrumentation factors that drive differences and variability, we used a standardized data analysis pipeline, including classical (CMN) and feature-based molecular networking (FBMN), to analyze data from a ring trial by 24 laboratories on identical sample sets of algal and DOM extracts that were mixed in predefined concentrations and spiked with standards. Our results showed that data sets from similar mass spectrometer types with unified instrument parameters were qualitatively comparable, resolving the same general trends and shared mass spectral features. Interlaboratory comparability was best for high-intensity features, while low-intensity features showed greater detection variability. Our analysis also highlights challenges when comparing data from instruments with different acquisition rates or operating with less standardized methods. Lastly, we provide recommendations for data integration, public data sharing, standardization, and best practices for standardized LC-MS/MS data acquisition, which will be critical for long-term time series and intercomparability of DOM chemical analyses.
    Keywords:  DOM; LC–MS/MS; dissolved organic matter; high resolution tandem mass spectrometry; interlaboratory comparison; non-targeted analysis; non-targeted metabolomics; structure-resolved chemical analysis
    DOI:  https://doi.org/10.1021/acs.est.5c12691
  17. Sci Rep. 2026 Feb 03.
    Determination of hydrocortisone and cortisone in artificial saliva by spray assisted fine droplet formation liquid phase microextraction coupled to LC-MS/MS.
    Selim Gürsoy, Süleyman Bodur, Arda Atakol, Sezgin Bakırdere.
      Hydrocortisone and cortisone are clinically informative steroids whose joint measurement at low concentrations is essential for ratio-reliable interpretation. A spraying-assisted fine droplet formation liquid-phase microextraction (SA-FDF-LPME) was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneous quantitation in saliva-type matrices as part of a method-development study. Method parameters were optimized to secure chromatographic resolution, stable ion ratios, and high sensitivity. Under direct injection, LOD/LOQ values were 1.05/3.50 µg kg-1 for cortisone and 0.80/2.68 µg kg-1 for hydrocortisone, with dynamic ranges of 3.65-150.77 and 2.59-268.45 µg kg-1, respectively. With SA-FDF-LPME coupled to LC-MS/MS, LOD/LOQ were 0.0164/0.0547 µg kg-1 for cortisone and 0.0734/0.245 µg kg-1 for hydrocortisone, and dynamic ranges were 0.049-4.983 and 0.210-5.50 µg kg-1, respectively. Accuracy was established by pre-extraction spiking at different concentrations in two artificial saliva formulations. Using external-standard calibration, recoveries were 105.3-129.4%/110.4-141.1% for cortisone and 110.0-134.9%/94.1-142.8% for hydrocortisone; with matrix-matched calibration, recoveries were 80.3-103.2%/98.1-117.8% for cortisone and 79.8-105.7%/97.3-114.0% for hydrocortisone. The developed SA-FDF-LPME-LC-MS/MS method enables sensitive, accurate, and simultaneous quantification of hydrocortisone and cortisone at saliva-relevant concentrations and offers a practical platform for selective bioanalysis of related small molecules.
    Keywords:  Artificial saliva; Cortisone; Hydrocortisone; Spraying assisted fine droplet formation liquid phase microextraction; Tandem mass spectrometry
    DOI:  https://doi.org/10.1038/s41598-026-38457-z
  18. J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Jan 16. pii: S1570-0232(26)00019-X. [Epub ahead of print]1273 124930
    Liquid chromatography-tandem mass spectrometry-based urinary steroid profiling applied to primary aldosteronism diagnosis.
    Jian Zhong, Xiaoli Ma, Danchen Wang, Wei Luo, Yicong Yin, Yutong Zou, Ying Zhu, Ming Li, Shaowei Xie, Songlin Yu, Ling Qiu.
      Steroid hormones are essential regulators of physiological homeostasis, which can help in diagnosing primary aldosteronism (PA). However, current methodologies for urinary steroid analysis face critical limitations, including narrow analyte coverage and tedious sample preparation workflows. Since the liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the preferred technique for steroid measurement, there is a great need to establish an LC-MS/MS method combined with simplified and efficient sample pretreatment for multi-steroid profiling. We establish a novel LC-MS/MS method for quantifying 35 steroid hormones within a 25-min runtime. 40 μL of urine samples can be efficiently hydrolyzed at room temperature in just 0.5 h using transgenic β-glucuronidase. Detection is performed on the Waters Acquity I-Class UPLC-tandem with a Waters TQ-S triple quadrupole MS/MS system, and the method performance is systematically evaluated. Linearity is excellent, and the recovery rates range from 80.0 to 120.6%. Acceptable intra-assay and inter-assay precisions are achieved, with the coefficients of variation ranging from 1.86 to 15.10% and 3.91 to 19.75%, respectively. For clinical application, patients with PA (n = 37) exhibit significantly higher levels of aldosterone, tetrahydroaldosterone, 18-hydroxycorticosterone, 18-oxocortisol, and 18-hydroxycortisol compared to non-PA patients (n = 104). Combined steroid profiling demonstrates strong diagnostic performance for PA, yielding an area under the curve of 0.914, with a sensitivity of 0.87 and a specificity of 0.88. In conclusion, this study establishes a technically advanced LC-MS/MS method that integrates efficient enzymatic hydrolysis to enable the accurate quantification of urinary steroids for the diagnosis of endocrine disorders.
