bims-metlip 2026-03-01 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–03–01
thirty-one papers selected by
Sofia Costa, Matterworks

Mass Spectrometry-Based Integrated Multiomics Platforms for Medicinal Plant Analysis.
Development and validation of a method for determinion of organophosphorus flame retardant di-ester and hydroxylated metabolites in human urine.
Development and validation of an efficient and sensitive LC-MS/MS assay for simultaneous determination of the high frequency used antibody drug conjugate payload MMAE and anti-vascular tyrosine kinase inhibitor lenvatinib in human plasma.
Definition of Metabolite: Size as a Critical Criterion.
A Simple and Sensitive LC-MS/MS Method for Quantification of Capsaicin in Human Plasma and Its Application to a Clinical Pharmacokinetic Study.
Simultaneous Quantification of Fumonisins and Their Hydrolyzed Metabolites in Donkey Matrices: A Tool for Exposure Assessment and Toxicokinetic Studies.
Unlocking the Bile Acid Universe: Advanced Workflows and a Multidimensional Library of 280 Unique Species.
QbD-based, greenness and whiteness-assessed LC-MS/MS method for simultaneous determination of sodium phenylbutyrate and Taurursodiol with pharmacokinetic application.
Chromatographic separation of vigabatrin, gabapentin and pregabalin on porous graphitic carbon: Application in therapeutic drug monitoring.
High-throughput reaction screening using acoustic ejection mass spectrometry.
A fully automated novel solid-phase extraction method for siRNA drug extraction and quantitation across multiple tissues using LC-MS.
Rapidly Switchable Dual Ion Source Combining ESI with iLTP/Tube Plasma for LC-MS, Multi-2D LC × LC, and SLIM-IM-MS Applications.
Development and Validation of a Confirmatory LC-MS/MS Method Using QuEChERS for Determination of Nitrofuran Metabolites in Eggs According to EU Regulation 2021/808.
Preparation of ZIF-8 grafted magnetic solid-phase extraction sorbent for sensitive determination of four cannabinoids in urine and oral fluid.
Learning from All Views: A Multiview Contrastive Framework for Metabolite Annotation.
Quantification of 24,25-Dihydroxyvitamin D3 in Serum Using LC-MS/MS With Derivatization and Lipid-Removal Filtration.
Dual Ionization Ion-Mobility Mass Spectrometry Hyphenated with Catalytic Oxygenation-Mediated Extraction.
LC-MS/MS-based simultaneous high-sensitivity quantification of clinically relevant high-potency androgens and estrogens in plasma and serum.
A Validated Mass Spectrometry Platform for Oxysterol Analysis of Single Human Gastruloids and Liver Organoids.
Best practices for cysteine analysis.
Simultaneous quantification of fat- and water-soluble vitamins in serum by valve-mediated dual LC-MS/MS.
Simultaneous Analysis of Δ9-THC, Δ8-THC, CBD, and CBN in Breath Aerosols Collected Using Cannabix Technologies Breath Collection Unit.
Enrichment and Preservation of Urine Metabolites Using Styrene Divinylbenzene Reversed Phase Sulfonate (SDB-RPS) Disks for Enhanced Biomarker Discovery and Disease Monitoring.
Development and validation of a HILIC-ESI/MS method for quantitation of three oxicams and 5-hydroxypiroxicam in human breast milk.
Development and Validation of a Liquid Chromatograph-Tandem Mass Spectrometry Method for Quantifying Ganciclovir in Dried Blood Spots for Therapeutic Drug Monitoring.
Development and Validation of an LC-MS/MS Method for the Determination of Alternaria Mycotoxins in Hepatic Tissue.
LC-MS/MS for Simultaneous Determination and Isomer Separation of 12 Glucocorticoid Residues in Multiple Aquatic Foods.
Determination of Gentamicin: Development and Validation of a Sensitive UPLC-MS/MS Assay According to the European Medicines Agency Guideline.
LCMS-Net: Deep Learning for Raw High Resolution Mass Spectrometry Data Applied to Forensic Cause-of-Death Screening.
Chemical reaction-enabled lipidomics: from sensitive structural analysis to biomedical applications.
Clinical Application of Microvolume LC-MS/MS for Therapeutic Drug Monitoring of Immunosuppressants in Solid-Organ Transplant Recipients.

Curr Protoc. 2026 Feb;6(2): e70334

Mass Spectrometry-Based Integrated Multiomics Platforms for Medicinal Plant Analysis.

Cemil Can Eylem, Sevilay Erdoğan Kablan, Tuba Reçber, Engin Koçak, Emirhan Nemutlu.

  Plant-based multiomics approaches provide powerful tools for elucidating metabolic regulation, biochemical diversity, and functional responses to genetic and environmental variation. However, plant matrices pose unique analytical challenges due to their chemical complexity, high levels of secondary metabolites, and strong matrix effects that can compromise reproducibility if workflows are not carefully standardized. This article presents a comprehensive and integrated set of protocols for untargeted plant metabolomics, lipidomics, and proteomics, coupled with robust data processing, statistical analysis, and multiomics integration strategies. The protocols describe harmonized workflows for sample collection, preparation, and analysis using gas chromatography-mass spectrometry (GC-MS)-based metabolomics, liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qTOF-MS)-based metabolomics, liquid chromatography-mass spectrometry (LC-MS)-based lipidomics, and microflow LC-MS/MS-based proteomics. Emphasis is placed on critical parameters specific to plant matrices, including complete solvent removal prior to GC-MS derivatization, optimized MS/MS acquisition strategies for high-confidence annotation, and quality control-driven experimental design. Detailed guidance is provided for instrument maintenance, QC strategies, and prevention of analytical artifacts. In addition, the article outlines best practices for data preprocessing, metabolite and lipid annotation, statistical analysis, pathway mapping, and integration of metabolomics with proteomics data to support biologically meaningful interpretation. Collectively, these protocols enable reproducible, high-quality plant multiomics studies and are suitable for both method development and large-scale comparative analyses across plant species, tissues, and experimental conditions. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Plant material collection, handling, and extraction processing Support Protocol 1: Soxhlet extraction Alternate Protocol 1: Dichloromethane (DME)-based sample preparation for lipidomics Alternate Protocol 2: Methyl-tert-butyl ether (MTBE)-based sample preparation for lipidomics Basic Protocol 2: GC-MS-based metabolomics analysis Basic Protocol 3: LC-qTOF-MS-based metabolomics analysis Basic Protocol 4: LC-MS-based lipidomics analysis Basic Protocol 5: Microflow LC-MS/MS-based proteomics analysis Basic Protocol 6: Multiomics data integration and statistical analysis.

Keywords:  GC‐MS; LC‐MS; lipidomics; metabolomics; multiomics integration; plant; proteomics

DOI:  https://doi.org/10.1002/cpz1.70334
Anal Chim Acta. 2026 Apr 08. pii: S0003-2670(26)00174-1. [Epub ahead of print]1394 345224

Development and validation of a method for determinion of organophosphorus flame retardant di-ester and hydroxylated metabolites in human urine.

Xiaohong Yu, Tian Qiu, Jingyu Wang, Qingqing Luo, Tong Shen, Yifu Lu, Xiaoming Shi.

