bims-metlip 2026-07-05 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–07–05
25 papers selected by
Sofia Costa, Matterworks

[Rapid determination of chlorfenapyr and its metabolites in plasma by high performance liquid chromatography-tandem mass spectrometry].
Simultaneous Determination of Levetiracetam, Sodium Valproate, and Olanzapine in Human Plasma by UPLC-MS/MS for Therapeutic Drug Monitoring.
Simultaneous Determination of Dechlorane-Related Compounds in Fish Muscle by Microwave-Assisted Extraction Combined With Enhanced Matrix Removal for Lipids Cleanup and Gas Chromatography-Tandem Mass Spectrometry.
[Development and Application of a Method for Determining 33 Aromatic Primary Amines in Medical-Grade Thermoplastic Polyurethane by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry].
Determination of the new psychoactive substances MDMB-4en-PINACA, ADB-BUTINACA and some of their metabolites in blood and urine using DLLE-LC-MS/MS: application to real forensic case samples.
A robust and validated method for the determination of 21 urinary metabolites of 15 plasticizers, including phthalates, DEHTP, and DINCH, by online SPE and liquid chromatography-tandem mass spectrometry.
Large Language Model-Generated Dietary Metabolite Biomarker Database Drives Deep Annotation of the Human Diet Metabolome.
The MassBank contributions of the mFam collaboration.
LC-MS/MS-Based Monitoring of Pregabalin and Gabapentin Reveals Marked Pharmacokinetic Variability and Limited Exposure-Response Relationships in Neuropathic Pain Patients.
Single-Cell Mass Spectrometry Imaging Using Desorption Electrospray Ionization Coupled To Orbitrap Mass Spectrometers.
Comparison of Information-Dependent Acquisition and Sequential Window Acquisition of All Theoretical Mass Spectra for Untargeted Drug Testing on a Linear Ion Trap-Pulsing Quadrupole-Time of Flight Mass Spectrometer.
A Robotic Sample Handling Platform for Fully Automated Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging.
Plasma formiminoglutamic acid shows potential as a functional marker of folate deficiency in cats and dogs: development and validation of a liquid chromatography-tandem mass spectrometry method.
An Efficient and Rapid HPLC-QQQ-MS Method for the Quantitation of Tropane Alkaloids in Medicinal Plants.
Metabolite Fraction Libraries for Quantitative NMR Metabolomics.
Single-cell mass spectrometry imaging: platform advances for multimodal spatial omics.
Recent advancements in sample pretreatment methods for glycoproteomics.
High-sensitive glycomics using seGRAP-mass spectrometry uncovers a conserved N-glycome in single human oocytes.
From tumors to neurons: mass spectrometry imaging in spatial lipidomics.
A single bioanalytical LC-MS/MS method for the quantification of pelitinib across biological matrices: An insight into in vitro metabolic stability, in vivo pharmacokinetics, and IVIVE.
MALDI Tandem Mass Spectrometry for Colony-Based Dereplication of Natural Products.
A simple, sensitive microsample LC-MS assay for quercetin and isorhamnetin in mouse and human plasma: application to EMIQ treatment in myotonic dystrophy type 1.
Simultaneous determination of 22 carbamate pesticides and their metabolites in biological samples using ultrasound-assisted dispersive liquid-liquid microextraction combined with LC-MS/MS: application to forensic cases.
Agentic AI for Structural Elucidation and Discovery of Drug Metabolites from Mass Spectrometry Data.
Chromatographic Method Validation for Simultaneous Determination of Bergenin and Alpha-Lipoic Acid in Skin Penetration Studies.

Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2026 Jun 20. 44(6): 490-493

[Rapid determination of chlorfenapyr and its metabolites in plasma by high performance liquid chromatography-tandem mass spectrometry].

B Zhou, H L Li, Y D Zhang, J Ma, F Dong.

  Objective: To establish a rapid method for the determination of chlorfenapyr and its metabolite tralopyril in human plasma by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) . Methods: In January 2025, after human plasma samples were pretreated by acetonitrile protein precipitation combined with liquid-liquid extraction, separation was performed using a Kinetex EVO C18 chromatographic column. Methanol-water was used as the mobile phase with isocratic elution at 75% B. Electrospray ionization in negative ion mode (ESI(-)) and multiple reaction monitoring (MRM) mode were adopted for detection, and the matrix-matched external standard method was applied for quantification. A comprehensive methodological validation was completed in accordance with the Guidelines for the Validation of Quantitative Analytical Methods for Biological Samples. Results: Chlorfenapyr in human plasma showed good linearity in the concentration range of 2-400 μg/L, with a correlation coefficient (r) of 0.99954. The average spiked recoveries ranged from 98.3%-112.8%, with both intra-batch and inter-batch relative standard deviations (RSD) below 10%. The limit of detection (LOD) was 0.67 μg/L, and the lower limit of quantification (LOQ) was 2.0 μg/L. For tralopyril, good linearity was observed in the range of 0.1-20.0 μg/L, with a correlation coefficient (r) of 0.99979. The average spiked recoveries ranged from 98.4%-113.0%, with both intra-batch and inter-batch RSD below 10%. The LOD was 0.003 μg/L, and the LOQ was 0.010 μg/L. Conclusion: This method features simple pretreatment, rapid analysis, high sensitivity, and satisfactory precision and accuracy. It meets the requirements for rapid quantitative detection of chlorfenapyr and tralopyril in plasma of clinically poisoned patients.

Keywords:  Chlorfenapyr; High performance liquid chromatography-tandem mass spectrometry; Plasma; Poisoning; Quantitative analysis; tralopyril

DOI:  https://doi.org/10.3760/cma.j.cn121094-20250329-00120
Biomed Chromatogr. 2026 Aug;40(8): e70536

Simultaneous Determination of Levetiracetam, Sodium Valproate, and Olanzapine in Human Plasma by UPLC-MS/MS for Therapeutic Drug Monitoring.

Feng Zhou, Xinjun Cai, Chongyang Wu.

