bims-metlip 2025-07-20 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–07–20
thirty-two papers selected by
Sofia Costa, Matterworks

Development and Validation of a UHPLC-MS/MS Method for Ciprofol Detection in Plasma: Application in Clinical Pharmacokinetic Studies.
Trimethylaminoethyl ester derivatization and stable isotope derivatization for enhanced analysis of fatty acids in biological samples by electrospray ionization tandem mass spectrometry.
Novel Equivalent Carbon Number Strategy for Large-Scale Lipidomics Data Analysis via Ultrahigh-Performance Liquid Chromatography-Orbitrap Astral Mass Spectrometry.
An evaluation methodology for machine learning-based tandem mass spectra similarity prediction.
Liquid chromatography-tandem mass spectrometry method for quantification of ertugliflozin in human plasma: Application to disposition kinetics.
Integrating LC-MS/MS and Molecular Networking for Advance Analysis and Comprehensive Flavonoids Annotation.
Accelerating biotherapeutics discovery: Blueprint for a universal LC-MALDI-MS offline approach.
Targeted metabolomics for cardiovascular disease: Validation of a high-throughput HPLC-MS/MS assay.
Mass Spectrometry Imaging.
Analytical Validation of an LC-MS/MS Method for Simultaneous Quantification of Multiple Immunosuppressants in Microvolume Whole Blood.
Recent applications of supercritical fluid chromatography in the analysis of polyphenols.
Development of LC-MS/MS and GC-MS/MS Methods for the Detection of Ethyl Glucuronide (EtG) and Ethyl Palmitate (EtPa) in Hair.
Untargeted Metabolomics of Epimastigote Forms of Trypanosoma cruzi.
Development of an HPLC-FLD Method for Estradiol and Metabolites: Application of Solid-Phase Microextraction.
A pilot study on LC-MS/MS quantification of remifentanil, etomidate, and rocuronium in maternal and fetal serum microsamples.
Targeted Gas Chromatography-Mass Spectrometry Analysis of 31 Phthalates and Replacements: Method Optimization and Application to Edible Oils and Silicone Wristbands.
Application of QSRR models for predicting the retention times of plant food bioactive compounds.
Development and validation of a RP-HPLC method for simultaneous determination of five COVID-19 antiviral drugs in pharmaceutical formulations.
SMART: An Approach for Accurate Formula Assignment in Spatially Resolved Metabolomics.
LC-MS/MS-Based Determination of Ambroxol in Human Plasma and Cerebrospinal Fluid: Validation and Applicability in a Phase II Study on GBA-Associated Parkinson's Disease Patients.
Constructing a consensus serum metabolome.
Evaluation of a New Chemical Label BASA: A Comparison with Gold Standards.
Development of a Liquid Chromatography-Tandem Mass Spectrometry Method for Quantifying Teicoplanin and Its Application in Critically Ill Patients.
Simultaneous Determination of Epimedin A1, Epimedin B, Epimedin C, Icariin, Icariside I, and Baohuoside I in Rat Plasma by UPLC-MS/MS After Oral Administration of Epimedium Total Flavonoids With Application to Pharmacokinetic Study.
UiO-66-NH2 Dispersed Solid-Phase Extraction Combined With Liquid Chromatography-Tandem Mass Spectrometry for Metabolomics Analysis in Cancer Patients and Healthy Individuals.
An innovative method for simultaneous separation and determination of monosaccharide and sugar alcohol isomers associated with glycometabolism in beagle plasma using gas chromatography-mass spectrometry.
Determination of nitrofuran metabolites in sausage casings and crawfish using LC-Q-Orbitrap HRMS: Method development and validation.
Lipidomic Profiling of Edible Japanese Sea Urchins by LC-MS.
Quantitative analysis of ethylene precursor ACC in plant samples by liquid chromatography-tandem mass spectrometry.
Sustainable Microextraction Using Switchable Solubility Solvent for the Liquid Chromatographic Determination of Three Profenoid Drugs in Urine Samples.
Machine Learning-Based Retention Time Prediction Tool for Routine LC-MS Data Analysis.
LipidCruncher: An open-source web application for processing, visualizing, and analyzing lipidomic data.

Drug Des Devel Ther. 2025 ;19 5821-5833

Development and Validation of a UHPLC-MS/MS Method for Ciprofol Detection in Plasma: Application in Clinical Pharmacokinetic Studies.

Jiaxi Zhu, Ying Cao, Xingan Zhang, Bo Xu.

   Objective: This study aimed to develop and validate an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the quantification of ciprofol in human plasma, with the goal of applying this method to pharmacokinetic studies in patients undergoing elective surgery under general anesthesia.
Methods: A methanol-based protein precipitation method was employed for sample preparation, using ciprofol-d6 as the internal standard. Chromatographic separation was achieved on a Shimadzu Shim-pack GIST-HP C18 column (3 µm, 2.1×150 mm) with a mobile phase consisting of 5 mmol·L⁻¹ ammonium acetate (A) and methanol (B). The flow rate was maintained at 0.4 mL·min⁻¹, and the column temperature was set at 40°C. Detection was performed using electrospray ionization (ESI) in negative ion mode with multiple reaction monitoring (MRM). The quantification ion pairs were m/z 203.100→175.000 for ciprofol and m/z 209.100→181.100 for the internal standard.
Results: Ciprofol exhibited excellent linearity across the concentration range of 5 to 5000 ng·mL⁻¹ (r > 0.999). The intra-batch and inter-batch precision values were within 4.30% to 8.28%, and the relative deviation ranged from -2.15% to 6.03%. The extraction recovery rate was 87.24% to 97.77%, and the matrix effect relative standard deviation (RSD) was less than 15%.
Conclusion: The developed UHPLC-MS/MS method is simple, rapid, accurate, and highly specific, making it suitable for the determination of ciprofol plasma concentrations and pharmacokinetic studies in clinical settings. This method provides a reliable analytical tool for future research on ciprofol in complex biological matrices.

Keywords:  UHPLC-MS/MS; ciprofol; pharmacokinetics

DOI:  https://doi.org/10.2147/DDDT.S518616
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jul 10. pii: S1570-0232(25)00287-9. [Epub ahead of print]1264 124733

Trimethylaminoethyl ester derivatization and stable isotope derivatization for enhanced analysis of fatty acids in biological samples by electrospray ionization tandem mass spectrometry.

Liu Yang, Qingchun Wang, Weiwei Chen, Ji Yang, Yu Lin, David M Lubman.