   BACKGROUND: As biomarkers for assessing human exposure to organophosphate flame retardants (OPFRs), the accurate measurement of urinary metabolites of OPFRs (mOPFRs) is critical for evaluating their health impacts.
RESULTS: We established a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique for the concurrent quantification of 14 mOPFRs in human urine, including diesters and hydroxylated metabolites. Urine samples were processed via solid-phase extraction, eluted with 2% ammoniated methanol solution, concentrated by nitrogen stream, and finally reconstituted in the original mobile phase before analysis using LC-MS/MS, executed in electrospray ionization and multiple reaction monitoring modes. High-sensitivity mass spectrometry signal acquisition was achieved based on ion fragmentation patterns. Baseline separation of isomeric compounds was accomplished by adjusting the mobile phase elution strength. Enhanced extraction efficiency and purification were based on quasi-molecular properties. The methodology exhibited method detection limits ranging from 0.018 to 0.16 μg/L. Spiked recovery rates varied between 72.2% and 109.6%, while relative standard deviations fluctuated from 5.1% to 14.7%, and matrix effects were noted to be within the range of 77.2%-112.3%. The reliability of method was validated through the analysis of certified reference materials and participation in proficiency evaluations conducted in various laboratories. This approach has been effectively utilized for analyzing urinary mOPFRs within framework of Chinese National Human Biomonitoring Program by conducting environmentally sustainable and applicable green assessment.
SIGNIFICANCE: This method thoroughly optimized essential elements concerning mass spectrometry settings, chromatographic parameters, and sample preparation procedures, providing reliable technical support for assessing population exposure to OPFRs and conducting risk evaluations.

Keywords:  Green assessment; Human urine; Liquid chromatography-tandem mass spectrometry; Organophosphorus flame retardant metabolites; Solid-phase extraction

DOI:  https://doi.org/10.1016/j.aca.2026.345224
J Pharm Biomed Anal. 2026 Feb 23. pii: S0731-7085(26)00103-2. [Epub ahead of print]274 117435

Development and validation of an efficient and sensitive LC-MS/MS assay for simultaneous determination of the high frequency used antibody drug conjugate payload MMAE and anti-vascular tyrosine kinase inhibitor lenvatinib in human plasma.

Yongqing Wen, Wenjuan Wang, Xu Ma, Hong Liu, Yue Yin, Ran Li, Yuanyuan Jiao.

  Antibody drug conjugates (ADCs)-based combination therapies are increasingly studied in clinics. Monomethyl auristatin E (MMAE) is the most frequently used payload in approved ADCs and lenvatinib is a classic anti-vascular tyrosine kinase inhibitor. This study aimed to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for simultaneous determining these two analytes in human plasma. Protein precipitation by methanol was used in sample preparation with deuterated agents as internal standards. A Kinetex C18 column (3.0 × 50 mm, 2.6 μm) was used with gradient elution (0.5 mL/min, 40 °C). Positive electrospray ionization in multiple reaction monitoring (MRM) mode was used for detection. Each run needs 50 μL sample and 3 min. The developed assay was linear over 0.20-100 ng/mL for MMAE and over 1.00-500 ng/mL for lenvatinib, which covered reported pharmacokinetic (PK) ranges for both agents. This assay was validated according to ICH M10 and was qualified in terms of selectivity, linearity, accuracy (-6.7 % to 4.5 %), precision (1.5-7.4 %), matrix effects, dilution test, carry-over and stability in human plasma. The in vitro studies suggested no significant in-process release for conjugated MMAE from two ADCs with protease-cleavable maleimidocaproylvaline-citrulline-p-aminobenzyloxycarbonyl linker. Clinical samples were efficiently analyzed and values fell in reported PK ranges. Overall, the developed method is robust and efficient and will potentially support clinical PK studies or therapeutic drug monitoring (TDM) for MMAE-containing ADCs and lenvatinib alone or in combinations. This method also serves as a starting point to build cassette assay by including more tyrosine kinase inhibitors.

Keywords:  Antibody-drug conjugate; Bioanalysis; Lenvatinib; Liquid chromatography-tandem mass spectrometry; MMAE; Pharmacokinetics

DOI:  https://doi.org/10.1016/j.jpba.2026.117435
J Proteome Res. 2026 Feb 24.

Definition of Metabolite: Size as a Critical Criterion.

Aldo Moreno-Ulloa.

  Metabolites have traditionally been defined as organic molecules smaller than 1500 daltons (Da). However, recent advances in analytical technologies and chemoinformatics have uncovered a wider chemical diversity, including biologically significant metabolites exceeding this conventional size cutoff─such as polypeptides, glycosphingolipids, and bacterial lipopolysaccharides. Detecting these larger metabolites challenges standard metabolomics approaches and necessitates optimized mass spectrometry acquisition parameters. In this perspective, the analysis of multiple databases (HMDB blood, GNPS, Plant Molecular Network [PMN], Natural Product Atlas [NPA] fungi, NPA bacteria, and MiMeDB) confirms that although most metabolites are below 1000 Da, notable populations exceed this threshold, particularly in bacterial data sets. Furthermore, reanalysis of liquid chromatography-mass spectrometry (LC-MS2) data sets from diverse biological samples, especially bacteria-rich matrices like feces and skin, reveals features (peaks with m/z and retention time) extending beyond 1500 m/z. These findings underscore that metabolites are often larger than commonly recognized in the literature. Therefore, the definition of metabolites should evolve to accommodate their size diversity, ensuring accurate knowledge dissemination to new generations of metabolomics researchers.

Keywords:  mass spectrometry; metabolite; metabolomics; size; spectral library

DOI:  https://doi.org/10.1021/acs.jproteome.5c00747
Biomed Chromatogr. 2026 Apr;40(4): e70405

A Simple and Sensitive LC-MS/MS Method for Quantification of Capsaicin in Human Plasma and Its Application to a Clinical Pharmacokinetic Study.

Xiaoyu Xu, Wenyuan Qi, Juan Wang, Lei Yang, Xiaohui Liu, Wei Xue, Kexin Li.

  Capsaicin, a transient receptor potential vanilloid 1 (TRPV1) agonist, is used topically for neuropathic pain. Its minimal systemic absorption necessitates highly sensitive quantification methods to accurately assess systemic exposure. This study aimed to develop and validate a simple, rapid, and highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of capsaicin in human plasma. Plasma samples (50 μL) were prepared by a simple protein precipitation using acetonitrile, with capsaicin-d3 as the internal standard (IS). Chromatographic separation was performed on a Waters Xbridge C18 column (2.1 × 50 mm, 3.5 μm) with a 3-min gradient elution using 1% formic acid in water and acetonitrile. Detection was conducted on a triple quadrupole tandem mass spectrometer in positive electrospray ionization (ESI) mode, utilizing multiple-reaction monitoring (MRM). The assay was validated over the linear range of 0.05-50 ng/mL. The method demonstrated excellent precision (CV% ≤ 11.62%) and accuracy (RE% within ±13.60%), with high recovery (>95%). Stability was confirmed under various conditions relevant to clinical sample handling. The validated method was successfully applied to a pharmacokinetic study involving 24 patients with knee osteoarthritis receiving a topical capsaicin liniment, demonstrating its suitability for clinical applications.

Keywords:  LC‐MS/MS; capsaicin; human plasma; pharmacokinetics; topical administration

DOI:  https://doi.org/10.1002/bmc.70405
Toxins (Basel). 2026 Feb 04. pii: 80. [Epub ahead of print]18(2):

Simultaneous Quantification of Fumonisins and Their Hydrolyzed Metabolites in Donkey Matrices: A Tool for Exposure Assessment and Toxicokinetic Studies.

Dongying Tian, Yunduo Zheng, Yandong Li, Qianwen Xing, Gang Lin, Ronghua Zhu, Quigang Ma, Peilong Wang, Ruiguo Wang.

  A novel, sensitive, and robust LC-MS/MS method was developed and fully validated for the simultaneous determination of fumonisins (FB1, FB2, FB3) and their hydrolyzed metabolites (HFB1, HFB2, HFB3) in donkey plasma, urine, and feces-three critical matrices for toxicokinetic studies. Sample preparation was optimized for each matrix: salting-out assisted liquid-liquid extraction (SALLE) with perchloric acidification for urine and feces, and a dilute-evaporate-shoot (DES) approach for plasma. Chromatographic separation was achieved on a BEH C18 column with water-ACN containing 0.5% formic acid. The method demonstrated excellent linearity (R2 ≥ 0.99), acceptable accuracy (mean recoveries: 73.3-111.5%), and good precision (intra- and inter-day RSDs < 20%). The limits of quantification (LOQ) for FBs and HFBs were 0.1-0.15 μg/L in plasma, 1.0 μg/L in urine, and 60 μg/kg in feces. To our knowledge, this is the first reported method capable of quantifying this comprehensive panel of analytes across multiple biological matrices in donkeys, providing an essential tool for future exposure assessments and pharmacokinetic research in this species.

