bims-metlip 2025-11-23 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–11–23
25 papers selected by
Sofia Costa, Matterworks

LC-MS/MS Assay for Quantification of DA-1241, a Novel GPR119 Agonist, in Animal and Human Plasma: Application to Preclinical Pharmacokinetic Studies.
Enhancing metabolite coverage using dedicated mobile phases for individual polarity modes in HILIC-MS.
Improving Metabolite Annotations in On-Tissue Chemical Derivatization Mass Spectrometry Imaging by Functional Group Filtering and Hydrogen-Deuterium Exchange.
MetScribeR: A Semiautomated Tool for Data Processing of In-House LC-MS Metabolite Reference Libraries.
Quantification of water-soluble vitamin profiles in human plasma via automated liquid chromatography-tandem mass spectrometry.
Magnetic bead-assisted extraction combined with LC-MS/MS for simultaneous quantification of aldosterone and related steroids in human plasma.
Simultaneous determination of 14 underivatized amino acids using ion-pairing chromatography and isotope dilution tandem mass spectrometry for a reference measurement procedure.
Improving the Detection of Analyte Degeneracies in Untargeted Liquid Chromatography-Tandem Mass Spectrometry Data.
Targeted Metabolomic Methods for 13C Stable Isotope Labeling with Uniformly Labeled Glucose and Glutamine Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
Signal enhancement of lipids and glycans using ammonium fluoride for IR-MALDESI mass spectrometry imaging.
Metabolic stability assessment and metabolite profiling of gunagratinib, a novel FGFR inhibitor, in rat, monkey and human liver microsomes by an integrated analysis method based on HPLC-MS/MS and HPLC-Orbitrap-HRMS.
Uracil in plasma: comparison of two in-house-developed LC-MS/MS methods.
Development and validation of ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to quantify monotropein in blueberries.
Surfactant-enhanced emulsification liquid-liquid microextraction combined with sweeping micellar electrokinetic chromatography-tandem mass spectrometry for therapeutic drug monitoring of alpelisib and fulvestrant in human plasma.
Improving metabolomics data comparability without long term quality controls using a post-acquisition correction strategy.
Exploration of Yeast Species Suitable for Preparation of Stable Isotope-Labeled Internal Standards Extracts (SILIS).
Deriving three one dimensional NMR spectra from a single experiment through machine learning.
Hyphenation of Trapped Ion Mobility to Two-Dimensional Mass Spectrometry for Protein Analysis in Complex Biomixtures.
Ultrasensitive Profiling of Arachidonic Acid Metabolites Based on 5‑(Diisopropylamino)amylamine Derivatization-Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.
Enhancing Chimeric Fragmentation Spectra Deconvolution Using Direct Infusion-Tandem Mass Spectrometry Across High-Resolution Mass Spectrometric Platforms.
The SIMPLIFY Protocol: A Monophasic Extraction System Suitable for Exposomics, Metabolomics, Lipidomics, and Proteomics Research.
High-speed characterization and quantification of polypropylene glycol based oligomeric mixtures by hydrophilic interaction liquid chromatography at critical conditions and mass spectrometry (LCCC/Q‑TOF MS).
Optimizing multiomics sample preparation: comparative evaluation of extraction protocols for HepG2 cells.
Trace-level quantitation of N-((2-isopropylthiazol-4-yl)methyl)-N-methylnitrous amide by liquid chromatography-tandem mass spectrometry.
A Validated UPLC-MS/MS Method for Monitoring Temozolomide and Perillyl Alcohol Metabolites in Biological Matrices: Insights From a Preclinical Pharmacokinetic Study.

J Mass Spectrom. 2025 Dec;60(12): e5194

LC-MS/MS Assay for Quantification of DA-1241, a Novel GPR119 Agonist, in Animal and Human Plasma: Application to Preclinical Pharmacokinetic Studies.

Seung Jin Kim, Won Seok Bang, Zhengri Li, Mi Hye Kwon, Dae Young Lee, Hee Eun Kang.

  DA-1241 is a novel GPR119 receptor agonist under development as a therapeutic candidate for metabolic dysfunction-associated steatohepatitis. To enable precise quantification of DA-1241 in mouse, rat, dog, and human plasma, a simple and highly sensitive LC-MS/MS approach was designed and thoroughly evaluated. Plasma samples were processed using protein precipitation with acetonitrile, followed by chromatographic separation on a reverse-phase C18 column (3.0-mm i.d. × 50 mm, 2.7 μm). Isocratic elution was achieved using a mobile phase composed of 1-mM ammonium formate and 2-mM formic acid in water and acetonitrile (13:87, v/v) operated at a flow setting of 0.3 mL/min. Mass spectrometry analysis was conducted using an Agilent 6460 triple quadrupole system operated in multiple reaction monitoring with positive electrospray ionization. Ion transitions were m/z 472.1 → 135.9 for DA-1241 and m/z 480.2 → 404.1 for DA-1241-d8 (internal standard). The total chromatographic run time was 4.5 min. Linearity of the calibration curves was confirmed throughout the range of 1-10 000 ng/mL, with no carry-over observed. Precision and accuracy for intra-assay and interassay were within 14.3%, except at the lower limit of quantification, where values remained within 16.0%. The method met all the other validation criteria for matrix effect, recovery, and stability. The established method was successfully implemented on plasma protein binding assessments and preclinical pharmacokinetic studies of DA-1241 after a single oral administration in mice, rats, and dogs.

Keywords:  DA‐1241; LC‐MS/MS; dog; human; mouse; pharmacokinetics; plasma; rat

DOI:  https://doi.org/10.1002/jms.5194
Anal Bioanal Chem. 2025 Nov 19.

Enhancing metabolite coverage using dedicated mobile phases for individual polarity modes in HILIC-MS.

Alena Langová, Malena Manzi, Jana Brejchová, Ondřej Kuda, Michal Holčapek, Robert Jirásko.