   BACKGROUND: Abnormal fluctuations in free fatty acids (FFAs) are associated with cardiovascular diseases. However, conducting a thorough analysis of individual FFAs via mass spectrometry has historically posed challenges due to their low ionization efficiency and the absence of distinctive fragment ions.
RESULTS: In this study, we introduce a method utilizing paired stable isotope derivatization coupled with liquid chromatography-triple quadrupole mass spectrometry (ID-LC-QQQ-MS) for thorough identification and relative quantification of fatty acids in serum samples. This method involves the derivatization of the carboxyl groups of FFAs using a pair of isotope reagents, resulting in the formation of FA trimethylaminoethyl ester (FA-TMAE-h3/d3), which can yield two distinct neutral fragments with masses of 59 and 62 Da during collision-induced dissociation (CID). Consequently, a quadruple neutral loss scan (QNLS) approach was utilized for the non-targeted profiling of FFAs in serum samples. The derivative peak pairs displaying matching retention times and distinct mass differences were extracted from the two QNLS spectra and recognized as potential FFAs. Subsequently, a multiple reaction monitoring (MRM) detection protocol was established for the relative quantification of fatty acids in the serum of Syrian Golden Hamsters subjected to various treatments, utilizing a pooled sample labeled with a heavy isotope as an internal standard. Partial least squares discriminant analysis (PLS-DA) revealed notable variations in these 23 fatty acids across the four groups.
SIGNIFICANCE: The current stable isotope derivatization (ID) method, in conjunction with tandem mass spectrometry (MS/MS) analysis, stands out as a promising approach for identifying and quantifying FFAs in real samples.

Keywords:  ESI-MRM-MS; ESI-QNLS-MS; Free fatty acids quantification; Serum samples; Stable isotope derivatization

DOI:  https://doi.org/10.1016/j.jchromb.2025.124733
Anal Chem. 2025 Jul 17.

Novel Equivalent Carbon Number Strategy for Large-Scale Lipidomics Data Analysis via Ultrahigh-Performance Liquid Chromatography-Orbitrap Astral Mass Spectrometry.

Yao Chen, Xinxin Wang, Rui Zhong, Zixuan Wang, Ziquan Fan, Tian Liu, Qi Li, Yaorui Ye, Chunxiu Hu, Guowang Xu.

Lipidomics enables studying lipid alterations in physiological or pathological states. The new Orbitrap mass spectrometry (MS), equipped with an Astral analyzer, significantly increases MS/MS acquisition rate. However, simultaneous analysis of lipid retention behavior and structural annotation from this data remains challenging. In this study, we propose a comprehensive strategy using the Equivalent Carbon Number (ECN) model to integrate the advantages of MS-DIAL (providing precise lipid retention behavior) and LipidSearch (offering accurate MS/MS spectral information). By investigating 34 lipid standards spiked into the NIST SRM 1950 plasma sample, the ECN strategy demonstrated high accuracy in retention time prediction with relative standard deviations below ± 5% for 90.0% of lipids in positive ion mode and 100% in negative ion mode. False-positive data from LipidSearch 5.1 were also significantly reduced; for example, in yeast, 68.8% and 80.1% of false positives were removed in the positive and negative ion modes, respectively. A total of 1933, 1539, 1969, 985, and 2786 lipids were annotated with the ECN strategy in HeLa cells, NIST plasma, mouse liver tissues, Saccharomyces cerevisiae yeast, and their pooled sample and were analyzed by Astral-MS, respectively. It was also found that the numbers of annotated lipids from Astral-MS data preprocessed with LipidSearch and MS-DIAL were 3-5 and 2-4 times higher than those from QE-MS data, respectively. This strategy enables efficient and accurate lipid identification with precise retention times and reliable MS/MS annotation, advancing large-scale lipidomics research and offering broad application in diverse biological contexts.

DOI: https://doi.org/10.1021/acs.analchem.5c01863
BMC Bioinformatics. 2025 Jul 11. 26(1): 174

An evaluation methodology for machine learning-based tandem mass spectra similarity prediction.

Michael Strobel, Alberto Gil-de-la-Fuente, Mohammad Reza Zare Shahneh, Yasin El Abiead, Roman Bushuiev, Anton Bushuiev, Tomáš Pluskal, Mingxun Wang.

   BACKGROUND: Untargeted tandem mass spectrometry serves as a scalable solution for the organization of small molecules. One of the most prevalent techniques for analyzing the acquired tandem mass spectrometry data (MS/MS) - called molecular networking - organizes and visualizes putatively structurally related compounds. However, a key bottleneck of this approach is the comparison of MS/MS spectra used to identify nearby structural neighbors. Machine learning (ML) approaches have emerged as a promising technique to predict structural similarity from MS/MS that may surpass the current state-of-the-art algorithmic methods. However, the comparison between these different ML methods remains a challenge because there is a lack of standardization to benchmark, evaluate, and compare MS/MS similarity methods, and there are no methods that address data leakage between training and test data in order to analyze model generalizability.
RESULT: In this work, we present the creation of a new evaluation methodology using a train/test split that allows for the evaluation of machine learning models at varying degrees of structural similarity between training and test sets. We also introduce a training and evaluation framework that measures prediction accuracy on domain-inspired annotation and retrieval metrics designed to mirror real-world applications. We further show how two alternative training methods that leverage MS specific insights (e.g., similar instrumentation, collision energy, adduct) affect method performance and demonstrate the orthogonality of the proposed metrics. We especially highlight the role that collision energy plays in prediction errors. Finally, we release a continually updated version of our dataset online along with our data cleaning and splitting pipelines for community use.
CONCLUSION: It is our hope that this benchmark will serve as the basis of development for future machine learning approaches in MS/MS similarity and facilitate comparison between models. We anticipate that the introduced set of evaluation metrics allows for a better reflection of practical performance.

Keywords:  Benchmark; Machine learning; Mass spectrometry; Metabolomics; Spectral similarity measure

DOI:  https://doi.org/10.1186/s12859-025-06194-1
Eur J Mass Spectrom (Chichester). 2025 Jul 15. 14690667251359789

Liquid chromatography-tandem mass spectrometry method for quantification of ertugliflozin in human plasma: Application to disposition kinetics.

Viritha Bezawada, Padma Mogili, Sireesha Dodda, Srinivasa Rao Polagani.

