bims-metlip 2026-08-16 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–08–16
thirty-six papers selected by
Sofia Costa, Matterworks

Analytical method development and validation of a serum LC-MS/MS assay for 2 emerging synthetic cannabinoids: 5F-MDMB-PICA and MDMB-4en-PINACA.
Secondary Amine-Based Chemical Isotope Labeling Coupled with Mass Spectrometry for Simultaneous Quantification of Carboxylic Compounds during Hepatocellular Carcinoma Progression.
Multi-class UHPLC-MS/MS method for the simultaneous determination of 32 endocrine-disrupting chemicals in the urine of pregnant women.
Development and Validation of a QuEChERS-LC-MS/MS Method for Simultaneous Determination of Four Direct Oral Anticoagulants in Citrate Plasma and Quantitative Dried Plasma Spots.
Development and Validation of an LC-MS/MS Method for Simultaneous Determination of 12 Antibiotics in Human Serum.
Chimeric structural isomer fragments as cost-efficient internal standards for amino acid quantification by mass spectrometry.
Development and validation of a novel LC-MS/MS method for simultaneous quantification of fidaxomicin and metabolite (OP-1118) from feces for gut pharmacobiome studies.
Analysis of the Chemical Composition of Inula hookeri f. major Ling Combining UHPLC-Q-Exactive-Orbitrap MS with Feature-Based Molecular Networking and SIRIUS.
LC-MS/MS quantification of endogenous taurine in plasma and urine using surrogate matrix calibration and background subtraction: Application to renal transporter phenotyping.
Simultaneous Quantification of Hundreds of Phosphorylated Metabolites in Multiple Biological Matrices with Ion-Pairing Reversed-Phase Ultrahigh-Performance Liquid Chromatography and Mass Spectrometry.
Comparative Metabolite Profiling of Different Solvent Extracts of Argemone ochroleuca Sweet and Argemone mexicana Linn Shoots and Roots Using Liquid Chromatography-Mass Spectrometry-Based Metabolomics and Molecular Networking.
Development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify protoporphyrin IX (PPIX) and zinc (II) protoporphyrin IX (Zn-PPIX), in human plasma.
Optimization and validation of a two-step extraction dispersive liquid-liquid microextraction (UA-DLLME) method for the determination of 17 antibiotics in influent wastewater.
The Application of LC-MS in Pharmaceutical Analysis.
Quantitative analysis of vitamin E, 5-methyltetrahydrofolate, and glutathione in seminal plasma by liquid chromatography-tandem mass spectrometry.
MetaboGraph: A Framework for Metabolomics and Lipidomics Annotation and Pathway Network Analysis.
A liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of vancomycin in human serum and plasma.
MALDI mass spectrometry imaging across spatial scales in toxicology: tissue evidence, cellular heterogeneity, and emerging single-cell opportunities.
Resolution and Quantitation of Ribonucleosides and Deoxyribonucleosides in Digested Genomic DNA by UHPLC-MS/MS.
Fragment Coverage: Unified Visualization and Quantitative Assessment of MS/MS Fragmentation Supports Metabolite Identification in Macromolecule Drug Metabolism Studies.
LC-HRMS suspect screening for expanded detection of (new) psychoactive substances in oral fluid: a practical approach for forensic casework.
Detection and confirmation of hypoxia-inducible factor activating agents in equine plasma and urine by liquid chromatography coupled to mass spectrometry.
Aza-Paternò-Büchi derivatization enables annotation of triacylglycerols by negative-ion-mode mass spectrometry at the double bond level.
Automated magnetic solid-phase extraction based on hydrophilic-lipophilic balanced materials for LC-MS/MS determination of macrolides in pork.
Rapid Chiral Analysis of Drugs with Multiple Stereocenters via High-Resolution Ion Mobility.
Separation of imidazolium-based ionic liquid cations with various substituents on a pentafluorophenylpropyl stationary phase using UHPLC-MS.
Protocol for untargeted LC-MS/MS metabolomics annotation and differential abundance analysis using the SIRIUS Python client.
Foundation Models for Liquid Chromatography-High-Resolution Mass Spectrometry: A New Era beyond Labeled Datasets.
Integrating liquid-liquid extraction and dispersive solid-phase extraction cleanup for high-throughput profiling of 11 neonicotinoids and 6 metabolites in human urine by HPLC-Q-Orbitrap HRMS.
A Rapid and Sensitive LC-MS/MS Method for Quantification of NDSRI in Melatonin Drug Product via Controlled In Situ Formation.
AQbD-guided development of a robust and green SI-RP-HPLC method for impurity profiling of an efonidipine hydrochloride ethanolate and chlorthalidone combination, with LC-MS and NMR-based identification of degradation product in efonidipine.
A Time-Gated Raman Spectroscopy and Mass Spectrometry Comparative Chemical Analysis of Plasticizers.
Simultaneous determination and pharmacokinetic characterization of 4-methyl-N-ethylcathinone and its metabolites in rat whole blood using a LC-MS/MS method.
Detection and quantification of 4F-metomidate and its metabolites, metomidate, in human blood and urine by ultraperformance liquid chromatography with triple quadrupole mass spectrometry (UPLC-MS/MS).
LC-MS/MS Quantification of Five Antihistamines in Antemortem Blood: Method Validation and Application.
Programmable One-Tube Sialic Acid Derivatization Enables Dual-Mode N-Glycomics of Low-Input Samples.

Medicine (Baltimore). 2026 Aug 14. 105(33): e50058

Analytical method development and validation of a serum LC-MS/MS assay for 2 emerging synthetic cannabinoids: 5F-MDMB-PICA and MDMB-4en-PINACA.

Murat Akbaba, Aysun Baransel Isir, Süleyman İbili, Can Erdem Yiğitsoy.

   BACKGROUND: This study aimed to develop and validate a highly sensitive and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 5F-MDMB-PICA and MDMB-4en-PINACA in human serum, ensuring suitability for both forensic and clinical toxicology applications.
METHODS: Drug-free serum samples were fortified with target analytes, extracted through solid-phase extraction, and analyzed using a triple-quadrupole LC-MS/MS system (Shimadzu LCMS-8045) equipped with a biphenyl column. Calibration was established within 1-100 ng/mL, and validation parameters - linearity, sensitivity, accuracy, and precision - were assessed according to international guidelines.
RESULTS: The calibration curves displayed excellent linearity (r2 = 0.999) for both analytes. The limits of detection were 0.113 ng/mL for 5F-MDMB-PICA and 0.145 ng/mL for MDMB-4en-PINACA, with limits of quantification of 0.339 ng/mL and 0.435 ng/mL, respectively. Recovery rates at 10 and 50 ng/mL ranged from 77.7% to 96.4%, while intra and inter-day precision (relative standard deviation %) remained below 15%.
CONCLUSION: The validated method provides reliable quantification of 5F-MDMB-PICA and MDMB-4en-PINACA in human serum. The observed linearity, sensitivity, recovery, and precision support its applicability for routine forensic toxicology analyses.

Keywords:  5F-MDMB-PICA; LC–MS/MS; MDMB-4en-PINACA; method validation; serum analysis; synthetic cannabinoids

DOI:  https://doi.org/10.1097/MD.0000000000050058
Anal Chem. 2026 Aug 11. 98(31): 23032-23040

Secondary Amine-Based Chemical Isotope Labeling Coupled with Mass Spectrometry for Simultaneous Quantification of Carboxylic Compounds during Hepatocellular Carcinoma Progression.

Jun Zhang, Yu-Han Dai, Yue Zhu, Xue-Yi Zhao, Guo-Jia Song, Chengjie Ma, Fei-Long Liu.

Carboxylic metabolites and drugs play essential roles in biological regulation and disease progression. Their simultaneous quantification remains analytically challenging due to their low ionization efficiency and poor chromatographic retention. Here, we synthesized a pair of secondary amine-based chemical isotope labeling reagents, N-(piperidin-4-ylmethyl)benzamide (PMBA) and d5-N-(piperidin-4-ylmethyl)benzamide (d5-PMBA), for the simultaneous labeling of carboxylic metabolites and drugs. The secondary amine moiety on PMBA/d5-PMBA can efficiently react with carboxyl groups on carboxylic compounds, substantially improving the reversed-phase (RP) chromatographic retention and separation, along with the enhancement of the MS sensitivity. Upon this strategy, the limits of detection (LODs) of 32 carboxylic metabolites and drugs were determined to range from 0.001 to 0.13 ng mL-1. The chemical isotope labeling approach with liquid chromatography-mass spectrometry (LC-MS) analysis enabled reliable quantification of diverse carboxylic compounds in serum samples and revealed significant alterations in their abundances during hepatocellular carcinoma progression. This method provides a sensitive and robust analytical platform for the simultaneous quantification of carboxylic metabolites and drugs and facilitates investigations of their biological and pharmacological functions in diseases.