  We have developed a new hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method with mobile phases optimized for high metabolite coverage in individual polarity modes. This dual mobile phase strategy expands the range of annotated metabolites and improves identification confidence, providing broader and more accurate metabolic profiles. The incorporation of a bioinert chromatographic system further enhances sensitivity. The bridged ethyl hybrid (BEH) amide column yields the best results for metabolomic analysis among the three chromatographic columns in this comparison. The method development involves investigating the effects of mobile phase composition, pH, and a medronic acid additive on the MS response under bioinert chromatographic conditions. The results highlight the important role of alkaline pH for the sensitive detection of polyphosphorylated metabolites, while demonstrating the redundancy of chelating additives in a fully bioinert system. Finally, the optimized method is applied to mouse plasma, pancreas, and liver samples to demonstrate its versatility and reliable performance in complex biological matrices, establishing it as a powerful tool for confident and reproducible metabolomics studies.

Keywords:  Bioinert chromatography; HILIC; Mass spectrometry; Metabolite annotation; Metabolomics

DOI:  https://doi.org/10.1007/s00216-025-06189-0
J Am Soc Mass Spectrom. 2025 Nov 19.

Improving Metabolite Annotations in On-Tissue Chemical Derivatization Mass Spectrometry Imaging by Functional Group Filtering and Hydrogen-Deuterium Exchange.

Anna Uhlmansiek, Josiah J Rensner, Young Jin Lee.

  Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) enables the direct visualization of metabolites from tissue sections with high spatial resolution. However, its application to untargeted spatial metabolomics is hindered by poor ionizing compounds and challenges in accurate metabolite annotation. On-tissue chemical derivatization (OTCD) is commonly employed to enhance the ionization of metabolites bearing specific functional groups, and platforms such as METASPACE facilitate high-throughput annotation of derivatized features. Nevertheless, distinguishing structural isomers for a large number of metabolites remains a major challenge, often resulting in incorrect annotations. To address this limitation, we developed an improved annotation workflow for OTCD-MALDI-MSI by integrating two filtering strategies. Functional group filtering leverages SMARTS-based substructure matching to retain only those metabolites that react with the applied OTCD reagent. In parallel, gas-phase hydrogen-deuterium exchange (HDX) in the MALDI source is used to determine the number of labile hydrogens for each feature, enabling the exclusion of annotations that are inconsistent with HDX behavior. We applied this workflow to MALDI-MSI of maize root sections using Girard's reagents T and P, along with the plant-specific COCONUT metabolite database. The combined filtering strategy reduced incorrect annotations by ∼67%, from ∼7.3 annotations per unique feature without filtering to ∼2.4 with filtering, substantially improving annotation accuracy and confidence. By coupling OTCD signal enhancement with structurally informed filtering, this workflow advances the utility of MALDI-MSI for untargeted spatial metabolomics, enabling more reliable and scalable metabolite profiling in complex biological tissues.

Keywords:  Hydrogen−Deuterium Exchange; METASPACE; Mass Spectrometry Imaging; Matrix-Assisted Laser Desorption Ionization; Metabolite Annotation; On-Tissue Chemical Derivatization; Spatial Metabolomics

DOI:  https://doi.org/10.1021/jasms.5c00293
J Proteome Res. 2025 Nov 17.

MetScribeR: A Semiautomated Tool for Data Processing of In-House LC-MS Metabolite Reference Libraries.

Adam M Tisch, Jason M Inman, Ewy A Mathé, Djawed Bennouna.

  One persistent challenge in untargeted metabolomics is the identification of compounds from their mass spectrometry (MS) signal, which is necessary for biological data interpretation. This process can be facilitated by building in-house libraries of metabolite standards containing retention time (RT) information, which is orthogonal and complementary to large, published MS/MS spectra repositories. Creating such libraries can require substantial effort and is time intensive. To streamline this process, we developed metScribeR, an R package with a Shiny application to accelerate the creation of RT and m/z libraries. metScribeR provides an easy, user-friendly interface for peak finding, filtering, and comprehensive quality review of the MS data. Uniquely, metScribeR does not require MS/MS spectral information and reports an identification probability estimate for each adduct. In our benchmarking, metScribeR required approximately 10 s of computational and manual effort per standard, showed a correlation of 0.99 between manual and metScribeR-derived RTs, and appropriately filtered out poor quality peaks. The metScribeR output is a.csv file including the identity, m/z, RT, and peak quality information for standards along with MS/MS spectra retrieved from MassBank of North America (MoNA). metScribeR is open source and available for download on GitHub at https://github.com/ncats/metScribeR.

Keywords:  LC-MS; R; mass spectrometry; metabolite identification; metabolite libraries; metabolomics; software

DOI:  https://doi.org/10.1021/acs.jproteome.5c00548
Anal Bioanal Chem. 2025 Nov 20.

Quantification of water-soluble vitamin profiles in human plasma via automated liquid chromatography-tandem mass spectrometry.

Xiaoyi Yi, Rongliang Liang, Yiqi Yang, Xiaojing Huang, Yingsong Wu.

  Water-soluble vitamins including thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), biotin (vitamin B7), 5-methyltetrahydrofolic acid (vitamin B9), and ascorbic acid (vitamin C) are essential micronutrients functionally important for human health. Reliable and accurate quantification of the water-soluble vitamin profiles is of vital importance for evaluating their deficiency and related diseases. However, simultaneous determination of these water-soluble vitamins in complex biological fluids such as plasma is a challenge. Herein, an automated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying water-soluble vitamins was developed using a robotic workstation. The optimized sample preparation was conducted through a two-step procedure, protein precipitation using trichloroacetic acid (TCA) and liquid-liquid extraction using methyl tert-butyl ether (MTBE). The entire sample preparation workflow was fully automated with a Hamilton robotic workstation. Eight water-soluble vitamins including B1, B2, B3, B5, B6, B7, B9, and C were simultaneously measured in 8 min by LC-MS/MS. The automated LC-MS/MS method was validated for measuring human plasma samples in clinical application. The water-soluble vitamins showed good linearity in their calibration ranges. The limits of detection (LOD) and quantification (LOQ) were 0.01-1.73 ng/mL and 0.13-7.30 ng/mL, respectively, for B-complex vitamins. For vitamin C, the LOD and LOQ were 36.2 ng/mL and 72.3 ng/mL, respectively. Satisfactory coefficients of variation (CVs) were obtained, with the intra-batch and inter-batch CVs of 0.9-5.3% and 2.5-14.8%, respectively. Recoveries were considered adequate in the range of 83.2-111.9%, 85.1-118.0%, and 76.5-112.2% for high, medium, and low concentration levels of the eight vitamins, respectively. Matrix effect factors were 80.3-116.0%, 73.2-101.7%, and 76.6-110.5% for high, medium, and low concentration levels, respectively. Robustness of the method in routine clinical practice was further demonstrated by quantifying clinical plasma samples in vitamin screening. The automated LC-MS/MS method for the simultaneous determination of water-soluble vitamins in plasma showed great promise for clinical application.