  Levetiracetam, sodium valproate, and olanzapine are widely used for epilepsy, but their therapeutic windows and pharmacokinetic variability require TDM. No existing method enables their simultaneous quantification. This study aimed to establish and validate a UPLC-MS/MS method for their simultaneous detection in human plasma. Plasma samples were processed by protein precipitation. Chromatographic separation was achieved with gradient elution (3.0 min total time), and MRM mode was used for detection. The method showed good linearity (R ≥ 0.9956), precision (CV ≤ 7.27%), and accuracy (bias: -8.95% ~ 9.17%). Extraction recoveries were 94.56%-103.98%, with negligible matrix effects. Application to 136 patients showed average concentrations within therapeutic ranges. This UPLC-MS/MS method is rapid, accurate, and suitable for routine TDM of the three drugs, providing a reliable tool for individualized pharmacotherapy.

Keywords:  UPLC‐MS/MS; antiepileptic drugs; human plasma; simultaneous quantification; therapeutic drug monitoring

DOI:  https://doi.org/10.1002/bmc.70536
J Sep Sci. 2026 Jul;49(7): e70480

Simultaneous Determination of Dechlorane-Related Compounds in Fish Muscle by Microwave-Assisted Extraction Combined With Enhanced Matrix Removal for Lipids Cleanup and Gas Chromatography-Tandem Mass Spectrometry.

Shuai Zhu, Ka Huang, Wei Pang.

  A robust and efficient analytical method was developed for the simultaneous determination of eight dechlorane-related compounds in fish muscle using microwave-assisted extraction combined with enhanced matrix removal for lipids cleanup and gas chromatography-tandem mass spectrometry. The method enables effective extraction of hydrophobic analytes from lipid-rich matrices and efficient removal of co-extracted lipids, resulting in improved analytical performance in terms of recovery and reproducibility. Under optimized conditions, the method exhibited good linearity (R2 = 0.9976-0.9997), low detection limits (method detection limits: 0.021-0.078 ng/g; limits of quantification: 0.067-0.248 ng/g), and satisfactory recoveries (77.6%-90.1%) with satisfactory precision (relative standard deviations: 2.9%-8.5%). Matrix effects were effectively controlled using matrix-matched calibration combined with isotope-labeled internal standards. The method was successfully applied to real fish samples, demonstrating reliable quantification of trace-level dechloranes. Owing to its simplified workflow and improved throughput, the proposed method provides a practical and reliable approach for the routine analysis of dechloranes in high-lipid biological matrices.

Keywords:  dechloranes; lipid‐rich matrices; matrix effects; sample cleanup

DOI:  https://doi.org/10.1002/jssc.70480
Sichuan Da Xue Xue Bao Yi Xue Ban. 2026 May 20. 57(3): 835-844

[Development and Application of a Method for Determining 33 Aromatic Primary Amines in Medical-Grade Thermoplastic Polyurethane by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry].

Yajing Yang, Jian Yang, Yu Xiao, Li Hu, Jie Cheng, Yanyan Guo, Yan Jiang, Daiguo Zhao.

   Objective: To develop an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of 33 aromatic primary amines in medical-grade thermoplastic polyurethane (TPU).
Methods: The target compounds were extracted using 80% methanol and PBS solution containing 10% ethanol as the solvents. The 80% methanol extract was filtered through and diluted to one-fifth of the original concentration. The PBS extract was processed through an HLB solid-phase extraction column (3 mL, 60 mg, methanol and water equilibrium), eluted with 5 mL methanol, filtered, and diluted to 5 mL with methanol. Then, analysis was performed using UPLC-MS/MS. Chromatographic separation was performed using an Agilent InfinityLab Poroshell 120 EC-C18 column (150 mm × 3.0 mm, 2.7 μm) with methanol (A) and deionized water containing 0.1% formic acid (B) as the mobile phases. Gradient elution was conducted with a column temperature of 40 ℃, a flow rate of 0.2 mL/min, and an injection volume of 1 μL. Mass spectrometry analysis was performed using an electro-spray ionization (ESI) source in multiple reaction monitoring (MRM) positive ion mode. The standard curves, quantification limits, precision, recovery rates, and matrix effects of the method under different extraction conditions were evaluated.
Results: The 33 aromatic primary amines analyzed in the study showed good linearity (r > 0.998) within the 2-100 ng/mL concentration range. Detection limits ranged from 0.1 to 0.5 ng/mL, and quantification limits ranged from 0.3 to 1.7 ng/mL. Recovery rates for the 33 aromatic primary amines in 80% methanol samples ranged from 70.7% to 116.0%. Recovery rates for 31 aromatic primary amines in PBS samples containing 10% ethanol ranged from 74.9% to 113.8%. The intra-day and inter-day precision for different samples was both less than 10%, indicating good sample stability. Analysis of aromatic primary amine content in sample extracts using this method revealed that methylenedianiline (MDA) was detected in all 6 initially tested samples. Under simulated extraction at 37 ℃ and accelerated extraction at 60 ℃, 3 samples exhibited continuous or delayed MDA release. 2,4'-MDA was detected in 2 samples during the initial limit extraction phase and remained detectable in 1 sample under 60 ℃ accelerated aging conditions, but no release was observed under the 37 ℃ simulated physiological environment. 2,2'-MDA was detected only in two samples during initial limit extraction and was not released in subsequent simulated or accelerated extraction processes.
Conclusion: The UPLC-MS/MS method demonstrates high sensitivity and accuracy, meeting the requirements for precise detection of 33 aromatic primary amines in medical-grade TPU and providing reliable technical support for risk assessment and quality control of aromatic primary amines in these materials and products.

Keywords:  Aromatic primary amines; Extraction and release; Medical-grade thermoplastic polyurethane; Risk assessment; Simultaneous determination; Ultra-high performance liquid chromatography-tandem mass spectrometry

DOI:  https://doi.org/10.12182/20260560401
Forensic Sci Int. 2026 Jun 25. pii: S0379-0738(26)00246-X. [Epub ahead of print]387 113059

Determination of the new psychoactive substances MDMB-4en-PINACA, ADB-BUTINACA and some of their metabolites in blood and urine using DLLE-LC-MS/MS: application to real forensic case samples.

Osman Can Çağılcı, Ümmühan Turgut Ocak, Miraç Ocak, Zeynep Sıla Gümüşcü.