DOI: https://doi.org/10.1021/acs.analchem.6c02580
Anal Bioanal Chem. 2026 Aug 11.

Multi-class UHPLC-MS/MS method for the simultaneous determination of 32 endocrine-disrupting chemicals in the urine of pregnant women.

Chao Zhang, Bolin Liu, Tingting Jiang, Chen Chen, Qi Meng, Shu Sun, Shuqin Li, Ziwei Zhao, Yanyu Dai, Dongsheng Di, Yuhui Wan, Jiahu Hao, Ji-An Xie.

  Endocrine-disrupting chemicals (EDCs) comprise structurally diverse contaminants that are frequently detected in human biological matrices and may interfere with endocrine homeostasis even at low exposure levels. Developing practical multi-residue methods for biomonitoring remains challenging because EDC classes differ markedly in polarity, ionization behavior, and urinary matrix susceptibility. In this study, we developed and validated an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of 32 target EDCs in urine from pregnant women, including 14 bisphenols, 8 per- and polyfluoroalkyl substances (PFAS), 5 organophosphate ester metabolites, and 5 preservatives. Sample preparation involved enzymatic hydrolysis followed by liquid-liquid extraction with ethyl acetate. The method was evaluated using matrix-matched calibration in artificial urine and isotope-labelled internal standards where available. Most analytes showed acceptable linearity, accuracy, precision, matrix effect, extraction efficiency, and stability according to commonly used bioanalytical validation criteria. Limits of quantification ranged from 0.001 to 0.4 ng/mL. The method was further applied to 188 urine samples from pregnant women, demonstrating its applicability for routine multi-class EDC biomonitoring. Although sensitivity for several phenolic analytes and extraction efficiency for selected compounds require further improvement, the proposed method provides a practical platform for high-throughput assessment of mixed EDC exposure in epidemiological studies.

Keywords:  EDCs; Pregnant women; UHPLC-MS/MS; Urine

DOI:  https://doi.org/10.1007/s00216-026-06690-0
J Sep Sci. 2026 Aug;49(8): e70505

Development and Validation of a QuEChERS-LC-MS/MS Method for Simultaneous Determination of Four Direct Oral Anticoagulants in Citrate Plasma and Quantitative Dried Plasma Spots.

Arkadiusz Kocur.

  Direct oral anticoagulants (DOACs), including apixaban, dabigatran, edoxaban, and rivaroxaban, may require quantitative measurement in selected clinical situations despite fixed-dose administration. This study developed and validated a quick, easy, cheap, effective, rugged, and safe (QuEChERS)-assisted liquid chromatography-tandem mass spectrometry method for their simultaneous determination in citrate plasma and quantitative dried plasma spot (qDPS) samples. Chromatographic separation was achieved on a Kinetex PS C18 column using a 4.0 min gradient, followed by positive electrospray ionization and scheduled multiple reaction monitoring. Sample preparation was optimized by comparing acetonitrile (ACN) protein precipitation, unbuffered and citrate-buffered QuEChERS extraction salts, and dispersive solid-phase extraction clean-up using primary-secondary amine, C18, EMR Lipid, Z-Sep+, or their combinations. The final procedure comprised ACN extraction with MgSO4, NaCl, and Bond Elut EMR Lipid, followed by clean-up with MgSO4, primary-secondary amine, and Z-Sep+. ACN provided the most consistent recovery across analytes, concentration levels, and matrices. The method was validated in accordance with International Council for Harmonization M10 using commercial plasma calibrators and independently prepared quality-control samples. Accuracy and precision met the predefined acceptance criteria in both matrices. Mean recovery ranged from 84.2% to 91.2% in citrate plasma and from 79.6% to 87.8% in dried plasma samples. Internal standard-normalized matrix factors ranged from 0.87 to 0.98, indicating limited ion suppression after the optimized clean-up procedure. External verification using materials from two independent proficiency-testing providers showed satisfactory agreement with assigned target values, with all results falling within the providers' acceptance ranges. The method provides a robust QuEChERS-assisted workflow for the quantitative determination of DOACs in conventional citrate plasma and qDPS samples. Its clinical interchangeability between matrices requires further evaluation using paired patient samples.

Keywords:  LC–MS/MS; QuEChERS; direct oral anticoagulants; microsampling; quantitative dried plasma spot; therapeutic drug monitoring

DOI:  https://doi.org/10.1002/jssc.70505
Infect Drug Resist. 2026 ;19 622481

Development and Validation of an LC-MS/MS Method for Simultaneous Determination of 12 Antibiotics in Human Serum.

Shuai Ruan, Jiayuan Wang.

   Purpose: To develop and validate a high-throughput LC-MS/MS method for simultaneous quantification of 12 clinically important antibiotics, addressing current limitations in multi-class therapeutic drug monitoring (TDM), particularly for β-lactam/β-lactamase inhibitor combinations.
Methods: A validated LC-MS/MS method was established using isotope-labeled internal standards. A total of 200 clinical serum samples were analyzed. Method performance was evaluated in accordance with CLSI C62 guidelines, including linearity, lower limits of detection (LLOD), lower limits of quantification (LLOQ), selectivity, accuracy, precision, matrix effects, carryover, interference, dilution effect and stability.
Results: All analytes showed excellent linearity (R2 ≥ 0.9938), with acceptable accuracy (87.01%-112.73%) and precision (<10% CV). Significant matrix-related interference was observed for imipenem, tigecycline, and vancomycin under hemolytic conditions, highlighting analyte-specific limitations. Clinical TDM results revealed that only 51.5% of patients achieved target therapeutic concentrations, while 48.5% were outside the recommended range.
Conclusion: This method provides a robust analytical platform for multi-antibiotic TDM. Importantly, clinical data demonstrate substantial variability in empirical dosing, supporting the need for concentration-guided antimicrobial therapy. However, further external validation and outcome-based studies are required before routine clinical implementation.

Keywords:  LC-MS/MS; TDM; antibiotics; liquid chromatography-tandem mass spectrometry; therapeutic drug monitoring

DOI:  https://doi.org/10.2147/IDR.S622481
Anal Methods. 2026 Aug 12.

Chimeric structural isomer fragments as cost-efficient internal standards for amino acid quantification by mass spectrometry.

Luis Alberto De León-Jiménez, Robert Winkler.

Amino acid (AA) profiles from body fluids such as blood and urine are clinical indicators for diagnosing metabolic and hepatic diseases. Current quantitative methods, such as liquid chromatography-mass spectrometry (LC-MS) with isotopically labelled internal standards (ISs), are costly and technically demanding. This study proposes a cost-efficient alternative using structural isomers as ISs in a direct liquid infusion (DLI) tandem mass spectrometry (MS/MS) approach. The method leverages chimeric spectra and fragment intensity ratios to quantify AAs, demonstrating high linearity and precision even with a 3D ion trap mass analyser. This approach offers a viable strategy for AA quantification in preventive medicine, particularly for screening metabolic diseases such as phenylketonuria, diabetes, and liver dysfunction.

DOI: https://doi.org/10.1039/d6ay01194b
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Aug 10. pii: S1570-0232(26)00338-7. [Epub ahead of print]1282 125249

Development and validation of a novel LC-MS/MS method for simultaneous quantification of fidaxomicin and metabolite (OP-1118) from feces for gut pharmacobiome studies.

Chenlin Hu, Taryn Eubank, Ann M McKelvey, Weiqun Wang, Julian G Hurdle, Kevin W Garey.

  Fidaxomicin is a first-line antibiotic for treating Clostridioides difficile infection. While it has low systemic absorption and reaches high colonic concentrations, it is hydrolyzed to a less active metabolite, OP-1118. Few studies have completely described critical experimental details of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantifying fecal fidaxomicin and OP-1118. This study developed and validated a simple, fast, and sensitive LC-MS/MS method to quantify fidaxomicin and OP-1118 in human and mouse feces. This method simplified fecal sample preparation without the use of solid phase extraction and optimized LC-MS/MS parameters. A broad working range (0.3-1000 ng/ml) in both diluted human and murine fecal matrices was achieved with good intra- and inter-day accuracy (93-107%), precision (1-7%), and recovery (70-105%) as well as little IS-normalized matrix effects. This method was utilized to quantify fidaxomicin and OP-1118 in human and murine fecal samples. This novel method was simple, fast, sensitive, and accurate in analyzing fecal fidaxomicin and OP-1118 and could be deployed to facilitate gut pharmacobiome research.

