bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2026–05–03
thirty papers selected by
Sofia Costa, Matterworks



  1. J Chromatogr A. 2026 Apr 15. pii: S0021-9673(26)00327-4. [Epub ahead of print]1779 466997
      Bifidobacterium, a key genus of the infant gut microbiome, produces d- and l- enantiomers of aromatic lactic acids that may influence early-life immune development through stereochemistry-dependent biological activity. No validated analytical methods currently enable their accurate enantio‑separation and quantification in human biological samples. We report the first validated, targeted, liquid chromatography-mass spectrometry method using a chiral column for the enantioselective separation and quantification of d- and l- forms of phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (4OH-PLA), and indolelactic acid (ILA) in faecal samples. The method achieves baseline separation of all enantiomers within 10 min, with resolution values of 2.66 (PLA), 1.77 (4OH-PLA), and 2.42 (ILA). Solid-phase extraction reduces matrix effects (>80 %) and improves analyte recovery (>80 %). Limits of quantification range from 2.9 to 6.7 ng mL-1, and calibration curves show excellent linearity (R² > 0.99). Inter- and intra-day precision expressed as %RSD are < 15 % for most analytes. The method was successfully applied to infant faecal samples, enabling sensitive and stereospecific quantification of aromatic lactic acids, thus establishing a foundation for exploring the biological relevance of these enantiomers in infant health.
    Keywords:  4-hydroxyphenyllactic acid; Aromatic lactic acids; Chiral chromatography; Gut microbiome; Indolelactic acid; Infants; LC-MS/MS; Method validation; Phenyllactic acid; Targeted metabolomics
    DOI:  https://doi.org/10.1016/j.chroma.2026.466997
  2. Methods Mol Biol. 2026 ;2996 71-78
      In this paper, an analytical method for the analysis of molecular lipids in algae samples is reported. The sample preparation is based on a modified Folch extraction, and the analysis is carried out with ultrahigh performance liquid chromatography combined with mass spectrometry (UPLC-MS). For the characterization of lipids, data dependent acquisition (DDA) analyses are carried out utilizing a high-resolution quadrupole-time-of-flight (Q-ToF) instrument. Throughput of the method is over 100 samples/day. The repeatability is good, and the relative standard deviation of spiked samples is <15%.
    Keywords:  Data dependent acquisition; Lipid; Liquid chromatography; Mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-5031-8_7
  3. J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Apr 22. pii: S1570-0232(26)00175-3. [Epub ahead of print]1278 125086
      To develop and validate a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of glucosylsphingosine (Lyso-GL1) and Lyso-GL3 (Lyso-GL3) in dried blood spots (DBS). Target analytes were extracted from DBS using methanol-water solution with isotopic internal standards for quantification. Chromatographic separation was performed on a C18 column (2.1 × 50 mm, 1.7 μm) at 50 °C, with mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) under gradient elution (0.4 mL/min). Extraction conditions (45 °C, 1300 rpm, 1.5 h) were optimized to enhance recovery, which is a simplified one-step extraction protocol replacing laborious conventional pretreatment methods (e.g., solid-phase extraction). Method validation included linearity, precision, sensitivity, and clinical verification using 20 DBS samples. The limits of quantification (LOQ) for Lyso-GL1 and Lyso-GL3 were 0.5 ng/mL and 0.8 ng/mL (S/N ≥ 10), respectively. Linear ranges were 2.716-51.088 ng/mL (R2 = 0.9997) for Lyso-GL1 and 2.416-49.846 ng/mL (R2 = 0.9991) for Lyso-GL3, with recoveries of 85%-115%, intra-/inter-batch precision CV <15%, and sensitivity down to 0.5 ng/mL. Clinical validation demonstrated robust method stability. This method is the first to apply DBS technology for simultaneous detection of Lyso-GL1 (Gaucher disease biomarker) and Lyso-GL3 (Fabry disease biomarker), featuring simplified sample pre-treatment, enables high-throughput screening for Gaucher and Fabry diseases, leveraging the stability and convenience of dried blood spots for rare disease screening.
    Keywords:  Dried blood spot; Isotope internal standard; LC-MS/MS; Lyso-GL1; Lyso-GL13
    DOI:  https://doi.org/10.1016/j.jchromb.2026.125086
  4. J Chromatogr A. 2026 Apr 24. pii: S0021-9673(26)00365-1. [Epub ahead of print]1779 467035
      Tryptophan metabolism plays a central role in host-microbiota interactions and immune regulation, yet the simultaneous quantification of metabolites from the kynurenine, serotonin, and indole pathways remains analytically challenging due to their diverse physicochemical properties and wide dynamic concentration ranges. Here, we report an efficient, derivatization-free HPLC-MS/MS workflow for the targeted quantification of eleven key tryptophan-derived metabolites in human plasma. The method employs a biphenyl stationary phase to achieve robust chromatographic resolution of structurally similar analytes, including compounds known to co-elute in C18 stationary phase, improving chromatographic selectivity and minimizing co-elution of structurally related analytes. Sample preparation requires only 50 µL of plasma and relies on a simple protein-precipitation step, allowing high throughput. The method demonstrated strong performance in terms of sensitivity, accuracy, and reproducibility, supported by extensive internal standardization. The combination of multi-pathway coverage, analytical cycle, and suitability for human plasma positions this workflow as a valuable tool for monitoring gut-related metabolic signatures in both research studies and potential clinical applications.