  MDMB-4en-PINACA and ADB-BUTINACA are indazole-derived synthetic cannabinoids that have increasingly been encountered in forensic toxicology, partly due to their potent cannabinoid receptor activity and their association with severe intoxication cases involving agitation, hallucinations, paranoia, and psychosis-like symptoms. In this study, a simplified and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method incorporating a simple and rapid sample preparation based on methanol-assisted protein precipitation followed by dispersive liquid-liquid extraction (DLLE) was developed and validated for the simultaneous determination of MDMB-4en-PINACA, ADB-BUTINACA, and their major metabolites, MDMB-4en-PINACA butanoic acid and ADB-BUTINACA N-butanoic acid, in blood and urine. The method provided effective separation and quantification of all analytes over a linear range of 1-100 ng/mL, with limits of detection between 0.51 and 1.20 ng/mL. No significant matrix interferences were observed, confirming adequate selectivity. The validated method was successfully applied to real forensic case samples, in which the inclusion of metabolite monitoring improved the confirmation of exposure particularly in cases where parent compounds were present at low concentrations or were no longer detectable. The results demonstrate that the proposed DLLE-LC-MS/MS method is a simple, robust, and reliable approach for the simultaneous determination of these synthetic cannabinoids and their metabolites in routine forensic toxicology, especially in investigations involving suspected synthetic cannabinoid-related neuropsychiatric manifestations such as acute psychosis.

Keywords:  ADB-BUTINACA; Dispersive liquid–liquid extraction; Forensic toxicology; LC–MS/MS; MDMB-4en-PINACA; Metabolites; Synthetic cannabinoids

DOI:  https://doi.org/10.1016/j.forsciint.2026.113059
Anal Bioanal Chem. 2026 Jul 04.

A robust and validated method for the determination of 21 urinary metabolites of 15 plasticizers, including phthalates, DEHTP, and DINCH, by online SPE and liquid chromatography-tandem mass spectrometry.

Rosa Mercadante, Simone Cafagna, Marco Rizzo, Elisa Roggia, Silvia Fustinoni.

  Phthalates and their alternative plasticizers terephthalates and hexahydrophthalates are widely used in consumer products, leading to continuous human exposure and potential risk for health. The aim of this study was to develop an analytical assay for the quantification of urinary metabolites of these plasticizers as biomarkers of exposure. Twenty-one urinary metabolites from 15 plasticizers, including 13 phthalates, one terephthalate (di-2-ethylhexyl terephthalate, DEHTP), and one hexahydrophthalate (di-isononyl cyclohexane-1,2-dicarboxylate, DINCH), were deconjugated by enzymatic hydrolysis, separated, and quantified using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) in the presence of 17 isotopically labeled internal standards. A full validation, according to international guidelines, was performed and an external proficiency test was undergone. Forty-six urine samples collected from adults belonging to the Italian general population were assayed to test the method. The analytes, after enzymatic deconjugation, were enriched by online solid phase extraction (SPE); the interferences from contaminants in the mobile phase were delayed with pre-analytical columns and the chromatography was successfully separating all analytes. The assay was precise (% relative standard deviation within 15.6%), accurate (% relative to theoretical value between 96.5 and 108.5%) and sensitive, with lower limits of quantification (LLOQ) in the range 0.10 to 0.78 µg/L. Biases arising from matrix effects and long-term storage had a negligible impact on accuracy, with recoveries remaining within 84-111%. The external proficiency exercise was successful across multiple rounds. In the study sample, the most abundant analytes were monoethyl phthalate (MEP), mono(2-ethyl-5-carboxypentyl) terephthalate (5-cx-MEPTP), monobutyl phthalate (MnBP) and monoisobutyl phthalate (MiBP) (38, 11, 8, and 8 µg/L), metabolites of diethyl phthalate (DEP), DEHTP, di-n-butyl phthalate (DnBP), and di-isobutyl phthalate (DiBP), respectively. The developed method demonstrates good performances, with accurate results and high throughput; it is well-suited for large-scale biomonitoring studies in the general population.

Keywords:  Biological monitoring; DINCH; LC-MS/MS; Metabolites of phthalates; Terephthalate; Urine

DOI:  https://doi.org/10.1007/s00216-026-06634-8
Anal Chem. 2026 Jul 01.

Large Language Model-Generated Dietary Metabolite Biomarker Database Drives Deep Annotation of the Human Diet Metabolome.

Zijun Nie, Fujian Zheng, Dejun Hu, Zhenzhen Fu, Chongjiang Cao.

The annotation of dietary biomarkers is crucial for nutritional epidemiology. While untargeted liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is a powerful analytical approach, the annotation of dietary biomarkers is hampered by the low specificity of existing public databases, which limits annotation coverage and accuracy. To address this limitation, we developed a novel database construction strategy and a dual-annotation workflow. We first employed an automated, large language model (LLM)-based text-mining pipeline to parse 7339 scientific articles and supplementary materials, creating the Dietary Metabolite Biomarker Database (DMBDB), which contains 4983 nonredundant biomarkers. The LLMs workflow demonstrated high performance, achieving an F1 score of 0.9269 for biomarker name recognition. Subsequently, two complementary annotation strategies were designed: (i) a specialized LC-MS database derived from DMBDB, incorporating predicted retention times and experimental MS/MS spectra for high-confidence matching, and (ii) a structure-guided molecular networking strategy (SGMNS) that uses DMBDB as background knowledge to annotate dietary biomarkers and their metabolites lacking spectral evidence. The framework was validated using untargeted LC-HRMS analysis of urine samples. LC-MS database directly annotated 566 metabolites, and the integration with SGMNS expanded the total number of annotations to 2078. The LLM-driven database construction combined with the dual-strategy annotation framework provides a powerful paradigm for achieving high-coverage and high-accuracy dietary metabolomics.

DOI: https://doi.org/10.1021/acs.analchem.6c01612
Metabolomics. 2026 Jul 02. pii: 114. [Epub ahead of print]22(4):

The MassBank contributions of the mFam collaboration.