Keywords:  LC-MS/MS; biological matrices; donkey; fumonisins; hydrolyzed fumonisins; method validation; toxicokinetics

DOI:  https://doi.org/10.3390/toxins18020080
bioRxiv. 2026 Feb 19. pii: 2026.02.18.706700. [Epub ahead of print]

Unlocking the Bile Acid Universe: Advanced Workflows and a Multidimensional Library of 280 Unique Species.

Guozhi Zhang, Emily C Vincent, Sadie M Disselkoen, James N Dodds, Quentin DuVal-Smith, Abubaker Patan, Ipsita Mohanty, Victoria Deleray, Jian Zhang, Paul A Thiessen, Evan E Bolton, Emma L Schymanski, Pieter C Dorrestein, Casey M Theriot, Erin S Baker.

Microbes and bile acids are tightly intertwined, especially in the gut. While the liver produces primary bile acids from cholesterol, gut bacteria transform these into diverse secondary forms which act as powerful signaling molecules, influencing host metabolism and immune function. Since bile acid changes are increasingly linked to health and disease, their accurate measurement in the gut and circulation is essential. Analytical evaluations, however, remain challenging as many bile acids co-elute in liquid chromatography (LC), share identical precursor masses in mass spectrometry (MS), and produce similar tandem mass spectrometry (MS/MS) spectra. As a result, conventional LC-MS/MS workflows struggle to differentiate bile acids, motivating the addition of orthogonal separations such as ion mobility spectrometry (IMS). Here, we assess optimal bile acid extraction parameters for stool, serum, and plasma; compare LC conditions; and assess electrospray ionization performance across polarities. Additionally, we created a multidimensional reference library containing LC retention times, IMS collision cross section values, and accurate precursor masses for 280 unique bile acids (264 endogenous and 16 deuterium-labeled species) including unconjugated, host-conjugated, and microbially conjugated bile acids. This multidimensional library empowers bile acid identification in complex samples and enables a more comprehensive exploration of their biological roles and disease associations.

DOI: https://doi.org/10.64898/2026.02.18.706700
J Pharmacol Toxicol Methods. 2026 Feb 21. pii: S1056-8719(26)00010-9. [Epub ahead of print]138 108417

QbD-based, greenness and whiteness-assessed LC-MS/MS method for simultaneous determination of sodium phenylbutyrate and Taurursodiol with pharmacokinetic application.

Thirumalavasu Palla, Venkata Kanaka Srivani Maddala, Kumaraswamy Gandla.

  Sodium phenylbutyrate (NaPB) and taurursodiol (TRS) are clinically important agents frequently investigated in combination for therapeutic applications. To facilitate pharmacokinetic studies, a sensitive, robust, and environmentally sustainable bioanalytical method for their simultaneous determination in plasma was developed. A Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method was systematically optimized using a Box-Behnken design within the Design of Experiments framework. The influence of organic solvent proportion, flow rate, and mobile phase pH was evaluated, yielding optimal conditions of 40% (v/v) organic modifier, a flow rate of 1.0 mL/min, and mobile phase pH 2.6. Detection was achieved via electrospray ionization in positive selected reaction monitoring (SRM) mode with transitions m/z 187.2 → 144.7 for NaPB, m/z 500.7 → 218.9 for TRS, and m/z 516.5 → 440.0 for taurocholic acid (internal standard). Method validation, performed according to EC 2002/657/EC guidelines, confirmed excellent linearity (r2 > 0.999), accuracy, precision within 15% CV, and analyte stability under various storage and handling conditions. The method was successfully applied to a pharmacokinetic study in Wistar rats. Greenness and whiteness assessment further demonstrated strong environmental compatibility. Overall, the developed LC-MS/MS protocol is precise, reproducible, and sustainable, providing a valuable tool for preclinical pharmacokinetic investigations of NaPB and TRS.

Keywords:  Design of expert; Green analytical chemistry; Liquid chromatography–tandem mass spectrometry; Pharmacokinetics; Sodium phenylbutyrate; Taurursodiol

DOI:  https://doi.org/10.1016/j.vascn.2026.108417
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Feb 21. pii: S1570-0232(26)00063-2. [Epub ahead of print]1274 124974

Chromatographic separation of vigabatrin, gabapentin and pregabalin on porous graphitic carbon: Application in therapeutic drug monitoring.

Jens Martens-Lobenhoffer, Stefanie M Bode-Böger.

  The antiepileptic drugs vigabatrin, gabapentin and pregabalin are of polar and hydrophilic nature, and thus difficult to retain on reversed phase stationary phases. In our study, we present the chromatographic separation of these substances on porous graphitic carbon as stationary phase by gradient elution using 0.1% trifluoroacetic acid and acetonitrile as mobile phases. Sample preparation from human plasma consisted of addition of the stable isotope labelled internal standard D4-pregabalin, dilution by 0.1% trifluoroacetic acid and protein precipitation by perchloric acid. Extraction efficiency was quantitative with no significant differences to 100% theoretical yield. The analytes were detected by tandem mass spectrometry. Selectivity was very good with no signals observed from endogenous substances nor other potentially co-administrated antiepileptic drugs. Calibration ranges were 0.5-25 μg/mL, 0.6-30 μg/mL and 0.2-10 μg/mL for vigabatrin, gabapentin and pregabalin, respectively, and covered the therapeutic concentrations of these substances in human plasma. Precision of the method was better than 10% for all analytes at all concentration levels, while accuracy was better than 15% at the LLOQ and better than 10% at all other levels. The method was successfully introduced in our laboratory for routine TDM in patients treated with these drugs.

Keywords:  Gabapentin; LC-MS/MS; Porous graphitic carbon; Pregabalin; Vigabatrin

DOI:  https://doi.org/10.1016/j.jchromb.2026.124974
Nat Protoc. 2026 Feb 23.

High-throughput reaction screening using acoustic ejection mass spectrometry.

Maowei Hu, Daniel J Blair.

High-throughput chemical synthesis plays a critical role in generating compound libraries and optimizing reaction conditions. Increasing adoption of robots and multiwell formats means that hundreds of reactions can be accessed with ease. However, conventional methods for quantitative detection of the product (for example, by liquid chromatography-mass spectrometry) create a bottleneck in the workflow because analyses need to be customized separately for each sample. Here we describe a tandem mass spectrometry approach where the samples are analyzed directly using acoustic ejection mass spectrometry. This approach features simple method development enabling accurate quantification for reactions where a characteristic neutral lost fragment is common to both the chemical starting material and the expected reaction products. Combining this precise fragmentation signature with acoustic ejection mass spectrometry allows whole 384-well plates of chemical reaction data to be collected at the same pace as two liquid chromatography-mass spectrometry samples, while maintaining the same levels of accuracy. To explain the principles involved in designing these experiments, we show four examples of common medicinal chemistry transformations for C-N and C-C bond formation that have been used in the synthesis of analogs of cereblon binding molecular glues, antifungals, antibiotics, and building blocks for automated small molecule synthesis. The whole procedure requires ~2 days of work to complete, including the 384-well plate reaction setup, analytical sample preparation, mass spectrometry data collection and analysis.

DOI: https://doi.org/10.1038/s41596-025-01320-y
Bioanalysis. 2026 Feb 26. 1-11

A fully automated novel solid-phase extraction method for siRNA drug extraction and quantitation across multiple tissues using LC-MS.