    Keywords:  Analytical methods; LC-MS/MS; Microbiota; Sample preparation; Tryptophan metabolites
    DOI:  https://doi.org/10.1016/j.chroma.2026.467035
  5. J Xenobiot. 2026 Apr 13. pii: 67. [Epub ahead of print]16(2):
      A comprehensive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous quantification of 50 pesticide biomarkers across nine current-use chemical classes in human urine. These classes include organophosphorus insecticides (which encompass dialkyl phosphates and specific metabolites), pyrethroid insecticides, fungicides, neonicotinoid insecticides, herbicides, insect repellents, organochlorine pesticide metabolites, and plant growth regulators. The method employs solid-phase extraction (SPE) for sample preparation, requiring only 0.2 mL of urine. Chromatographic separation was optimized using a Hypersil Gold AQ column, achieving a total run time of 18 min. Mass spectrometric detection utilized polarity switching in electrospray ionization mode with multiple reaction monitoring. Method validation demonstrated satisfactory linearity (R2 > 0.99), high sensitivity with limits of detection ranging from 0.01 to 0.88 ng/mL, and extraction efficiencies between 85% and 113%. Precision and accuracy were within acceptable ranges, with relative standard deviations generally below 15%. The method's robustness was confirmed through participation in external quality assessment schemes. Application to real samples revealed significant inter-individual variability in pesticide biomarker concentrations, with total measured biomarker levels ranging from 89 to 1242 ng/mL across the 10 individuals analyzed. This method offers comprehensive coverage of current-use pesticide chemical classes, including 30 biomarkers from the U.S. National Health and Nutrition Examination Survey (NHANES) biomonitoring program, and demonstrates improved sensitivity and broader analyte coverage compared to existing methods. The developed assay provides a valuable tool for large-scale biomonitoring studies and environmental health research.
    Keywords:  Exposomics; UHPLC–MS/MS; human biomonitoring; multi-class analysis; pesticide biomarkers; solid-phase extraction
    DOI:  https://doi.org/10.3390/jox16020067
  6. BMC Chem. 2026 Apr 29.
      With the global spread of antimicrobial resistance (AMR), infections caused by multidrug-resistant bacteria have become a major challenge in clinical treatment. 'Imipenem, Cilastatin Sodium and Relebactam for Injection' is a critical therapeutic agent for treating infections caused by multidrug-resistant Gram-negative bacteria. However, it is associated with gastrointestinal adverse reactions, necessitating therapeutic drug monitoring (TDM) for precise dosing. Currently, there is no method for simultaneously detecting the concentrations of all three components in 'Imipenem, Cilastatin Sodium and Relebactam for Injection'. Therefore, this study established an analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous quantification of imipenem, relebactam, and cilastatin in serum, urine, and peritoneal drainage fluid (PD). After protein precipitation, samples were separated using a CORTECS® HILIC column with 5 mM ammonium acetate aqueous solution-acetonitrile as the mobile phase for gradient elution, detected in positive electrospray ionization (ESI+) mode. This LC-MS/MS method showed excellent linearity (R² > 0.99) for all three drugs, and the linearity ranges had well met the clinical requirements. The limits of quantification (LOQ) were 40 ng/mL (serum/ PD) and 80 ng/mL (urine), and the carryover contamination rate was < 0.1%. All intra-day and inter-day coefficients of variation (CVs) were < 15%, the recovery rates ranged from 89.7% to 103.2%, and the matrix effects were acceptable (87.7%-110.8%). In conclusion, this LC-MS/MS method is simple to operate, exhibits high sensitivity and accuracy, and meets the requirements for the TDM of 'Imipenem, Cilastatin Sodium and Relebactam for Injection', providing support for individualized treatment and reducing the risk of adverse reactions.
    Keywords:  Cilastatin; Imipenem; Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Relebactam; Therapeutic drug monitoring (TDM)
    DOI:  https://doi.org/10.1186/s13065-026-01791-4
  7. Lab Med. 2026 Apr 03. pii: lmag017. [Epub ahead of print]57(3):
       INTRODUCTION: Treosulfan is an alkylating agent administered before hematopoietic stem cell transplantation. Because pharmacokinetic parameters exhibit interindividual variability, therapeutic drug monitoring may be necessary. In this study, a liquid chromatography-tandem mass spectrometry method for monitoring treosulfan was developed and validated.
    METHODS: Treosulfan was separated from 25 µL plasma after protein precipitation with acetonitrile. Treosulfan-D4 was used as the internal standard. Separation was performed using an ACQUITY UPLC Ι-Class PLUS System (Waters Corporation) coupled with a Xevo TQ-XS Mass Spectrometer (Waters Corporation). Limit of quantification, selectivity, linearity, imprecision, carryover, matrix effect, and stability were validated according to Clinical and Laboratory Standards Institute guidelines.
    RESULTS: The mass-to-charge transitions were treosulfan 296.2 > 87.1 and treosulfan-D4 300.2 > 91.1. The total run time was 4 minutes. The limit of quantification of treosulfan was 3.13 µg/mL. The calibration curve was linear in the 3.13 to 100-µg/mL range. The method was adequately selective and without carryover or matrix effect. Treosulfan was stable in plasma for 2 hours at room temperature and for up to 3 days and 1 week when stored at 10 °C and ‒20 °C, respectively.
    DISCUSSION: Our liquid chromatography-tandem mass spectrometry method for measuring treosulfan met Clinical and Laboratory Standards Institute validation requirements and exhibited robust performance, suggesting its utility for therapeutic drug monitoring in clinics treating patients with treosulfan.
    Keywords:  CLSI; LC-MS/MS; alkylating agent; hematopoietic stem cell transplant; therapeutic drug monitoring; treosulfan
    DOI:  https://doi.org/10.1093/labmed/lmag017
  8. Ital J Food Saf. 2026 Apr 29.
      Emerging mycotoxins produced by Fusarium and Alternaria species, such as enniatins (ENN), beauvericin (BEA), alternariol (AOH), and alternariol monomethyl ether (AME), are increasingly detected along the feed-food chain and raise growing concerns for animal health and food safety. Despite their widespread occurrence, regulatory limits are currently lacking, mainly due to insufficient occurrence and exposure data, highlighting the need for robust analytical methods applicable to both feed and edible animal tissues. In this study, a multi-residue ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) method was developed and validated for the simultaneous determination of seven emerging mycotoxins (AOH, AME, BEA, ENA, ENA1, ENB, and ENB1) in pork, liver, and pig feed. Sample preparation was based on solvent extraction followed by a defatting step, while chromatographic separation was achieved using a reversed-phase column coupled to Orbitrap high-resolution mass spectrometry operating in full-scan and product ion scan modes. Method validation was performed according to Commission Implementing Regulation (EU) 2023/2782, evaluating selectivity, linearity, precision, recovery, and sensitivity. Excellent linearity was obtained for all analytes in all matrices, with coefficients of determination >0.99. Recoveries ranged from 70 to 91% in liver, from 72 to 95% in muscle and from 73 to 120% in feed, while intra- and inter-day precision values were consistently below 15%. The limit of quantification was established at 1 ng g⁻¹ for all compounds. Overall, the validated UHPLC-HRMS method proved to be sensitive, selective, and reproducible, providing a reliable analytical tool for monitoring emerging mycotoxins in swine production and associated feedstuffs and supporting future occurrence studies, exposure assessments, and risk evaluation activities along the agri-food chain.