Anusha Ahlendorf, Asaph Aharoni, Khabat Vahabi, Tillmann G Fischer, Pierre-Marie Allard, Megan Augustin, Ulschan Bathe, David I Broadhurst, Corey Broeckling, Joerg Buescher, Adrian Covaci, Katyeny Manuela da Silva, Ric C H de Vos, Micha Gracianna Devi, Stefanie Döll, Maximilian Frey, Andrej Frolov, Emmanuel Gaquerel, Vasuk Gautam, Esteban Charria-Girón, Alain Goossens, Jeremy Grosjean, Maria Halabalaki, Elias Iturrospe, Kim Kultima, Stephanie Herman, Toni M Kutchan, Romain Larbat, René Meier, Eleni V Mikropoulou, Gregory Mouille, Luca Nicolotti, Nir Shahaf, François Perreau, Pierre Pétriacq, Michael Reichelt, Stacey N Reinke, Rani Robeyns, Alena Soboleva, Otmar Spring, Akshai Parakkal Sreenivasan, Alain Tissier, Jean Chrisologue Totozafy, Hiroshi Tsugawa, Josep Valls-Fonayet, Maria van de Lavoir, Justin J J van der Hooft, Fredd Vergara, David Wishart, Ludger A Wessjohann, Jean-Luc Wolfender, Jörg Ziegler, Gerd Ulrich Balcke, Steffen Neumann.

   INTRODUCTION: The analysis of metabolic profiles using high resolution mass spectrometry (MS) data provides deep insights into biological processes. In metabolomics, MS analysis generates a large number of features that represent metabolites. However, identifying specific metabolites from these features can be challenging. One of the major bottlenecks in the metabolomics field is the identification of MS features, which is a prerequisite for any biochemical interpretation. By identifying similarities and differences within a metabolite family (mFam), evaluating MS features at the metabolite family level can help assigning functional roles to individual MS features. These data can help interpreting metabolic pathways and processes within a biological system. For the assignment of metabolite families to MS features, it is important to have good quality, reliable, and comprehensive spectral libraries.
OBJECTIVE: We initiated a global effort to collect high-resolution MS/MS spectra of metabolites from labs working in different fields, including metabolomics of animals, microorganisms, and plants. The mFam-MS/MS collection delivers valuable training data to assign machine-readable classified information on the unknown metabolites.
RESULTS: The mFam collaboration used a standardized metadata template and has developed a globally curated MS/MS spectral library of 7,872 spectra with 2,126 unique metabolites. This library was compiled from 47 datasets contributed by 25 laboratories measured on 12 instrument types, including QTOF, Orbitrap, and Ion Mobility-QTOF systems. It comprises 4,646 spectra in positive mode and 3,226 in negative mode. This standardized resource significantly enhances metabolite identification capabilities, supports the development of machine learning-based annotation tools, and accelerates the discovery of novel metabolites. All spectra are available under the collective contributor label mFam in the MassBank system, including the web interface and the 2025.10 data release available at GitHub and Zenodo.

Keywords:  Data processing; FAIR data; Metabolomics; Open science; Reference spectra; Spectral libraries; Tandem mass spectrometry

DOI:  https://doi.org/10.1007/s11306-026-02480-y
Biomed Chromatogr. 2026 Aug;40(8): e70539

LC-MS/MS-Based Monitoring of Pregabalin and Gabapentin Reveals Marked Pharmacokinetic Variability and Limited Exposure-Response Relationships in Neuropathic Pain Patients.

Duygu Eryavuz Onmaz, Fettah Eren, Kadir Gulsen, Firdevs Sak, Mohammad Ahmad Bik, Rabia Coban, Sedat Abusoglu, Ali Unlu.

  This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantification of pregabalin (PGB) and gabapentin (GBP) and to explore real-world pharmacokinetic variability. A bioanalytical LC-MS/MS method was validated using serum samples prepared by acetonitrile protein precipitation. Chromatographic separation was achieved on a C18 column with gradient elution and positive electrospray ionization. The validated method was applied to serum samples obtained from 65 patients, and associations between serum drug concentrations and clinical measures were evaluated. The assay demonstrated excellent linearity (R² > 0.998), acceptable precision and accuracy, recovery ranging from 96.9% to 101.5%, and matrix effects below 7%. Considerable inter-individual variability in serum concentrations was observed (PGB: median 700 ng/mL [IQR: 500-1000]; GBP: median 4500 ng/mL [IQR: 3200-5700]). No significant dose-concentration relationships were identified (PGB: ρ = 0.03; GBP: ρ = -0.08), and no significant associations were observed between serum concentrations and the clinical outcome measures assessed. In contrast, both drugs showed moderate inverse correlations with post-dose sampling interval (PGB: ρ = -0.46; GBP: ρ = -0.45). These findings indicate substantial pharmacokinetic variability of gabapentinoids under routine clinical conditions. Although no clear dose-concentration or exposure-response relationships were identified in this cohort, interpretation should be made cautiously because of non-standardized sampling conditions and the relatively narrow range of renal function among participants.

Keywords:  LC–MS/MS; bioanalytical method validation; gabapentin; pregabalin; therapeutic drug monitoring

DOI:  https://doi.org/10.1002/bmc.70539
J Vis Exp. 2026 Jun 12.

Single-Cell Mass Spectrometry Imaging Using Desorption Electrospray Ionization Coupled To Orbitrap Mass Spectrometers.

Nathan Colwell, Dan Chen, Deepti Bhusal, Zongkai Peng, Zhibo Yang.

Desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) enables ambient, matrix-free molecular imaging with minimal sample preparation. This protocol describes a workflow for single-cell MSI of OVCAR-8 cells using DESI coupled to Orbitrap mass spectrometers. The system integrates a DESI sprayer, a modified mass spectrometer interface, a motorized XYZ stage, and an optical breadboard for high-spatial-resolution imaging. Cells are cultured on gridded glass coverslips, washed with ammonium formate solution to reduce salt interference, dried, and analyzed under ambient conditions. Solvent is delivered continuously using a nano-liquid chromatography system, and nebulizing nitrogen gas is regulated to maintain spray stability. The protocol also describes raster imaging, pixel control, and optical-to-mass spectrometry image registration for single-cell localization. High mass resolution and mass accuracy provided by Orbitrap detection enable differentiation of closely related molecular species and support single-cell metabolomic analysis. This protocol provides an adaptable strategy for implementing high-spatial-resolution single-cell MSI using multiple Orbitrap mass spectrometer platforms.

DOI: https://doi.org/10.3791/71691
Clin Chem. 2026 Jul 01. pii: hvag079. [Epub ahead of print]

Comparison of Information-Dependent Acquisition and Sequential Window Acquisition of All Theoretical Mass Spectra for Untargeted Drug Testing on a Linear Ion Trap-Pulsing Quadrupole-Time of Flight Mass Spectrometer.