Keywords:  Antibiotics; Assay development; Chromatography; Clostridioides difficile infection; Fidaxomicin; LC-MS/MS; OP-1118; Pharmacobiome

DOI:  https://doi.org/10.1016/j.jchromb.2026.125249
Molecules. 2026 Jul 30. pii: 2658. [Epub ahead of print]31(15):

Analysis of the Chemical Composition of Inula hookeri f. major Ling Combining UHPLC-Q-Exactive-Orbitrap MS with Feature-Based Molecular Networking and SIRIUS.

Liangyin Shu, Wenqing Xiao, Jihui Zhao, Jieyao Ma.

  For the evaluation of the chemical composition of Inula hookeri f. major Ling, ultra-high-performance liquid chromatography quadrupole-Exactive Orbitrap mass spectrometry (UHPLC-Q-Exactive-Orbitrap MS) was integrated with feature-based molecular networking (FBMN) and SIRIUS. The chromatographic separation of the chemical components from Inula hookeri f. major Ling was conducted on a Hypersil GOLD™ aQ C18 (100 mm × 2.1 mm, 1.9 μm) using gradient elution, with a 0.1% formic acid aqueous solution and acetonitrile as the mobile phase. Mass spectrometric analysis was performed in both positive and negative ion modes using an electrospray ionization (ESI) source. Based on the obtained high-resolution fragment ions, FBMN was conducted to enable the clustering and visualization of structurally similar compounds. Molecular formulas were predicted and MS/MS fragment ions were interpreted with the assistance of SIRIUS. A total of 162 compounds were tentatively annotated from Inula hookeri f. major Ling, including terpenoids, phenylpropanoids, fatty acids, organic acids, and other types of compounds. The application of the strategy incorporating UHPLC-Q-Exactive-Orbitrap MS, FBMN, and SIRIUS enables the systematic characterization of the chemical composition of Inula hookeri f. major Ling.

Keywords:  FBMN; Inula hookeri f. major Ling; SIRIUS; UHPLC-Q-Exactive-Orbitrap MS; tentative annotation

DOI:  https://doi.org/10.3390/molecules31152658
J Pharm Biomed Anal. 2026 Aug 12. pii: S0731-7085(26)00372-9. [Epub ahead of print]282 117704

LC-MS/MS quantification of endogenous taurine in plasma and urine using surrogate matrix calibration and background subtraction: Application to renal transporter phenotyping.

Kathley Lanna Rezende de Azevedo, Álef Machado Gomes Pego, Adriana Rocha, Jhohann Richard de Lima Benzi, Patrícia Pereira Dos Santos Melli, Ricardo Carvalho Cavalli, Geraldo Duarte, Vera Lucia Lanchote, João Paulo Bianchi Ximenez.

  Taurine is an abundant endogenous amino acid-related compound involved in several physiological processes, including osmoregulation, antioxidative defense, and cytoprotection. Its quantification in biological matrices is analytically challenging due to its high polarity, endogenous background levels, and the absence of a true blank biological matrix. In addition, taurine has emerged as a potential endogenous biomarker for renal transporter phenotyping, particularly for organic anion transporters 1 and 3 (OAT1/3). In this study, a rapid and robust LC-MS/MS method was developed and validated for the quantification of endogenous taurine in human plasma and urine using surrogate matrix calibration combined with background subtraction. Chromatographic separation was achieved on a Luna NH₂ 100 Å column (5 µm, 150 × 4.6 mm) with a total run time of 5.5 min. Calibration curves prepared in 2% bovine serum albumin and diluted urine were linear over concentration ranges of 0.05-50 µg/mL and 0.05-100 µg/mL, respectively (r² > 0.99). Precision and accuracy met regulatory acceptance criteria (≤15%, or ≤20% at the lower limit of quantification). No significant matrix effects were observed across independent plasma and urine sources, including lipemic and hemolyzed plasma samples. Matrix and dilutional parallelism were confirmed, and taurine remained stable under all tested conditions. The method was successfully applied to clinical plasma and urine samples, enabling taurine pharmacokinetic profiling and renal clearance estimation. This validated LC-MS/MS assay provides a reliable analytical platform for taurine quantification in biological matrices and supports future pharmacokinetic, biomarker, nutritional, and transporter phenotyping studies.

Keywords:  Background subtraction; Endogenous biomarker; LC-MS/MS; Renal transporters; Taurine

DOI:  https://doi.org/10.1016/j.jpba.2026.117704
Anal Chem. 2026 Aug 11. 98(31): 22666-22673

Simultaneous Quantification of Hundreds of Phosphorylated Metabolites in Multiple Biological Matrices with Ion-Pairing Reversed-Phase Ultrahigh-Performance Liquid Chromatography and Mass Spectrometry.

Huan Wang, Dandan Yang, Qi Wang, Qingyu Hu, Qinsheng Chen, Huiru Tang.

Many phosphorylated analytes are present in complex mixtures and have diverse essential functions involving biology, chemistry, food, and environmental sciences; their simultaneous quantification is vital for efficiently revealing their crucial functions but remains challenging. Here, we developed a high-coverage method for quantifying many phosphorylated analytes in one run using ion-pairing reversed-phase ultrahigh-performance liquid chromatography and tandem mass spectrometry. Good sensitivity (limit-of-detection <0.95 pmol), linearity (R2 > 0.99), recoveries (80%-120%), precision (CV < 20%), and intertechnician consistency (CV < 20%) were demonstrated for simultaneously quantifying 125 such analytes including nucleotides, nucleotide sugars, phosphorylated amino acids and sugars, and the enzyme-cofactors derived from vitamin B1 (TMP, TPP, TTP), B2 (FMN, FAD), B3 (NAD, NADP), B5 (acyl-CoAs), and B6 (PLP, PMP). The method applicability was also confirmed by quantifying 43-78 phosphorylated metabolites in five typical biological matrices including human urine, plasma, cells, feces, and rabbit liver tissue, illustrating their significant molecular phenotypic differences in the phosphometabolome. By quantifying the phosphometabolomic differences using the method, we further revealed some metabolic characteristics associated with the drug resistances for human non-small cell lung cancer cells. This offers a reliable method for the quantitative investigation of phosphorylated metabolites and their functions.

DOI: https://doi.org/10.1021/acs.analchem.5c08214
Molecules. 2026 Aug 06. pii: 2734. [Epub ahead of print]31(15):

Comparative Metabolite Profiling of Different Solvent Extracts of Argemone ochroleuca Sweet and Argemone mexicana Linn Shoots and Roots Using Liquid Chromatography-Mass Spectrometry-Based Metabolomics and Molecular Networking.

Nezelo Trizer Mlombo, Fikile Nelly Makhubu, Zakheleni Palane Dube, Ntakadzeni Edwin Madala, Thilivhali Emmanuel Tshikalange.

  Argemone ochroleuca and Argemone mexicana are widespread weed species known for being rich in various secondary metabolites. However, a comprehensive understanding of their chemical diversity remains limited. Insufficient information exists on metabolite variation between plant parts and the influence of solvent polarity on metabolite recovery. Therefore, this study aimed to characterize the metabolomic profiles of the shoots and root extracts of both species using solvents of varying polarity to evaluate plant part-specific metabolite distribution and solvent effects on metabolome coverage. Untargeted ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS)-based metabolomics and molecular networking were employed for metabolite analysis. Principal component analysis (PCA) did not reveal clear clustering of A. ochroleuca and A. mexicana, suggesting similarities in their metabolomic profiles. A total of 15 and 13 metabolite classes, yielding 59 and 54 metabolites, were identified in A. ochroleuca and A. mexicana, respectively, including flavonoids, terpenoids, phenolic compounds, fatty acids, and monoterpenoids. Argemone ochroleuca exhibited higher metabolite abundance, particularly in methanol and acetone extracts, and with shoots showing higher abundance than roots, with flavonoids being the dominant class. The findings show that LC-MS metabolomics, molecular networking, and careful solvent selection are effective for identifying key metabolites with potential applications in crop protection.

Keywords:  Argemone; LC-MS; metabolites; metabolomics; molecular network

DOI:  https://doi.org/10.3390/molecules31152734
Anal Biochem. 2026 Aug 13. pii: S0003-2697(26)00178-8. [Epub ahead of print] 116222

Development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify protoporphyrin IX (PPIX) and zinc (II) protoporphyrin IX (Zn-PPIX), in human plasma.

Bhavik Shah, Edmond Huang, Uma Sinha, Frank Hsieh, Pete Schmidt.