Keywords:  Automation; Human plasma; Liquid chromatography-tandem mass spectrometry; Robotic workstation; Water-soluble vitamins

DOI:  https://doi.org/10.1007/s00216-025-06211-5
Anal Bioanal Chem. 2025 Nov 18.

Magnetic bead-assisted extraction combined with LC-MS/MS for simultaneous quantification of aldosterone and related steroids in human plasma.

Yudong Chu, Qin Su, Ziyuan Huang, Chenyi Yuan, Shazhou Ye, Xueyan Bian.

  The quantitative analysis of aldosterone and its associated steroid hormone profile including aldosterone (ALD), 18-hydroxycorticosterone (18-OHB), 18-hydroxycortisol (18-OHF), 18-oxocortisol (18-OXOF) and cortisol (F) is critical for the screening, confirmatory testing, and subtype classification of primary aldosteronism (PA). Conventional hormone extraction methods, such as solid-phase extraction and liquid-liquid extraction, are labor-intensive and time-consuming, thereby limiting their applicability and widespread adoption in clinical laboratories. This study developed a novel high-efficiency methodology integrating automated magnetic solid-phase extraction (MSPE) pretreatment with liquid chromatography-tandem mass spectrometry (LC-MS/MS), enabling simultaneous and accurate quantification of ALD, 18-OHB, 18-OHF, 18-OXOF, and F. To ensure the reliability and practicality of the new method, a comprehensive validation of its linearity, accuracy, precision, matrix effect, carryover, stability, and specificity was conducted, and a comparison was made with the traditional solid-phase extraction method. Under optimized conditions, the method demonstrated a satisfactory linear range (18-OHB/18-OHF/18-OXOF: 5-3000 pg/mL, ALD: 0.02-10 ng/mL, F: 2-1000 ng/mL) and extremely low limits of quantification (18-OHB/18-OHF/18-OXOF: 3.75 pg/mL, ALD: 0.02 ng/mL, F: 1.5 ng/mL). Moreover, the recoveries of the five analytes ranged from 90.23% to 104.27%, and the repeatability was between 0.23% and 5.45%. Notably, the magnetic bead-assisted extraction method did not show obvious matrix effects or carryover problems. The target analytes in the processed sample remained stable for 24 h at room temperature and under autosampler conditions. There was a good correlation between this method and traditional solid-phase extraction method. MSPE serves as a critical technological link between complex manual procedures and high-throughput clinical MS. By leveraging its inherent compatibility with automation to streamline workflows and enhance efficiency, it facilitates the broader adoption and application of clinical MS technologies. In conclusion, we have developed a novel method combining magnetic bead extraction with LC-MS/MS that enables simultaneous quantification of ALD, 18-OHB, 18-OHF, 18-OXOF and F for the screening and diagnosis of primary aldosteronism (PA), with potential for further application in clinical research.

Keywords:  Liquid chromatography-tandem mass spectrometry; Magnetic solid-phase extraction (MSPE); Primary aldosteronism; Steroids

DOI:  https://doi.org/10.1007/s00216-025-06228-w
J Chromatogr A. 2025 Nov 17. pii: S0021-9673(25)00885-4. [Epub ahead of print]1766 466541

Simultaneous determination of 14 underivatized amino acids using ion-pairing chromatography and isotope dilution tandem mass spectrometry for a reference measurement procedure.

Dong Wook Seok, Seohyun Choi, Ji-Seon Jeong.

  We describe the simultaneous quantification of 14 underivatized amino acids (AAs) in various matrices using ion-pairing liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with isotope dilution mass spectrometry (IDMS) as a newly developed reference measurement procedure. The underivatized AAs were successfully separated on a reversed-phase column using mixed ion-pairing reagents of trifluoroacetic acid and heptafluorobutyric acid within 6 min through optimized gradient elution. The method was applied to different matrices, including buffer and plasma with appropriate sample treatments, followed by dilution with water to adjust the concentration range from 1 to 20 mg/kg, ensuring high measurement accuracy and precision. To facilitate exact-matched double IDMS, isotope-labeled AAs were added to the sample prior to treatment as internal standards. The optimized procedure was rigorously validated for linearity, sensitivity, accuracy, and precision, while its reliability was confirmed using commercially available certified reference materials for the AAs. Finally, the method was applied to certify AAs in candidate certified reference material batches. The optimized protocol proved to be suitable for the accurate quantification of 14 AAs in various matrices, and thus may serve as a reliable primary method for AA measurement across different applications.

Keywords:  Amino acids; Certified reference material; Ion-pairing chromatography; LC-MS; Reference measurement procedure; Underivatized amino acids

DOI:  https://doi.org/10.1016/j.chroma.2025.466541
Anal Chem. 2025 Nov 21.

Improving the Detection of Analyte Degeneracies in Untargeted Liquid Chromatography-Tandem Mass Spectrometry Data.

Caitlin N Cain, Cheng-Hua Ma, Pavlo Popov, Robert T Kennedy.

Advances in liquid chromatography-tandem mass spectrometry (LC-MS/MS) and chemometrics have driven the field of untargeted metabolomics forward. However, interpretation of these studies can be challenging, since these data sets often return thousands of features with only a small fraction identifiable. Most of these unidentified features arise from degeneracy, where a single analyte produces multiple features from adduct formation, in-source fragments, and isotopes. This work improves the detection and clustering of these degenerate features with a new peak shape consistency metric, termed lack-of-fit (LOF). This metric quantifies the residual error between two features within a time window, where a LOF <20% suggests degeneracy. To first evaluate metric performance, 21 analytes were spiked into brain dialysate and features were discovered using tile-based Fisher ratio (F-ratio) analysis. Incorporating the proposed LOF metric not only reduced the feature list and retained all the spiked analytes in the top 25 hits but also outperformed other data-driven degeneracy methods by ∼18-48%. LOF clustering was then applied to reduce the number of features detected in brain dialysate at different preconcentration levels and mobile phase gradient lengths. LOF clustering provided a 90% degenerate feature classification accuracy, surpassing existing LC-MS/MS data processing methods. Despite the chromatographic complexity, this metric could resolve features from coeluting compounds at lower resolutions (Rs ≤ 0.3) than the standard correlation-based method. LOF clustering reduced this untargeted data set by ∼79-86%, ultimately revealing ∼583 unique metabolites in brain dialysate. These results collectively demonstrate that the LOF metric can improve and strengthen the interpretation of untargeted metabolomics data.