    DOI:  https://doi.org/10.4081/ijfs.2026.14740
  9. Anal Bioanal Chem. 2026 May 01.
      Wash protocols are a simple, commonly used approach to enhance the detectability of low-abundant or poorly ionisable compounds in mass spectrometry imaging (MSI). The washing procedures aim to enhance analyte ionisation by removing interfering metabolites that affect the ionisation efficiency and detection of the target metabolites. However, despite the widespread use of wash protocols in MSI, their impact on small molecule metabolites (SMM) has not been systematically evaluated. In this study, 12 different aqueous and organic wash solvents were investigated to assess their impact on the signal intensities of SMMs in tumour tissue using desorption electrospray ionisation mass spectrometry imaging (DESI-MSI). The added wash steps proved to be a promising tool for increasing detection sensitivity for targeted metabolites, with >90% of analytes investigated here showing increased sensitivity following the optimum wash solvent step. While chloroform was found most efficient in removing lipids overall, the most versatile solvent to significantly enhance the detection of polar and semi-polar metabolites, including amino acids, nucleic acid compounds, sugars, and organic acids, was found to be ethyl acetate. In contrast, water-based washes enhanced fatty acids and lipids while removing hydrophilic metabolites. This study emphasises the importance of adjusting pretreatment protocols to the molecular class of interest and provides a targeted guide for increasing ion detection sensitivity across a broad range of metabolites.
    Keywords:  Ionisation efficiency; MS imaging; Sample preparation; Spatial metabolomics; Tumour; Wash protocols
    DOI:  https://doi.org/10.1007/s00216-026-06534-x
  10. Biomed Chromatogr. 2026 Jun;40(6): e70465
      Monitoring medication compliance in mentally disordered offenders is crucial for effective rehabilitation and reducing recidivism. While LC-MS/MS is established as a primary technique for forensic urine drug testing, manual data evaluation using vendor-specific software reduces efficiency and introduces subjectivity. To address this, we developed a fully automated, vendor-neutral data reporting workflow using open-source mzML and R for urine drug screening and quantitative confirmation. The initial screening step automated the detection of 59 psychotropic drugs and metabolites by applying rigorous logic-based criteria, including absolute intensity thresholds, coelution tolerances, and qualifier-to-quantifier ratio limits. The subsequent confirmation process executed quantitative analysis using internal standard normalization and weighted linear regression for quetiapine and its primary active metabolite, norquetiapine. Following validation using forensic urine samples from probationers, the automated system demonstrated robust analytical reliability, successfully meeting established international bioanalytical guidelines. By ensuring compatibility across different platforms, this standardized approach enhanced laboratory throughput, overcame vendor-dependent limitations, and fortified evidentiary integrity. Furthermore, this open-source workflow demonstrates high versatility, streamlining data evaluation from various instrument vendors and facilitating its broad application in diverse bioanalytical studies.
    Keywords:  LC–MS/MS; antipsychotics; automated workflow; drug screening and confirmation; medication compliance monitoring; mzML
    DOI:  https://doi.org/10.1002/bmc.70465
  11. Metabolites. 2026 Mar 27. pii: 219. [Epub ahead of print]16(4):
       BACKGROUND: Among atmospheric-pressure mass spectrometry imaging (MSI) methods, desorption electrospray ionization (DESI) and desorption electro-flow focusing ionization (DEFFI) represent cost-effective, high-throughput approaches that utilize pneumatically assisted charged solvent droplets to directly desorb and ionize analytes from sample surfaces.
    METHODS AND RESULTS: In this study, we systematically compare the performance of conventional DESI-MSI with previously reported DEFFI-MSI configurations on the Orbitrap mass spectrometer platform, focusing on evaluating the lateral spatial resolution, signal intensity, and imaging speed. By scanning a standard patterned sample which has sharp edges, DESI-MSI achieved a spatial resolution of 70 µm, while DEFFI-MSI achieved 15 µm (approximately 4.7-fold improvement). For the representative ion at m/z 782.5621, DEFFI-MSI demonstrated significantly higher signal intensity across solvent flow rates ranging from 0.5 to 1.5 µL min-1. The enhanced ion yield directly translates to improved Orbitrap-based MSI efficiency: in both negative- and positive-ion modes, DEFFI generates rich full-scan mass spectra within the maximum 10 ms ion injection time, whereas DESI produces weaker mass spectra under the same conditions.
    CONCLUSIONS: Taken together, these results quantify the key performance metrics between DESI-MSI and DEFFI-MSI, demonstrating that DEFFI is the preferred method on Orbitrap-based MSI, because it simultaneously enhances spatial resolution, signal intensity, and imaging speed.