Tony Karlsborn, Carl L Stenbratt, Amalyn Nain-Perez, Mikko Hölttä.

   BACKGROUND: Approaches such as liquid-liquid extraction and two-step liquid-liquid extraction combined with SPE has been widely used for extraction of oligonucleotide therapeutics. It has been more challenging to develop a generic one-step SPE method that can be used across multiple tissue types.
MATERIALS & METHODS: An SPE method was developed for extraction of oligonucleotide therapeutics from tissue homogenates utilizing an ASO internal standard and a siRNA analyte. The method was fully automated using a liquid handler and an automated SPE station. Quantification of the siRNA antisense was performed using LC-MS, with calibration samples prepared in a surrogate matrix.
RESULTS: The fully automated SPE method enabled quantification of the siRNA antisense across nine different tissues from three different species. The SPE method utilizing surrogate matrix calibration was compared to liquid-liquid extraction by analyzing in-vivo samples using both workflows, showing good agreement between the two different extraction methods.
CONCLUSIONS: This manuscript presents a one-step SPE method greatly reducing blank tissue requirements and sample preparation workload by enabling use of surrogate matrix calibration and full automation. The presented SPE method simplifies and reduces analytical time in bioanalysis of tissue biodistribution studies and adheres to the principles of 3R.

Keywords:  LC-MS/MS analysis; Solid phase extraction (SPE); automation; oligonucleotide therapeutics; siRNA quantification; surrogate matrix calibration; tissue homogenates

DOI:  https://doi.org/10.1080/17576180.2026.2636553
J Am Soc Mass Spectrom. 2026 Feb 25.

Rapidly Switchable Dual Ion Source Combining ESI with iLTP/Tube Plasma for LC-MS, Multi-2D LC × LC, and SLIM-IM-MS Applications.

Marvin Häßler, Katharina Wetzel, Cedric Thom, Sebastian Löbbecke, Jaqueline Leddin, Florian Uteschil, Dustin Linke, Aristotelis Charchantis, Lidia Montero, Juan F Ayala-Cabrera, Oliver J Schmitz.

  High-throughput analysis is becoming increasingly important in liquid chromatography-mass spectrometry coupling to minimize downtime for efficient and cost-effective operations in the analytical laboratory. In response to this need, a dual ion source has been designed to provide two ionization modes under atmospheric conditions: electrospray ionization (ESI) and dielectric-barrier discharge (DBD)-based plasma. This dual ion source allows for the rapid switching of the ionization mode during a chromatographic run. Therefore, substances with different ionization behaviors can be detected within one measurement if their retention times differ. The performance of the dual ion source was evaluated using single- and multidimensional liquid chromatography coupled to a quadrupole-time-of-flight (Q-TOF) mass spectrometer and a structures for lossless ion manipulations ion mobility mass spectrometer to analyze mixed standards and real samples. Most notably, the dual ion source maintained strong ion signals and a stable performance while switching ionization modes during a run. Instrumental limits of detection reached down to the sub 1 nM range for both ESI and DBD. This work describes the design and construction of the dual ion source, which provides electrospray- and plasma-based ionization that can be switched within a chromatographic run.

Keywords:  SLIM; TPI; iLTP; ion mobility; mass spectrometry; plasma-based ionization

DOI:  https://doi.org/10.1021/jasms.6c00015
Molecules. 2026 Feb 17. pii: 700. [Epub ahead of print]31(4):

Development and Validation of a Confirmatory LC-MS/MS Method Using QuEChERS for Determination of Nitrofuran Metabolites in Eggs According to EU Regulation 2021/808.

Elmira Marku, Kozeta Vaso, Martin Danaher, Erinda Pllaha, Suela Teqja, Jonida Canaj, Ina Pasho, Ilir Ajdini.

  Nitrofurans are banned veterinary medicinal products due to their carcinogenic and mutagenic properties; however, their protein-bound metabolites (AOZ, AMOZ, AHD, SEM, and DNSAH) may persist in food-producing animals, particularly in eggs. Reliable confirmatory methods are therefore essential for residue monitoring under the stringent requirements of Commission Implementing Regulation (EU) 2021/808. This study reports the development and validation of a sensitive and selective LC-MS/MS method combining acid hydrolysis, 2-nitrobenzaldehyde derivatization, and QuEChERS extraction for the determination of nitrofuran metabolites in eggs. Chromatographic separation was carried out using a phenyl-hexyl column, and detection using a tandem mass spectrometer, supported by isotope-labeled internal standards, ensured robust identification and quantification. Linearity was satisfactory over the investigated concentration range (R2 > 0.99), with recoveries between 82 and 109%. The method's precision was acceptable, with repeatability RSD values below 10% and within-laboratory reproducibility RSD values below 22%. Matrix effects were effectively controlled, remaining within ±20% following internal standard normalization. Decision limits (CCα) ranged from 0.29 to 0.37 µg/kg, well below the EU reference point for action of 0.5 µg/kg. The method's performance was further confirmed through participation in an accredited proficiency test scheme. Overall, the validated method provides a reliable analytical tool for routine official control laboratories, enabling the sensitive confirmatory detection of banned nitrofuran residues in eggs and supporting food safety and regulatory compliance.

Keywords:  2-nitrobenzaldehyde derivatization; LC-MS/MS; eggs; food safety; metabolites; nitrofurans; validation

DOI:  https://doi.org/10.3390/molecules31040700
Anal Chim Acta. 2026 Apr 08. pii: S0003-2670(26)00144-3. [Epub ahead of print]1394 345194

Preparation of ZIF-8 grafted magnetic solid-phase extraction sorbent for sensitive determination of four cannabinoids in urine and oral fluid.

Hongyu Ning, Yilei Fan, Haodong Wang, Huijun Liu, Zhongping Huang, Haixing Wang, Xing Ke, Yu Xu.

   BACKGROUND: The global increase in cannabis use has created a strong demand for reliable and efficient detection methods. Traditional urine-based assays are widely used and standardized, but suffer from complex hydrolysis requirements, long detection windows, and an inability to monitor recent use. In contrast, oral fluid testing enables non-invasive, real-time detection of parent cannabinoids, but is limited by low sample volume, matrix interference, and insufficient sensitivity in common rapid tests. It is clear that a sample preparation and detection approach is needed that is robust, highly sensitive, streamlined, and compatible with high-throughput confirmatory analysis such as LC-MS/MS.
RESULTS: In this study, a magnetic solid-phase extraction (MSPE) method was developed using a novel magnetic metal-organic framework composite, Fe3O4@poly(GMA/DVB-ZIF-8), for the efficient extraction of four cannabinoids from urine and oral fluid. The sorbent uses multiple interactions-including hydrophobic, π-π, electrostatic, and Zn-O coordination-to achieve high analyte recovery (exceeding 85%). Coupled with UHPLC-MS/MS analysis, the method demonstrates wide linearity (0.05-25 ng/mL, r2 > 0.9945), low detection limits (as low as 0.017 ng/mL), and good repeatability (RSDs <13.8%). Comparative evaluation with conventional liquid-liquid extraction showed minimal deviation (relative percent difference <6.0%), confirming high trueness and reliability for cannabinoid monitoring in biological samples.
SIGNIFICANCE: This MSPE approach provides a simple, efficient and high-throughput sample preparation method for reliable and rapid forensic analysis of cannabinoids in complex biological samples.

Keywords:  Biological samples; Cannabinoids; Magnetic solid-phase extraction; Metal–organic frameworks; Sensitive detection; UHPLC–MS/MS

DOI:  https://doi.org/10.1016/j.aca.2026.345194
Anal Chem. 2026 Feb 23.

Learning from All Views: A Multiview Contrastive Framework for Metabolite Annotation.

Yan Zhou Chen, Soha Hassoun.