  Protoporphyrin IX (PPIX) is a photoreactive heme precursor and is a key pathogenic driver in erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP). In both EPP and XLP, excess PPIX in plasma circulates to the skin and hepatobiliary system, resulting in acute, painful cutaneous photosensitivity and in some patients, gallstones, or hepatic failure. A key distinguishing feature between EPP and XLP is proportions of PPIX that become metalated with zinc (Zn-PPIX) or remain metal-free. Thus, ability to accurately measure both PPIX and Zn-PPIX in plasma is especially useful for assessing emerging therapies for EPP and XLP. This manuscript presents a fully validated, sensitive liquid chromatography-tandem mass spectrometry method for the quantification of PPIX and Zn-PPIX across a dynamic range in human plasma, which is especially suited for measuring drug-induced effects on plasma PPIX and Zn-PPIX levels. It also presents the first reported accurate measurements of plasma PPIX in healthy individuals and begins to establish a preliminary baseline range for this major form of PPIX in plasma of individuals without protoporphyria, which can facilitate future development of therapies for EPP and XLP.

Keywords:  Erythropoietic protoporphyria; Liquid chromatography-tandem mass spectrometry; Plasma; Protoporphyrin IX; X-linked erythropoietic protoporphyria; X-linked protoporphyria

DOI:  https://doi.org/10.1016/j.ab.2026.116222
J Chromatogr A. 2026 Sep 27. pii: S0021-9673(26)00643-6. [Epub ahead of print]1785 467315

Optimization and validation of a two-step extraction dispersive liquid-liquid microextraction (UA-DLLME) method for the determination of 17 antibiotics in influent wastewater.

Eirini Siandri, Christoforos Bouzoukas, Dimitrios-Triantafyllos Gerokonstantis, Marios G Kostakis, Nikolaos S Thomaidis.

  A rapid and efficient method based on two-step dispersive liquid-liquid microextraction (DLLME) was developed for the simultaneous determination of antibiotics belonging to six different subclasses (fluoroquinolones, macrolides, sulfonamides, lincosamides, nitroimidazoles and trimethoprim) in influent wastewater samples. Analysis was performed using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). During method optimization, several parameters affecting DLLME performance were evaluated: the type and volume of extraction and dispersive solvents, sample pH, sample volume, salt content (sodium chloride) for the salting-out effect, and ultrasonication time. Under optimized conditions, the method's performance characteristics exhibited linearity between 100 and 4000 ng/L (R² ≥ 0.98). The matrix effect (ME) ranged from 32.5% to 93.3%. Recovery values were satisfactory, mostly ranging between 80% and 120%. Relative standard deviation (RSD) values indicated good repeatability and intermediate precision (< 20%), while limits of quantification (LOQs) ranged from 10.7 to 369 ng/L. Finally, the validated method was applied to the analysis of influent wastewater samples collected from the Psyttalia Wastewater Treatment Plant.

Keywords:  Antibiotics; Dispersive liquid- liquid microextraction (DLLME); Liquid chromatography- tandem mass spectrometry (LC-MS/MS); Method optimization; Method validation; Wastewater- based epidemiology (WBE)

DOI:  https://doi.org/10.1016/j.chroma.2026.467315
Molecules. 2026 Jul 28. pii: 2624. [Epub ahead of print]31(15):

The Application of LC-MS in Pharmaceutical Analysis.

Wing Lam, Wei Cai.

Liquid chromatography-mass spectrometry (LC-MS) has evolved from a specialized analytical tool into an indispensable cornerstone of pharmaceutical analysis over the past three decades [...].

DOI: https://doi.org/10.3390/molecules31152624
Front Chem. 2026 ;14 1900122

Quantitative analysis of vitamin E, 5-methyltetrahydrofolate, and glutathione in seminal plasma by liquid chromatography-tandem mass spectrometry.

Tianwei Zhang, Bin Liu, Yang Fu, Yan Jin, Fang Luan, Yong Wang.

   Background: Oxidative stress contributes to male infertility, but quantifying non-enzymatic antioxidants in seminal plasma remains challenging. This study aimed to establish liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods for measuring vitamin E (VE), glutathione (GSH), and 5-methyltetrahydrofolate (5-MTHF) in human seminal plasma.
Methods: Analyte-specific pretreatments were established: liquid-liquid extraction for VE, 96-well phospholipid removal plate for 5-MTHF, and sulfosalicylic acid precipitation for GSH without derivatization, with stable isotope-labeled internal standards. LC-MS/MS analysis was performed on AB SCIEX 4500 MD and Waters Xevo TQ-S systems. The method was validated in accordance with the CLSI C62-A guidelines. Preliminary method-specific reference intervals (RIs) were established in 120 healthy reproductive-aged men. Serum-seminal plasma correlations were assessed in 22 paired samples, and exploratory clinical associations were evaluated in 47 infertile men.
Results: High linearity (R > 0.99) was achieved, with lower measuring interval limits of 78.10, 1.31, and 15.37 ng/mL for VE, 5-MTHF, and GSH, respectively. The intra- and inter-assay coefficients of variation were <6.8% and <5.5%, respectively. Recovery rates ranged from 88.78% to 108.10%. No significant matrix effects, carryover, or interference were observed. No significant seminal plasma-serum correlations detected. Seminal 5-MTHF and GSH levels were significantly lower in infertile men than in healthy controls [5-MTHF: 139.50 (68.02-198.50) vs. 387.80 (152.50-623.80) ng/mL, P < 0.001; GSH: 4314.00 (3051.00-5974.00) vs. 9049.00 (6128.00-11828.00) ng/mL, P < 0.001], whereas VE levels showed no significant difference [199.00 (138.00-252.00) vs. 168.50 (129.00-237.80) ng/mL, P = 0.211].
Conclusion: The validated LC-MS/MS methods enable reliable quantification of seminal VE, 5-MTHF, and GSH and provide preliminary method-specific RIs in healthy Chinese men. Lower seminal 5-MTHF and GSH levels in infertile men suggest their potential relevance to male infertility. Further multicenter studies are needed to validate their clinical applicability.

Keywords:  5-methyltetrahydrofolate; glutathione; liquid chromatography-tandem mass spectrometry (LC-MS/MS); male infertility; oxidative stress; vitamin E

DOI:  https://doi.org/10.3389/fchem.2026.1900122
Anal Chem. 2026 Aug 11. 98(31): 22755-22770

MetaboGraph: A Framework for Metabolomics and Lipidomics Annotation and Pathway Network Analysis.

Oluwatosin Daramola, Judith Nwaiwu, Odunayo Oluokun, Mojibola Fowowe, Yehia Mechref.

Untargeted metabolomics and lipidomics generate high-dimensional data sets whose biological interpretation remains challenging, particularly at the pathway and network levels. Here, we present MetaboGraph, a standalone Python-based workflow for end-to-end metabolomics and lipidomics analysis, enabling pathway-level interpretation from small-molecule data. MetaboGraph integrates automated data cleaning, comprehensive multidatabase metabolite and lipid annotation, pathway mapping, and direction-aware pathway inference. A central feature of the platform is its ability to predict pathway direction by integrating metabolite/lipid-level fold changes with pathway membership structure, supporting biologically interpretable pathway and network analyses beyond conventional enrichment approaches. MetaboGraph supports multiomics integration and comparative analysis, enabling consistent pathway-level interpretation across metabolomics, lipidomics, and multiple studies. We demonstrate the platform using untargeted LC-MS/MS metabolomics and lipidomics data comparing two breast cancer cell lines with distinct metastatic potential, MCF7 (HTB22; less metastatic) and MDA-MB-453 (HTB131; more metastatic). Relative to HTB22, the HTB131 cells exhibited coordinated metabolic remodeling, including altered amino acid and nitrogen metabolism, increased nucleotide biosynthetic demand, lipid remodeling, and changes in energy-associated pathways. These pathway-level alterations are consistent with established metabolic adaptations associated with increased cancer aggression. MetaboGraph expands the analytical toolbox for small-molecule biology and facilitates reproducible, biologically grounded insights from metabolomics and lipidomics data sets.

DOI: https://doi.org/10.1021/acs.analchem.6c01446
Clin Chem Lab Med. 2026 Aug 12.

A liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of vancomycin in human serum and plasma.

Sandra Fleischer, Neeraj Singh, Friederike Bauland, Andrea Geistanger, Judith Taibon, Elie Fux.

   OBJECTIVES: Vancomycin is a glycopeptide antibiotic used to treat severe Gram-positive infections; its narrow therapeutic window makes therapeutic drug monitoring (TDM) critical. We report the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) candidate reference measurement procedure (RMP) to quantify vancomycin in human plasma and serum.
METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was used to determine the absolute content of the reference material, ensuring direct traceability to the International System of Units (SI). Sample preparation used a protein precipitation protocol followed by high dilution. LC-MS/MS parameters, including selectivity, precision, accuracy, equivalence to the listed RMP, and measurement uncertainty (MU) were evaluated. MU determination was performed according to the guide to the expression of uncertainty in measurement (GUM).
RESULTS: The RMP was validated over a measuring interval of 0.690-58.7 μmol/L (1.00-85.0 μg/mL). Intermediate precision and repeatability were <2.9 % and <2.1 %, respectively. Direct comparison with the current JCTLM-listed RMP revealed an 18.3 % downward shift in results. This bias was proven to be entirely attributable to the transition from activity-based to mass-based standardization. Expanded MU (k=2) ranged from 5.1 % to 6.0 % for single measurements and 2.1-3.1 % for target value assignment.
CONCLUSIONS: This candidate RMP provides a superior metrological foundation for vancomycin quantification by establishing direct SI-traceability and a fully characterized uncertainty budget. While its implementation necessitates a re-evaluation of clinical decision thresholds, it significantly enhances the global standardization of TDM assays.