DOI: https://doi.org/10.1021/acs.analchem.5c03704
J Proteome Res. 2025 Nov 20.

Targeted Metabolomic Methods for 13C Stable Isotope Labeling with Uniformly Labeled Glucose and Glutamine Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Erin Q Jennings, W Kimryn Rathmell, Jeffrey C Rathmell.

  Heavy carbon labeling has emerged as a popular way to study metabolic diseases. However, most carbon labeling techniques use untargeted mass spectrometry, which typically requires dependence on a research core and specialized software. By combining published 13C labeling patterns and known enzyme reactions, an optimized targeted mass spectrometry method was generated to measure stable isotope labeling with carbon-13 through glycolysis, the tricarboxylic acid cycle, the hexosamine biosynthetic pathway, and glutaminolysis using uniformly labeled glucose or glutamine. This method provides a novel and adaptable approach to investigate pointed hypotheses on the utilization of glucose or glutamine in disease states and models.

Keywords:  carbon tracing; stable isotope labeling; tandem mass spectrometry; targeted metabolomics

DOI:  https://doi.org/10.1021/acs.jproteome.5c00514
Anal Bioanal Chem. 2025 Nov 18.

Signal enhancement of lipids and glycans using ammonium fluoride for IR-MALDESI mass spectrometry imaging.

Seth M Eisenberg, Alora R Dunnavant, Tana V Palomino, David C Muddiman.

  Mass spectrometry imaging (MSI) is an analytical technique that enables the simultaneous detection and localization of hundreds to thousands of molecules across a tissue surface. The addition of ammonium fluoride to the electrospray or to tissue samples has been shown to increase the signal of lipids in both nano-DESI-MSI and MALDI-MSI, respectively. Herein, this strategy is applied to infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) which demonstrated signal enhancement for both lipids and N-linked glycans using 70 µM and 350 µM NH4F in 50% ACN with 1 mM acetic acid as the ESI solvent, respectively. Up to a 93-fold increase in signal was observed for these biomolecules without any change in sample preparation or to the mass spectrometer parameters, including detecting lipids not seen with the control ESI solvent. On average, lipids were enhanced by eightfold, and the glycan signal increased by 1.2-fold. The enhancement factor for lipids was significantly impacted by lipid class, while glycan enhancement was affected by charge state and the observed adduct.

Keywords:  Ammonium fluoride; Electrospray doping; Glycans; IR-MALDESI; Mass spectrometry imaging

DOI:  https://doi.org/10.1007/s00216-025-06197-0
J Pharm Biomed Anal. 2025 Nov 13. pii: S0731-7085(25)00594-1. [Epub ahead of print]269 117253

Metabolic stability assessment and metabolite profiling of gunagratinib, a novel FGFR inhibitor, in rat, monkey and human liver microsomes by an integrated analysis method based on HPLC-MS/MS and HPLC-Orbitrap-HRMS.

Lei Zhang, Jiabiao Ji, Zhen Hu, Shuai Qu, Yayun Zhou, Hongjian Zhang, Jianming Yang.

  Gunagratinib is a potent fibroblast growth factor receptor (FGFR) inhibitor currently in clinical development for head and neck cancers and cholangiocarcinoma. However, information on its hepatic metabolism remains limited. In this study, we developed and validated an ultra-high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for quantifying gunagratinib in liver microsomes. Chromatographic separation used a Waters ACQUITY BEH C18 column with a gradient elution system of 0.1 % formic acid in water and acetonitrile. Quantification employed positive electrospray ionization with selective reaction monitoring (SRM) for gunagratinib (m/z 424.2→407.3) and the internal standard (IS; m/z 419.2→296.1). The assay demonstrated excellent linearity over the concentration range of 2.0-2000 nM, suitable for in vitro high-throughput screening. Gunagratinib exhibited moderate projected hepatic extraction ratio in rat (EH = 0.39), contrasting with high ERH in monkey (EH = 0.70) and human (EH = 0.71). Metabolite characterization was performed using HPLC coupled with benchtop Orbitrap high-resolution mass spectrometry (HPLC-Orbitrap-HRMS) in full MS/dd-MS2 scan mode, enabling accurate mass measurement, chemical formula assignment, and MS2 fragmentation interpretation. Seven metabolites were structurally identified, with M7 (N-demethylation) being the most abundant. Demethylation and oxygenation were the primary metabolic pathways. Cytochrome P450 3A4 was the predominant enzyme responsible for gunagratinib metabolism based on recombinant human P450 enzyme analysis and chemical inhibition study. This study presents the first integrated analytical method based on HPLC-MS/MS and HPLC-Orbitrap-HRMS for the in vitro metabolic assessment of gunagratinib. We anticipate the clinical application of this method in future pharmacokinetic or metabolism studies.

Keywords:  CYP3A4; Fibroblast growth factor receptor; Gunagratinib; High-resolution mass spectrometry; Metabolic stability; Metabolite characterization

DOI:  https://doi.org/10.1016/j.jpba.2025.117253
Clin Chem Lab Med. 2025 Nov 17.

Uracil in plasma: comparison of two in-house-developed LC-MS/MS methods.

Svend H Hansen, Eva Greibe.