Metabolomics, enabled by high-throughput mass spectrometry, promises to advance our understanding of cellular biochemistry and guide new discoveries in disease mechanisms, drug development, and personalized medicine. However, as the assignment of molecular structures to measured spectra is challenging, annotation rates remain low and hinder potential advancements. We present MultiView Projection (MVP), a novel framework for learning a joint embedding space between molecules and spectra by leveraging multiple data views: molecular graphs, molecular fingerprints, spectra, and consensus spectra. MVP builds on contrastive multiview learning to capture mutual information across views, leading to more robust and generalizable representations for spectral annotation. Unlike prior approaches that consider multiple views via concatenation or as targets of auxiliary tasks, MVP learns from all views jointly, resulting in improved molecular candidate ranking. Notably, MVP supports annotation using either individual spectra or consensus spectra, enabling flexible use of multiple measurements. On the MassSpecGym benchmark, we show that annotation using query consensus spectra significantly outperforms rank aggregation strategies based on constituent spectrum annotation. Using the consensus spectrum view, MVP achieves 36.0 and 14.0% rank@1 when retrieving candidates by mass and formula, respectively. When ranking using individual spectra, MVP demonstrates performance that is superior to or on par with existing methods, achieving 26.4 and 11.1% rank@1 for candidates by mass and formula, respectively. MVP offers a flexible, extensible foundation for learning from multiple molecule/spectra data views.

DOI: https://doi.org/10.1021/acs.analchem.5c05675
Int J Anal Chem. 2026 ;2026 5736140

Quantification of 24,25-Dihydroxyvitamin D3 in Serum Using LC-MS/MS With Derivatization and Lipid-Removal Filtration.

Marta Studecká, Eliška Fousková, Josefa Provalilová, Roman Viták, Zdeněk Tůma, Michal Jirásko, Markéta Králová, Kateřina Oulehle, Jan Vachek, Ladislav Pecen, Radek Kučera, Richard Pikner.

Accurate assessment of vitamin D metabolism is crucial not only for the diagnosis and treatment of disorders related to bone health and calcium homeostasis but also for understanding its broader physiological roles in immunity, cellular differentiation, cardiovascular regulation, and endocrine function. Although 25-hydroxyvitamin D3 (25(OH)D3) is routinely measured in clinical practice, the low-abundance metabolite 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) provides complementary insight into vitamin D catabolism. Reduced or undetectable 24,25(OH)2D3 levels may signal impaired CYP24A1 function or insufficient conversion of 25(OH)D3 to maintain appropriate intracellular concentrations of biologically active 1,25(OH)2D3. However, quantification of 24,25(OH)2D3 remains analytically challenging and requires highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. In-house developed LC-MS/MS approaches are being increasingly employed, offering greater specificity and sensitivity over conventional immunoassays, allowing accurate detection of low-abundance vitamin D metabolites. Additionally, when interpreted together with 25(OH)D3, 24,25(OH)2D3 allows the calculation of the vitamin D metabolite ratio (VMR), which offers a more accurate assessment of vitamin D sufficiency and catabolism. The aim of this study was to develop and validate a robust LC-MS/MS method using dynamic multiple reaction monitoring (dMRM) and sample derivatization for quantifying 24,25(OH)2D3 in human serum. The method described was evaluated according to EMA guidelines and DEQAS controls. The matrix effect was minimized through lipid-removal filtration. The assay demonstrated excellent linearity (R 2 = 0.9982), intra- and inter-assay precision below 14%, and LOQ of 0.64 ng/mL. Recovery from DEQAS samples ranged from 80% to 118%, with a matrix-induced ion enhancement of ∼17%. Additionally, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatization enhanced the sensitivity 100-fold. This highly sensitive LC-MS/MS method is suitable for clinical and research laboratories equipped with an electrospray ionization (ESI) source. Precise quantification of 24,25(OH)2D3 can complement routine 25(OH)D3 analysis, support VMR determination, and serve as a reliable biomarker for disorders associated with altered vitamin D metabolism.

DOI: https://doi.org/10.1155/ianc/5736140
ACS Meas Sci Au. 2026 Feb 18. 6(1): 204-213

Dual Ionization Ion-Mobility Mass Spectrometry Hyphenated with Catalytic Oxygenation-Mediated Extraction.

Tzu-Ching Tsai, Chamarthi Maheswar Raju, Pawel L Urban.

  Catalytic oxygenation-mediated extraction (COME) is an environmentally friendly liquid-gas extraction technique that generates oxygen microbubbles via the catalytic decomposition of hydrogen peroxide. While corona discharge atmospheric pressure chemical ionization (APCI) is widely used for analyzing moderately polar and lower-polarity analytes with low molecular weights, secondary electrospray ionization (SESI) is a soft ionization technique that effectively ionizes polar volatile analytes. This study aims to integrate APCI and SESI with COME drift-tube ion-mobility (IM) triple quadrupole mass spectrometry (MS) to analyze volatile organic compounds (VOCs) with different physicochemical properties present in liquid matrices. The coupling of a house-built ion-mobility spectrometer with a commercial triple quadrupole mass spectrometer provides 2D separation at low cost. The user can choose one of the two ionization modes to achieve high signals with VOCs of different polarity. COME was applied to extract ethyl acetate from complex matrices (Taiwanese millet wine and whiskey) for immediate IM-MS analysis. An isotopically labeled internal standard was used to compensate for drift time and intensity shifts across multiple analyses. The system operates automatically with a graphical user interface enabling immediate ion-mobility spectrum visualization for targeted m/z.

Keywords:  atmospheric pressure chemical ionization; green analytical chemistry; ion-mobility spectrometry; ionization methods; sample preparation; secondary electrospray ionization; volatile organic compounds

DOI:  https://doi.org/10.1021/acsmeasuresciau.5c00160
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Feb 22. pii: S1570-0232(26)00070-X. [Epub ahead of print]1274 124981

LC-MS/MS-based simultaneous high-sensitivity quantification of clinically relevant high-potency androgens and estrogens in plasma and serum.

Patrick Caron, Michèle Rouleau, Chantal Guillemette.

  We present a robust assay coupling liquid chromatography with tandem mass spectrometry (LC-MS/MS) that enables the quantification of 19 steroids with high sensitivity in 300 μL of human plasma or serum. It includes a broad array of classical adrenal precursors, progesterone, estrogens, androgens, and their metabolites - dehydroepiandrosterone (DHEA), androstenediol (5-diol), androstenedione (A4), dihydrotestosterone (DHT), testosterone (T), androsterone (AST), 5α-androstan-3α-17β-diol (3α-diol), 5α-androstan-3β-17β-diol (3β-diol), estrone (E1), estradiol (E2), and progesterone (PROG). It also includes eight 11‑oxygenated androgens, comprising the abundant 11β-hydroxyandrostenedione (11OHA4) and its 11β-hydroxylated metabolites: 11-keto-androstenedione (11KA4), 11-hydroxytestosterone (11OHT), 11-keto-testosterone (11KT), 11-hydroxydihydrotestosterone (11OHDHT), 11-keto-dihydrotestosterone (11KDHT), 11β-hydroxyandrosterone (11OHAST), and 11-keto-androsterone (11KAST). It directly quantifies PROG and A4 whereas other steroids are derivatized to enhance sensitivity. Rigorous validation and cross-validation against established assays ensure its reliability and accuracy in steroid quantification. The assay exhibits high sensitivity, achieving a lower limit of quantification as low as 3.7 pM, enabling reliable detection at minimal concentrations. These capabilities are crucial for accurate quantification in hormone-related clinical settings. Clinical applicability is demonstrated using plasma and serum specimens of men with prostate cancer undergoing hormone therapy, underscoring the assay's relevance in contexts of low circulating steroid concentrations. The high sensitivity of this assay warrants its utility in diverse clinical contexts where precise quantification is critical.