Keywords:  SI units; liquid chromatography-tandem mass spectrometry; qNMR characterization; reference measurement procedure; traceability; vancomycin

DOI:  https://doi.org/10.1515/cclm-2026-0509
Anal Bioanal Chem. 2026 Aug 10.

MALDI mass spectrometry imaging across spatial scales in toxicology: tissue evidence, cellular heterogeneity, and emerging single-cell opportunities.

Yuang Qin, Jie Lian, Honggang Nie.

  Toxicological responses are inherently spatially heterogeneous, yet conventional analytical strategies generally rely on homogenized samples and therefore provide limited information on where xenobiotics accumulate, which tissue regions or cellular populations are preferentially affected, and how local molecular perturbations relate to pathology and mechanism. Mass spectrometry imaging (MSI) addresses this limitation by enabling label-free, multiplexed, and spatially resolved detection of xenobiotics, metabolites, lipids, peptides, and proteins directly in biological specimens. In this review, we examine the role of MSI in spatial toxicology, with particular emphasis on matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) as a practical core platform for many current tissue-level and emerging cellular-scale toxicology studies. We first discuss why MSI is analytically well suited for spatial toxicology and compare the major MSI platforms in terms of molecular coverage, spatial resolution, and toxicological applicability. We then summarize representative applications at the tissue level, where MSI has already shown clear value in mapping xenobiotic localization, lesion-associated molecular remodeling, and organ-specific toxicity. Next, we discuss cellular-scale MSI and the transition toward true single-cell analysis, emphasizing that current progress is driven more by workflow development, multimodal integration, and computational advances than by routine toxicological deployment. Finally, using per- and polyfluoroalkyl substances as a focused case study, we illustrate how MSI can connect tissue burden, regional bioaccumulation, and localized biochemical response. Overall, MSI is becoming a key analytical framework for mechanistic spatial toxicology, although broader impact will depend on further advances in standardization, annotation confidence, quantification, and multimodal interpretation.

Keywords:  MALDI-MSI; Mass spectrometry imaging; Single-cell analysis; Spatial metabolomics; Spatial toxicology; Xenobiotic localization

DOI:  https://doi.org/10.1007/s00216-026-06705-w
Biomed Chromatogr. 2026 Oct;40(10): e70599

Resolution and Quantitation of Ribonucleosides and Deoxyribonucleosides in Digested Genomic DNA by UHPLC-MS/MS.

Joshua J Deppas, Reyna Jones, Robert A Parise, Pinakin Pandya, Christopher J Bakkenist, Jan H Beumer.

  Deoxyuridine (dUrd) is often misincorporated into genomic DNA in cancer patients receiving antimetabolite chemotherapy. This activates base excision repair and recruits ataxia telangiectasia-mutated and Rad3-related (ATR), which coordinates DNA repair with nucleotide metabolism by facilitating ribonucleotide reductase (RNR) activity. Ceralasertib (AZD6738) is the most clinically advanced ATR inhibitor (ATRi) and may abrogate this regulatory pathway and diminish RNR-mediated deoxynucleotide triphosphate synthesis. To further understand ATRi-induced dUrd misincorporation, we developed a highly sensitive LC-MS/MS method to quantitate: deoxyuridine, uridine, guanosine, adenosine, cytidine, thymidine, deoxyguanosine, deoxyadenosine, and deoxycytidine from digested genomic DNA. Assay application was demonstrated with genomic DNA digested from multiple murine cell lines treated with ceralasertib. Chromatographic separation was achieved with an Inertsil ODS-3 (150 × 2.1 mm, 3 μm) column and a gradient elution program of 0.1% formic acid in water and 0.1% formic acid in methanol over an 18-min run time. Detection was performed on a SCIEX 6500+ tandem mass spectrometer. The method proved to be accurate (90.8%-114.2%) and precise (< 7.73% CV) across analytes. Freeze-thaw stability (106.4%-114.3%), stability for 12 months at -80°C (88.7%-113.7%), and stability for 4 h at room temperature (104.1%-114.0%) were acceptable across QCs.

Keywords:  LC–MS/MS; ceralasertib; deoxynucleosides; nucleoside quantitation; ribonucleosides

DOI:  https://doi.org/10.1002/bmc.70599
Rapid Commun Mass Spectrom. 2026 Nov 15. 40(21): e70163

Fragment Coverage: Unified Visualization and Quantitative Assessment of MS/MS Fragmentation Supports Metabolite Identification in Macromolecule Drug Metabolism Studies.

Paula Cifuentes, Albert Garriga, Fabien Fontaine, Ismael Zamora.

   RATIONALE: Peptides and oligonucleotides are increasingly used as therapeutic agents, making the identification and structural elucidation of their metabolites a critical step in drug development. Liquid chromatography-mass spectrometry (LC-MS/MS) is the primary analytical technique for this purpose; however, extensive backbone fragmentation generates highly complex MS/MS spectra, making spectral interpretation challenging and limiting the exploitation of the structural information contained in MS/MS fragmentation data.
METHODS: This study introduces two fragment-based metrics, fragment coverage and complete fragment coverage, to quantify structural coverage derived from MS/MS fragmentation at metabolite and parent compound levels. Their performance was evaluated across 33 LC-MS/MS experiments comprising peptide and oligonucleotide datasets. Ranking analysis based on mean average precision (MAP) assessed discrimination between true and false metabolite assignments. Additionally, a fragment viewer was developed to map MS/MS fragment ions onto structures, facilitating visualization and spectral interpretation.
RESULTS: A total of 255 metabolites were identified, including 159 true metabolites and 96 false positives. True metabolites consistently exhibited higher fragment coverage and complete fragment coverage values than false positives. This trend was supported by ranking analysis using mean average precision (MAP), where complete fragment coverage achieved the highest performance (0.9737), followed by fragment coverage (0.9676), MassMetaSite score (MMS score) (0.9390), and isotopic similarity (0.9090). Two representative case studies demonstrated the applicability of the proposed metrics.
CONCLUSIONS: The proposed fragment coverage metrics enable quantitative assessment of MS/MS fragmentation, providing complementary information to support manual interpretation of LC-MS/MS data in MetID workflows. Together with the fragment viewer tool, these approaches facilitate more efficient and interpretable analysis of complex MS/MS spectra in macromolecules.

Keywords:  LC‐MS/MS; MS/MS fragmentation; macromolecule metabolite identification; oligonucleotides; peptides

DOI:  https://doi.org/10.1002/rcm.70163
Forensic Sci Int. 2026 Aug 07. pii: S0379-0738(26)00290-2. [Epub ahead of print]388 113102

LC-HRMS suspect screening for expanded detection of (new) psychoactive substances in oral fluid: a practical approach for forensic casework.

Miriam Blanco-Ces, Rosa Montes, Elena Lendoiro, María Cobo-Golpe, Ángela López-Rabuñal, Angelines Cruz, Ana de-Castro-Ríos.

  Liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are routinely used in forensic toxicology to confirm illicit drugs in oral fluid (OF). However, they are inherently limited to predefined analytes and may fail to detect newly emerging psychoactive substances (NPS) or compounds not included in routine panels. This study evaluates the applicability of a liquid chromatography-high resolution mass spectrometry (LC-HRMS) suspect screening workflow for the reanalysis of oral fluid extracts previously processed using a routine targeted LC-MS/MS method, with focus on its implementation in forensic casework. OF samples (0.25 mL) were extracted using a solid-phase extraction procedure routinely employed for the confirmation of traditional illicit drugs and psychopharmaceuticals. The same extracts were reanalyzed by LC-HRMS operating in data-dependent acquisition mode. Data processing was performed using a Personal Compound Database Library (PCDL) containing 2376 entries (1693 with MS/MS spectra), generated from HighResNPS and expanded with in-house acquired spectra and retention times. Qualitative validation was performed for 105 available reference standards, with limits of detection ≤ 10 ng/mL for 64.8% of them. The applicability of the workflow was demonstrated through the analysis of 25 authentic OF samples from drivers (n = 15), chemsex users (n = 6) and emergency unit patients (n = 4). LC-HRMS screening confirmed compounds previously detected by targeted LC-MS/MS, while enabling the identification of additional psychoactive substances not included in routine panels. This strategy provides a complementary approach for forensic toxicology laboratories, expanding screening capabilities from existing sample extracts and supporting the improved monitoring of emerging NPS in routine forensic casework.