   OBJECTIVES: To prevent intoxication with 5-fluorouracil (5-FU) during chemotherapy, the initial measurement of plasma uracil levels has recently been recommended by the European Medicines Agency. Here we present and compare two new chromatographic methods for analysis of uracil in plasma. Two independent laboratories analyzed the same set of samples using in-house-developed analytical methods. The aim of this comparison is to demonstrate that our methods are harmonized, providing comparable concentrations and consistent clinical interpretations.
METHODS: The approaches presented employ mass spectroscopic detection in negative ion mode but differ in sample preparation and chromatographic techniques. Method 1: Protein precipitation using acetonitrile, followed by injection of a small sample volume for hydrophilic interaction liquid chromatography (HILIC). Method 2: Protein precipitation with acetonitrile combined with filtration through a 96-well filter plate, followed by evaporation and reconstitution in the chromatographic mobile phase for C18 reverse-phase chromatography.
RESULTS: Both methods are demonstrated robust and accurate quantification of uracil. Results obtained from patient samples (n=161) show a strong correlation between the two methods, with a relative mean deviation of 2 %. The same clinical interpretation is obtained for the majority of samples (150/161). The remaining samples (11/161) were close to the lower clinical decision limit (16 μg/L).
CONCLUSIONS: For the analysis of uracil, both presented methods, which are based on simplified sample preparation procedures, provide accurate results without significantly impacting the clinical interpretation. However, the results indicate a potential gray area of analytical imprecision from 14 to 20 μg/L, encompassing the lower clinical decision limit (16 μg/L).

Keywords:  5-fluorouracil toxicity; HILIC and reverse phase C18 chromatography; analytical harmonization; gray area; uracil

DOI:  https://doi.org/10.1515/cclm-2025-0782
PLoS One. 2025 ;20(11): e0329723

Development and validation of ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to quantify monotropein in blueberries.

Ishveen Kaur, Courtney P Leisner, Dennis P Cladis.

Blueberries (Vaccinium species) are an economically important fruit crop rich in bioactive compounds like polyphenols and flavonoids. Interestingly, some blueberry cultivars also produce monotropein, which has bioactive properties, including anti-inflammatory and neuroprotective effects. However, methods to quantify monotropein in blueberries have not been optimized. To address this gap, an optimized analytical method for monotropein extraction and quantification using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed. Different extraction strategies were compared, including variations in temperature, time, and ultrasonication treatments. Optimal extraction was achieved by heating samples to 60 °C for 15 mins in methanol. The method had high percent recovery and good repeatability. This protocol was then applied to 28 blueberry cultivars, 14 of which had not been previously analyzed for monotropein. Monotropein ranged from 0-1807 ng/mg dry weight. The developed method provides a robust tool that can be applied to future evaluations of monotropein in diverse blueberry cultivars.

DOI: https://doi.org/10.1371/journal.pone.0329723
Anal Bioanal Chem. 2025 Nov 17.

Surfactant-enhanced emulsification liquid-liquid microextraction combined with sweeping micellar electrokinetic chromatography-tandem mass spectrometry for therapeutic drug monitoring of alpelisib and fulvestrant in human plasma.

Zvonimir Mlinarić, Lu Turković, Biljana Nigović, Miranda Sertić.

  A novel bioanalytical method combining surfactant-enhanced emulsification liquid-liquid microextraction (SE-LLME) with sweeping micellar electrokinetic chromatography-tandem mass spectrometry (MEKC-MS/MS) was developed and validated for therapeutic drug monitoring (TDM) of alpelisib (ALP) and fulvestrant (FUL) in human plasma. This method addresses the need for sensitive, selective quantification in patients with PIK3CA-mutated, HR+/HER2- breast cancer. Sample preparation involved protein precipitation followed by SE-LLME using pentadecafluorooctanoic acid (PFOA) and chloroform, yielding recoveries greater than 88.4% for ALP and 78.7% for FUL. Optimised MEKC conditions were 50 mM ammonium pentadecafluorooctanoate at pH 9.75 with 25% methanol, 30 kV separation voltage, 30 °C capillary temperature, and 100 mbar additional pressure during the analysis. Sweeping preconcentration significantly enhanced sensitivity-109-fold for ALP and 11.2-fold for FUL. The method was validated per ICH guidelines, demonstrating excellent linearity ( r≥ 0.9963) across the calibration ranges (200-2000 ng/mL for ALP, 10-100 ng/mL for FUL), accuracy (mean biases -10.3% to 7.5%), and precision (RSD < 12.6%). Despite notable matrix effects for ALP, consistency across six different plasma sources (RSD ≤ 12.7%) ensured reliability. Analytes were stable under benchtop, autosampler, freeze-thaw, and long-term conditions (bias ≤ 11.1%). No carry-over was detected, and dilution integrity was confirmed (bias ≤ 2.7%). Application to patient samples validated the method's clinical relevance, with measured concentrations aligning with the expected ones. This is the first capillary electrophoresis method for ALP in biological matrices and the only method for simultaneous TDM of ALP and FUL, offering a robust, cost-effective, and eco-friendly alternative to traditional chromatographic approaches.

Keywords:  Bioanalytical method; Breast cancer; Capillary electrophoresis; Mass spectrometry; Personalised treatment; Surfactant-enhanced emulsification liquid-liquid microextraction

DOI:  https://doi.org/10.1007/s00216-025-06233-z
Anal Chim Acta. 2025 Dec 22. pii: S0003-2670(25)01147-X. [Epub ahead of print]1380 344753

Improving metabolomics data comparability without long term quality controls using a post-acquisition correction strategy.

Elfried Salanon, Blandine Comte, Delphine Centeno, Stéphanie Durand, Julien Boccard, Estelle Pujos-Guillot.

   BACKGROUND: Recent advances in analytical techniques for metabolomics allowed generating data of increasing quality in terms of sensitivity and robustness, thus opening the door to its large-scale application. However, the integration of separately collected metabolomic data is currently limited by the lack of methods able to correct for the analytical bias without long-term quality controls. This significant bottleneck prevents inter-comparisons across studies and limits metabolomics impact in precision biology. Overcoming these major challenges is therefore of great importance in many application fields to improve interoperability across studies and offer more reliable and reproducible conclusions.
RESULTS: In this work, we propose a post-acquisition strategy (PARSEC) to improve metabolomics data comparability that consists in a three-step workflow starting from the combined extraction of raw data from the different studies or cohorts analyzed, through standardization, to the filtering of features based on analytical quality criteria. This workflow was applied to two case studies to evaluate the performance of the developed correction approach and to compare it with the classically used locally estimated scatterplot smoothing (LOESS) method. The PARSEC strategy allowed reducing the inter-group variability, and producing a more homogeneous sample distribution. In addition, results showed an improvement in the comparability of the data in both case studies, allowing biological information initially masked by unwanted sources of variability to be revealed more clearly than with the LOESS method.
SIGNIFICANCE: The proposed post-acquisition correction strategy, which combines batch-wise standardization and mixed modeling, enhances data comparability and scalability for metabolomics studies. By addressing both batch and group effects, this approach minimizes the influence of analytical conditions while preserving biological variability. Therefore, it offers a valuable tool for harmonizing datasets across different studies or cohorts without common long-term quality control samples.