    Keywords:  desorption electro-flow focusing ionization; desorption electrospray ionization; imaging speed; mass spectrometry imaging; spatial resolution
    DOI:  https://doi.org/10.3390/metabo16040219
  12. J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Apr 25. pii: S1570-0232(26)00185-6. [Epub ahead of print]1278 125096
      Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants whose accurate quantification in human serum demands sensitive, rigorously validated analytical methods. A refined solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) workflow was developed and validated for the simultaneous quantification of four legacy PFAS (PFOA, L-PFOS, PFNA, and PFHxS) in 100 μL of human serum. Chromatographic conditions were systematically optimized on a Kinetex® C18 column (2.6 μm, 2.1 × 50 mm) to achieve baseline resolution of linear and branched PFOS isomers within a 6-min total run time. Four critical SPE parameters (protein precipitation solvent, acid additive, evaporation temperature, and reconstitution solvent) were optimized to maximize extraction efficiency and signal reproducibility from the limited-volume matrix. Calibration linearity was established over 0.3-40 ng/mL (PFOA) and 0.2-40 ng/mL (L-PFOS, PFNA, PFHxS), with all correlation coefficients (r2) exceeding 0.99. Validation conducted according to current FDA and EMA guidelines demonstrated within- and between-batch accuracy (RE, -5.3% to +7.0%) and precision (RSD ≤8.0%), IS-normalized matrix effects within 95.6-111.4% (RSD <15.0%), and confirmed analyte stability under bench-top, long-term, freeze-thaw, and autosampler conditions. The validated method was applied to serum samples from 200 participants in a rural Korean cohort; all four PFAS were quantifiable in every sample, yielding mean (± SD) concentrations of 2.9 ± 1.3, 15.9 ± 10.9, 2.3 ± 1.4, and 2.9 ± 2.7 ng/mL for PFOA, L-PFOS, PFNA, and PFHxS, respectively (total PFOS: 21.5 ± 15.7 ng/mL, calculated as the sum of linear and branched fractions). Concentration ranges were consistent with Korean National Environmental Health Survey and National Health and Nutrition Examination Survey reference datasets. Stratified analyses confirmed the method's capability to resolve biologically meaningful sex- and age-dependent PFAS differences. By combining volume efficiency with rigorous chromatographic and bioanalytical performance, this method provides a practical platform for large-scale PFAS biomonitoring under sample-limited conditions.
    Keywords:  Human biomonitoring; Liquid chromatography–tandem mass spectrometry; Per– and polyfluoroalkyl substances; Serum; Solid–phase extraction
    DOI:  https://doi.org/10.1016/j.jchromb.2026.125096
  13. Anal Bioanal Chem. 2026 Apr 29.
      The accurate and precise measurement of endogenous steroid hormone levels in serum is essential for their use as biomarkers of endocrine and metabolic diseases in patient care and translational science. Herein, we describe a newly developed, highly accurate, and precise isotope-dilution liquid chromatography-mass spectrometry (LC-MS/MS) method capable of simultaneously measuring eight clinically relevant polar and non-polar steroid hormones in 200 µL of serum. This steroid hormone panel method uses sequential liquid-liquid extractions for the isolation of total testosterone (TT), estradiol (E2), progesterone (P4), 17-hydroxyprogesterone (17-OHP), androstenedione (AD), estrone (E1), estrone sulfate (E1S), and dehydroepiandrosterone sulfate (DHEAS) without derivatization or hydrolysis. The method demonstrated a broad analytical measurement range for all eight hormones as a result of improved selectivity and sensitivity, making it suitable for the analysis of general population serum samples, including postmenopausal women and children. The total imprecision, expressed as coefficients of variation, was evaluated at three levels for each analyte and ranged from 3.5 to 10.7%. The mean bias to well-established secondary reference materials was -1.00% for TT and 1.91% for E2. When applied to 268 commercially sourced serum samples from men, women, and children, only 2% of measurement results were below the limits of detection; therefore, this method was deemed suitable for measurement of eight steroid hormones across the general population. The establishment of this new steroid hormone panel LC-MS/MS method will facilitate investigations of associations between health disorders and individualized steroid profiles, which can be utilized for evaluations at the individual or population levels.
    Keywords:  Androgens; Estrogens; LC-MS/MS; Liquid-liquid extraction; Progestogens; Steroid hormones
    DOI:  https://doi.org/10.1007/s00216-026-06504-3
  14. J Sep Sci. 2026 May;49(5): e70430
      Recently, bile acids (BAs) have garnered significant attention due to their crucial roles in various metabolic diseases. However, the simultaneous quantification of BAs in biological samples is challenging due to the structural similarities, complex biological matrix, and tissue-specific distribution. We developed a sensitive UHPLC-MS/MS method for quantitation of 19 BAs in mouse liver (mainly conjugated BAs) and feces (mainly unconjugated BAs). We compared protein precipitation, liquid-liquid extraction, and solid-phase extraction (SPE) methods for sample preparation, and SPE was chosen due to the low background noise and high extraction efficiency for both conjugated and unconjugated BAs. The method was extensively validated by evaluating the linearity (R2 ≥ 0.991), extraction recovery (82%-112%), limits of detection (1-2 ng/mL) and low limits of quantification (2-5 ng/mL). Then, the method was applied to the detection of BAs in liver and feces of metabolic dysfunction-associated steatotic liver disease mice, and the correlations between BA levels and the mRNA expression levels of BA metabolic enzymes, transporters, and receptors were analyzed. This study provides a direct and reliable determination method for the in-depth investigation of the enterohepatic circulation of BAs.
    Keywords:  UHPLC–MS/MS; bile acid; metabolic dysfunction‐associated steatotic liver disease; quantification; solid‐phase extraction
    DOI:  https://doi.org/10.1002/jssc.70430
  15. Anal Chem. 2026 Apr 29.
      Spatially resolved profiling of lipid carbon-carbon double bond (C═C) isomers, especially for poorly ionizable glycosphingolipids, remains a significant challenge in mass spectrometry imaging (MSI). Herein, we report a novel MSI method that leverages a diselenide-functionalized covalent organic framework (COF-Se2) by integrating its heterogeneous catalysis and selective surface capture capabilities for on-tissue lipid capture-epoxidation, enabling the differentiation of C═C position isomers with significantly enhanced coverage for glycosphingolipids. In a finely tuned solvent environment, neutral glycosphingolipids were selectively captured and catalytically epoxidized on the COF-Se2 surface with near-quantitative efficiency (∼100%), thereby achieving their separation from epoxidized phospholipids and largely reducing ion suppression. The subsequent desorption of epoxidized glycosphingolipids for mass spectrometry (MS) analysis was allowed by switching the solvent to ammonia-methanol. Epoxidized lipids generate diagnostic fragments for the C═C position in MS/MS, with glycosphingolipids showing distinct patterns from the reported phospholipids and fatty acids. By coating COF-Se2 onto slides for tissue thaw-mounting, on-tissue capture-epoxidation was completed within 5 min. With a two-step ambient liquid extraction MS/MS imaging workflow, epoxidized phospholipids and glycosphingolipids on tissue were imaged separately in each step. Compared with the conventional method, this strategy substantially improved the lipid coverage of isomer-differentiated MSI by 7-fold in brain tissue, especially for glycolipids or sphingolipids (27 vs 0). This work illuminates the inaccessible spatial landscape of glycosphingolipid C═C isomers, opening new avenues for understanding their roles in brain function and disease.