Keywords:  11‑oxygenated androgens; Clinical samples; Estrogens; LC-MS/MS; Mass spectrometry; Steroid

DOI:  https://doi.org/10.1016/j.jchromb.2026.124981
Anal Chem. 2026 Feb 23.

A Validated Mass Spectrometry Platform for Oxysterol Analysis of Single Human Gastruloids and Liver Organoids.

Kristina Sæterdal Kømurcu, Malgorzata Elzbieta Zawadzka, Igor Meszka, Aleksandra Aizenshtadt, Helena Hrušková, Lydia Emilie Aakervik, James L Thorne, Steven Ray Wilson, Stefan Johannes Karl Krauss, Hanne Røberg-Larsen.

Oxysterols, i.e., hydroxylated cholesterol metabolites, are associated with various signaling pathways and diseases. Their low abundance and structural complexity create analytical challenges, particularly in small sample sizes. We here present an optimized and validated miniaturized sample preparation method that enables oxysterol detection and quantification in single stem cell-derived 3D cell aggregates, as exemplified in human liver organoids (stem cell-based 3D liver models) and human gastruloids (stem cell-based embryo models) using liquid chromatography-mass spectrometry (LC-MS). The method, utilizing enzyme-assisted derivatization with Girard-T reagent, allowed a 10-fold decrease in starting material compared to conventional methodology while maintaining sensitivity and precision. A validation based on Eurachem guidelines confirmed quantitative performance and reproducibility across days and operators. In addition, we introduce a tailored normalization method, allowing same-sample measurements of oxysterols and the total protein content. The miniaturized method enabled successful detection and quantification of oxysterols of expected presence (e.g., 26-hydroxycholesterol), as well as unexpected (24S-hydroxycholesterol) and unknown oxysterols. Using our updated method, we could reveal significant heterogeneity among individual organoids and gastruloids, both between and within cell sources/protocols. Overall, we provide a reliable and high-sensitivity method for analyzing oxysterols in limited biological samples, opening opportunities for further insights into their roles in, e.g., liver function and early embryogenesis.

DOI: https://doi.org/10.1021/acs.analchem.5c07140
Ferroptosis Oxid Stress. 2026 ;pii: 202508. [Epub ahead of print]2

Best practices for cysteine analysis.

Feroza K Choudhury, Gina M DeNicola.

  Accurate measurement of cysteine and related thiol-containing metabolites is essential for understanding cellular redox regulation. However, the intrinsic reactivity and instability of cysteine present substantial analytical challenges. This review summarizes the biochemical context of cysteine and glutathione metabolism, emphasizing their dynamic redox equilibria and physiological relevance. We critically examine existing analytical approaches, including mass spectrometry-based, enzyme-coupled, and colorimetric methods, and discuss their respective strengths and limitations. Particular attention is given to sample preparation, derivatization strategies, and reagent selection, as these steps are crucial for preserving native thiol-disulfide status. Among various alkylating agents, N-ethylmaleimide is identified as the most reliable for thiol stabilization in liquid chromatography-mass spectrometry (LC-MS) workflows, while specific reagents such as monobromobimane or β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) are required for persulfide and polysulfide detection. The review also highlights the pitfalls of using indirect surrogates-such as glutathione or cystathionine levels-to infer cysteine availability, which can lead to significant misinterpretation of metabolic states. We conclude that direct LC-MS-based quantification of cysteine and glutathione, combined with careful derivatization and sample handling, remains the most reliable and accurate approach currently available for the assessment of thiol metabolism and redox homeostasis.

Keywords:  Cysteine; LC-MS; N-ethylmaleimide; derivatization; glutathione; redox homeostasis; thiol analysis

DOI:  https://doi.org/10.70401/fos.2025.0010
J Pharm Biomed Anal. 2026 Feb 23. pii: S0731-7085(26)00108-1. [Epub ahead of print]274 117440

Simultaneous quantification of fat- and water-soluble vitamins in serum by valve-mediated dual LC-MS/MS.

Weifeng Li, Ka Luo, Hou Qian, Yike Wu, Yufan Wu, Yao Gao, Dayong Gu.

  Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the benchmark platform for multiplex vitamin quantification, yet simultaneous determination of chemically disparate fat-soluble (FSVs) and water-soluble vitamins (WSVs) remains analytically challenging. We introduce a pragmatic dual-stream workflow that converts the intrinsic waste segments of any LC gradient into productive analytical time, delivering complete FSV and WSV profiles. Two 5-min chromatographic separations, optimized for fat-soluble and water-soluble vitamins respectively, are staggered through a six-port divert valve: while one column eluted its analytes to the mass spectrometer, the other was shunted to waste. This seamless overlap effectively doubles the throughput within 5 min. And every WSV elution window remained fully enclosed within the waste segment of the FSV gradient, with CVs < 10 % for the internal-standard peak areas across both vitamin classes. Moreover, method comparison between dual LC-MS/MS and single LC-MS/MS across twenty patient sera yielded Spearman's rho values ranging from 0.950 to 0.997, Passing-Bablok slopes spanning 0.884-1.128, and Bland-Altman biases below 8.3 %, confirming clinical concordance. Owing to its simple equipment requirements and setup process, this method should be highly accessible for other laboratories.

Keywords:  Dual LC-MS/MS; Fat- and Water-Soluble Vitamins; Simultaneous quantification; Valve-mediated Switching

DOI:  https://doi.org/10.1016/j.jpba.2026.117440
J Anal Toxicol. 2026 Feb 22. pii: bkag016. [Epub ahead of print]

Simultaneous Analysis of Δ9-THC, Δ8-THC, CBD, and CBN in Breath Aerosols Collected Using Cannabix Technologies Breath Collection Unit.

Shaza Deeb, Zaire E Fabian, Mikko Määttä, Pedro Fraccarolli, David A Engelhart.

  A simple, accurate, and cost-effective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the simultaneous quantification of Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-tetrahydrocannabinol (Δ8-THC), cannabidiol (CBD), and cannabinol (CBN) in breath aerosols collected using the Cannabix Technologies' Breath Collection Unit (BCU). The method is suitable for use in workplace drug testing and in the investigation of driving under the influence of drugs (DUID) cases. The method was validated according to ANSI/ASB Standard 036, First Edition 2019 guidelines. A lower limit of quantification (LLOQ) of 2.5 pg/L was achieved. Calibration curves were linear (R2 > 0.995) and quality control (QC) accuracy between 91.3% and 100.5% with precision (%CV) less than 17.7%. No major matrix effects or drug interferences were observed. Samples were stable at room temperature and refrigerated at 2-8 °C up to 7 days. Breath samples were collected using the BCU from volunteer regular cannabis users before cannabis consumption and at multiple timepoints after smoking cannabis up to 90 minutes. The results verified the methods ability to capture and quantitate the cannabinoids in breath. The data indicated similar concentration versus time trends for THC metabolism assessed from breath to that previously reported from blood. Breath is an alternative, non-invasive sample matrix that holds promise for identifying recent cannabis use.

Keywords:  Cannabis; Cannabix Breath Collection Unit; breath aerosols; liquid chromatographytandem mass spectrometry analysis

DOI:  https://doi.org/10.1093/jat/bkag016
J Proteome Res. 2026 Feb 26.

Enrichment and Preservation of Urine Metabolites Using Styrene Divinylbenzene Reversed Phase Sulfonate (SDB-RPS) Disks for Enhanced Biomarker Discovery and Disease Monitoring.

Aiwei Wang, Jiyu Xu, Jiameng Sun, Jiayue Ma, Li Wang, Xiaoyan Liu, Haidan Sun, Zhengguang Guo, Yajie Wang, Wei Sun.