Keywords:  Batch-effect; Group-effect; Metabolomics; Post-acquisition standardization

DOI:  https://doi.org/10.1016/j.aca.2025.344753
Mass Spectrom (Tokyo). 2025 ;14(1): A0177

Exploration of Yeast Species Suitable for Preparation of Stable Isotope-Labeled Internal Standards Extracts (SILIS).

Taisuke Seike, Kahar Prihardi, Chiaki Ogino, Fumio Matsuda.

  Isotope dilution mass spectrometry is a widely used method for measuring intracellular metabolite concentrations, relying on the ratio of peak areas between the target compound and its stable isotope-labeled internal standard. For metabolome analysis of microorganisms, comprehensive concentration measurements have been achieved through the preparation of stable isotope-labeled internal standard extracts (SILIS). Methods have been developed to prepare SILIS by extracting crude metabolites from fully 13C-labeled bacteria Escherichia coli and yeasts Saccharomyces cerevisiae and Pichia pastoris (Komagataella phaffii). For cost-effective preparation of SILIS, ideal characteristics of host yeasts include rapid cell growth, high biomass production, and significant metabolite accumulation. In this study, suitable yeast species for SILIS production were investigated from diverse candidates. Batch cultures of 15 yeast species from 12 genera were performed in synthetic defined medium, with cells harvested at different growth phases and metabolites extracted using the methanol/chloroform/water method. Metabolomic analysis by liquid chromatography-tandem mass spectrometry revealed the relative concentrations of 65 metabolites. The results demonstrated that S. cerevisiae and Kluyveromyces marxianus in the stationary phase were the most effective for SILIS production of central metabolic intermediates. SILIS production using S. cerevisiae and K. marxianus can be widely applied in standard laboratories because these species are safe, the media are commercially available, and the extraction methods are easily implementable.

Keywords:  Kluyveromyces marxianus; Saccharomyces cerevisiae; isotope dilution mass spectrometry; metabolomics; stable isotope–labeled standards

DOI:  https://doi.org/10.5702/massspectrometry.A0177
Nat Commun. 2025 Nov 19. 16(1): 10159

Deriving three one dimensional NMR spectra from a single experiment through machine learning.

Alessia Vignoli, Stefano Cacciatore, Leonardo Tenori.

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for analyzing complex mixtures due to its ability to manage matrix complexity, provide detailed molecular insights, and preserve sample integrity. In metabolomics, NMR enables the identification, quantification, and characterization of metabolites with minimal sample preparation, a broad dynamic detection range, and high reproducibility. Various NMR experiments, such as Nuclear Overhauser Effect SpectroscopY (NOESY), Carr-Purcell-Meiboom-Gill (CPMG), diffusion-edited, and J-resolved spectroscopy (JRES), offer complementary insights into biofluids like serum and plasma. However, acquiring multiple spectra for high-throughput applications can be resource-intensive and time-consuming. This study proposes a machine learning approach to predict CPMG, diffusion-edited, and JRES spectra directly from acquired NOESY spectra, leveraging serum samples as a case study to streamline analysis and improve efficiency in NMR-based metabolomics.

DOI: https://doi.org/10.1038/s41467-025-65294-x
J Am Soc Mass Spectrom. 2025 Nov 18.

Hyphenation of Trapped Ion Mobility to Two-Dimensional Mass Spectrometry for Protein Analysis in Complex Biomixtures.

Callan Littlejohn, Meng Li, Christopher A Wootton, Mark P Barrow, Peter B O'Connor.

The analysis of complex biological mixtures remains a significant challenge in mass spectrometry (MS), particularly when using conventional direct infusion MS/MS approaches due to inherent limitations in resolving power and spectral complexity. Here, we demonstrate the integration of trapped ion mobility spectrometry (TIMS) with two-dimensional mass spectrometry (2DMS) to enable high-resolution TIMS-MS/2DMS experiments for detailed protein characterization within mixtures. TIMS provides separation based on the ion's size-to-charge ratio, effectively reducing the occurrence of chimeric tandem mass spectra containing fragments from more than one precursor ion. This coupling allows for an improved peak capacity and reduced ambiguity in tandem spectral interpretation. When applied to a model protein mixture, the TIMS-MS/2DMS method allows resolution of near m/z species, including isomeric and isonucleonic species, and it was possible to assign secondary fragmentation with greater confidence.

DOI: https://doi.org/10.1021/jasms.5c00292
ACS Pharmacol Transl Sci. 2025 Nov 14. 8(11): 4095-4106

Ultrasensitive Profiling of Arachidonic Acid Metabolites Based on 5‑(Diisopropylamino)amylamine Derivatization-Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

Peiyan Zheng, Xiaowen Huang, Qing Wang, Yida Zhang, Mingtao Liu, Manyun Jiang, Sitong Liu, Huimin Huang, Wenting Luo, Jian-Lin Wu, Baoqing Sun.

  Arachidonic acid (AA) and its metabolites play critical roles in inflammation and immune regulation, modulating the initiation, amplification, and resolution of inflammation. However, their comprehensive quantification remains a challenging endeavor owing to complex metabolic pathways and biological matrix effects. This study introduces a novel metabolomics method involving 5-(diisopropylamino)-amylamine (DIAAA) derivatization coupled with ultraperformance liquid chromatography-tandem mass spectrometry to address these issues. The method demonstrated high sensitivity and specificity, with limits of quantification meeting stringent criteria (relative standard deviation <20%; recovery rate, 85-115%, signal-to-noise ratio >10). It effectively quantified 14 key AA metabolites, including hydroxyeicosatetraenoic acids, prostaglandins, and leukotrienes, across a wide linear range (R 2 > 0.98). The results of intra- and interassay precision tests exhibited low coefficients of variation (≤15%), underscoring the reproducibility of the method. DIAAA derivatization also mitigated matrix variability, improving the accuracy of metabolite detection in serum samples. The hallmark of allergic diseases is a disrupted AA metabolism, where elevated specific metabolites (AA, HETEs, LTB4, and PGD2) show strong diagnostic promise, and a unique metabolite signature in polysensitized patients indicates a link to inflammatory severity. This advanced analytical approach offers significant potential for elucidating the role of AA metabolism in allergic diseases and holds promise for applications in clinical diagnostics and therapeutic monitoring.