    Keywords:  Adrenal; Enzymatic hydrolysis; High performance liquid chromatography; Mass spectrometry; Steroids and steroid hormones
    DOI:  https://doi.org/10.1016/j.jchromb.2026.124930
  19. J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Jan 29. pii: S1570-0232(26)00031-0. [Epub ahead of print]1272 124942
    Anabasine analysis in human plasma using liquid chromatography coupled to tandem mass spectrometry to Verify tobacco use: empirical challenges and limitations.
    Christof Manuel Schönenberger, Aurelie Berthet, Matthias Briel, Alain Amstutz, Matthias Cavassini, Loïc Sartori, Camille Rime, Davide Staedler, Fiorella Lucarini.
      Anabasine is an alkaloid frequently quantified by LC-MS/MS to differentiate tobacco use from nicotine replacement therapy. While urine provides reliable measurement, plasma remains a poorly validated and analytically challenging matrix. This study assessed widely used sample preparation strategies for anabasine determination in human plasma. Across all methods, recovery in undiluted plasma was highly variable and largely outside acceptable analytical ranges, with marked ion suppression and poor reproducibility. Matrix dilution increased apparent signal but substantially worsened variability. Experiments in albumin-enriched saline excluded protein binding as the main determinant of analyte loss, indicating broader plasma-related matrix effects. In human plasma, including time-course sampling during and after smoking, anabasine remained consistently below quantifiable levels. None of the tested workflows met the robustness criteria required for quantitative LC-MS/MS analysis, indicating that current preparation approaches do not enable reliable anabasine measurement in plasma, a critical limitation for studies using anabasine as a biomarker of tobacco exposure.
    Keywords:  Anabasine; Analytical variability; Biomonitoring; LC-MS/MS; Matrix effect; Plasma; Recovery; Sample preparation; Tobacco biomarkers
    DOI:  https://doi.org/10.1016/j.jchromb.2026.124942
  20. Bioanalysis. 2026 Feb 04. 1-12
    Rapid quantification of bile acids in serum by LC-MS/MS and application to serum bile acid profile in voriconazole administered patients with invasive fungal infections.
    Xuping Yang, Haoran Qin, Jing Ling, Lulu Dong, Yan Jiang, Sulan Zou, Nan Hu.
       OBJECTIVES: Disruption of bile acid homeostasis is implicated in the pathogenesis and progress of several liver diseases, including drug-induced liver injury (DILI). A rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying 30 bile acids in human serum was developed and applied to analyze serum bile acid profile in voriconazole administered patients with invasive fungal infections.
    METHODS: The stable isotope-coded bile acids were used as internal standards. The analytes in serum samples (50 μL) were extracted by protein precipitation and isolated by a Kinetex EVO C18 column (50 × 2.1 mm, 2.6 μm). The detection was performed using multiple reaction monitoring (MRM) in electrospray negative ionization mode.
    RESULTS: All of the 30 bile acids were sufficiently separated within 10 min with good linearity (r > 0.99) over tested calibration ranges. The accuracy and precision values were in the range of 85.42-114.3% and 1.8-13.8%, respectively. The matrix effect, recovery, stability, and dilution integrity all met the acceptance criteria. Serum bile acid profile changed in patients with supratherapeutic voriconazole concentration (>10 μg/mL).
    CONCLUSIONS: This LC-MS/MS method for the quantification of 30 bile acids in human serum might apply a new analytical approach for liver function evaluation.
    Keywords:  Bile acids; hepatotoxicity; liquid chromatography-tandem mass spectrometry (LC-MS/MS); serum; voriconazole
    DOI:  https://doi.org/10.1080/17576180.2026.2625865
  21. Anal Methods. 2026 Feb 06.
    Dynamic multiple reaction monitoring for high throughput detection and quantitation of polycyclic aromatic compounds.
    Zhe Xia, Thor Halldorson, Nipuni Vitharana, Marcus Kim, Dami Daramola, Chris Marvin, Philippe J Thomas, Reyd A Dupuis-Smith, Jennifer F Provencher, Gregg T Tomy.
      This study presents a dynamic multiple reaction monitoring (dMRM) method for the simultaneous analysis of 122 polycyclic aromatic compounds (PACs), including polycyclic aromatic hydrocarbons (PAHs), alkylated PAHs, halogenated PAHs, heterocyclic PACs, and halogenated HPACs using gas chromatography-tandem mass spectrometry (GC-MS/MS). Conventional MRM methods for these complex mixtures (including the one used as our benchmark) require multiple GC injections and time segments to maintain sufficient MS dwell and cycle times. The dMRM developed here captured all our analytes in a single GC-injection. The analytical performance characteritsitc of the dMRM method was compared to our conventional time-segmented MRM methods using matrices of increasing complexity, including calibration standards, certified sediment and mussel reference materials, and an in-house fortified egg reference material. Instrument detection limits were similar for both methods and ranged from 0.1 to 1.3 pg µL-1. The dMRM method achieved comparable or improved precision and accuracy compared to conventional MRM for less complex matrices, such as standard solutions and biota. Negative systematic biases were observed for a subset of analytes in the sediment matrix for both approaches and are attributed to to non-exhaustive extractions rather than limitations of the MS methods. Provided that sample preparation is carefully optimized for challenging matrices, the dMRM technique offers a powerful tool for high-throughput environmental analysis of PACs, enabling a single GC injection to streamline laboratory workflows and enhance analytical efficiency.