    DOI:  https://doi.org/10.1021/acs.analchem.6c00547
  16. Anal Bioanal Chem. 2026 Apr 30.
      Cystinosis is a rare, lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cystine transporter, resulting in lysosomal cystine accumulation, the phenotypic hallmark of cystinosis, and progressive cellular dysfunction. Accurate quantification of cystine levels is therefore essential for assessing lysosomal transport deficiency and treatment response. In vitro cell models provide a controlled platform to investigate disease mechanisms and to evaluate emerging therapeutic strategies. To determine intracellular cystine concentrations in these models, adequate sample preparation, storage, and highly sensitive analytical methods are essential. In this work, a rapid hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed for the direct determination of cystine in cellular extracts. Use of a PEEK-lined HILIC-Z column proved essential to minimize metal-induced peak tailing and improve chromatographic performance. The total run time of 5 min enabled high-throughput analysis, facilitating efficient screening of novel therapeutic approaches in cellular systems. Validation demonstrated excellent linearity (R2 ≥ 0.9986) and a lower limit of quantification (LLOQ) of 12.5 nM, representing a > 8-fold improvement over reported reversed-phase LC methods. Addition of N-ethylmaleimide (NEM) prior to cell lysis effectively limited cysteine oxidation and maintained sample stability at 4 °C. Applicability was demonstrated in an isogenic laboratory HEK293T cell model, where CTNS-knockout (KO) cells exhibited significantly elevated intracellular cystine levels compared to wild-type (WT) cells. As additional controls, elevated levels were successfully restored following cysteamine treatment or lentiviral-vector (LV)-mediated CTNS protein re-expression. The developed method hence provides a sensitive and reliable analytical platform for in vitro evaluation of novel therapeutic strategies in cystinosis research.
    Keywords:  Bioanalysis; HILIC-MS/MS; High-throughput; PEEK-lined HILIC column
    DOI:  https://doi.org/10.1007/s00216-026-06526-x
  17. Methods Mol Biol. 2026 ;2995 67-76
      To decipher the complex tissue microenvironment for comprehensively elucidating the biological machinery, spatial proteomics is becoming popular. Laser microdissection (LMD) technology combined with integrated sample preparation and high-sensitive liquid chromatography-mass spectrometry (LC-MS) is the most powerful strategy. In this chapter, we describe the protocols for tissue slice preparation and staining, LMD-based histological regions collection, simple and integrated spintip-based proteomics technology (SISPROT) based sample preparation, and highly sensitive LC-MS analysis.
    Keywords:  LC-MS; LMD; SISPROT; Spatial proteomics
    DOI:  https://doi.org/10.1007/978-1-0716-5027-1_5
  18. Methods Mol Biol. 2026 ;2996 79-88
      Constituents of microalgae and sample preparation for UPLC-ELSD and GC-MS analyses are described. Bound fatty acids from acylglycerols, alkylacylglycerols, galactosyldiacylglycerols, glycerophospholipids, and sterol esters are derivatized by using transesterification with sodium methoxide to form fatty acid methyl esters. Compounds containing free hydroxyl groups, either present originally or formed during previous step, like free fatty acids, sterols, α-tocopherol, phytol, and nonesterified alkoxyglycerols, are trimethylsilylated. The compounds in algal lipid extract are subsequently derivatized by these two steps.
    Keywords:  Evaporative light-scattering detection; Fatty acids; Gas chromatography mass spectrometry; Lipid classes; Transesterification; Trimethylsilylation; Ultra performance liquid chromatography
    DOI:  https://doi.org/10.1007/978-1-0716-5031-8_8
  19. Clin Chim Acta. 2026 Apr 25. pii: S0009-8981(26)00208-1. [Epub ahead of print]589 121026
      Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a severe complication of chronic kidney disease (CKD), linked to the systemic accumulation of protein-bound uremic toxins (PBUTs). Current methods for PBUT analysis are often constrained by limited analyte coverage and lack of integrated high-throughput quantification of multiple toxins. A high-throughput targeted Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) method was developed and validated for the quantitative determination of PBUTs in human serum, including Nδ-(carboxymethyl)lysine (CML) and pentosidine (PE). Reproducible sample preparation and reliable quantification were achieved using 96-well protein/phospholipid removal plates for sample preparation and optimizing chromatographic and mass spectrometric conditions. The method was validated, demonstrating acceptable linearity over the concentration range of 10-2000 ng/mL for all 15 analytes (R2 > 0.99), with lower limits of quantification (LLOQ) of 10-20 ng/mL. The limits of detection (LOD) ranged from 5 to 10 ng/mL, and the precision expressed as relative standard deviation (RSD) was below 15%. Application to serum samples from 40 uremic patients and 20 healthy controls revealed significantly elevated levels of 10 PBUTs, with p-cresyl sulfate (PCS) concentrations approximately 400-fold higher in patients. Five PBUTs were undetectable in healthy individuals. This study provides a reliable and accurate method for PBUTs quantification, offering valuable insights into the critical role of PBUTs in the pathogenesis of CKD-MBD, and advancing PBUT-related metabolomic research.