Keywords:  5-(diisopropylamino)amylamine derivatization; arachidonic acid metabolites; metabolite quantification; ultraperformance liquid chromatography−tandem mass spectrometry

DOI:  https://doi.org/10.1021/acsptsci.5c00498
Rapid Commun Mass Spectrom. 2026 Feb 15. 40(3): e10170

Enhancing Chimeric Fragmentation Spectra Deconvolution Using Direct Infusion-Tandem Mass Spectrometry Across High-Resolution Mass Spectrometric Platforms.

Arina Ivanova, Wei Tang, Carsten Simon, Kai Dührkop, Sebastian Böcker, Gerd Gleixner.

RATIONALE: Direct infusion mass spectrometry (DI-MS) is a rapid analytical technique widely used in omics research and other fields. However, the complexity of DI-MS spectra frequently leads to co-fragmentation of analytes with similar m/z, resulting in chimeric fragmentation spectra that complicate compound identification. A DI-based tandem mass spectrometric method (DI-MS2), which modulates the intensity of precursors and fragments by the stepwise movement of the quadrupole isolation window, has been shown to successfully deconvolute chimeric fragmentation spectra. Yet, its applicability to different instruments and optimisation has not been evaluated.
METHOD: We evaluate the performance of DI-MS2 on two high-resolution instruments: a linear ion trap-Orbitrap (LIT-Orbitrap) and a quadrupole-Orbitrap (Q-Orbitrap). We examined the impact of six instrumental settings, including mass resolving power, isolation window width, step size between MS2 scans, number of microscans, collision energy and automatic gain control (AGC) target, on the analysis of isobaric mixtures with varying m/z differences.
RESULTS: The LIT-Orbitrap consistently achieved high-quality chimeric spectra deconvolution with an average similarity score of 0.98 despite unexpected intensity modulation patterns. The Q-Orbitrap provided four times faster measurements but showed more variable results: It achieved a similarity score of 0.96 for isobars with a m/z difference larger than 0.02, but only 0.56 for m/z differences of 0.006.
CONCLUSIONS: These findings indicate that the DI-MS2 is a robust and flexible method applicable across different MS platforms, though the Q-Orbitrap may be less suited for highly complex samples with multiple peaks per nominal mass. This highlights the potential of the DI-MS2 for structural elucidation of complex biological mixtures. Additionally, we provide initial setting optimisation guidelines to improve spectra deconvolution and measurement speed.

DOI: https://doi.org/10.1002/rcm.10170
Anal Chem. 2025 Nov 19.

The SIMPLIFY Protocol: A Monophasic Extraction System Suitable for Exposomics, Metabolomics, Lipidomics, and Proteomics Research.

Anh Hoang Nguyen, Victor Castro-Alves, Emilia Holmström Flores, João Marcos G Barbosa, Matilda Kråkström, Päivikki Reinivuori, Otto Kauko, Alex Dickens, Matej Orešič, Tuulia Hyötyläinen.

Advancing our understanding of human health and disease requires comprehensive analytical approaches capable of capturing the complex interplay between endogenous metabolism and environmental exposures. A major challenge in clinical research is the ability to capture multidimensional data, particularly a broad range of biochemical profiles, due to limitations of biological resources, time, and budget. In this study, we introduce the SIMPLIFY Protocol, a unified monophasic extraction method that enables the simultaneous extraction of chemical exogenous products and endogenous molecules. The method was evaluated against in-house extraction techniques, including protein precipitation with methanol (MeOH) and acetonitrile (ACN), and the Folch method using various sample types, particularly certified reference materials. We demonstrate that the SIMPLIFY Protocol not only performs comparably to our in-house methods but also offers enhanced versatility for additional applications such as derivatization and proteomics. The analyte abundances and reproducibility with this method strongly correlate with those from in-house-established techniques across diverse sample types. The method encompasses a broad spectrum of compounds, effectively profiling approximately 800 identified compounds, including polar compounds (e.g., amino acids), semipolar compounds (e.g., polyfluorinated compounds, bile acids, and lysophophatidylcholine), and nonpolar compounds (e.g., cholesteryl ester), with some limitations in extracting triacylglycerols. By maintaining simplified workflow and minimizing biological and resource consumption of multiple extractions, this method supports high-throughput exposomics/metabolomics and lipidomics studies. Furthermore, its streamlined design facilitates (semi)automation, making it highly suitable for large-scale clinical studies, where efficiency, cost-effectiveness, and sample availability are critical factors.

DOI: https://doi.org/10.1021/acs.analchem.5c05322
J Chromatogr A. 2025 Nov 15. pii: S0021-9673(25)00893-3. [Epub ahead of print]1766 466549

High-speed characterization and quantification of polypropylene glycol based oligomeric mixtures by hydrophilic interaction liquid chromatography at critical conditions and mass spectrometry (LCCC/Q‑TOF MS).

Sebastian Hagenhoff, Antje Wegener, Matthias Pursch.

  A rapid method for determination and characterization of polypropylene glycol diglycidylether and related oligomers (dGE-PPG) has been developed. The method is based on liquid chromatography at critical conditions coupled to high-resolution mass spectrometry (LCCC/MS). The LCCC method is operated in hydrophilic interaction liquid chromatography (HILIC) mode with acetonitrile/water at isocratic conditions using a core-shell silica column. The developed method was found to be superior to conventional size exclusion chromatographic as well as reversed phase (RP) chromatographic techniques when coupled to mass spectrometry. Mass spectral overlap between different polypropylene glycol based compound classes that leads to over- and underestimation of such could be avoided by using the new method. In contrast to most published LCCC separations, this method is based on HILIC instead of RP or normal phase (NP) liquid chromatography. It provided higher chromatographic resolution between PPG based compound classes bearing different polar end groups. A run time of less than 2 minutes is achieved with high MS sensitivity at high organic modifier content, which facilitated compound identification and quantitation. Multiple compound classes could be identified, each having a distribution of propylene glycol oligomers. The methodology was straightforward to implement, and critical conditions could be maintained over the course of several months.