    DOI:  https://doi.org/10.1039/d5ay01950h
  22. Forensic Sci Int. 2026 Jan 31. pii: S0379-0738(26)00024-1. [Epub ahead of print]381 112837
    The mystery of post-mortem Gamma-hydroxybutyrate formation - method development and validation for the detection of endogenous GHB and its related compounds.
    Jana Kietzerow, Mario Thevis, Hilke Andresen-Streichert.
      Interpreting γ-hydroxybutyrate (GHB) concentrations in post-mortem samples remains challenging, due to the potential for formation both in corpore after death and in vitro after sampling. The possible influence of metabolically-related endogenous substances on post-mortem GHB levels has not yet been clarified. To address this, an analytical method was developed and validated for the simultaneous detection of GHB, γ-butyrolactone (GBL), and eight related endogenous compounds: succinic semialdehyde, γ-aminobutyric acid, putrescine, α-hydroxybutyrate (AHB), β-hydroxybutyrate (BHB), L-glutamic acid, succinic acid, and GHB-glucuronide (GHB-Gluc). Sample preparation involved protein precipitation using acetonitrile/methanol (85:15), followed by solid-phase extraction. Chromatographic separation was achieved using a reversed-phase C-18 analytical column with a 33-minute gradient run employing water and acetonitrile, both containing 0.1 % formic acid, as mobile phases. Human whole blood served as the matrix for calibration and quality controls, with endogenous levels corrected using matrix blanks. The method achieved limits of detection and limits of quantification of 0.5 µg/mL for all analytes. Calibration ranges extended up to 75 µg/mL, depending on the substance. Linear regression was applicable for most analytes, except BHB, GHB, putrescine, and succinic acid. Accuracy and precision were satisfactory (< +/- 10 %) across all concentration levels. The LC-MS/MS method allows for comprehensive quantification of GHB and related endogenous substances potentially involved in its post-mortem increase. Application to real post-mortem samples will help clarifying the role of these compounds in GHB formation after death and support more accurate interpretation of forensic toxicological findings.
    Keywords:  GHB; LC-MS/MS; Metabolites; Method-development; Post-mortem increase
    DOI:  https://doi.org/10.1016/j.forsciint.2026.112837
  23. J Am Soc Mass Spectrom. 2026 Feb 04.
    Efficient and Cost-Effective Enzyme Deposition onto Tissues for Mass Spectrometry Imaging of N-Glycans Using a Mini-Humidifier.
    Erik Sveen, Alyssa M Moore, Miranda R Weigand, Julia Laskin.
      Mass spectrometry imaging (MSI) is a powerful technique for studying the spatial localization of N-linked glycans in biological tissues, which are important biomarkers for various diseases. Analyzing N-linked glycans requires the deposition of an enzyme onto a biological tissue section to release them from proteins. However, existing equipment for enzyme deposition is relatively expensive and may not be readily accessible to some laboratories. To address this challenge, we developed and evaluated a cost-effective approach for enzyme application onto tissue sections using a mini-humidifier. We demonstrate the capabilities of this approach by applying peptide N-glycosidase F (PNGase F) for MSI of N-linked glycans present within mouse brain tissue sections using nanospray desorption electrospray ionization (nano-DESI). The performance of the mini-humidifier was comparable to that of a widely used HTX-TM Sprayer, establishing it as a cost-effective and efficient alternative for enzyme deposition onto tissue samples in MSI experiments. This work offers an accessible approach for enzyme application on biological samples to study the spatial distribution of N-linked glycans using MSI.
    DOI:  https://doi.org/10.1021/jasms.5c00435
  24. Anal Bioanal Chem. 2026 Feb 02.
    Analytical validation of an advanced U-HPLC-MS/MS method for lactose detection in food supplements and pharmaceuticals.
    Alessandro Nencioni, Kristian Comelli, Michela Bulfoni, Emanuele Nencioni.
      Lactose intolerance is common, so accurate detection of lactose in supplements and pharmaceuticals is critical. We validated an ultra-high performance liquid chromatography-tandem mass spectrometry (U-HPLC-MS/MS) method with high sensitivity and specificity for trace lactose in products labeled "lactose-free." The workflow mitigates matrix effects through solid-phase extraction and filtration and explicitly accounts for α/β mutarotation to ensure correct identification and quantification. Validation per ISO and ICH Q2 confirmed robustness and performance superior to enzymatic/colorimetric assays for quality control and regulatory compliance. The method achieved excellent linearity (R2 > 0.995) over 0.05-10 ppm, recoveries of 95-105%, precision with RSD < 2%, LOQ 0.05 ppm, and matrix effect < 15%. These results enable reliable verification of "lactose-free" claims across diverse matrices, reducing false positives, enhancing reproducibility, and improving labeling transparency.