    Keywords:  Biomarkers; Bone disorder; Chronic kidney disease; Protein-bound uremic toxins; Ultra high-performance liquid chromatography-tandem mass spectrometry; Vascular calcification
    DOI:  https://doi.org/10.1016/j.cca.2026.121026
  20. Development. 2026 Apr 15. pii: dev205350. [Epub ahead of print]153(8):
      Metabolic processes are essential for regulating and maintaining developmental transitions. However, the distinct metabolite-driven mechanisms that are crucial for development remain poorly characterized due to inherent challenges in measuring their localization and function in situ. We applied desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to generate near single-cell resolution (50-80 µm) images of metabolites in the maize root tip, which has a well-characterized longitudinal developmental gradient. We developed a new computational tool, called Developmental Imaging Mass Spectrometry Pipeline for Linear Evaluation (DIMPLE), which processes mass signatures along linear gradients and clusters metabolites based on their developmental enrichment patterns. We employed this method to compare developmental enrichment of metabolites in Oaxacan Green, a salt-resilient maize variety, to B73, which is salt sensitive. DIMPLE uncovers specific differences in individual mass signatures and overall enrichment patterns between these varieties. Further characterization of these differences revealed meristem enrichment of D-erythrose, a metabolite that can improve stress tolerance in maize. Overall, DIMPLE enables comprehensive and rapid analysis of metabolite patterns along a linear gradient, informing biological hypotheses related to plant growth and stress response.
    Keywords:  Bioinformatics; Cell differentiation; Erythrose; Root development; Spatial metabolomics; Stress resilience
    DOI:  https://doi.org/10.1242/dev.205350
  21. Environ Sci Pollut Res Int. 2026 May 01.
      Neonicotinoids (NEOs) are the most widely used class of insecticides worldwide. Consequently, human exposure occurs through multiple pathways, including dietary intake and environmental contact. Previous analytical methods have primarily focused on parent compounds, often excluding key metabolites due to matrix effects and poor quantification in complex biological samples. This gap severely limits the ability to accurately assess total NEO exposure and internal dose, highlighting the urgent need for more comprehensive and reliable biomonitoring approaches. This study developed a comprehensive LC-MS/MS method to quantify 21 NEOs and metabolites in human urine, uniquely including key transformation products such as clothianidin-n-desmethyl, clothianidin-urea, imidacloprid-urea, imidacloprid-olefin, 5-hydroxy-imidacloprid, thiamethoxam-urea, and thiamethoxam-n-desmethyl, thereby enabling a more thorough assessment of human exposure to NEOs. The method was validated using both synthetic and pooled human urine, providing a critical assessment of matrix-dependent variations in detection limits, accuracy, and precision. Method performance demonstrated high sensitivity, specificity, accuracy, and precision, with limits of quantification (LOQ) from 0.017 to 0.46 ng/mL and limits of detection (LOD) from 0.0050 to 0.14 ng/mL. The method was applied to urine samples from 246 pregnant women and 47 farmers in eastern Iowa to assess exposure levels. Results indicated widespread NEO exposure with detection frequencies of 99% of pregnant women and 100% in farmers, with 18 different NEOs and metabolites detected across both populations. Metabolite monitoring revealed that imidacloprid metabolites (IMI-O, 5-OH-IMI) were stronger predictors of total neonicotinoid burden than parent compounds alone (rs = 0.735 vs < 0.6 for parents), with some metabolites showing higher concentrations in pregnant women than farmers despite farmers having higher overall exposure. These findings demonstrate that parent-only biomonitoring substantially underestimates internal neonicotinoid exposure and fails to capture critical exposure pathways, particularly for vulnerable populations where metabolite patterns may differ significantly from occupational exposure profiles.
    Keywords:  Clothianidin-n-desmethyl; Human exposure assessment risk; Imidacloprid olefin; LC–MS/MS; Neonicotinoid insecticides; Thiamethoxam urea; Urine
    DOI:  https://doi.org/10.1007/s11356-026-37779-9
  22. Anal Chem. 2026 Apr 29.
      Preparing samples for analysis by mass spectrometry (MS) can be laborious and challenging. Hyphenated separation tools are convenient but costly and require much attention. Here, we present a facile low-cost online MS method for analysis of complex samples. It relies on a standard unmodified electrospray ionization (ESI) source, which incorporates polyether ether ketone tubing connected to a grounded metal union or diverter valve. The sample is infused by a syringe pump, and the flow is stopped. During the stopped-flow stage, ionic species are spontaneously separated due to the electric field between the ESI capillary and the grounded element. The flow is then restarted, and the separated species pass to the ion source. The signals of analytes are enhanced with respect to the signals obtained during direct infusion. This enhancement is attributed to the microseparation of analyte and matrix ions occurring in the sample flow line section. The signal change factors ranged from 0.74 to 295. The reported intrinsic property of ESI─operated in a run-stop-run sequence─has broad applicability in analysis of metabolites, peptides, and proteins in complex matrices. Given the tens of thousands of ESI-MS systems used worldwide, this fundamental finding could have broad applicability.
    DOI:  https://doi.org/10.1021/acs.analchem.5c08265
  23. Cells. 2026 Apr 21. pii: 736. [Epub ahead of print]15(8):
      Recent advancements in neuroproteomics have enabled detailed analysis of protein expression in the human brain, yet resolving synaptic dysfunction-a central feature of many neurological and psychiatric disorders-requires careful methodological consideration. Leveraging the high sensitivity of modern liquid chromatography-tandem mass spectrometry (LC-MS/MS), we evaluated the utility of whole-tissue lysates versus enriched synaptosome preparations for detecting synaptic protein signatures. First, we optimized and standardized a sample preparation protocol for frozen human gray matter (GM) by refining the suspension trapping (sTRAP) digestion method using thin human tissue sections. We accomplished low technical variation by minimizing sample handling and achieved a highly reproducible sample preparation workflow by rigorously applying standardization and randomization across dissection, processing, and LC-MS/MS runs. Second, comparative LC-MS/MS analysis showed that while whole-tissue lysates provide a high-throughput survey of the synaptic proteome, synaptosome isolation is required to investigate synapse-specific proteins to detect alterations at the terminal that are obscured in the soma. Because these methods offer distinct but synergistic levels of information, we recommend a tiered neuroproteomics strategy. This approach utilizes whole-tissue lysates for broad disease-associated screening and consistent quantification in large cohorts, followed by targeted synaptosome proteomics to provide a unique window of insight into synaptic composition and stability. This integrated workflow respects the biological necessity of spatial resolution while maintaining the reproducibility required for robust human brain proteomics. Furthermore, initial tissue-level analysis provides the necessary context to correctly interpret synaptosome data in cases of global synapse loss or gain.