  A high-performance liquid chromatography-tandem mass spectrometry method has been developed for the quantification of ertugliflozin in human plasma, employing ertugliflozin D5 as the internal standard. Methyl tertiary butyl ether-based liquid-liquid extraction technique was employed, followed by chromatographic separation on Kromasil-C18 (100 × 4.6 mm, 5 µm) column using a mixture of methanol and 10 mM ammonium formate buffer (80:20, v/v) as the mobile phase at a flow rate of 1 mL/min. The mass transitions were observed from m/z 437.4 to 329.2 for ertugliflozin and from m/z 442.2 to 334.3 for ertugliflozin D5 by multiple reaction monitoring in a positive ion electro spray ionization source. The linearity was established in the concentration range of 1-500 ng/mL, with the correlation coefficient, r2 > 0.99. Validation of the method was performed as per US FDA guidelines, and the results were found well within the acceptance limits. The method was applied successfully for the pharmacokinetic study of ertugliflozin 15 mg after a single oral dose under fasting conditions in healthy male volunteers. The Cmax and tmax values obtained were 288.28 ng/mL and 1.32 h, respectively. Authentication of the results was further done by the incurred sample reanalysis.

Keywords:  Ertugliflozin; bioanalysis; incurred sample reanalysis; liquid chromatography-tandem mass spectrometry/mass spectrometry; liquid-liquid extraction; pharmacokinetic study

DOI:  https://doi.org/10.1177/14690667251359789
J Sep Sci. 2025 Jul;48(7): e70230

Integrating LC-MS/MS and Molecular Networking for Advance Analysis and Comprehensive Flavonoids Annotation.

Priscillia Ann David, Anis Irfan Norazhar, Mohamad Shazeli Che Zain.

  Flavonoids, a subclass of phenolic compounds, exhibit diverse therapeutic properties, including antioxidant, anti-inflammatory, and wound healing properties. These health-promoting effects of flavonoids are greatly dependent on the variation in their structural diversity, which are generally perceived as complex metabolomic datasets. Among detection techniques used, state-of-the-art high-resolution liquid chromatography hyphenated with tandem mass spectrometry (LC-MS/MS) has become a popular analytical method of choice for the analysis of flavonoids from various plant tissue extracts. Despite its broad applicability, the vast amount and complexity of fragmentation data produced have made the comprehensive identification of flavonoids remains a key challenge. An offshoot of metabolomics, currently, molecular networking (MN), a computational approach based on MS/MS data, has emerged as a revolutionary technique for identifying and characterizing numerous flavonoid molecular families. By visualizing the spectral similarities of flavonoid fingerprints, MN enables rapid dereplication, efficient in assisting annotation of unknown features with known chemical scaffolds, and demonstrates high precision in resolving structurally diverse flavonoid isomers. Various MN tools, i.e., classical molecular networking (CLMN), feature-based molecular networking (FBMN), and substructure-based MN (MS2LDA), streamline the identification process and improve the understanding of flavonoids biosynthesis. This review aimed to describe the recent advancement in MS-based strategy for flavonoids characterization, starting with an overview on the application of LC-MS/MS and its limitation in the typical dereplication workflow, followed by specific sections on MN techniques, highlighting the aspects of general principles, workflow, and its application in flavonoid research.

Keywords:  flavonoids; isomers; mass spectrometry; molecular networking

DOI:  https://doi.org/10.1002/jssc.70230
Anal Chim Acta. 2025 Sep 22. pii: S0003-2670(25)00706-8. [Epub ahead of print]1368 344312

Accelerating biotherapeutics discovery: Blueprint for a universal LC-MALDI-MS offline approach.

Elizabeth F Bayne, Xiong-Zhuo Gao, Yuan Jiang, Raffeal Bennett, Erik L Regalado, Gregory F Pirrone, Alexey A Makarov.

   BACKGROUND: Biotherapeutics are a prominent class of molecules used to treat a wide range of diseases, including cancer, infections, inflammatory and immune disorders. The discovery workflow involves high-throughput screening of critical quality attributes using liquid chromatography (LC) to optimize cell line development and facilitate drug candidate selection. However, several LC techniques, such as size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC), are not directly compatible with electrospray ionization (ESI), hindering mass spectrometry (MS)-based identification of impurity peaks. Consequently, significant time and effort are often required to adapt these method conditions to ESI-MS, which slows down discovery efforts.
RESULTS: Matrix-assisted laser desorption ionization (MALDI) is a surface-based ionization technique that offers an attractive alternative to ESI for high-throughput screening due to its rapid analysis times and resistance to buffer/matrix interferences. MALDI-MS is highly compatible with automated liquid handling tools for sample transfer and requires minimal sample volume. To address the increasingly complex needs and challenging timelines of next-generation biologics development, we have developed an LC-MALDI-MS offline approach for rapid peak identification using an LC-fraction collector and MALDI-TOF-MS. This streamlined approach is adaptable to multiple chromatographic techniques, regardless of their inherent MS compatibility, and is suitable for automation. The workflow is versatile and can be applied universally in combination with various chromatographic techniques and analytes, including monoclonal and bispecific antibodies, fusion proteins, and peptides. Importantly, fraction collection and MS data acquisition can be completed within hours without the need for additional method development or sample preparation for MS-based measurements.
SIGNIFICANCE: Herein, we systematically demonstrate the use of an automated LC-MALDI-MS workflow for rapid peak identification involving SEC, HIC, and Reversed Phase LC for proteins and peptides, as well as a use case with a bispecific antibody and fusion protein using SEC.

Keywords:  Antibody-drug conjugates; Biotherapeutics; Bispecific antibodies; LC Fraction collector; MALDI-TOF MS; Peptides

DOI:  https://doi.org/10.1016/j.aca.2025.344312
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jul 09. pii: S1570-0232(25)00286-7. [Epub ahead of print]1264 124732

Targeted metabolomics for cardiovascular disease: Validation of a high-throughput HPLC-MS/MS assay.

Sabina N Baskhanova, Natalia E Moskaleva, Ksenia M Shestakova, Maria V Kozhevnikova, Ekaterina O Korobkova, Victor M Samoylov, Svetlana A Appolonova.