    Keywords:  Allergens; Dietary supplements; ISO standards; Lactose; Method validation; Quality control; U-HPLC–MS/MS technology
    DOI:  https://doi.org/10.1007/s00216-026-06327-2
  25. Methods Mol Biol. 2026 ;3015 95-108
    Low-Input Proteomics Protocols for Laser-Captured FFPE Samples with Orbitrap Astral DIA-MS.
    Daniele Musiani, Alessandro Cuomo.
      Laser microdissection (LMD) combined with high-resolution mass spectrometry (MS) has become a powerful approach for analyzing targeted regions of fixed tissues, enabling detailed investigation of their heterogeneity. Here, we present two protocols optimized for low-input samples: a solid-phase enhanced preparation method (SP3) and a detergent/organic solvent-based approach (DDM/ACN). Both workflows are designed to minimize sample loss and are suitable for manual processing when automation platforms are not available. We provide practical guidance to reduce input requirements down to 1500 μm2 of 10 μm thick FFPE tissue while maintaining reproducibility and proteome depth. These protocols, when coupled with fast instruments such as the Orbitrap Astral mass spectrometer working in DIA acquisition mode, enable sensitive, high-resolution proteomic analysis of microdissected tissue regions. Together, they establish a robust approach for investigating spatial heterogeneity in clinical samples with high coverage and precision.
    Keywords:  FFPE tissue; Laser microdissection; Low-input sample preparations for LC-MS/MS; Orbitrap Astral
    DOI:  https://doi.org/10.1007/978-1-0716-5154-4_8
  26. J Pharm Biomed Anal. 2026 Feb 02. pii: S0731-7085(26)00056-7. [Epub ahead of print]273 117388
    Determination of antigen components in inactivated SARS-CoV-2 vaccine using ultra-high-performance liquid chromatography tandem mass spectrometry.
    Jiexia Shi, Fenfang Deng, Yongxian Li, Juntao Li, Rongfei Peng, Jun Yuan, Lei Tan.
      Accurate and reliable quantification of antigen components in vaccines is critical in vaccine quality control and evaluation of immunogenic consistency. However, conventional immunoassays often suffer from limited specificity, trace-level antigen concentrations, and indirect quantification. In this study, we demonstrated an ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the determination of effective antigen components in inactivated SARS-CoV-2 vaccines. Specifically, the vaccine samples were denatured and digested with trypsin to generate tryptic peptides. Then, the signature peptides derived from the nucleocapsid protein and their stable isotope-labeled internal standards were selectively captured and separated using anti-peptide antibody-conjugated magnetic beads. The signature peptides were characterized by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, which confirmed their amino acid sequence and multi-charged ionization states. Quantitative analysis was then performed using UHPLC-MS/MS in positive electrospray ionization mode with multiple reaction monitoring. Chromatographic separation of the signature peptides was achieved on an ACQUITY Premier Peptide BEH C₁₈ column using 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile as the mobile phases. The method was validated and exhibited excellent linearity for the signature peptides over the concentration range of 1-60 μg/L, with correlation coefficients higher than 0.999. The recovery ranged from 75.1 % to 86.3 %, with intra-day precision (RSD) of 0.8-1.0 % and inter-day precision of 1.3-3.6 %. Finally, the method was successfully applied to determine the effective antigen components in inactivated SARS-CoV-2 vaccine samples. The concentrations of the signature peptide ADETQALPQR ranged from 4.95 to 12.95 µg/L across the three vaccine batches analyzed, corresponding to 4.38-11.47 nmol/L of nucleocapsid protein. The results indicated that the method exhibited great promise for the determination of active antigenic proteins in inactivated SARS-CoV-2 vaccine samples and provided an alternative analytical platform for vaccine quality control.
    Keywords:  Antigen components; Inactivated vaccine; SARS-CoV-2; Signature peptides; UHPLC-MS/MS
    DOI:  https://doi.org/10.1016/j.jpba.2026.117388
  27. Anal Chim Acta. 2026 Mar 08. pii: S0003-2670(26)00059-0. [Epub ahead of print]1390 345109
    A high-throughput detection platform for dried blood spots constructed with DESI-MS/MS.
    Siqi Gao, Jinhui Zhao, Yue Guan, Liyan Liu.
       BACKGROUND: Dried blood spot (DBS) technology, as a method of blood collection and preservation, has the advantages of small blood collection, convenient transportation and avoiding the degradation of metabolites. In particular, it has broad clinical application prospects in newborn screening and therapeutic drug monitoring. Desorption electrospray mass spectrometry (DESI-MS) is a simple, rapid and in situ spatial metabolomics detection technique, but its throughput, accuracy and coverage for DBS remain to be investigated compared with traditional methods.
    RESULTS: In the UPLC-MS/MS platform, the optimal extraction solvent, extraction solvent volume and extraction method were ACN: H2O (v:v/3:1), 300 μL and ultrasonic extraction, respectively. Based on the DESI-MS/MS platform, the best spray solvent was MeOH: H2O (v:v/3:1), the spray velocity was set at 2 μL/min, the spray needle angle was set at 55°, and the capillary voltage was set at 4.5 kv (DESI+), respectively. The approach delivered consistent data assurance, broad metabolite coverage, and acceptable reproducibility. Finally, these methods were applied to detect the metabolic profiles of subjects before and after drinking sugar-sweetened soy milk, and the metabolic map was well isolated, and the difference metabolites were successfully found.