Keywords:  Critical conditions; Functionality type distribution; HILIC; LC/MS; Liquid chromatography

DOI:  https://doi.org/10.1016/j.chroma.2025.466549
Anal Bioanal Chem. 2025 Nov 19.

Optimizing multiomics sample preparation: comparative evaluation of extraction protocols for HepG2 cells.

Tilman F Arnst, Selina Hemmer, Claudia Fecher-Trost, Lea Wagmann, Markus R Meyer.

  Multiomics approaches enable a comprehensive characterization of complex biological systems by simultaneously investigating multiple molecular layers. Generating multiple omics datasets from a single sample is crucial to minimize biological variability and ensure cross-layer consistency, which is critical for robust downstream data analysis. However, existing workflows often require adaptation to the specific experimental context and instrumental setup. This study systematically compared two established protocols for the simultaneous extraction of metabolites, lipids, and proteins from HepG2 cells: (i) a biphasic extraction with subsequent overnight protein digestion from the interphase pellet, and (ii) a monophasic extraction involving on-bead protein digestion. For the monophasic approach, we further investigated the effects of bead size and digestion conditions. Metabolomics samples were analyzed using liquid chromatography coupled to high-resolution tandem mass spectrometry; lipidomics and proteomics samples were analyzed by nano-scale liquid chromatography coupled with ion mobility separation and high-resolution tandem mass spectrometry. Each method was evaluated in terms of total feature count, selectivity, reproducibility, handling complexity, and overall performance. While neither protocol was optimal across all criteria, the monophasic extraction using paramagnetic beads with shortened incubation time proved to be the most reproducible, efficient, and cost-effective solution for in-house multiomics workflows in HepG2 cells.

Keywords:  HepG2 cells; Mass spectrometry; Metabolomics; Multiomics; Proteomics; Sample preparation

DOI:  https://doi.org/10.1007/s00216-025-06235-x
J Pharm Biomed Anal. 2025 Nov 17. pii: S0731-7085(25)00609-0. [Epub ahead of print]270 117268

Trace-level quantitation of N-((2-isopropylthiazol-4-yl)methyl)-N-methylnitrous amide by liquid chromatography-tandem mass spectrometry.

Sarju Adhikari, Chad Catbagan, Gavin Carr, Khawla Mustafa, Yu Zhang, Dan Li, Yi Li, Chris Foti, Alban R Pereira.

  Nitrosamines, a class of N-nitroso compounds, have raised significant health concerns due to their status as highly potent mutagenic carcinogens. Recently, the potential occurrence of the nitrosamine drug substance-related impurity, N-((2-isopropylthiazol-4-yl)methyl)-N-methylnitrous amide (NITMA), also known as N-nitroso-2,4-thiazole amine (NNTA), has been reported in some finished drug products containing ritonavir. Stringent acceptable intake (AI) limits, as low as 18 ng/day, have been recommended for NITMA by various regulatory agencies, requiring a need for highly sensitive quantitative methods. In this study, a versatile and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the detection and quantitation of NITMA in ritonavir drug substance and test formulation blends of ritonavir. NITMA was ionized and quantified in positive mode APCI using multiple reaction monitoring. The formation of NITMA from its precursor, 1-(2-isopropylthiazol-4-yl)-N-methylmethanamine (ITMA), was studied under accelerated conditions to ultimately prevent its artifactual formation during sample preparation and analysis. Adequate peak selectivity for NITMA was also studied to demonstrate the versatility of the method in the presence of high concentrations of APIs, impurities, degradation products, and excipients. The method was successfully validated as per ICH Q2(R2) guidelines over a wide concentration range (0.0225-0.27 ng/mL). The validated quantitation limit (0.0225 ng/mL) aligns with current regulatory requirements for NITMA. Overall, the newly developed method was found suitable to support risk assessment strategies and regulatory compliance.

Keywords:  LC-MS/MS; NDSRI; NITMA; NNTA; Nitrosamine; Ritonavir

DOI:  https://doi.org/10.1016/j.jpba.2025.117268
Biomed Chromatogr. 2025 Dec;39(12): e70253

A Validated UPLC-MS/MS Method for Monitoring Temozolomide and Perillyl Alcohol Metabolites in Biological Matrices: Insights From a Preclinical Pharmacokinetic Study.

Ariane Krause Padilha Lorenzett, Tatiane Patricia Babinski, Samila Horst Peczek, Ana Paula Tartari, Jeferson Ziebarth, Andressa Zago, Thais Carla Brussulo Pereira, Rubiana Mara Mainardes.

  Glioblastoma multiforme (GBM) is among the most aggressive brain tumors, largely due to the restrictive blood-brain barrier (BBB) and limited drug bioavailability. Temozolomide (TMZ), an alkylating agent, and perillyl alcohol (POH), a monoterpene with antitumor properties, have shown promise in GBM therapy. This study developed and validated a UPLC-MS/MS method for the simultaneous quantification of TMZ, its active metabolite 5-aminoimidazole-4-carboxamide (AIC), and perillic acid (PA), the primary POH metabolite, in rat plasma and tissues. The method met FDA and EMA validation criteria, showing high selectivity, linearity (R2 > 0.995), accuracy (80%-120%), precision (RSD < 15%), and matrix-specific stability across plasma, brain, liver, kidney, spleen, and lung. Following single oral doses of TMZ (2 mg/kg) and POH (47 mg/kg), pharmacokinetic analysis revealed that TMZ had the highest systemic exposure (AUC0-24h: 8173.64 ng·h/mL) and Cmax (1448.64 ng/mL), while PA showed the fastest absorption (tmax: 0.5 h). AIC levels confirmed efficient TMZ metabolism. Biodistribution analysis showed TMZ accumulation in the brain (~1000 ng/mL), supporting its CNS efficacy. PA was mainly distributed to liver and kidneys, with limited brain penetration. The validated method enables preclinical pharmacokinetic and tissue distribution studies, offering valuable insights into TMZ and POH behavior for GBM treatment.

Keywords:  bioanalytical method; bioavailability; biodistribution; glioblastoma

DOI:  https://doi.org/10.1002/bmc.70253