Keywords:  Illicit drugs; NPS; Oral fluid; Psychopharmaceuticals; Routine toxicological workflow; Suspect screening

DOI:  https://doi.org/10.1016/j.forsciint.2026.113102
J Chromatogr A. 2026 Aug 05. pii: S0021-9673(26)00654-0. [Epub ahead of print]1786 467326

Detection and confirmation of hypoxia-inducible factor activating agents in equine plasma and urine by liquid chromatography coupled to mass spectrometry.

Fuyu Guan, Matthew A Adreance, Savannah Fay, Leif K McGoldrick, Bethany Keen, Youwen You, Mary A Robinson.

  Hypoxia-inducible factor (HIF) activating agents can promote the transcription of the erythropoietin gene, enhancing an athlete's performance, and are therefore prohibited in sports. A liquid chromatography coupled to high-resolution mass spectrometry (LCHRMS) method was developed for the detection and confirmation of 11 HIF-activating substances in equine plasma and urine. Ammonium formate added to the mobile phase system suppressed the responses of all the analytes in either positive or negative ion mode and thus was not used. The starting organic percentage in the mobile phase gradient and sample reconstitution solution were optimized to minimize the carryover of the analytes. The analytes were extracted with solid-phase extraction. No interference from plasma or urine matrix was observed in the detection of the analytes by negative ion HRMS. Ten calibrators containing all 11 analytes at 10 to 10,000 pg/mL in plasma or urine were used to evaluate calibration ranges and limits of detection (LODs) by HRMS, and limits of confirmation (LOCs) by parallel reaction monitoring. LOD was 10 to 50 pg/mL for all analytes in plasma and 10 to 200 pg/mL in urine. LOC was 50 to 200 pg/mL for all analytes in plasma, except daprodustat and IOX 3 at 500 pg/mL, and 20 to 500 pg/mL in urine, except daprodustat and IOX 3 at 1000 pg/mL. The developed LC-MS method was successfully applied to the analysis of plasma and urine samples from a research horse following oral administration of JNJ 42041935.

Keywords:  Enarodustat; Hypoxia-inducible factor; JNJ 42041935; TP 0463518; prolyl-hydroxylase inhibitors

DOI:  https://doi.org/10.1016/j.chroma.2026.467326
Talanta. 2026 Aug 07. pii: S0039-9140(26)01052-0. [Epub ahead of print]312(Pt A): 130396

Aza-Paternò-Büchi derivatization enables annotation of triacylglycerols by negative-ion-mode mass spectrometry at the double bond level.

Andrea Cerrato, Aldo Laganà, Carmela Maria Montone, Enrico Taglioni, Daniel Weissman, Anna Laura Capriotti.

  Triacylglycerols (TGs) represent one of the most abundant lipid classes in biological and food matrices; however, their structural characterization by mass spectrometry remains challenging because conventional workflows often provide only sum composition information. In the present study, an aza-Paternò-Büchi (aPB) derivatization strategy based on 6-azauracil was optimized and applied for negative-ion-mode, isomer-resolved analysis of TGs by high-resolution mass spectrometry. Reaction conditions were systematically investigated through solvent optimization, mixture design modeling, and kinetic studies. Moreover, the ionization and fragmentation behavior of aPB-derivatized TGs was comprehensively investigated, revealing distinct fragmentation pathways associated with charge localization effects in neutral lipids. Application to complex matrices demonstrated the feasibility of the approach, providing direct access to TG regioisomer distributions in human plasma and vegetable seed oils. Overall, the proposed strategy enables untargeted and double-bond-resolved annotation of TGs in complex matrices while maintaining straightforward sample preparation and compatibility with routine lipidomics workflows. For the first time, negative-ion mode was employed for TG annotation, expanding the molecular information accessible in TG lipidomics and food analysis.

Keywords:  Carbon-carbon double bonds; High-resolution mass spectrometry; Lipidomics; Plasma; Regioisomer; Seed oil

DOI:  https://doi.org/10.1016/j.talanta.2026.130396
J Chromatogr A. 2026 Aug 10. pii: S0021-9673(26)00676-X. [Epub ahead of print]1786 467348

Automated magnetic solid-phase extraction based on hydrophilic-lipophilic balanced materials for LC-MS/MS determination of macrolides in pork.

Ruiguo Wang, Shulin Wei, Peilong Wang, Wei Wei, Ming Yan, Kang Ma, Songyan Liu, Yandong Li.

  A fully automated method based on magnetic solid-phase extraction (MSPE) using commercial hydrophilic-lipophilic balanced magnetic particles (MHLB) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of nine macrolides in pork. Key parameters of the automated MSPE procedure, including elution solvent, adsorbent amount, and ultrasonic time, were systematically optimized, achieving complete elution with only 2 mL of methanol. Processing one sample consumed only 30 mg of MHLB and 3 mL of organic solvent, and the automated protocol enabled batch processing of up to 20 samples. The method showed good linearity (R² > 0.99), low limits of quantification (0.03-0.33 μg/kg), and satisfactory recoveries (77.8-90.0% with RSDs < 10.3%). Compared with conventional HLB cartridges (200 mg sorbent, 14 mL organic solvent, mandatory defatting), MHLB offered superior solvent economy, material efficiency, and operational simplicity, with better matrix effect tolerance and more consistent recoveries. This automated MHLB-LC-MS/MS approach provides a reliable, high-throughput, and more environmentally friendly alternative for monitoring macrolide residues in livestock products.

Keywords:  Animal-derived foods; Automated sample preparation; LC-MS/MS; Macrolide antibiotics; Magnetic solid-phase extraction (MSPE)

DOI:  https://doi.org/10.1016/j.chroma.2026.467348
Anal Chem. 2026 Aug 11. 98(31): 22627-22635

Rapid Chiral Analysis of Drugs with Multiple Stereocenters via High-Resolution Ion Mobility.

Benjamin K Blakley, Jody C May, Valeria Guidolin, Bao Nguyen, John A McLean.

The increasing structural complexity of small-molecule therapeutics requires sensitive analytical techniques that facilitate rapid chiral analysis of drugs with multiple chiral centers. Here, we demonstrate ultrafast chiral separations for compounds possessing 3 chiral centers (8 stereoisomers, 4 enantiomer pairs) using gas-phase ion mobility spectrometry-mass spectrometry (IM-MS). Separations are achieved using spontaneous noncovalent copper-tyrosine complexation.This complexation strategy converts all stereoisomers into diastereomers that are structurally distinguishable by IM-MS. Notably, this strategy is over 3 orders of magnitude faster than traditional liquid-phase chiral chromatography (ms vs min, respectively). Using IM-MS, we achieved direct baseline separation of enantiomer pairs and progress toward differentiation of all 8 stereoisomers. Results suggest that higher-order complexes (i.e., copper-tyrosine-bound drug dimers) enhance chiral differentiation at the expense of increased spectral complexity. Exchanging the chirality of the amino acid for complexation resulted in an inversion of separation elution ordering. Peak fitting analysis suggests that an IM resolving power of several thousand is necessary to resolve all stereoisomers for quantitative purposes, which is approximately an order of magnitude higher than the current state-of-the-art. Collectively, these findings demonstrate the speed and versatility of gas-phase ion mobility spectrometry for complex chiral analysis.

DOI: https://doi.org/10.1021/acs.analchem.6c03422
J Chromatogr A. 2026 Sep 27. pii: S0021-9673(26)00650-3. [Epub ahead of print]1785 467322

Separation of imidazolium-based ionic liquid cations with various substituents on a pentafluorophenylpropyl stationary phase using UHPLC-MS.

Hannah Lawson, Shuvro Chakraborty, Amanda L Patrick.