    SIGNIFICANCE: Two protocols of metabolic profiles for DBS were constructed and optimized. The good performance of DESI-MS/MS was obtained when comparing to the UPLC-MS/MS. The application of sweetened soy milk showed that the two protocols developed have the practicability of detecting differential changes in metabolic profiles of DBS.
    Keywords:  Desorption electrospray mass spectrometry; Dry blood spot; Metabolic profiles; Ultra performance liquid chromatography
    DOI:  https://doi.org/10.1016/j.aca.2026.345109
  28. Bioanalysis. 2026 Feb 05. 1-11
    Development of a chromatographic method for the analysis of risdiplam in serum extracts.
    Natalia Balińska, Anna Lemska, Maria Mazurkiewicz-Bełdzińska, Sylwia Studzińska.
       BACKGROUND: Risdiplam has been used to treat spinal muscular atrophy for 3 years. There are limited number of papers devoted to its analytics. Until now, risdiplam and its metabolites have only been analyzed using a C18 column, while the sample preparation method involved protein precipitation.
    RESEARCH DESIGN AND METHODS: Risdiplam was analyzed using reversed-phase UHPLC. The experiment was designed to compare the retention of risdiplam on five columns using various mobile phases. The protein precipitation was used as the sample preparation method.
    RESULTS: Risdiplam shows greater retention on phenyl columns, where π-π interactions take part in retention. The increase of mobile phase pH caused increased risdiplam retention, while salt concentration had no significant effect. An octadecyl column with pentafluorophenyl groups was selected with a mobile phase containing 10 mM ammonium formate (pH 4) and acetonitrile. The method was characterized by good linearity, repeatability, and short analysis time. It was applied to risdiplam analysis in serum samples after protein precipitation with different solvents. Finally, proteins were effectively precipitated using 10% TFA solution, providing 90% recovery.
    CONCLUSIONS: The developed procedure of extraction and determination of risdiplam is simple, fast and reliable. It may find application for routine monitoring of risdiplam or for quality control.
    Keywords:  Risdiplam; liquid chromatography; protein precipitation; retention of risdiplam; stationary phases
    DOI:  https://doi.org/10.1080/17576180.2026.2624594
  29. J Chromatogr A. 2026 Jan 28. pii: S0021-9673(26)00082-8. [Epub ahead of print]1771 466752
    Accurate quantification of quinolones in marine sediment by UPLC-MS/MS after ultrasonic extraction and automated SPE clean-up.
    Zhenhua Li, Qiaoling Zhao, Haoji Zhao, Tiejun Li, Hongmei Hu, Yuanming Guo, Heyong Cheng.
      As a class of the most prevalent broad-spectrum antibiotics, quinolones (QNs) are ubiquitous in various environmental matrices (in particular marine sediment). However, determination of multiple QNs in marine sediment is a challengeable task due to matrix complexity and strong QNs-sediment adsorption. Herein we presented an efficient quantification strategy for accurate measurement of 15 QNs in marine sediment based on ultrasonic extraction with 0.2 M Na2HPO4 in 50 v/v % acetonitrile, followed by automated solid-phase clean-up and ultra-performance liquid chromatography-tandem mass spectrometry with isotope dilution calibration. The method demonstrated negligible matrix effect, satisfactory recoveries (85 %-119 %), and low limits of detection (2-15 pg/g). Five QNs were detected in marine sediment from Yueqing Bay and Daiquyang with total concentrations of 5.6-12.1 μg/kg and 0.2-1.0 μg/kg, respectively, where four QNs including norfloxacin (NOR), enrofloxacin, ciprofloxacin, and ofloxacin were found in all samples, and NOR was the predominant compound both in Yueqing Bay and Daiquyang. Overall, the presented method is highly sensitive and reliable for routine quinolone analysis in marine sediment.
    Keywords:  Antibiotics; Isotope dilution; Sample preparation; Sediment; UPLC-MS/MS
    DOI:  https://doi.org/10.1016/j.chroma.2026.466752
  30. Biomed Chromatogr. 2026 Mar;40(3): e70389
    Analytical Method Development and Validation of Treosulfan and Its Impurities by Ultra Performance Liquid Chromatography (UPLC) and Identification of Degradation Products by LC-MS/MS.
    Sudha Divya Madhuri Kallam, Mithun Rudrapal.