    Keywords:  frozen tissue; human; sTRAP; shotgun neuroproteomics; synapse proteomics
    DOI:  https://doi.org/10.3390/cells15080736
  24. Wei Sheng Yan Jiu. 2026 Mar;55(2): 290-296
       OBJECTIVE: To establish a non-derivatization detection method based on solid-phase extraction-ultra performance liquid chromatography-triple quadrupole mass spectrometry for the simultaneous and quantitative determination of cadaverine, putrescine, histamine, tyramine, tryptamine and phenethylamine in livestock, poultry and aquatic prefabricated food.
    METHODS: Samples were extracted with 5% aqueous perchloric acid-acetonitrile(2∶8, V/V), and purified with Oasis PRiME HLB solid-phase extraction column. The target compounds were separated on ZORBAX RRHD Eclipse Plus C18 column(3.0 mm×150 mm, 1.8 μm) by gradient elution using 0.1% formic acid aqueous solution and methanol as mobile phase. The detection was performed with electrospray ionization(ESI) in positive mode under multiple reaction monitoring(MRM) mode, and quantified by internal standard method.
    RESULTS: Six target compounds of cadaverine, putrescine, histamine, tyramine, tryptamine and phenethylamine were well reserved on the chromatographic column, and the linear range was 10-2000 μg/L(R~2&gt;0.999). The limit of detection and limit of quantitation were 5 μg/kg and 10 μg/kg, respectively. The recovery rates of the six compounds ranged from 71.6% to 105.2%, and the relative standard deviations were 0.73% to 12.64%(n=6). A total of 103 livestock, poultry and aquatic prefabricated food were tested for the target compounds. The detection rates of putrescine, tyramine, tryptamine, cadaverine, and phenethylamine were 96.1%, 60.2%, 22.3%, 34.0%, and 58.2%, respectively.
    CONCLUSION: This method has the advantages of simple pretreatment, fast analysis speed, high accuracy and sensitivity, which is suitable for the determination of six biogenic amines in livestock, poultry, and aquatic prefabricated food.
    Keywords:  biogenic amines; mass spectrometry; prefabricated food; solid-phase extraction; ultra-performance liquid chromatography
    DOI:  https://doi.org/10.19813/j.cnki.weishengyanjiu.2026.02.018
  25. J Chromatogr Sci. 2026 Apr 30. pii: bmag015. [Epub ahead of print]64(5):
      The co-administration of ibuprofen (IBU) and phenyramidol HCl (PHE) is frequently preferred for the management of musculoskeletal disorders to enhance therapeutic efficacy. This study aimed to develop and validate a rapid, sensitive and cost-effective high-performance liquid chromatography-photodiode array (HPLC-PDA) method for the simultaneous determination of IBU and PHE in human plasma and pharmaceutical preparations, and to demonstrate its applicability in pharmacokinetic studies. Chromatographic separation was achieved on a C18 column using a mobile phase consisting of phosphate buffer (pH 6.0 ± 0.05) and methanol (30:70, v/v) at a flow rate of 1.0 mL·min -1. The retention times of IBU and PHE were 3.7 and 2.7 min, respectively, with a total analysis time of 6 min. The method showed excellent linearity within the range of 0.05-40.0 μg·mL -1 for both drugs, with LLOQ values of 0.05 μg·mL-1 for IBU and 0.0025 μg·mL-1 for PHE. The plasma samples were collected at four time points (0.0, 1.0, 4.0 and 8.0 h) after oral administration of IBU and PHE. Cmax, Tmax and AUC0-8 were calculated, with Cmax of 16.14 μg·mL-1 for IBU and 3.71 μg·mL-1 for PHE, while Tmax was 1.0 h for both. Despite limitations in the number of sampling time points, the method proved highly applicable for determining pharmacokinetic profiles and for routine quality control analyses. Its short analysis time, high sensitivity and low sample volume requirement make it a powerful tool for clinical research and therapeutic monitoring. This is the first validated HPLC-PDA method applied to the simultaneous pharmacokinetic evaluation of IBU and PHE in human plasma.
    DOI:  https://doi.org/10.1093/chromsci/bmag015
  26. Biomed Chromatogr. 2026 Jun;40(6): e70470
      Hepatocellular carcinoma, the third leading cause of cancer-related deaths globally, presents a critical public health burden in China due to its high incidence and mortality. While targeted therapies and immunotherapies have improved survival in advanced HCC, drug resistance remains a major therapeutic challenge. Recent studies suggest that gefitinib, an EGFR inhibitor, overcomes lenvatinib resistance, yet its mechanistic underpinnings are incompletely understood. To investigate gefitinib's metabolic effects in HCC, we conducted untargeted metabolomic profiling using two separate platforms: gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) with both hydrophilic interaction liquid chromatography (HILIC) and reversed-phase modes. Raw data were processed by Mass Hunter, normalized with internal standards, and analyzed via SIMCA for pattern recognition. Principal component analysis (PCA) of quality control samples and experimental groups (n = 6 each) confirmed system stability and clear inter-group separation. Orthogonal projections to latent structures discriminant analysis models were validated by 200 permutation tests. Analysis identified 42 metabolites with VIP > 1, of which 25 showed significant alterations (p < 0.05) post-gefitinib treatment. KEGG/RaMP-DB enrichment revealed perturbations in four key pathways: arginine-proline metabolism, nitrogen metabolism, branched-chain amino acid biosynthesis, and taurine metabolism. These results delineate gefitinib-induced metabolic reprogramming in HCC cells, providing a foundation for targeting metabolic vulnerabilities to overcome therapy resistance.