  Urine metabolomics plays a crucial role in biomarker discovery and disease monitoring, but challenges in metabolite preservation remain. This study evaluates the use of styrene divinylbenzene reversed phase sulfonate (SDB-RPS) disks for enriching and preserving urine metabolites utilizing ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) for analysis. We compared SDB-RPS-enriched urine samples with untreated urine across three experimental parts: (1) metabolic profiling using C18 and HILIC chromatography under both positive and negative ion modes; (2) degradation kinetics, where SDB-RPS and untreated urine samples were incubated at 55, 65, and 75 °C with constant humidity (75%); and (3) disease classification using hepatitis (n = 72) and cirrhosis (n = 72) samples. The results revealed that metabolite identification was highly consistent between SDB-RPS and urine samples, with an overlapping rate of 88.26%. Additionally, in the disease classification task, the SDB-RPS panel demonstrated consistent performance, with AUC values of 0.867 and 0.828 in training and validation data sets, respectively, outperforming the urine panel (AUC: 0.765 and 0.691, respectively). These findings suggest that SDB-RPS disks significantly enhance the enrichment and long-term preservation of urine metabolites, offering a promising tool for clinical sample analysis and biomarker discovery.

Keywords:  UPLC−MS analysis; disease classification; metabolite preservation; reversed-phase sulfonate disks; urine metabolomics

DOI:  https://doi.org/10.1021/acs.jproteome.5c00367
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Feb 18. pii: S1570-0232(26)00062-0. [Epub ahead of print]1274 124973

Development and validation of a HILIC-ESI/MS method for quantitation of three oxicams and 5-hydroxypiroxicam in human breast milk.

Natalia Kamperi, Ariadni Geballa-Koukoula, Sophia Komninou, Iraklis Salvanos, Ioanna Ioannou, Foteini Theiakou, Irene Panderi.

  Human breast milk is a complex biological matrix that plays a central role in infant nutrition and development. It may also serve as a route of exposure to pharmaceutical residues following maternal drug use. Quantitative data on the transfer of non-steroidal anti-inflammatory drugs (NSAIDs) into breast milk are essential to support their safe use during lactation, however, available analytical methods remain limited. This study reports the development and validation of a rapid and sensitive hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry (HILIC-ESI/MS) method for the simultaneous quantitation of tenoxicam, meloxicam, piroxicam, and the metabolite 5'-hydroxypiroxicam in human breast milk. Sample preparation involves simple protein precipitation procedure using only 25 μL of human breast milk. Chromatographic separation is achieved within 8 min. The method was validated over the range of 20 to 2000 ng mL-1, with a limit of quantitation of 20 ng mL-1 and correlation coefficients greater than 0.998. This is the first HILIC-ESI/MS method for the simultaneous quantitation of the targeted analytes in human breast milk. The validated HILIC-ESI/MS method was subsequently applied to screen breast milk samples collected from ten lactating women donating to a hospital milk bank. Although oxicam NSAIDs were not detected in this cohort, the method is well-suited for targeted screening of human milk in clinical or surveillance settings, including milk banks and pharmacovigilance studies.

Keywords:  Breast milk; HILIC; LC-MS; NSAIDs; Oxicams; Protein precipitation

DOI:  https://doi.org/10.1016/j.jchromb.2026.124973
Biomed Chromatogr. 2026 Apr;40(4): e70411

Development and Validation of a Liquid Chromatograph-Tandem Mass Spectrometry Method for Quantifying Ganciclovir in Dried Blood Spots for Therapeutic Drug Monitoring.

Naoki Tamura, Kotaro Itohara, Suguru Kouyama, Yumi Kitahiro, Kazuhiro Yamamoto, Tomohiro Omura, Toshiyasu Sakane, Jun Saegusa, Ikuko Yano.

  Valganciclovir (VGCV) is the first-line drug for preemptive therapy of cytomegalovirus (CMV) infection. However, even at standard doses, plasma concentrations of the active metabolite ganciclovir (GCV) show substantial inter-individual variability. To ensure therapeutic efficacy and minimize adverse effects, therapeutic drug monitoring (TDM) based on the area under the concentration-time curve (AUC) is essential. Yet, conventional TDM via venous blood sampling is invasive and unsuitable for frequent monitoring. In this study, we aimed to develop a simple and minimally invasive method for GCV quantification using dried blood spots (DBS) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method demonstrated a good linearity over a concentration range of 0.25-16 μg/mL and satisfied the validation criteria for accuracy and precision. It showed acceptable stability for up to 7 days under refrigerated conditions. Methanol was identified as the optimal extraction solvent, allowing for a simplified sample pretreatment without the need for ultrasonic processing. While hematocrit levels affected spot size and quantification accuracy, reliable measurements were obtained within the 30%-50% hematocrit range. The established DBS-based LC-MS/MS method provides a promising, minimally invasive approach for TDM of GCV in the management of CMV infections.

Keywords:  cytomegalovirus; dried blood spots; ganciclovir; therapeutic drug monitoring; valganciclovir

DOI:  https://doi.org/10.1002/bmc.70411
Toxins (Basel). 2026 Feb 02. pii: 77. [Epub ahead of print]18(2):

Development and Validation of an LC-MS/MS Method for the Determination of Alternaria Mycotoxins in Hepatic Tissue.

María García-Nicolás, Alicia Navarro-Botia, Natalia Arroyo-Manzanares, Pilar Viñas.

  The presence of Alternaria mycotoxins in hepatic tissue of both human and animal origin remains unexplored. This work describes the development of an analytical method based on salt-assisted liquid-liquid extraction (SALLE) and ultrahigh-performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS) for the determination of six main Alternaria mycotoxins and related metabolites. Sample treatment was fully optimized, including sample mass, extraction solvent, and volume and sodium chloride mass. The method was validated, achieving calibration curve R2 values above 0.99 and limits of detection between 0.01 and 1.46 µg kg-1. Moreover, satisfactory trueness (apparent recoveries between 84% to 111%) and precision (RSD values below 10%) were achieved, complying with EU requirements. Matrix effects in terms of signal suppression/enhancement varied between 53% for TeA and 78% for AME. Applied to real liver samples (20 human and 20 animal), alternariol monomethyl ether (AME) was found in pig liver, while alternariol (AOH) and tentoxin (TEN) were found in human forensic liver tissues. No other Alternaria mycotoxin metabolites were detected. This methodology is the first validated approach for determining Alternaria mycotoxins in liver tissue.

Keywords:  Alternaria toxins; animals; biomonitoring; human; liver; mycotoxins

DOI:  https://doi.org/10.3390/toxins18020077
Foods. 2026 Feb 11. pii: 652. [Epub ahead of print]15(4):

LC-MS/MS for Simultaneous Determination and Isomer Separation of 12 Glucocorticoid Residues in Multiple Aquatic Foods.

Siman Li, Feng Han, Dongmei Huang, Jingnan Zhang, Yunyu Tang.

  Glucocorticoid (GC) residues present in aquatic products raise food safety concerns, as their chronic dietary intake may pose potential risks of endocrine and metabolic disruption. For the first time, a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated herein for the simultaneous determination of 12 GCs residues, including critical isomeric pairs and acetate ester derivatives, in a variety of aquatic foods, employing deuterated isotopic internal standards. Key optimizations included using a pentafluorophenyl column for effective isomer separation, a synergistic extraction system for high recovery, and QuEChERS purification to mitigate matrix effects. The method exhibited excellent linearity (r2 > 0.996) and high accuracy (recoveries 97.3-99.3%), and the intra- and inter-day precision values were below 3% in five representative aquatic matrices, with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.5 μg/kg and 0.75 μg/kg, respectively. Animal experiments confirmed the in vivo retention of acetate derivatives, justifying their inclusion in monitoring. Real sample analysis of 18 market samples revealed the presence of cortisone and hydrocortisone in 17 samples. This represents the first reported LC-MS/MS method that provides a sensitive, reliable tool for regulatory monitoring of GC residues in diverse aquatic products, thereby supporting food safety assurance.