      Treosulfan is a bifunctional alkylating agent widely used as a conditioning drug in haematopoietic stem cell transplantation. In this study, a robust reverse-phase UPLC method was developed and validated for the quantitative determination of treosulfan and its related substances. The optimised method employed a Waters Acquity UPLC BEH shield RP-18 (50 × 1.0 mm, 1.7 μm) column with an isocratic mobile phase of acetonitrile and ammonium acetate (pH 2.5)/formic acid in a 40:60 ratio, a flow rate of 0.2 mL/min, injection volume of 5 μL and detection at 239 nm. Validation, performed according to ICH Q2 (R2), confirmed excellent system suitability, specificity, precision (%RSD < 1%), linearity (r2 > 0.999), accuracy (recoveries 99%-101%) and sensitivity, with LODs and LOQs meeting acceptable criteria. Forced degradation studies under acid, alkali, oxidative, reductive, photolytic, hydrolytic and thermal conditions confirmed the method's stability-indicating capability, with all purity angles below their respective purity thresholds and acceptable mass balance. LC-MS/MS analysis further enabled structural characterisation of major degradation products. Overall, the developed RP-UPLC method is selective, reproducible and suitable for routine quality control and stability evaluation of treosulfan in bulk drugs.
    Keywords:  ICH guidelines; LC–MS/MS; UPLC; impurity profiling; stability‐indicating method; treosulfan
    DOI:  https://doi.org/10.1002/bmc.70389
  31. Food Res Int. 2026 Mar 01. pii: S0963-9969(25)02600-6. [Epub ahead of print]227 118260
    Highly sensitive separation and characterization of multiple mycotoxins by supercritical fluid chromatography combined with ion mobility quadrupole time-of-flight mass spectrometry.
    Yi-Wen Wu, Qi-Zhong Jin, Kai-Yang Fan, Jun Cao.
      Addressing the growing demand for efficient and green analytical techniques in food safety, this study established a novel supercritical fluid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry (SFC-IM-QTOF/MS) approach for rapid identification of multiple mycotoxins in complex matrices, which demonstrated clear advantages in analysis speed and solvent consumption over conventional methods. This method was systematically optimized and enabled the rapid determination of 20 mycotoxins, providing collision cross-section values as a stable and reliable identification marker, and the fragmentation patterns of typical compounds were analyzed. The developed SFC-IM-QTOF/MS method demonstrated good linearity (r2 > 0.9985), precision (RSD < 4.883 %), and low limits of detection (0.0012-0.1685 μg/mL). Moreover, the approach was successfully applied to the simultaneous determination of typical mycotoxins in corn and walnut, with recoveries ranging from 78.72 % to 113.12 %, which was acceptable within analytical standards.
    Keywords:  Corn; Ion mobility mass spectrometry; Mycotoxins; Quadrupole time-of-flight mass spectrometry; Supercritical fluid chromatography; Walnut
    DOI:  https://doi.org/10.1016/j.foodres.2025.118260
  32. J Anal Toxicol. 2026 Feb 06. pii: bkag008. [Epub ahead of print]
    Micro-QuEChERS extraction method optimization for quantification of designer benzodiazepines in forensic postmortem blood samples.
    Karla Aparecida de Oliveira Souza, Janaína Aparecida Reis Teodoro, Débora Zorron Berlinck, Ana Paula Knak Rezendes, Daniela Mendes Louzada de Paula, Fabricio Souza Pelição, Elvis Medeiros de Aquino, Victor Alexandre Percinio Gianvecchio, Mauricio Yonamine, Jose Luiz Costa.
      The aim of this study was to develop and validate a quantitative method for the analysis of designer benzodiazepines in postmortem blood samples using micro-QuEChERS extraction and liquid chromatography tandem mass spectrometry (LC-MS/MS). A comprehensive optimization of the method was performed using a multivariate statistical approach, incorporating validation criteria in line with established practices for method validation in forensic toxicology. The method showed linearity between 1 and 200 ng/mL (r2>0.990), with good imprecision (<9.8%) and inaccuracy (<11.1%) evaluated at three different quality control concentrations. Matrix effects and recovery rates were found to be better than 58% and 77.5%, respectively. No carryover or interferences were detected during the analysis. The method was effectively utilized on two real forensic postmortem blood samples, both of which tested positive for bromazolam, showing concentrations of 31 ng/mL and 40 ng/mL. The micro-QuEChERS extraction method demonstrated satisfactory analytical performance and is an environmentally sustainable option, minimizing the use of solvents and reagents, with potential for application in both clinical and forensic analyses, aligning with green analytical toxicology principles.
    Keywords:  LC-MS/MS; designer benzodiazepines; green analytical toxicology; micro-QuEChERS; postmortem blood samples
    DOI:  https://doi.org/10.1093/jat/bkag008
  33. J Pharm Anal. 2026 Jan;16(1): 101385
    FPS_P/N: A two-dimensional mass spectrometry utilization program with precursor ion determination for accurately distinguishing anthocyanin from other flavonoids.
    Ya-Hui Ge, Lili Zhang, Shilin Gong, Wen Miao, Li Zhang, Weibin Bai, Jian-Lin Wu, Na Li.