  Cardiovascular diseases (CVD) remain a leading cause of mortality worldwide, necessitating innovative diagnostic tools to improve early detection and management. This study represents the optimization and validation of a high-throughput HPLC-MS/MS method for simultaneous quantification of 98 metabolites in human plasma, including amino acids and its derivatives (n = 29), tryptophan pathway metabolites (n = 17), nucleosides (n = 4), water-soluble vitamins (n = 3), acylcarnitines (n = 39), and others (n = 6). The method utilizes chemical derivatization to enhance retention and sensitivity of polar metabolites providing accurate analysis across diverse physicochemical properties. The presented method was validated in accordance with EMA guidelines and included assessment of linearity, accuracy, precision, matrix effects, recovery, and stability. Parallelism testing confirmed the suitability of a surrogate matrix for calibration. The method was applied for the analysis of plasma samples from 399 patients with cardiovascular diseases and 75 healthy controls, revealing significant metabolic alterations in pathways associated with inflammation, nitric oxide metabolism, and mitochondrial function. The presented comprehensive approach may serve as a rapid screening method for the identification of selective CVD biomarkers using targeted metabolomic profiling.

Keywords:  Cardiovascular diseases; Liquid chromatography - tandem mass spectrometry (LC-MS/MS); Metabolomics; Validation

DOI:  https://doi.org/10.1016/j.jchromb.2025.124732
Anal Chem. 2025 Jul 17.

Mass Spectrometry Imaging.

Aljoscha Körber, Ian G M Anthony, Ron M A Heeren.

Mass spectrometry imaging (MSI) maps the spatial distributions of chemicals on chemically complex surfaces. MSI offers unrivaled sensitivity and information density with each pixel comprising a mass spectrum. Over the past three decades, numerous technological developments have enabled MSI to evolve into a mainstream technique for untargeted molecular and elemental imaging with wide-spread applications ranging from material analysis to life sciences and clinical diagnostics. Here, we review the field of MSI with a focus on key technological advancements. We examine different image acquisition modes and the most popular ionization methods in MSI, including matrix-assisted laser desorption/ionization (MALDI), laser ablation inductively coupled plasma (LA-ICP), laser ablation electrospray ionization (LAESI), secondary ion mass spectrometry (SIMS), and desorption electrospray ionization (DESI). For each method, we discuss figures of merit, such as spatial resolving power and sensitivity, the ionization mechanism, sample preparation, advantages, and disadvantages, including ways to overcome them wherever applicable. We subsequently discuss more aspects of MSI instrumentation, such as commonly used mass analyzers, tandem mass spectrometry, ion mobility, and advancements in imaging throughput. Based on these technological developments, targeted MSI strategies are explained, including imaging mass cytometry (IMC), multiplexed ion beam imaging (MIBI), and stable isotope labeling (SIL), as well as approaches for multimodal imaging. Last, we present selected application examples of MSI in cancer research, single cell analysis, and drug distribution studies. We target this review to provide researchers with an interest in recent developments in MSI with a concise technological understanding of the different main approaches to MSI.

DOI: https://doi.org/10.1021/acs.analchem.4c05249
Int J Mol Sci. 2025 Jul 01. pii: 6358. [Epub ahead of print]26(13):

Analytical Validation of an LC-MS/MS Method for Simultaneous Quantification of Multiple Immunosuppressants in Microvolume Whole Blood.

Kenichi Aizawa, Natsuka Kimura, Takahiro Goda, Sho Nishida, Yasunaru Sakuma, Daiki Iwami, Ryozo Nagai.

  Immunosuppressants are essential for preventing allograft rejection; however, they require therapeutic drug monitoring to maintain efficacy and to prevent severe complications such as opportunistic infections. Calcineurin inhibitors (CIs) are primarily distributed in red blood cells, whereas mycophenolic acid (MPA) and its metabolites are found in plasma. These differences necessitate separate analyses for each drug, increasing laboratory workload, analytical complexity, and patient burden. We developed a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of CIs such as tacrolimus (Tac), everolimus (Eve), sirolimus (Sir), cyclosporine A (CycA) and MPA in 2.8-µL whole-blood samples, with a hematocrit-based correction to estimate plasma-equivalent MPA concentrations. Performance of this method was assessed by comparison with conventional immunoassay results using linear regression and Bland-Altman analyses, demonstrating excellent agreement, with strong linearity (R2 > 0.995) at <2 to 35 ng/mL for three CIs, 26.0 to 1866 ng/mL for CycA, and 0.1 to 50 μg/mL for MPA. Furthermore, MPA and tacrolimus concentrations closely aligned with routine clinical results (R2 > 0.900), indicating high accuracy and reproducibility. This new approach may be particularly beneficial for hospitalized patients with limited venous access, pediatric populations, and in remote care settings where frequent blood sampling is challenging because of simultaneous quantification and fewer sample volume requirements.

Keywords:  LC-MS/MS immunosuppressants; hematocrit correction; microvolume whole blood; simultaneous analysis

DOI:  https://doi.org/10.3390/ijms26136358
J Chromatogr A. 2025 Jul 07. pii: S0021-9673(25)00544-8. [Epub ahead of print]1758 466198

Recent applications of supercritical fluid chromatography in the analysis of polyphenols.

Enrico Taglioni, Anna Laura Capriotti, Andrea Cerrato, Aldo Laganà, Carmela Maria Montone, Susy Piovesana, Chiara Cavaliere.

  Polyphenols are a large group of plant secondary metabolites, synthesized mostly through the phenylpropanoid pathway, and they comprise flavonoids, stilbenes, lignans, and simple phenolic acids. The characterization of these natural compounds is challenging, due to the complexity of plant extracts, the variety of polyphenols forms in the various plant organs, and the limited availability of authentic standards. The analysis of polyphenols is mostly achieved by reversed-phase liquid chromatography (LC), but poor retention and resolution are often observed for the polar ones. Supercritical fluid chromatography (SFC) represents a versatile chromatographic technique, and a valid and greener alternative to traditional LC, suitable for a heterogeneous class of compounds as polyphenols, where very polar or chiral compound separation is requested. The advancements in instrumental technology allowed the coupling with mass spectrometry (MS) and the introduction of sub-2 µm stationary phases, and paved the way to the use of SFC for the analysis of these compounds. The present review provides an overview of the latest SFC methods for the characterization of polyphenols, over the period 2019-2024. Coupling with MS and applications in plants are mostly discussed in this review, along with other selected applications suitable to critically describe the state-of-the-art and potential of this technique in the field of polyphenol characterization. The survey indicated that SFC is still underutilized in this field, particularly in combination with MS, mainly because of the need for extensive optimization of several critical parameters. Most of the described works are quantitative targeted studies, and only a few reports use metabolomics approaches for large-scale analysis of polyphenols. As a result, SFC applications have been limited to a small range of polyphenols rather than complex mixtures, making this technique still not competitive with LC. Nonetheless, SFC has potential, especially in filling the existing gap of LC for analysis, as better characterizations can be obtained for polar and chiral polyphenols.