Morgan W Mann, Lilly Lim, Ruben Y Luo, Kara L Lynch.

BACKGROUND: Comprehensive drug testing (CDT) by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is a valuable method for evaluating clinical samples for unknown toxicological agents. However, the sensitivity of CDT methods is generally lower than targeted approaches. A novel MS technology, linear ion trap (LIT)-pulsing, has demonstrated improved sensitivity in proteomics and metabolomics applications, but its utility and interactions with different HRMS acquisition types, such as information-dependent acquisition (IDA) and sequential window acquisition of all theoretical fragment ion spectra (SWATH), have not been explored in small molecule analysis.
METHODS: CDT methods utilizing both IDA and SWATH acquisition were developed on a quadrupole time-of-flight (QTOF) instrument featuring LIT pulsing. Limits of detection (LODs) and process efficiencies were estimated for 150 toxicological agents. Additionally, 101 patient urine specimens were used to compare both methods against an established clinical CDT without LIT-pulsing. Selectivity was assessed using a targeted LC-HRMS method.
RESULTS: LIT-pulsed SWATH acquisition improved detection of toxicological agents compared to LIT-pulsed IDA, with a median 5-fold reduction to measured LODs and an average 5.9% reduction to matrix suppression. In authentic patient samples, LIT-pulsed IDA and SWATH acquisition identified 889 and 1083 toxicological agents compared to 835 using IDA without LIT-pulsing. The estimated selectivity of LIT-pulsed IDA was improved compared to conventional IDA acquisition, but SWATH acquisition demonstrated significant nonselectivity.
CONCLUSION: LIT-pulsing improves the sensitivity and selectivity of CDT methods. The largest improvements to sensitivity are observed using SWATH acquisition; however, these gains are accompanied by reduced selectivity. This highlights the need for careful validation of SWATH CDT methods to ensure high clinical performance.

DOI: https://doi.org/10.1093/clinchem/hvag079
J Am Soc Mass Spectrom. 2026 Jul 02.

A Robotic Sample Handling Platform for Fully Automated Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging.

Li-Xue Jiang, Emerson Hernly, Benjamin W Cerjan, Mushfeqa Iqfath, Tommy Zhang, Julia Laskin.

  Recent advances in mass spectrometry imaging (MSI) have enabled high-throughput experiments, making it possible to image multiple samples and perform three-dimensional molecular mapping. For these applications, automated sample handling is critical. Here, we present the first fully automated nanospray desorption electrospray ionization (nano-DESI) MSI platform that integrates robotic sample handling with automated sample recognition system. A robotic arm, controlled by our custom LabVIEW program, automatically places samples onto a sample holder and removes them after the analysis. The sample holder, mounted on a motorized XYZ stage, is automatically transferred to a mass spectrometer for imaging. A newly developed on-site sample region recognition system enables on-the-fly identification of sample coordinates and transfers them to LabVIEW, which plans and executes the imaging experiment. A microfluidic nano-DESI probe (MFP) was used due to its robustness and high-throughput capability. Shear force feedback distance control was incorporated into the MFP to address height variations across multiple glass slides. Using this system, five mouse brain tissue sections on two glass slides were imaged sequentially with high reproducibility and consistency, without human intervention. These results demonstrate the feasibility and performance of a fully automated nano-DESI MSI workflow, paving the way for multisample and three-dimensional MSI experiments.

Keywords:  3D mass spectrometry imaging (MSI); automation; microfluidic probe; nanospray desorption electrospray ionization (nano-DESI); robotic arm

DOI:  https://doi.org/10.1021/jasms.6c00089
Am J Vet Res. 2026 Jul 01. 1-9

Plasma formiminoglutamic acid shows potential as a functional marker of folate deficiency in cats and dogs: development and validation of a liquid chromatography-tandem mass spectrometry method.

Margaret Cohn-Urbach, Craig G Ruaux.

   Objective: To evaluate formiminoglutamic acid (FIGLU) as a potential marker of folate deficiency in cats and dogs by (1) establishing a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the quantification of FIGLU in feline and canine plasma, and (2) reporting plasma FIGLU concentrations in clinically normal cats and dogs.
Methods: An LC-MS-MS method for FIGLU quantitation was developed and analytically validated. Clinically normal cats and dogs were prospectively included for plasma FIGLU and serum folate measurement (via LC-MS-MS and chemiluminescence immunoassay, respectively). Statistical analysis was performed to assess for correlations between plasma FIGLU and serum folate concentrations.
Results: Accuracy, precision, linearity, and recovery experiments met validation requirements. Dilution integrity experiments did not meet the acceptance criteria, potentially due to ion suppression effects. Plasma FIGLU ranged from 4.5 to 21.0 ng/mL in 11 clinically normal dogs and 9.1 to 57.0 ng/mL in 10 clinically normal cats. There was no evidence of a correlation between serum folate and plasma FIGLU concentrations in clinically normal cats and dogs.
Conclusions: The amount of FIGLU present in the plasma of clinically normal cats and dogs is quantifiable via LC-MS-MS, and reference interval determination of plasma FIGLU is therefore feasible. Future studies should include both normofolatemic and hypofolatemic animals to assess the correlation between serum folate and plasma FIGLU and to determine the optimal serum folate cutoff limit.
Clinical Relevance: Plasma FIGLU shows promise as a practical research tool and a potential functional marker of folate availability in cats and dogs.

Keywords:  LC-MS-MS; analytical validation; folate deficiency; formiminoglutamic acid; functional marker

DOI:  https://doi.org/10.2460/ajvr.26.03.0112
J Vis Exp. 2026 Jun 12.

An Efficient and Rapid HPLC-QQQ-MS Method for the Quantitation of Tropane Alkaloids in Medicinal Plants.

Zihan Li, Zhiqi Liu, Liqi Feng, Qi Zheng, Tao Zhou, Binjie Xu, Yuntong Ma, Yuan Pan.