  Ionic liquid cations are inherently fixed-charge and, when paired with smaller or polar substituents, not well-suited for reverse phase columns; they are also bulky organic species which can make methods traditionally used for monoatomic cation analysis less than ideal. For aromatic IL cations, such as those built upon the popular imidazolium scaffold, the pentafluorophenylpropyl (PFPP) column allows π-π stacking to improve retention. The PFPP stationary phase also has the potential to better retain polar substituents via additional dipole-dipole and/or H-bonding interactions. Previous related studies have shown good retention of alkyl and benzyl imidazolium cations on the PFPP stationary phase, using UV/Vis as a detector. The aim here is to (1) evaluate the PFPP stationary phase for methyl-imidazolium cations with a more chemically diverse set of substituents, specifically including polar groups, that mimic task-specific IL design, and (2) prioritize compatibility of mobile phases with electrospray-mass spectrometric detection to facilitate improved selectivity and sensitivity. Elution orders for six IL cations were determined across several mobile phase compositions. It was found that all six IL cations were adequately retained on the PFPP column; that acetonitrile performed better than methanol as a mobile phase organic modifier; and that 20/80 acetonitrile/water with 0.1% formic acid allowed for acceptable separation of all six analytes. Further, it was established that, under these experimental conditions, the nature of the anion contributed negligibly to the observed retention times. This provides promising results for the broad suitability of PFPP-based separations of imidazolium IL cations across the scope of synthetic tunability.

Keywords:  Electrospray ionization; Ionic liquids; Mass spectrometry; Ultra high-pressure liquid chromatography

DOI:  https://doi.org/10.1016/j.chroma.2026.467322
STAR Protoc. 2026 Aug 07. pii: S2666-1667(26)00424-7. [Epub ahead of print]7(3): 104771

Protocol for untargeted LC-MS/MS metabolomics annotation and differential abundance analysis using the SIRIUS Python client.

Jonas A Emmert, Sebastian Böcker, Markus Fleischauer.

  We present a protocol for programmatic untargeted LC-MS/MS metabolomics using PySirius, the Python client for SIRIUS. We describe steps for importing data with LC-MS feature alignment, and annotating features with molecular formulas, structures, and compound classes. We then detail procedures for analyzing differential abundance by fold change between groups and visualizing results. As proof of concept, we replicate the finding that rosmarinic acid is more abundant in old than young rosemary (Rosmarinus officinalis) leaves.

Keywords:  Bioinformatics; Metabolomics; Protocols in Metabolomics and Lipidomics; Special Issue

DOI:  https://doi.org/10.1016/j.xpro.2026.104771
Anal Chem. 2026 Aug 11. 98(31): 22586-22594

Foundation Models for Liquid Chromatography-High-Resolution Mass Spectrometry: A New Era beyond Labeled Datasets.

Andrea Junior Carnoli, Federico Padilla-Gonzalez, Leonieke M van den Bulk, Daan Korporaal, Martin Alewijn, Marco H Blokland, Bas H M van der Velden.

Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) is a widely used analytical technique for characterizing the chemical composition of organic samples. Due to its high sensitivity and ability to detect thousands of chemical features in a single run, untargeted LC-HRMS experiments generate highly complex and data-rich datasets that typically require advanced computational methods, including machine learning, for meaningful interpretation. While traditional machine learning approaches have been applied to LC-HRMS data, their performance remains limited for complex tasks. Deep learning has demonstrated improved performance, but both machine and deep learning are often constrained by the complexity and scarcity of labeled LC-HRMS data. Foundation models present a promising new horizon for LC-HRMS data analysis, given their ability to learn transferable representations from large-scale unlabeled data and adapt efficiently to downstream tasks with limited labeled samples. Recent studies have shown that foundation models can outperform conventional machine learning approaches in chemical annotation and molecular property prediction. We envision that foundation models for LC-HRMS data will benefit from the expansion of curated sample repositories and spectral libraries, developing privacy-preserving training strategies, enabling simultaneous modeling of multiple LC-HRMS data types, and improving model explainability.

DOI: https://doi.org/10.1021/acs.analchem.6c01825
Anal Bioanal Chem. 2026 Aug 12.

Integrating liquid-liquid extraction and dispersive solid-phase extraction cleanup for high-throughput profiling of 11 neonicotinoids and 6 metabolites in human urine by HPLC-Q-Orbitrap HRMS.

Feng Liu, Decong Yu, Kun He, Yanan Song, Yichao Li, Haoxuan Gao, Bing Wu, Xiaoyu Li, Ji Zhao, Fei Xu, Huifang Yang.

  The widespread use of neonicotinoids has raised growing concerns about their potential health risks. In this study, we developed a robust and high-throughput analytical method based on liquid-liquid extraction (LLE) and dispersive solid-phase extraction (dSPE), followed by high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (HPLC-Q-Orbitrap HRMS) for the simultaneous determination of 11 neonicotinoids and their 6 metabolites in human urine. After pretreatment of LLE and dSPE, qualitative and confirmatory analysis was achieved using tMS2 acquisition of HPLC-Q-Orbitrap HRMS, and quantitative analysis was performed using isotope-labeled internal standards and matrix-matched calibration curves. The method was validated with good linearity (R2 > 0.9975), recoveries of 75.8%~118.9% (RSDs < 15%), and detection limits of 0.001~0.06 μg/L. The established method was successfully applied to urine samples of community-dwelling elderly population individuals (n = 150) and to a suspected case of acute acetamiprid poisoning in Ningxia, China. The results showed that thirteen neonicotinoids and metabolites were detected in the elderly cohort, and the poisoning incident was confirmed to be caused by acetamiprid and its metabolite N-desmethyl-acetamiprid. Meanwhile, Spearman correlation analysis indicated significant positive correlations between parent compounds and their metabolites for all analytes except acetamiprid, and a strong correlation between clothianidin and thiamethoxam, suggesting shared exposure sources or common metabolic pathways. The established method is sensitive, accurate, and high-throughput, and has proven reliable for human biomonitoring and health risk assessment of neonicotinoid exposure.

Keywords:  Dispersive solid-phase extraction; High-performance liquid chromatography-Q-Orbitrap high resolution mass spectrometry; Liquid-liquid extraction; Neonicotinoids; Urine samples

DOI:  https://doi.org/10.1007/s00216-026-06732-7
Biomed Chromatogr. 2026 Oct;40(10): e70591

A Rapid and Sensitive LC-MS/MS Method for Quantification of NDSRI in Melatonin Drug Product via Controlled In Situ Formation.

Jaya Yalamanchili, Ganja Himavathi, Salakolusu Suresh.

  Nitrosamine drug substance-related impurities (NDSRIs) pose a significant safety risk due to their mutagenic and carcinogenic potential, prompting strict regulatory oversight under ICH M7. The propensity for in situ formation of NDSRIs during analytical procedures, particularly in active pharmaceutical ingredients containing reactive amines, emphasizes the need for highly accurate and scrupulously validated methodologies. Melatonin, containing a vulnerable amine, was chosen as a model drug product. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantifying N-Nitroso melatonin. To prevent in situ formation, a scavenger was incorporated during sample preparation. Validation followed regulatory standards, assessing specificity, linearity, accuracy, precision, sensitivity, and robustness. The method showed excellent linearity (1.8-22.5 ng/mL, R2 = 0.9983), sensitivity with LOD of 0.9 ng/mL and LOQ of 1.8 ng/mL, and accuracy with recoveries between 85.3% and 95.9%. Precision was confirmed with %RSD below 2%, whereas robustness testing demonstrated consistent performance under varied conditions. Crucially, the scavenger eliminated false positives by suppressing in situ nitrosamine formation, ensuring data integrity. This validated approach strengthens analytical accuracy and provides a transferable framework for monitoring NDSRIs in other drug products containing reactive amines, supporting regulatory compliance and safeguarding patient health.

Keywords:  liquid chromatography‐tandem mass spectrometry (LC–MS/MS); melatonin; multiple reaction monitoring (MRM); nitrosoamine drug substance–related impurities (NDSRIs)

DOI:  https://doi.org/10.1002/bmc.70591
J Chromatogr A. 2026 Sep 27. pii: S0021-9673(26)00649-7. [Epub ahead of print]1785 467321

AQbD-guided development of a robust and green SI-RP-HPLC method for impurity profiling of an efonidipine hydrochloride ethanolate and chlorthalidone combination, with LC-MS and NMR-based identification of degradation product in efonidipine.

Pranav S Bang, Vishnu P Choudhari, Vikram S Gharge.

  Efonidipine Hydrochloride Ethanolate (EHE) and Chlorthalidone (CTD) are co-formulated for the management of hypertension owing to their complementary pharmacological actions. Ensuring accurate impurity profiling in such combination formulations is vital for maintaining drug stability, safety and regulatory compliance. However, conventional analytical techniques often face challenges related to sustainability and robustness. Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) remains a key analytical approach for impurity determination, yet the integration of Quality by Design (QbD) principles into its development is still evolving. The present study focuses on developing a novel, sustainable RP-HPLC method for impurity profiling of EHE and CTD tablets using a QbD approach and green chemistry. Chromatographic separation was achieved on a Zorbax SB C8 column (150 mm × 4.6 mm, 5 µm). Critical method parameters such as mobile phase composition and pH were systematically optimized through QbD to enhance method robustness. A photolytic degradation product of EHE was identified as Efonidipine Related Compound A, and a plausible photolytic degradation pathway was proposed. A photolytic degradation product was identified in the LC-MS and its structure was confirmed through NMR spectroscopy. The developed green method exhibited excellent resolution, precision and reproducibility across multiple batches, allowing for effective separation and quantification of impurities. This green QbD-optimized, environmentally sustainable RP-HPLC method provides a reliable and scalable solution for impurity profiling in complex pharmaceutical formulations, aligning with modern quality control and regulatory expectations.