Keywords:  Polar natural compounds; SFC; convergence chromatography; flavonoids; phenolic acids; polyphenols

DOI:  https://doi.org/10.1016/j.chroma.2025.466198
Molecules. 2025 Jun 21. pii: 2681. [Epub ahead of print]30(13):

Development of LC-MS/MS and GC-MS/MS Methods for the Detection of Ethyl Glucuronide (EtG) and Ethyl Palmitate (EtPa) in Hair.

Sharnette Ashiru, Ethan Webster, Benjamin Barrett, Mathew Wade, Brian Rooney.

  Alcohol abuse is a widespread addiction globally, leading to long-term health issues and social consequences. Ethyl glucuronide (EtG) and ethyl palmitate (EtPa) are frequently requested by local authorities, solicitors, or private individuals to assess long-term chronic excessive alcohol consumption. In this paper, we present a validation process aimed at developing sensitive methods for detecting EtG and EtPa in hair samples. EtG was extracted by overnight sonication in water followed by sample clean-up using solid phase extraction (SPE) and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). EtPa was extracted using a simple ultrasonication extraction followed by analysis using gas chromatography-tandem mass spectrometry (GC-MS/MS). The analytical method was validated by assessing linearity, precision, accuracy, recovery, sensitivity, and selectivity. Both EtG and EtPa methods obtained a coefficient of determination (r2) above 0.999 across concentration ranges of 4, 8, 16, 24, 48, and 96 pg/mg and 120, 240, 360, 480, 600, and 720 pg/mg. Extraction recoveries were both close to 100% with stable retention times and proven sensitivity and selectivity. These methods were validated according to the standards set by the United Kingdom Accreditation Service (UKAS) Lab51 and ISO 17025.

Keywords:  ethyl glucuronide (EtG); ethyl palmitate (EtPa); hair testing; method validation

DOI:  https://doi.org/10.3390/molecules30132681
Bio Protoc. 2025 Jul 05. 15(13): e5368

Untargeted Metabolomics of Epimastigote Forms of Trypanosoma cruzi.

Michel Augusto Silva, Mario Izidoro, Bruno Souza Bonifácio, Sergio Schenkman.

  Trypanosoma cruzi, the causative agent of Chagas disease, faces significant metabolic challenges due to fluctuating nutrient availability and oxidative stress within its insect vector. Metabolomic techniques, such as gas chromatography-mass spectrometry (GC-MS), have been widely used to study the adaptive mechanisms of the parasite. This article describes a standardized method for the untargeted metabolomics analysis of T. cruzi epimastigote, covering parasite cultivation, sample deproteinization with methanol, metabolite extraction, derivatization with BSTFA, and GC-MS analysis. To ensure robustness and reproducibility, statistical analysis uses univariate tests, as well as multivariate approaches such as principal component analysis (PCA) and partial least squares (PLS) regression. The protocol offers a reliable and sensitive method to study metabolic responses in T. cruzi under environmental stress, with low biological variability and high reproducibility. Key features • GC-MS was used to conduct a standardized metabolomics investigation of Trypanosoma cruzi epimastigote, assuring reproducibility and minimum biological variability. • Includes sample deproteinization, metabolite extraction, and derivatization with BSTFA for accurate metabolite profiling under different biological conditions. • Employs robust statistical approaches (PCA, PLS) to investigate differences among experimental groups and detect significant alterations in metabolism. • Internal standards and multiple replicates ensure high sensitivity and repeatability, which is excellent for investigating metabolic processes in protozoan parasites.

Keywords:  Bioinformatics; GC-MS; Metabolism; Metabolite; Metabolomics; Protozoan; T. cruzi

DOI:  https://doi.org/10.21769/BioProtoc.5368
Int J Mol Sci. 2025 Jun 27. pii: 6194. [Epub ahead of print]26(13):

Development of an HPLC-FLD Method for Estradiol and Metabolites: Application of Solid-Phase Microextraction.

Anna Kaliszewska, Piotr Struczyński, Tomasz Bączek, Lucyna Konieczna.

  Estrogens are potent hormones involved in numerous physiological and pathological processes. Their typically low concentrations in biological samples necessitate highly sensitive analytical methods for accurate quantification. This study presents a high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method for quantifying estradiol and its metabolites in blood serum and saliva. Analytes were extracted using solid-phase microextraction with a divinylbenzene sorbent and methanol as the desorption agent. FLD was performed after the derivatization of the analytes with dansyl chloride. Separation was achieved on a Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 µm) at 50 °C using water with 0.1% formic acid and methanol as the mobile phase at 0.5 mL/min. A gradient elution increased the methanol concentration from 76% to 100% over 0-8 min, then it returned to 76% at 8.1 min and was held until 11 min had passed. Detection was at λEX 350 nm and λEM 530 nm. Good linearity was observed for estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol (10-300 ng/mL; R2 = 0.9893-0.9995). The LOQ for all analytes was 10 ng/mL. Solid-phase microextraction (SPME) offered advantages over liquid-liquid extraction. The method is suitable for quantifying estrogens in the 10 ng/mL-1 µg/mL range.

Keywords:  derivatization procedure; estrogen; fluorescence detection

DOI:  https://doi.org/10.3390/ijms26136194
Sci Rep. 2025 Jul 16. 15(1): 25876

A pilot study on LC-MS/MS quantification of remifentanil, etomidate, and rocuronium in maternal and fetal serum microsamples.

Hao Liu, Meng Cai, Yong Peng, Jing-Kun Miao, Jin Yu.

  While remifentanil, etomidate, and rocuronium are increasingly used for cesarean section due to their favorable hemodynamic stability and fetal safety profile, their pharmacokinetics and potential effects on neonates remain poorly understood. This study developed and validated a rapid, sensitive LC-MS/MS method for simultaneous quantification of the three anesthetics in microsamples of 10 μl serum, followed by a paired maternal and umbilical cord serum investigation. After protein precipitation with acetonitrile, analytes were separated within 4 min using positive electrospray ionization in MRM mode. Method validation demonstrated excellent linearity (R2 > 0.99) for all compounds, with LLOQs of 0.15 ± 0.02 ng/mL (remifentanil), 16.87 ± 0.51 ng/mL (etomidate), and 106.73 ± 8.63 ng/mL (rocuronium). Precision (intra-/inter-day < 15%) and minimal carry-over (< 5%) met bioanalytical standards. Applied to 20 maternal-newborn pairs, the method quantified differential drug distribution: maternal arterial concentrations (remifentanil 4.75 ± 0.19 ng/mL; etomidate 412.71 ± 35.29 ng/mL; rocuronium 7.08 ± 0.48 μg/mL) exceeded umbilical vein levels (2.43 ± 0.13 ng/mL; 302.15 ± 29.03 ng/mL; 0.86 ± 0.16 μg/mL), which were higher than umbilical artery concentrations (1.33 ± 0.15 ng/mL; 166.24 ± 21.53 ng/mL; 0.44 ± 0.77 μg/mL). Calculated placental transfer rates significantly differed among anesthetics (remifentanil 0.52 ± 0.02; etomidate 0.75 ± 0.04; rocuronium 0.13 ± 0.02; all P < 0.001), reflecting distinct pharmacokinetic behaviors. The validated method enables reliable microvolume analysis for perinatal pharmacokinetic studies, particularly valuable when sample availability is limited. Its rapid throughput and sensitivity make it suitable for clinical research applications investigating maternal-fetal drug transfer dynamics.