Anisodus tanguticus is an important Tibetan medicinal plant. The tropane alkaloids (TAs), such as hyoscyamine, anisodamine, and scopolamine, exhibit significant analgesic and sedative pharmacological activities, making this species a valuable natural source for extracting these bioactive compounds. However, the chemical composition of A. tanguticus is highly complex. It includes a large number of compounds without elucidated structures, rendering systematic identification and structural characterization of its active constituents particularly important. Although high-performance liquid chromatography (HPLC) has been widely used for the separation of extracts from this plant, many unknown components remain challenging to accurately identify, even with spectral database matching. This study established an analytical method based on high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC-QQQ-MS) for the determination of tropane alkaloids in A. tanguticus. The method encompasses a series of systematic procedures, including standardized sample preparation, mass spectrometric parameter configuration, LC pre-equilibration, method establishment, acquisition of MS data, multistage mass spectrometry (MSⁿ) scanning, and manual data interpretation. Using this strategy, we successfully identified three representative tropane alkaloids in A. tanguticus: hyoscyamine, anisodamine, and scopolamine. Additionally, it validates the precision of the established HPLC-QQQ-MS method, confirming that the method is rapid, sensitive, feasible, and accurate for the quantitative determination of tropane alkaloids (TAs) in A. tanguticus. The established approach demonstrates good versatility and reliability, and is suitable for the high-throughput identification and quantification of tropane alkaloids in A. tanguticus and other medicinal plants.

DOI: https://doi.org/10.3791/70510
Anal Chem. 2026 Jul 01.

Metabolite Fraction Libraries for Quantitative NMR Metabolomics.

Christopher Esselman, Kara Garrison, Leandro Ponce, Ricardo M Borges, Frank Delaglio, Arthur S Edison.

Nuclear magnetic resonance (NMR) has unique strengths in metabolomics studies, particularly in quantifying mixtures and elucidating the structures of unknown molecules. One-dimensional (1D) proton (1H) NMR is the most common method; however, spectral overlap is significant, making analysis challenging. We present a new approach that utilizes chromatographically separated fractions from a pooled sample, henceforth called a metabolite fraction library (mFL). We developed an algorithm to extract highly correlated peaks from the mFL, collectively forming a metabolite basis set (mBS). The mBS can be fit to NMR profiling data, enabling comprehensive quantification. Applied to 10 mixtures of 53 metabolites, our approach accurately quantified 50 metabolites, quantified one impurity and one oxidation product, and described between 91 and 96% of the total spectral intensity. The method is demonstrated using the fungus Neurospora crassa, resulting in the identification of 45 metabolites with high confidence and 45 with medium confidence, accounting for 94% of the total spectral intensity.

DOI: https://doi.org/10.1021/acs.analchem.6c01279
Anal Bioanal Chem. 2026 Jul 02.

Single-cell mass spectrometry imaging: platform advances for multimodal spatial omics.

Lauren E Hill, Lyndsay E A Young, James W Dressman, Peggi M Angel, Anand S Mehta, Richard R Drake.

  Spatial omics has transformed biomedical research by uncovering the molecular characterization of biological systems while preserving spatial context. Among these approaches, mass spectrometry imaging (MSI) provides a label-free, in situ visualization of diverse molecular classes, including metabolites, lipids, proteins, and glycans. Recent advances in instrumentation, sample preparation, and data acquisition have pushed MSI into the field of single-cell analysis, providing unprecedented access to cellular heterogeneity and molecular states across biological contexts. Here, we review current single-cell MSI platforms and highlight key innovations that have improved spatial resolution, sensitivity, and throughput. Presented examples from published workflows highlight the variability in strategies for cell isolation, capture, and data acquisition. The three main ionization techniques of desorption electrospray ionization (DESI), secondary ion mass spectrometry (SIMS), and matrix-assisted laser desorption ionization (MALDI) are highlighted for their capabilities to generate robust single-cell multi-omics profiling. We outline future directions for the field and the potential of single-cell MSI to impact translational spatial omic research and precision medicine.

Keywords:  MALDI-MSI; Mass spectrometry imaging; Single-cell imaging; Spatial omics

DOI:  https://doi.org/10.1007/s00216-026-06644-6
Biophys Rep. 2026 Jun 30. 12(3): 175-192

Recent advancements in sample pretreatment methods for glycoproteomics.

Wei Zhang, Siyuan Kong, Weiqian Cao.

  Comprehensive glycoprotein analysis is essential for exploring the role of protein glycosylation in diverse biological processes and disease mechanisms. Yet it remains challenging due to the structural complexity and heterogeneity of glycans. Bottom-up glycoproteomics utilizing liquid chromatography-mass spectrometry (LC-MS)-based techniques has emerged as a powerful tool for in-depth protein glycosylation analysis. Sample pretreatment is the first and critical step that significantly influences subsequent chromatographic separation and MS analysis. This review provides an overview of the key steps in current sample pretreatment workflows for glycoproteomics, emphasizing recent advancements in sample preparation and enrichment strategies developed over the past decade. It highlights improvements in enrichment efficiency, compatibility with high-throughput analyses, and applications to biological samples, and also discusses the remaining challenges and future directions for these technologies.

Keywords:  Enrichment strategies; Glycoproteomics; High-throughput analysis; Sample pretreatment

DOI:  https://doi.org/10.52601/bpr.2025.240072
Nat Commun. 2026 Jul 03. pii: 5836. [Epub ahead of print]17(1):

High-sensitive glycomics using seGRAP-mass spectrometry uncovers a conserved N-glycome in single human oocytes.

Nafisa Tursumamat, Qiannan Liu, Jihong Lu, Shuhong Guo, Shuangshuang Du, Shengyang Liu, Shuye Wu, Heming Li, Peng Wu, Bin Li, Juan Wei.

High-sensitivity glycomic analysis is essential for advancing both basic and translational biomedical research, yet remains methodologically challenging for limited-quantity samples, primarily due to complex workflows and reliance on specialized instrumentation. Here, we introduce solution-enhanced glycan reduction and permethylation (seGRAP), a streamlined, accessible, and high-sensitive method that enables glycan profiling from sub-nanogram protein inputs and picoliter-scale human plasma using widely available mass spectrometry (MS) platforms. seGRAP-MS demonstrates consistently robust and reproducible glycomic performance and enables comprehensive N-glycome characterization of human oocytes at single-cell resolution. Our single-oocyte analysis uncovers a highly conserved N-glycome across both individuals and developmental stages, revealing a previously uncharacterized layer of molecular stability in human reproduction. By significantly lowering technical and logistical barriers, seGRAP-MS redefines the benchmark for high-sensitive glycomics, expanding accessibility and empowering broader applications in fundamental biology, clinical research, and precision medicine.