      Anthocyanins, a unique class of flavonoids with flavylium skeletons, are valued for antioxidant properties. However, distinguishing anthocyanins from co-existing flavonoids using conventional automated tandem mass spectrometry (MS) analysis methods remains challenging. This difficulty arises from low specificity of MS features and confusion of precursor ions, leading to substantial false confidence annotations. To address it, we have developed the strategy of positive (POS)-to-negative (NEG) primary MS (MS1) intensity ratios detecting with fast polarity switching (FPS), termed FPS-POS/NEG, to determine their specific precursor ions. Moreover, we developed an automated program leveraging FPS-POS/NEG strategy (FPS_P/N) streamlining screening candidate pool with molecular networking analysis from MS1 and secondary MS (MS2), determining precursor ions with FPS-POS/NEG, and annotation with MS2. This program enables simultaneous capture of positive and negative signals in a single run and accurate determination of precursor ions for anthocyanins (5.98-9.28) and other flavonoids (-2.52 to 2.08). The underlying mechanisms were elucidated by difference in protonated and deprotonated Gibbs free energy (ΔG) and in-source fragmentation (ISF). FPS-POS/NEG strategy was validated across a broad pH range (0.1%-2% formic acid (FA)) and demonstrated high alignment accuracy (retention time difference, 0.011 min) and consistency (relative standard deviation (RSD), 0.38%-4.62%). Using blueberry, 20 anthocyanins (nonacylated and acylated) and 14 additional flavonoids were annotated. With two-dimensional integration of positive and negative MS1 intensities with intensity ratios, FPS_P/N program provides a novel way to identify anthocyanins from other flavonoids. We anticipate this innovative method will enhance the high-throughput qualification of anthocyanins and other flavonoids in complex samples.
    Keywords:  Anthocyanins; Fast polarity switching; Flavonoids; High-throughput metabolomics analysis; Precursor ion determination
    DOI:  https://doi.org/10.1016/j.jpha.2025.101385
  34. Data Brief. 2026 Apr;65 112464
    Multi-analytical dataset on Lekhaniya Mahakashaya: HRLC-MS/MS Orbitrap profiling, HPTLC fingerprinting with marker estimation, and FTIR spectroscopy.
    Narayan Singh, Anjali Upadhyay, Debajyoti Chakraborty, Girimalla Patil, Pramod Yadav, Galib R, Pradeep Kumar Prajapati.
      This dataset provides a comprehensive, multidimensional phytochemical characterization of Lekhniya Mahakashaya (LMK), a classical Ayurvedic formulation used for the Treatment of obesity and metabolic disorders. Three complementary analytical platforms were employed: High-Resolution Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (HRLC-MS/MS) Orbitrap, High-Performance Thin Layer Chromatography (HPTLC), and Fourier-Transform Infrared (FTIR) spectroscopy. For HRLC-MS/MS analysis, Hydroalcoholic extracts of LMK were prepared and analysed in both Positive and negative ionisation modes using an Orbitrap mass spectrometer. The dataset includes 2034 metabolomics-identified compounds: 1712 in positive ion mode and 322 in negative ion mode, with detailed retention times, molecular weights, and fragmentation patterns, suitable for compound annotation, metabolite networking, and cheminformatics-based correlation studies. HPTLC fingerprinting was performed using methanolic extracts (2-10 µL) on silica gel 60 F₂₅₄ plates, which yielded 7-8 reproducible peaks across the Rf range 0.12-0.89 under 254 nm, 366 nm, and 540 nm, confirming LMK's polyherbal complexity. Marker-based quantification revealed that berberine (0.24 % w/w) and curcumin (0.31 % w/w) were performed using validated HPTLC protocols, and calibration curves are included for reproducibility. FTIR Spectroscopic data encompass 19 absorption peaks (3278-0468 cm⁻¹), representing hydroxyl, aliphatic, unsaturated, sulfur-, nitrogen-, and halogen-containing functional groups, which highlights LMK's diverse phytochemical matrix. This dataset is structured for pharmacological exploration, quality control, and phytochemical standardisation of LMK and associated Ayurvedic formulations. This dataset is a reference resource. Additionally, the dataset can be used for molecular docking validation, network pharmacology mapping, metabolomics comparisons, and future drug discovery. To promote transparency, encourage computational or experimental reuse, and support integrative research on traditional medicine, all raw chromatograms, spectrum files, and processed data tables are made available in widely accessible formats.
    Keywords:  Ayurveda formulation; Berberine; Bioactive compounds; Curcumin; Metabolomics; Phytochemical analysis
    DOI:  https://doi.org/10.1016/j.dib.2026.112464
  35. Analyst. 2026 Feb 06.
    Initial testing procedure based on microextraction followed by LC-HRMS analysis to determine 107 xenobiotics and their metabolites in serum/plasma.
    Monica Mazzarino, Koen Deventer, Laurie De Wilde, Kris Roels, Peter Van Eenoo.
      A simple and sensitive microextraction protocol based on the use of unmodified cellulose was developed for the simultaneous extraction of 107 prohibited compounds and their metabolites from 20 µL of serum and plasma. Sample preparation consisted of spotting 20 µL of serum/plasma onto a cellulose card, followed by extraction of the analytes with 500 µL of a methanol/acetonitrile (1 : 1, v/v) mixture for 20 min. The extracts were analyzed by liquid chromatography coupled to high-resolution mass spectrometry. The entire workflow was validated in terms of selectivity (no interferences were detected at the retention times of the target analytes), sensitivity (limits of detection in the range of 0.08-7.50 ng mL-1) carry-over (no signals in the negative sample injected after the positive sample at high concentration), matrix effect (10-28%), extraction yield (42-89%), and extract stability (the analytes were stable for at least 72 h in the autosampler at 10 °C). The method was successfully applied to the analysis of samples containing the compounds at low nanogram per milliliter range, demonstrating its effectiveness for doping control purposes. Stability studies showed that the compounds were stable for at least 3 months at -20 and 4 °C in serum and plasma samples. In contrast, at 22 °C several thiazide-based compounds were completely degraded after 4 weeks; FG2216 was no longer detectable after 7 weeks; S6 and RAD140 were no longer detectable after 9 weeks, whereas trenbolone was completely degraded after 14 weeks. The other compounds were still visible for the entire study period, with variations in the range of 37-56%.