Keywords:  Anesthetics; Caesarean section; LC–MS/MS; Newborn

DOI:  https://doi.org/10.1038/s41598-025-09454-5
J Sep Sci. 2025 Jul;48(7): e70227

Targeted Gas Chromatography-Mass Spectrometry Analysis of 31 Phthalates and Replacements: Method Optimization and Application to Edible Oils and Silicone Wristbands.

Kaley T Adams, Caoilinn Haggerty, Richard P Scott, Steven O'Connell, Kim A Anderson.

  Interest in phthalate detection of foods and other environmental media has grown rapidly in the past decade. However, current analytical and separation techniques are often limited in the breadth of chemistry targeted, most often targeting less than 15 compounds. Challenges to successful methods with this compound group include chromatographic resolution, quantitation across diverse concentration ranges, and sample preparation due to the chemical similarity of these compounds. This project describes the development of a selective ion monitoring gas chromatography mass spectrometry method for quantitation of 29 phthalates and two phthalate replacements along with considerations for quantitation, sample cleanup, and standard storage. Our range of phthalates includes less-studied ones like bis(2-propylheptyl), diundecyl, didecyl, and ditridecyl. Analytical performance included limits of detection ranging from 17-230 ng/mL and robust reproducibility with relative percent differences below 8% for complex matrices. Two calibration ranges were used to accommodate the wide dynamic range of phthalate concentrations observed in real samples. Method application was demonstrated with edible oils (n = 12) and silicone wristbands (n = 18), representing dietary and personal exposure pathways. Sample preparation strategies, including solid phase extraction were evaluated to mitigate matrix interferences. In addition, compound storage stability was assessed over 133 days to inform best practices for standard preparation and handling. The finalized method demonstrates the uniquely large compound ranges for some phthalates and the importance of analyzing a wide variety of these compounds, making it a valuable foundation for comprehensive environmental monitoring of phthalates and their alternatives.

Keywords:  GC‐MS; SPE; alternatives; gas chromatography; mass spectrometry; phthalates; silicone wristbands

DOI:  https://doi.org/10.1002/jssc.70227
J Chromatogr A. 2025 Jul 05. pii: S0021-9673(25)00540-0. [Epub ahead of print]1758 466194

Application of QSRR models for predicting the retention times of plant food bioactive compounds.

Bakhtyar Sepehri, Viviana Consonni, Davide Ballabio, Enmanuel Cruz Muñoz, Ehsan Abbasi, Roberto Todeschini.

  Liquid chromatography (LC) coupled to mass spectrometry (MS) is a powerful and versatile technique with several applications in analytical chemistry such as the untargeted analysis of complex mixtures of plant food bioactive compounds. In this framework, identification of compounds mainly relies on mass and fragmentation patterns derived from MS spectra of available databases. However, to develop automated tools for a rapid compound identification, prior knowledge of retention times (RTs) has been demonstrated to be a valid support to MS data to reduce the number of possible candidate structures. Unlike experimental methods, which are time-consuming and limited to a few classes of compounds, data-driven computational approaches can predict retention times for a wide range of compounds using only their molecular structures and physicochemical properties. In this research, Genetic Algorithms (GAs) coupled to Multiple Linear Regression (MLR) were applied to select the most relevant molecular descriptors to establish quantitative structure-retention relationships (QSRRs) aimed at predicting the retention times of plant food bioactive compounds across three different LC chromatographic systems. The statistical parameters showed model robustness and satisfactory predictive ability. Particular attention was paid to measuring the uncertainty of predictions and assessing their reliability based on the model applicability domain. Interpretation of the selected molecular descriptors provided valuable insight into the separation mechanism. Finally, the developed models were applied to predict the unknown retention times, for the three studied LC chromatographic systems, of a large library of plant food bioactive compounds, which were made freely available to further assist the research in the field of natural products.

Keywords:  Data sharing; FooDB database; HPLC; Molecular descriptors; Plant food bioactive compounds; PredRet database; QSRR; Retention time

DOI:  https://doi.org/10.1016/j.chroma.2025.466194
Sci Rep. 2025 Jul 15. 15(1): 25470

Development and validation of a RP-HPLC method for simultaneous determination of five COVID-19 antiviral drugs in pharmaceutical formulations.

Mohamed W Nassar, Ahmed Serag, Mohamed A Hasan, Mohamed Kamel.

A rapid, sensitive, and selective reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous determination of five COVID-19 antiviral drugs: favipiravir, molnupiravir, nirmatrelvir, remdesivir, and ritonavir. The chromatographic separation was achieved on a Hypersil BDS C18 column (4.5 × 150 mm, 5 μm particle size) using an isocratic mobile phase consisting of water and methanol (30:70 v/v, pH 3.0 adjusted with 0.1% ortho-phosphoric acid) at a flow rate of 1 mL/min with UV detection at 230 nm. The optimized method demonstrated good chromatographic resolution with retention times of 1.23, 1.79, 2.47, 2.86, and 4.34 min for favipiravir, molnupiravir, nirmatrelvir, remdesivir, and ritonavir, respectively. The method was validated according to ICH guidelines, showing linearity in the concentration range of 10-50 µg/mL with correlation coefficients (r²) ≥ 0.9997 for all analytes. The limits of detection were 0.415-0.946 µg/mL, while the limits of quantification were 1.260-2.868 µg/mL. The method demonstrated high trueness (99.59-100.08%) and precision (RSD < 1.1%). The validated method was successfully applied to the determination of these drugs in their pharmaceutical formulations, with recovery values ranging from 99.98 to 100.7% and no significant interference from excipients. Comprehensive greenness and practicality evaluations using five assessment tools yielded favorable scores: AGREE (0.70), AGREEprep (0.59), MoGAPI (70%), BAGI (82.5), and CACI (79), indicating good environmental performance and excellent practical applicability for routine pharmaceutical quality control analysis. The multi-tool assessment confirmed the method's environmental friendliness through strategic solvent selection and minimal sample preparation requirements, while demonstrating superior practical implementation characteristics including cost-effectiveness and accessibility in standard analytical laboratories.