Keywords:  Chlorthalidone; Chlorthalidone impurity B; Efonidipine hydrochloride ethanolate; Efonidipine related compound A

DOI:  https://doi.org/10.1016/j.chroma.2026.467321
ACS Polym Au. 2026 Aug 12. 6(4): 1134-1146

A Time-Gated Raman Spectroscopy and Mass Spectrometry Comparative Chemical Analysis of Plasticizers.

Julie M Rieland, Yamil Simón-Manso, Adam Zuber, Anthony P Kotula, Kalman B Migler, William E Wallace, Kathryn L Beers.

  Quantitative chemical characterization of postconsumer plastics is challenging because of the large number of polymer chemistries and additives used in formulating plastic products. Plasticizers are an important family of additives used to lower the glass transition temperature and enhance flexibility in polymer formulations. However, their identification and quantification are difficult in recycling and end-of-life processes. This study critically assesses two analytical techniques for their suitability in plasticizer identification: mass spectrometry (high resolution, selectivity, and sensitivity) and time-gated Raman spectroscopy (nondestructive, quicker, and minimal sample preparation). Here, we generate searchable libraries for the identification of 91 common plasticizers using these two techniques, along with the development of reliable tools to measure their spectral similarity. We then analyze the reliability of identifications based on time-gated Raman and/or liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization, explicitly highlighting the advantages and disadvantages of each technique for a given plasticizer chemical family. We have applied these tools to identify and quantify plasticizers in a NIST Standard Reference Material (SRM 2860, Phthalates in Polyvinyl Chloride).

Keywords:  chemometrics; mass spectrometry; plasticizers; polymers; time-gated Raman spectroscopy

DOI:  https://doi.org/10.1021/acspolymersau.6c00065
J Pharm Biomed Anal. 2026 Aug 01. pii: S0731-7085(26)00357-2. [Epub ahead of print]282 117689

Simultaneous determination and pharmacokinetic characterization of 4-methyl-N-ethylcathinone and its metabolites in rat whole blood using a LC-MS/MS method.

Yue Shi, Fangqi Cao, Xin Bai, Xia Ran, Xiaoliang Yuan, Daiwen Zhang, Zhiru Xu, Chaojun Li.

  4-Methyl-N-ethylcathinone (4-MEC) is a novel psychoactive substance and its in vivo exposure characterization remains unknown. In this study, a sensitive and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for the simultaneous quantification of 4-MEC and its two major metabolites in rat whole blood. Then it was applied to pharmacokinetic studies of 4-MEC in rats by intravenous and intragastric administration, characterizing the concentration-time profiles of the parent compound and its metabolites. The results revealed distinct exposure patterns between 4-MEC and its metabolites. Notably, M2 (N-deethylated metabolite) exhibited a prolonged detection window compared with 4-MEC, supporting metabolite-based monitoring strategies and thereby extending its application in forensic toxicology and drug abuse identification.

Keywords:  4-MEC; LC-MS/MS; Pharmacokinetics

DOI:  https://doi.org/10.1016/j.jpba.2026.117689
Leg Med (Tokyo). 2026 Aug 06. pii: S1344-6223(26)00178-1. [Epub ahead of print]85 102950

Detection and quantification of 4F-metomidate and its metabolites, metomidate, in human blood and urine by ultraperformance liquid chromatography with triple quadrupole mass spectrometry (UPLC-MS/MS).

Yinyin Dai, Xiaofei Hao, Jiajun Sun, Amin Wurita, Koutaro Hasegawa.

   PURPOSE: The purpose of this study is to develop and validate a sensitive and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous detection and quantification of 4F-metomidate and its metabolite metomidate in human blood and urine, and to apply this method to authentic specimens obtained from suspected drug abusers.
METHODS: A liquid-liquid extraction method was employed for sample preparation, followed by analysis using UPLC-MS/MS. In addition, in vitro human liver microsomes (HLMs) incubation experiments were conducted to confirm the metabolic conversion of 4F-metomidate to metomidate.
RESULTS: The developed method exhibited excellent linearity (R2 > 0.9989) within the concentration range of 10-1000 pg/mL for both analytes in blood and urine. The LOD and LLOQ were 5 pg/mL and 10 pg/mL, respectively. Both 4F-metomidate and metomidate were detected in all authentic blood and urine samples from suspected abusers. In the seized e-cigarette liquids, only 4F-metomidate was identified, with no detectable metomidate. HLMs incubation experiments confirmed that 4F-metomidate undergoes metabolic conversion to metomidate in vitro.
CONCLUSION: This study presents the first validated UPLC-MS/MS method for the simultaneous quantification of 4F-metomidate and its metabolite metomidate in human blood and urine. The detection of metomidate in biological samples does not necessarily indicate direct intake of metomidate itself, as it may originate from the metabolism of 4F-metomidate or other etomidate analogs. These findings provide a scientific basis for forensic interpretation and highlight the importance of considering metabolic conversion in drug abuse cases.

Keywords:  4F-metomidate; Human blood and urine; Metabolite; Metomidate; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.legalmed.2026.102950
J Anal Toxicol. 2026 Aug 13. pii: bkag074. [Epub ahead of print]

LC-MS/MS Quantification of Five Antihistamines in Antemortem Blood: Method Validation and Application.

Kenson Jean, Kristin W Kahl, Lisa J Reidy.

  First-generation H1 antihistamines have central nervous system penetration and anticholinergic activity, with potential side effects including sedation, cognitive impairment, anterograde amnesia. First-generation antihistamines pose risks in driving under the influence of drugs (DUID) and drug-facilitated crimes (DFC) forensic casework. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for five first-generation H1 antihistamines: chlorpheniramine, diphenhydramine, doxylamine, hydroxyzine, and promethazine. The calibration range was 1-200 ng/mL with a 1/x linear calibration model for quantitative compounds, and the limit of detection was determined to be 0.5 ng/mL for all compounds. Bias and precision were acceptable, with minor but acceptable suppression observed at low and high concentrations. No exogenous or endogenous interferences were observed. Carryover was not detected after a 400 ng/mL sample. Processed samples were determined to be stable 24 hours after initial injection when stored in the refrigerated multi-sampler. Authentic forensic specimens from DUID and DFC investigations showed a wide range of concentrations for diphenhydramine. Selected case reports are presented to demonstrate the applicability of the method. The high percentage of polysubstance cases, range of concentrations, and delayed sample collection in DFC cases underscore the importance of a sensitive and reproducible method for these first-generation antihistamines, especially in human-performance casework.

Keywords:  Antihistamines; driving under the influence; drug facilitated crimes; human-performance; method validation

DOI:  https://doi.org/10.1093/jat/bkag074
Anal Chem. 2026 Aug 11. 98(31): 23084-23095

Programmable One-Tube Sialic Acid Derivatization Enables Dual-Mode N-Glycomics of Low-Input Samples.

Jihong Lu, Nafisa Tursumamat, Shengyang Liu, Qiannan Liu, Heming Li, Xiaopeng Su, Shuye Wu, Shuhong Guo, Xingyu Shen, Wenjie Peng, Ping Wang, Juan Wei.

Low-input glycomics remains particularly challenging for sialylated N-glycans because rigorous linkage-specific derivatization often increases sample handling, disperses signal across multiple linkage isomers, and compromises sensitivity. Here, we report a programmable one-tube workflow that integrates cell lysis, N-glycan release, sialic acid derivatization, and purification in a single microcentrifuge tube for highly sensitive N-glycome profiling. By controlling ammonia activity and reaction time after ethyl esterification, the workflow enables optional sialic acid linkage-specific, linkage-nonspecific, and sequential dual-mode analyses on the same low-input sample. On a standard mass spectrometry platform, the method demonstrated direct N-glycomic profiling from nanogram-scale glycoprotein inputs, nanoliter-scale human serum, and only a few thousand primary T cells. Applied to splenic B cells from an LPS-induced inflammatory model, a marked remodeling of the N-glycome from high-mannose species toward sialylated complex glycans was revealed, while maintaining an unchanged α2,3/α2,6 overall ratio. These results establish a chemistry-programmable route to dual-mode N-glycomics for low-input samples and expand the practical scope of linkage-resolved glycomic analysis in mass-limited biospecimens.

DOI: https://doi.org/10.1021/acs.analchem.6c02757