    Keywords:  GC/MS; LC/MS; gefitinib; hepatocellular carcinoma; metabolomics
    DOI:  https://doi.org/10.1002/bmc.70470
  27. Anal Bioanal Chem. 2026 Apr 28.
      An interlaboratory comparison study was conducted among five laboratories for determining 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in human serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. The Centers for Disease Control and Prevention (CDC), Imperial College Healthcare NHS Trust, University College Cork, University of Liège, and University of Washington analyzed 50 single-donor samples and two new Standard Reference Materials (SRMs®). The results from each laboratory were compared with target values assigned by the National Institute of Standards and Technology (NIST) using a reference measurement procedure (RMP) and evaluated using Ordinary Deming linear regression and Bland-Altman analysis. Three of the five laboratory methods provided results that were in good agreement with the NIST RMP results showing linear regression slopes ranging from 0.972 to 1.003 and Bland-Altman mean bias of -0.092 nmol/L, 0.025 nmol/L, and 0.035 nmol/L. Two laboratories demonstrated a significant positive bias with linear regression slopes of 1.158 and 1.214 and Bland-Altman mean bias of 0.162 nmol/L and 0.708 nmol/L. The CDC method is currently used to assign "information only" values for 24,25(OH)2D3 in the quarterly distributions of the Vitamin D External Quality Assessment Scheme (DEQAS). Given the agreement (linear regression slope = 0.984, R2 = 0.996 and mean bias of -0.092 nmol/L) between the CDC method and the NIST RMP observed in this study, the CDC-assigned 24,25(OH)2D3 values in DEQAS may provide a more accurate reference than the current participant consensus mean values.
    Keywords:  24,25-Dihydroxyvitamin D3 ; 25-Hydroxyvitamin D2 ; 25-Hydroxyvitamin D3 ; Certified reference materials (CRMs); Standard reference materials (SRMs); Vitamin D External Quality Assessment Scheme (DEQAS)
    DOI:  https://doi.org/10.1007/s00216-026-06480-8
  28. Anal Chem. 2026 May 01.
      Comprehensive xenometabolome characterization is essential for understanding the effects of xenobiotics in biological systems. This study presents a multidimensional analytical workflow integrating orthogonal chromatographic separations, trapped ion mobility spectrometry (TIMS), high-resolution mass spectrometry and biotransformation-informed data processing to address xenometabolome assessment challenges. Zebrafish larvae exposed to 4-Methylbenzotriazole (4-MeBT) were used as a challenging case study. TIMS dimension provided orthogonal experimental evidence for isomer annotation, with inverse reduced mobility (1/K0) supporting conjugation site assignment for the dominant O-S-4MeBT and O-G-4MeBT isomers. The combination of TIMS with the Parallel Accumulation Serial Fragmentation (PASEF) acquisition further reduced spectral complexity, enhanced signal-to-noise ratio, and improved MS/MS coverage (70%), generating high-quality analytical evidence crucial for structural elucidation. To leverage these analytical dimensions, we developed a data processing strategy that leverages in-silico-based suspect screening and biotransformation-informed nontarget screening. In this regard, we introduce two novel frameworks; the "Building Blocks" (BB) concept which interprets unknown bio-TPs as modular assemblies of parent- and pathway-derived substructures, and the "Spectral Characteristics Knowledgebase" (SCKB), which use known biotransformation MS/MS motifs to provide structural insights and facilitate unknown identification. Our results demonstrated the identification of all previously known 4-MeBT bio-TPs with enhanced confidence (O-Sulfate- and O-Glucuronide-4MeBT) and the discovery of 29 new bio-TP features across 12 bio-TP classes, highlighting its efficacy in unraveling complex xenobiotic metabolism. Among these, a putative dimerization product (4-MeBT-263) was reported for the first time in zebrafish. Overall, this workflow has the potential to advance the understanding of bio-TP formation and detoxification processes in xenometabolome studies.
    DOI:  https://doi.org/10.1021/acs.analchem.5c08213
  29. Toxins (Basel). 2026 Apr 03. pii: 172. [Epub ahead of print]18(4):
      Highly sensitive methods for trace-level aflatoxin determination are indispensable for cereal food safety and public health protection. This study developed a ZrO2-assisted QuEChERS-UHPLC-MS/MS method for the simultaneous determination of AFB1, AFB2, AFG1, and AFG2 in maize, wheat, rice, and soybean. Systematic optimization identified acetonitrile as the optimal extraction solvent and 10 mg ZrO2 in combination with PSA, C18, and GCB as the optimal cleanup formulation, providing recoveries of 107.33-111.60%. Chromatographic baseline separation was achieved within 8.0 min using a moderate gradient program. The method exhibited excellent linearity (R2 > 0.999) with LODs of 0.15-0.25 µg/kg and LOQs of 0.50-0.75 µg/kg. Negligible matrix effects (0.85-1.02) validated the efficacy of ZrO2-assisted cleanup in eliminating co-extractive interferences in maize. Satisfactory accuracy (recoveries of 86.66-111.04%) and precision (RSDs < 14%) were obtained across all matrices. The method demonstrated consistent performance across diverse cereal and soybean matrices, fulfilling international regulatory requirements for routine aflatoxin monitoring in agricultural commodities.
    Keywords:  QuEChERS; UHPLC-MS/MS; aflatoxins; cereal and soybean matrices; food safety; zirconium dioxide
    DOI:  https://doi.org/10.3390/toxins18040172
  30. J Am Soc Mass Spectrom. 2026 Apr 27.
      Two-dimensional mass spectrometry (2D MS) is a method for tandem mass spectrometry that enables the correlation between precursor and fragment ions without the need for ion isolation. On a Fourier transform ion cyclotron resonance mass spectrometer, the phase correction functions for absorption mode data processing were found to be linear in the precursor ion dimension and quadratic in the fragment ion dimension. Phase-corrected absorption mode data processing on limited data sets has previously shown improvements in signal-to-noise ratio (SNR) and resolving power by a factor of 2. Here, we have expanded phase-corrected absorption mode data processing to 2D mass spectra regardless of size and frequency range. We have applied phase-corrected absorption mode 2D MS to top-down analysis of variously oxidized ubiquitin proteoforms generated by fast photochemical oxidation of proteins (FPOP) and to an extract of ergot alkaloids. We show that phase-corrected absorption mode data processing significantly improves both the SNR and the resolving power of the 2D mass spectrum compared to standard magnitude mode in terms of sequence coverage in top-down proteomics, as well as the accuracy of precursor-fragment correlation in metabolomics.
    Keywords:  FT-ICR MS; metabolomics; proteomics; tandem mass spectrometry; two-dimensional mass spectrometry
    DOI:  https://doi.org/10.1021/jasms.6c00007