DOI: https://doi.org/10.1038/s41598-025-09904-0
Anal Chem. 2025 Jul 14.

SMART: An Approach for Accurate Formula Assignment in Spatially Resolved Metabolomics.

Yinghao Cao, Shengxi Li, Zhaoyi Liu, Zixian Jia, Weidong Zhuang, Xu Pan, Jinyu Zhou, Lifeng Yang, Lin Wang.

Spatially resolved metabolomics plays a critical role in unraveling tissue-specific metabolic complexities. Despite its significance, this profound technology generates thousands of features, yet accurate annotation significantly lags behind that of LC-MS-based approaches. To bridge this gap, we introduce SMART, an open-source platform designed for precise formula assignment in mass spectrometry imaging. SMART constructs a KnownSet database containing 2.8 million formulas linked by ChemEdges derived from repositories such as HMDB, ChEMBL, PubChem, and BioEdges from KEGG biological reactant pairs. Using a multiple linear regression model, SMART extracts formula networks associated with the m/z of interest and scores potential candidates based on several criteria, including linked formulas, database existence score, ChemEdges/BioEdges, and ppm values. Benchmarking against reference data sets demonstrates that SMART achieves prediction accuracy rates of up to 95.8%. Applied to mass spectrometry imaging, SMART successfully annotated 986 formulas in developing mouse embryos. This robust platform enables systematic formula annotation within tissues, enhancing our understanding of metabolic heterogeneity.

DOI: https://doi.org/10.1021/acs.analchem.4c06210
Int J Mol Sci. 2025 Jun 25. pii: 6094. [Epub ahead of print]26(13):

LC-MS/MS-Based Determination of Ambroxol in Human Plasma and Cerebrospinal Fluid: Validation and Applicability in a Phase II Study on GBA-Associated Parkinson's Disease Patients.

Valentina Franco, Michela Palmisani, Fabiana Colucci, Rosa De Micco, Simone Aloisio, Federico Cazzaniga, Silvia Cerri, Francesca Crema, Francesca Dattrino, Barbara Garavaglia, Matteo Gastaldi, Pierfrancesco Mitrotti, Fabio Moda, Paola Rota, Rita Stiuso, Cristina Tassorelli, Roberto Eleopra, Alessandro Tessitore, Enza Maria Valente, Micol Avenali, Roberto Cilia.

  Heterozygous mutations in the GBA1 gene, encoding the enzyme glucocerebrosidase (GCase), are major risk factors for Parkinson's Disease (PD). Ambroxol, a small chaperone originally used as a mucolytic agent, has been shown to cross the blood-brain barrier, enhance GCase activity, and reduce α-synuclein levels, making it a promising therapeutic candidate for disease-modifying effects in GBA1-associated PD (GBA1-PD). This study aimed to develop a method to quantify ambroxol levels in human plasma and cerebrospinal fluid (CSF) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ambroxol was determined by online solid-phase extraction (SPE), coupled with LC-MS/MS, by gradient elution on a monolithic column. Detection employed a 3200 QTRAP tandem mass spectrometer in the positive electrospray ionization mode. Calibration curves exhibited linearity across the analyzed ranges in both plasma and CSF. The recovery rate ranged from 106.7% to 113.5% in plasma and from 99.0% to 103.0% in CSF. No significant matrix effect was observed. Intra-day and inter-day precisions were below 11.8% in both matrices, and accuracy ranged from 89.9% to 103.1% in plasma and from 96.3% to 107.8% in CSF. We evaluated and confirmed the stability of the analyte in plasma and CSF across various storage conditions. The method was successfully validated according to European Medicine Agency (EMA) guidelines and its applicability was confirmed in the context of a multicenter, randomized, double-blind, placebo-controlled, phase II study, designed to monitor the ambroxol levels in the plasma and CSF of GBA1-PD.

Keywords:  LC-MS/MS; Parkinson’s Disease; ambroxol; cerebrospinal fluid; glucocerebrosidase; plasma

DOI:  https://doi.org/10.3390/ijms26136094
bioRxiv. 2025 May 11. pii: 2025.05.07.652782. [Epub ahead of print]

Constructing a consensus serum metabolome.

Yuanye Chi, Joshua M Mitchell, Maheshwor Thapa, Shujian Zheng, Zackary Frohock, Yi Li, Aleksandr Smirnov, Xiuxia Du, Shuzhao Li.

Blood analysis is the most common in biomedical applications and a reference metabolome will be critical for effective annotation and for guiding scientific investigations. However, compiling such a reference is hindered by many technical challenges, despite the availability of large amount of metabolomics data today. Based on a new set of data structures and tools, we have assembled a consensus serum metabolome (CSM) from over 100,000 mass spectrometry acquisitions of more than 200 million spectra. This provides a comprehensive survey of human blood chemistry, revealing the frequency dependent nature of metabolome and exposome. Major gaps are found between CSM and the current databases. The CSM enables community-level data alignment and significantly improves annotation quality of LC-MS metabolomics.
Highlights: A reference of human biochemistry linked to observation frequencyMajor gaps revealed in current databases and experimental methodsEnabling cross-laboratory, cross-platform data alignmentAccelerated and cumulative metabolite annotation.

DOI: https://doi.org/10.1101/2025.05.07.652782
Anal Chem. 2025 Jul 13.

Evaluation of a New Chemical Label BASA: A Comparison with Gold Standards.

Jérémy Monteiro, Karen Ple, Antoine Lefèvre, Hamid Marzag, Benjamin Blondeau, Camille Dupuy, Adeline Oury, Sylvain Routier, Lydie Nadal-Desbarats, Patrick Emond.