DOI: https://doi.org/10.1038/s41467-026-73050-y
Anal Methods. 2026 Jun 29.

From tumors to neurons: mass spectrometry imaging in spatial lipidomics.

Bharath S Kumar.

Lipids are fundamental biomolecules that regulate cellular structure, energy metabolism, and signaling, and their dysregulation is increasingly implicated in the pathogenesis of cancer and neurological disorders. Although conventional lipidomics has provided valuable insights into global lipid composition, it lacks spatial information essential for understanding tissue-level heterogeneity. Mass spectrometry imaging (MSI) has emerged as a transformative, label-free analytical platform that enables spatially resolved molecular mapping of lipids directly within biological tissues. Utilizing complementary ionization techniques such as matrix-assisted laser desorption/ionization (MALDI), desorption electrospray ionization (DESI), and secondary ion mass spectrometry (SIMS), MSI allows simultaneous detection of hundreds to thousands of lipid species while preserving their two- and three-dimensional spatial context. Recent technological advances have significantly improved spatial resolution, lipid identification, and biological interpretation through integration with multimodal imaging and machine learning approaches. In cancer research, spatial lipidomics have revealed heterogeneous lipid distributions within tumor microenvironments, providing insights into metabolic reprogramming, tumor progression, and therapeutic resistance. In neuroscience, MSI-based neurolipidomics has enabled region-specific characterization of lipid alterations associated with neurodegeneration, neuroinflammation, and myelin pathology. Despite ongoing challenges related to standardization, isomeric lipid discrimination, and data integration, MSI continues to reshape our understanding of lipid biology. This review highlights recent methodological innovations and biological applications of MSI, underscoring its growing impact on spatial lipidomics in cancer and neuroscience.

DOI: https://doi.org/10.1039/d6ay00170j
J Pharm Biomed Anal. 2026 Jun 23. pii: S0731-7085(26)00290-6. [Epub ahead of print]280 117622

A single bioanalytical LC-MS/MS method for the quantification of pelitinib across biological matrices: An insight into in vitro metabolic stability, in vivo pharmacokinetics, and IVIVE.

Harshada Anil Bhalerao, Pooja Dhurjad, Anish Bharate, Rajesh Sonti.

  Pelitinib's (PTB) strong, irreversible inhibition of EGFR continues to pique scientific curiosity and warrants investigation of potential therapeutic uses, necessitating reliable analytical methods for its pharmacokinetic and metabolic assessment. Here, we have developed and validated a single, sensitive, and reliable LC-MS/MS (liquid chromatography-tandem mass spectrometry) method in accordance with USFDA guidelines for the quantification of PTB in plasma, human, and rat liver microsomes, where greenness assessment by (Analytical GREEnness metric approach and software) AGREE and (Methodological Overall Green Analytical Procedure Index) MoGAPI tools exhibited a good to moderate environmental performance. Chromatographic separation was achieved on an Agilent Eclipse Plus C18 column using a gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile. Pelitinib exhibited dose-dependent systemic exposure and slow systemic elimination in vivo, along with low intrinsic clearance in microsomal incubations. Hepatic clearance was underestimated relative to the observed pharmacokinetic clearance, according to in vitro-in vivo extrapolation (IVIVE). Further, SwissADME (Swiss Absorption, Distribution, Metabolism, and Excretion) provided additional insights regarding the pharmacokinetic characteristics using in silico predictions.

Keywords:  Bioanalytical method; In vitro-in vivo extrapolation; LC-MS/MS; Metabolic stability; Pelitinib; Pharmacokinetics

DOI:  https://doi.org/10.1016/j.jpba.2026.117622
bioRxiv. 2026 Jun 22. pii: 2026.06.21.733640. [Epub ahead of print]

MALDI Tandem Mass Spectrometry for Colony-Based Dereplication of Natural Products.

Robert A Shepherd, Limar Y Gad, Michael Strobel, Gordon T Luu, Jin Feng, Carlo De Silva, Shaun M K McKinnie, Mingxun Wang, Laura M Sanchez.

Microbial libraries remain an important resource for natural product discovery; however, constructing taxonomically and chemically diverse collections remains a challenge. Advances in dereplication strategies, including molecular networking, have reduced the rediscovery of known bioactive molecules and facilitated the identification of novel chemical scaffolds, but these approaches are typically applied after library construction or to existing repositories. Furthermore, many dereplication workflows require scaled fermentation and extraction, increasing the time needed to assess a microbe's metabolite profile. Here, we integrate matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) into the bioinformatics platform IDBac, enabling streamlined characterization of microbial taxonomic identity, metabolite production potential, and preliminary metabolite annotation through GNPS2 molecular networking. This miniaturized high-content workflow facilitates strain prioritization by providing metabolite annotations directly from single microbial colonies prior to scale-up and extraction. Application of this approach to marine actinomycetes enabled the annotation of lavanducyanin and multiple napyradiomycin analogs. Subsequent investigation led to the discovery of napyradiomycin B8 from marine Streptomyces sp. CNZ-289, which was confirmed by 1D and 2D NMR spectroscopy and MALDI-MS/MS. Expanding this workflow to an untargeted analysis of 25 commensal marine vertebrate-derived bacterial isolates resulted in the annotation of several known bioactive natural products, including surugamides, antimycins, desferrioxamine siderophores, and the isolation and elucidation of harmane derivatives using NMR.
Abstract Figure:

DOI: https://doi.org/10.64898/2026.06.21.733640
Bioanalysis. 2026 Jul 03. 1-12

A simple, sensitive microsample LC-MS assay for quercetin and isorhamnetin in mouse and human plasma: application to EMIQ treatment in myotonic dystrophy type 1.

Purnajai Srivijay Saravanan Vanaja, Subodh Mishra, John Douglas Cleary, Masayuki Nakamori, J Andrew Berglund, Nicholas E Johnson, Phillip M Gerk.