    DOI:  https://doi.org/10.1039/d5an01329a
  36. Mikrochim Acta. 2026 Feb 02. 193(2): 123
    Tailoring of metal organic framework with electrospun polyacrylonitrile for on-chip electromembrane extraction and determination of anticancer drugs in biological samples.
    Mehdi Bagheri, Yadollah Yamini, Razieh Zamani.
      An on-chip electromembrane extraction integrated with solid-phase microextraction (EME-SPME) was developed for the determination and monitoring of anticancer drugs, including imatinib, irinotecan, and mitomycin C, in various biological fluids. The extraction device incorporated an electrospun polyacrylonitrile (PAN)/MOF-303 nanocomposite fiber, which functioned as the extraction phase. Simultaneous analyte migration under an applied electric field and adsorption onto the nanocomposite surface enabled efficient preconcentration within the compact microfluidic platform. Under optimized conditions, the proposed EME-SPME-HPLC-UV method exhibited low limits of detection ranging from 0.1 to 1.5 µg L-1. Satisfactory linearity was achieved across the ranges 0.5-1000 ng mL-1 for imatinib and 5-1000 ng mL-1 for both irinotecan and mitomycin C, with coefficients of determination (R2) ≥ 0.9938. The method also demonstrated acceptable precision, with relative standard deviations (RSDs) ≤ 7.5%. The applicability of the system was investigated through the extraction of target analytes from human urine and plasma samples, yielding relative recoveries between 78 and 110%. These results highlight the potential of the developed EME-SPME platform as a sensitive, precise, and environmentally friendly technique for therapeutic drug monitoring of anticancer agents in complex biological matrices.
    Keywords:  Anticancer drugs; Chemotherapy drugs; Electromembrane extraction; MOF-303; On-chip EME; Solid phase microextraction
    DOI:  https://doi.org/10.1007/s00604-025-07747-0
  37. BMC Chem. 2026 Feb 03.
    Green evaluation of human plasma levels of metformin, linagliptin, and empagliflozin using HPLC and HPTLC methods: a pharmacokinetic study.
    Osama I Abdel Sattar, Hamed H M Abuseada, Mohamed Saleh Emara, Islam Selim, Mohamed A Ali.
      Two simple, rapid, cost-effective, and environmentally friendly chromatographic methods were developed and validated for the simultaneous determination of metformin (MEF), linagliptin (LIN), and empagliflozin (EMP) in human plasma, with successful application to pharmacokinetic study. Plasma sample preparation was performed using a straightforward protein precipitation technique employing acetonitrile: methanol: trichloroacetic acid (50:49:1, by volume), which provided high extraction recovery and minimal matrix interference. The first method was based on high-performance liquid chromatography with diode array detection (HPLC-DAD) using an ODS Hypersil C18 column and isocratic elution with a mobile phase consisting of acetonitrile, methanol, and phosphate buffer (pH 3) in a ratio of (40:40:20, by volume), at a flow rate of 1.3 mL/min, with detection at 230 nm. The second method employed high-performance thin-layer chromatography (HPTLC) with densitometric detection at 225 nm, using silica gel 60 F254 plates and n-hexane: methanol: glacial acetic acid (6:3:1, by volume) as the developing system. Excellent linearity was achieved over concentration ranges of 85-1650 ng/mL for MEF, 50-1100 ng/mL for EMP, and 45-950 ng/mL for LIN using the HPLC method, and 500-2800, 100-800, and 50-550 ng/band, respectively, using the HPTLC method, with correlation coefficients exceeding 0.998. The lower limits of quantitation for the HPLC method were 85, 50, and 45 ng/mL for MEF, EMP, and LIN, respectively. Both methods demonstrated satisfactory accuracy, precision, recovery (> 92%), stability, and negligible matrix effects in accordance with European Medicines Agency guidelines. The validated methods were successfully applied to a pharmacokinetic study in healthy volunteers, yielding mean Cmax values of 877.5 ± 162.2 ng/mL (MEF), 576 ± 87.5 ng/mL (EMP), and 680.8 ± 7.9 ng/mL (LIN), with Tmax values of 2.42 ± 0.38, 1.5 ± 0.61, and 5.3 ± 0.52 h, respectively. The obtained pharmacokinetic parameters were consistent with reported literature, confirming the reliability and clinical applicability of the proposed green bioanalytical methods.
    Keywords:  Empagliflozin; HPLC; Linagliptin; Metformin; Pharmacokinetic; Plasma
    DOI:  https://doi.org/10.1186/s13065-026-01726-z
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