Keywords:  LC-MS/MS; QuEChERS; aquatic food; glucocorticoid; isomer

DOI:  https://doi.org/10.3390/foods15040652
Antibiotics (Basel). 2026 Jan 28. pii: 130. [Epub ahead of print]15(2):

Determination of Gentamicin: Development and Validation of a Sensitive UPLC-MS/MS Assay According to the European Medicines Agency Guideline.

Raquel Diez, Eva M Vazquez, Beatriz Romero, Raul de la Puente, Nelida Fernandez, Ana M Sahagun, M Jose Diez, Cristina Lopez.

  Background/Objectives: Gentamicin (GEN) is an aminoglycoside antibiotic used in veterinary medicine to treat infections caused mainly by Gram-negative bacteria. GEN is a mixture of pharmacologically active components, known as isoforms. The objective was to develop and validate a sensitive, accurate, and precise Ultra-Performance Liquid Chromatography with triple quadrupole mass detector (UPLC-MS/MS) method to quantify the different GEN isoforms in pig plasma and feces using streptomycin as an internal standard. Methods: Solid-phase extraction (SPE) was carried out. A high-strength silica (50 × 2.1 mm, 1.8 µm) column was used for chromatographic separation and a mobile phase of 0.26% HFBA in water (A) and acetonitrile (B) was delivered in a gradient with a flow rate of 0.5 mL/min. The column temperature was 40 °C and the sample injection volume was 30 µL. Results: The method showed good selectivity and specificity, with no interfering peaks. Calibration curves were linear in the range from 0.05 to 0.3 µg/mL for all isoforms in both matrices. Within- and between-run precision and accuracy were satisfactory for the lower limit of quantification (LLOQ), with coefficients of variation (CV) ≤ 13.4% and deviations ≤ 116.5% in plasma and CV ≤ 12.3% with deviations ≤ 101.7% in feces. No carry-over was observed, and analyte stability was confirmed under different storage conditions. Conclusions: The method development fulfilled all validation criteria established by the European Medicine Agency Guideline (EMA/CHMP/ICH/172948/2019). Moreover, the applicability of the method in clinical practice was demonstrated by the quantification of GEN in plasma and feces samples from pigs.

Keywords:  EMA guideline; UPLC; feces; gentamicin; plasma; validation

DOI:  https://doi.org/10.3390/antibiotics15020130
Anal Chem. 2026 Feb 26.

LCMS-Net: Deep Learning for Raw High Resolution Mass Spectrometry Data Applied to Forensic Cause-of-Death Screening.

Lisa M Menacher, Liam J Ward, Fredrik Heintz, Henrik Green, Oleg Sysoev.

Current preprocessing workflows for untargeted metabolomics using liquid chromatography-high resolution mass spectrometry (LC-HRMS) are time-consuming and require significant domain knowledge. Furthermore, they lack reproducibility or may fail to detect some metabolites entirely. We introduce LCMS-Net, an end-to-end deep learning model for the analysis of LC-HRMS data, to address these challenges. LCMS-Net mitigates the need for manual data preprocessing by operating directly on the raw LC-HRMS data and explicitly modeling its spatial properties. The effectiveness of this fully automated workflow is shown through two case-studies, cause-of-death (CoD) screening and colon cancer detection. For the cause-of-death screening task, LCMS-Net achieved a 9% improvement in F1-score compared to the previous state-of-the-art model (OPLS-DA). For the colon cancer detection task, LCMS-Net achieved an F1-score improvement of 1.8% compared to the previous state-of-the-art model (DeepMSProfiler). Furthermore, LCMS-Net significantly reduces batch effects that are a common source of bias in metabolomics data analyses. This was shown by using a training and test set from different measurement instruments, where the performance only differed by at most 3% as to using data from the same instrument. Compared to other end-to-end deep learning methods for LC-HRMS data, LCMS-Net is also structurally simpler and does not rely on pretraining, which makes it faster and computationally more efficient.

DOI: https://doi.org/10.1021/acs.analchem.5c05404
Analyst. 2026 Feb 27.

Chemical reaction-enabled lipidomics: from sensitive structural analysis to biomedical applications.

Qirui Yu, Xiaoxiao Ma.

Lipidomics has emerged as a vital discipline for understanding cellular metabolism and disease pathology. However, the immense structural diversity, wide dynamic range, and varying ionization efficiencies of lipids present significant analytical challenges. The MS analysis workflow often falls short in detecting low-abundance species and resolving complex structural isomers. To address these limitations, chemical derivatization has been widely adopted to manipulate the chemical properties of lipids prior to analysis. This review summarizes the significant progress in chemical derivatization-enabled lipidomics over the past decades, highlighting its pivotal role in bridging the gap between analytical capability and biological complexity. We critically discuss three core dimensions of improvement: (1) enhancement of detection sensitivity through derivatization strategies that increase the ionization efficiency of lipids; (2) refinement of structural elucidation, specifically using selective reactions to pinpoint carbon-carbon double bond locations and differentiate isomers; and (3) advancement of spectrometric specificity and quantification via mass-shift profiling, which enables precise quantification or high-throughput multiplex analysis. Finally, we discuss how these chemical tools are facilitating the discovery of novel lipid biomarkers and providing deeper insights into lipid metabolism in biomedical research.

DOI: https://doi.org/10.1039/d5an01334h
J Clin Med. 2026 Feb 16. pii: 1565. [Epub ahead of print]15(4):

Clinical Application of Microvolume LC-MS/MS for Therapeutic Drug Monitoring of Immunosuppressants in Solid-Organ Transplant Recipients.

Daiki Iwami, Natsuka Kimura, Sho Nishida, Makiko Mieno, Takehiro Ohyama, Kyoko Minamisono, Yasunaru Sakuma, Joji Kitayama, Yasushi Imai, Ryozo Nagai, Kenichi Aizawa.

  Background/Objectives: Therapeutic drug monitoring (TDM) is essential for optimizing immunosuppressive therapy in solid-organ transplant recipients by maintaining efficacy, while minimizing adverse effects. However, conventional TDM relies on venous sampling and separate assays for tacrolimus (TAC) in whole blood and mycophenolic acid (MPA) in plasma, thereby increasing patient burden and procedural complexity. To address these limitations, we investigated the clinical utility of a microvolume, liquid-phase microsampling device (MSW2™) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methods: We established and applied an LC-MS/MS method for simultaneous quantification of TAC, MPA, and mycophenolic acid β-D-glucuronide (MPAG) using only 2.8 µL of whole blood collected with MSW2™, which eliminates drying or extraction steps. Hematocrit-based correction was applied to estimate plasma MPA concentrations from whole-blood measurements. The method was evaluated in 60 renal transplant recipients with paired venous samples for comparison. Analytical performance was assessed using regression, Bland-Altman analyses, predictive metrics, and stability testing under different storage conditions. Results: Microsampled and venous concentrations were strongly correlated (R2 > 0.95). Estimated plasma MPA concentrations derived from whole blood closely approximated plasma concentrations (bias < 5%). Reducing the sample volume from 5.6 µL to 2.8 µL improved precision and increased the success rate of blood collection from 72.9% to 94.0%. All analytes remained stable for up to 72 h at ≤25 °C. Conclusions: This approach enables accurate, simultaneous quantification of multiple immunosuppressants from trace blood volumes. By reducing sampling burden and simplifying logistics, it provides a clinically feasible and patient-centered strategy for precision TDM, supporting broader implementation of limited sampling strategies and expanding applicability to pediatric, home-based, and telemedicine settings.

Keywords:  LC-MS/MS; hematocrit correction; microsampling; mycophenolic acid; pharmacokinetics; solid-organ transplantation; tacrolimus; therapeutic drug monitoring

DOI:  https://doi.org/10.3390/jcm15041565