   BACKGROUND: Quercetin, a dietary flavonoid with emerging therapeutic relevance in myotonic dystrophy type 1 (DM1), has low solubility and poor oral bioavailability. Enzymatically modified isoquercitrin (EMIQ), a water-soluble prodrug, raises systemic quercetin exposure. Pharmacokinetic studies require a sensitive assay that uses minimal sample volume.
RESEARCH DESIGN AND METHODS: We developed a single-quadrupole liquid chromatography-mass spectrometry (LC-MS) assay for free quercetin, total quercetin (after enzymatic hydrolysis of glucuronide and sulfate conjugates), and the methylated metabolite isorhamnetin in mouse and human plasma. The method used protein precipitation, 10 µL of plasma, reversed-phase C18 separation, and single-ion recording of [M+H]+ adducts. Validation followed a fit-for-purpose approach consistent with M10 guidelines, and the assay was applied to plasma from EMIQ-treated DM1 and wild-type mice (15 g/L for 6 and 12 weeks).
RESULTS: Calibration curves showed r2 > 0.99, with an LLOQ of 0.070 µM for quercetin in both matrices. The assay was successfully validated for quercetin in mouse and human plasma. Total quercetin and isorhamnetin were quantifiable in all treated mice. Exploratory analysis suggested glucuronidation as the major conjugation pathway.
CONCLUSIONS: This simple, cost-effective microsampling assay suits preclinical and translational studies of EMIQ in DM1, though the conjugation findings remain exploratory.

Keywords:  Quercetin; enzymatically modified isoquercitrin; liquid chromatography-mass spectrometry; microsampling; myotonic dystrophy type 1

DOI:  https://doi.org/10.1080/17576180.2026.2695233
Anal Methods. 2026 Jul 01.

Simultaneous determination of 22 carbamate pesticides and their metabolites in biological samples using ultrasound-assisted dispersive liquid-liquid microextraction combined with LC-MS/MS: application to forensic cases.

Xian Ju, Di Liang, Zhuoyi Wang, Huan Gao, Xiaoyuan Yang, Dan Zhang, Zhongyuan Guo, Keming Yun, Zhe Chen.

Carbamate pesticides are extensively employed in agricultural and public health, but their toxicity results from the inhibition of acetylcholinesterase, which can lead to poisoning in both humans and animals. In this study, a simple and rapid ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of 22 carbamate pesticides and their metabolites in biological samples. The method exhibited excellent sensitivity, with limits of quantification (LOQ) determined to be 1.00 ng mL-1, and showed good linearity (R2 > 0.99) over the concentration range of 1.00-1000.00 ng mL-1. Precision and accuracy met international bioanalytical guidelines, as indicated by intra- and inter-day relative standard deviation (RSD) below 15% and recoveries within acceptable limits. The method was successfully applied to authentic forensic cases involving human and animal poisonings, confirming its suitability for determining carbamate pesticides and their metabolites in complex matrices such as blood, liver, and urine. This robust and efficient analytical approach is well suited for applications in forensic investigations and clinical toxicology.

DOI: https://doi.org/10.1039/d6ay00503a
bioRxiv. 2026 Jun 26. pii: 2026.06.23.734138. [Epub ahead of print]

Agentic AI for Structural Elucidation and Discovery of Drug Metabolites from Mass Spectrometry Data.

Xianghu Wang, Abubaker Patan, Haoqi Nina Zhao, Vincent Charron-Lamoureux, Yourae Shin, Daniel Petras, Yuhui Hong, Benjamin P Bowen, Trent R Northen, Pieter C Dorrestein, Mingxun Wang.

The majority of chemical signals detected in public metabolomics repositories remain structurally undefined. Large language models (LLMs) are probabilistic systems whose capacity to generate outputs beyond their training data, which can cause hallucinations, makes them also potentially suited to hypothesize structures for molecules that have never been described. We aimed to build a system that could harness this LLM generative capacity combined with domain specific tools/framework to constrain hallucination and produce validated discoveries. We developed a GNPS2 agentic AI system that interprets LC-MS/MS data by integrating spectral alignment, molecular formula inference, rule-based structural enumeration, machine learning-based spectrum prediction, and translates natural language hypotheses from domain experts into dynamically generated analytical workflows. We demonstrate the annotation of unknown drug metabolites from public data guided by chemical hypotheses. The agent predicted, and we experimentally confirmed, a phosphorylated hydroxyzine, an acetaminophen-p-coumaric acid ester, and identified two new oxidative ibuprofen-carnitine conjugates from public repositories. These results demonstrate that LLM-driven agentic reasoning, when combined with domain expertise, can indeed generate experimentally testable structural hypotheses for previously uncharacterized metabolites leveraging pan repository data.

DOI: https://doi.org/10.64898/2026.06.23.734138
Biomed Chromatogr. 2026 Aug;40(8): e70538

Chromatographic Method Validation for Simultaneous Determination of Bergenin and Alpha-Lipoic Acid in Skin Penetration Studies.

Samuel Dutra-Portes, Clara S Alves, Breno N Matos, Tais Gratieri, Marcilio Cunha-Filho, Emerson S Lima, Guilherme M Gelfuso.

  This study validates a selective, robust high-performance liquid chromatography (HPLC) method for the simultaneous determination of bergenin (BER) and alpha-lipoic acid (ALA) in skin permeation studies. Separation used a Discovery C18 column (250 × 4.6 mm, 5 μm) and a mobile phase of acetonitrile and aqueous phosphoric acid (pH 3.0). A multistep gradient (10%-30% acetonitrile from 0-2 min; 30%-40% from 2-6 min; and 40%-60% from 6-9 min) at 1.0 mL/min enabled a 20-min run time with UV detection at 210 nm. Following ICH M10 bioanalytical guidelines, the method showed linearity (r > 0.999) from 0.5 to 15 μg/mL for both analytes. Limits of detection were 0.120 and 0.083 μg/mL, and limits of quantification were 0.363 and 0.250 μg/mL for BER and ALA, respectively. Precision and accuracy were satisfactory, with a relative standard deviation (RSD) < 3.5%, while recoveries ranged between 80.8% and 96.2% (RSD < 10.0%) from porcine stratum corneum and viable skin. The method proved robust against variations in temperature, flow rate, and column batch, without endogenous skin interference. In conclusion, this validated method is a reliable tool for supporting the development of topical formulations containing the association of BER and ALA.

Keywords:  antioxidants; liquid chromatography; skin permeation; topical formulation

DOI:  https://doi.org/10.1002/bmc.70538