Personalized medicine involves tailoring medical treatments to the unique characteristics of each individual. For example, genomic insights enable precise diagnoses and targeted therapies in oncology. However, significant challenges remain in understanding the molecular mechanisms underlying many diseases. Metabolomics, the study of small molecules within biological systems, holds great promise for advancing personalized medicine through molecular-level insights. By combining derivatization and LC-MS, it is possible to achieve metabolite identification, enhancing both the sensitivity and coverage of analyses. In this study, we introduce BASA as a derivatization reagent. After identifying the specific fragmentation patterns of BASA-derivatized compounds, a library of approximately one hundred molecules was derivatized to compare BASA with four commercial chemical labels. In addition to its versatility, BASA allows for sample analysis in negative mode, further increasing analytical sensitivity. Negative mode presents an opportunity for untargeted analysis of biological samples due to reduced background noise. Combined with specific fragments, BASA-derived compounds seem promising for metabolomic exploration. Further studies are needed to fully demonstrate their contributions to exploring complex biological environments.

DOI: https://doi.org/10.1021/acs.analchem.5c00096
Infect Dis Clin Microbiol. 2025 Jun;7(2): 195-207

Development of a Liquid Chromatography-Tandem Mass Spectrometry Method for Quantifying Teicoplanin and Its Application in Critically Ill Patients.

Hasan Memiş, Ahmet Çakır, Zeynep Ülkü Gün, Hatice Saraçoğlu, Çiğdem Karakükcü, Aliye Esmaoğlu, Zafer Doğan.

   Objective: Teicoplanin, a glycopeptide antibiotic, is used to treat infections caused by Gram-positive pathogens. Trough-level monitoring of teicoplanin is recommended in specific -patient populations, including critically ill patients. This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify teicoplanin in human plasma and adapt the method to a critically ill patient sample.
Materials and Methods: Teicoplanin trough levels were measured using a newly validated LC-MS/MS method. Analysis was conducted using a C18 column with an inner diameter of 2.7 μm (50.0 x 3.0 mm), and vancomycin hydrochloride was used as the internal standard. The method's run time per sample was 5.5 minutes. Non-parametric tests were used for statistical analysis. Univariate and multivariate logistic regression were performed to identify teicoplanin target attainment factors. A p-value of <0.05 was considered statistically significant.
Results: The method demonstrated linearity between 1.56-100 mg/L teicoplanin concentration and had a lower limit of detection and quantification of 0.33 mg/L and 1.00 mg/L, respectively. Precision, accuracy, recovery rate, and carry-over effects were all within acceptable limits, according to the U.S. Food and Drug Administration (FDA) guidance. Twenty patients were included in the study. The target teicoplanin trough level (≥10 mg/L) attainment rate was 50%. The patient's laboratory values did not significantly change after teicoplanin treatment (p>0.05), except for erythrocyte count, haemoglobin, and haematocrit values, which decreased significantly (p<0.05). Multivariate analysis revealed no significant factors affecting target attainment (p>0.05).
Conclusion: The LC-MS/MS assay validated in this study is high-throughput, robust, and quick enough to be implemented in clinical therapeutic drug monitoring (TDM) laboratories. More large-scale studies are needed to understand better the relationship between teicoplanin trough levels and patient-related factors.

Keywords:  clinical pharmacists; critical illness; drug monitoring; intensive care units; liquid chromatography-tandem mass spectrometry; teicoplanin

DOI:  https://doi.org/10.36519/idcm.2025.528
Biomed Chromatogr. 2025 Aug;39(8): e70167

Simultaneous Determination of Epimedin A1, Epimedin B, Epimedin C, Icariin, Icariside I, and Baohuoside I in Rat Plasma by UPLC-MS/MS After Oral Administration of Epimedium Total Flavonoids With Application to Pharmacokinetic Study.

Shuang Liang, Wentao Zhang, Jinlin Li, Danlei Li, Hongwei Zhang, Yijia Ma, Ziwei Luo, Xiaodong Huang.

  In this study, a UPLC-Orbitrap-HRMS method was employed to analyze the components of epimedium total flavonoids (ETF), as well as the prototypes and metabolites absorbed into rat plasma following oral administration. Furthermore, a rapid and sensitive UPLC-MS/MS method was developed for simultaneous quantification of epimedin A1, epimedin B, epimedin C, icariin, icariside I, and baohuoside I in rat plasma following oral administration of ETF. Plasma samples were precipitated using methanol-acetonitrile (1:1, v/v), with extraction recovery > 80% for all analytes. Chromatographic separation was achieved on an ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm) with gradient elution comprising water containing 0.1% formic acid (A) and methanol-acetonitrile (1:1, v/v, B), delivered at a flow rate of 0.3 mL/min. The method demonstrated excellent linearity (1.0-500 ng/mL) with correlation coefficients (r) more than 0.9950. Method validation revealed satisfactory precision and accuracy: intraday precision (RSD < 12.32%) with accuracy (-8.83% to 10.00%) and interday precision (RSD < 10.30%) with accuracy (-13.00% to 11.09%). The validated method was successfully applied to pharmacokinetic investigations, revealing critical disposition patterns of these flavonoids post-ETF administration. This study provides novel insights into the in vivo disposition of ETF, aiding in explaining the mechanisms underlying its effectiveness and toxicity of this herbal preparation.

Keywords:  Epimedium total flavonoids; absorbed components; method validation; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.70167
J Sep Sci. 2025 Jul;48(7): e70217

UiO-66-NH2 Dispersed Solid-Phase Extraction Combined With Liquid Chromatography-Tandem Mass Spectrometry for Metabolomics Analysis in Cancer Patients and Healthy Individuals.

Piao Liu, Changhong Li, Minqi Ou, Chang Yin, Xinyue Kang, Jun Chen, Ran Liu, Jialang Zhuang.

  A dispersive solid-phase extraction process was established using UiO-66-NH2 as an adsorbent, in combination with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technology, and the performance of this combination for the metabolomic analysis of biological specimens was assessed. Differential metabolites in plasma samples from oral squamous cell carcinoma and hepatocellular carcinoma patients and healthy volunteers were screened, and metabolic pathway enrichment analysis was performed via pseudo-targeted metabolomics. Based on the significantly enriched metabolic pathways and related literature, 20 amino acids were selected and targeted for quantitative analysis. The key parameters for extraction recovery, including the type of liquid in the adsorption system, pH of the eluent, amount of UiO-66-NH2, and time of extraction and elution, were optimized based on the selected amino acids. The optimized method demonstrated a remarkable performance: correlation coefficients > 0.99, limits of detection ranging from 0.002 to 0.080 µg/mL (signal-to-noise ratio = 10), and recoveries of 63.9%-110.8% in plasma samples. Thus, via targeted metabolomics analysis, 10 amino acids (including lysine and 3-iodo-L-tyrosine) and 10 amino acids (including arginine and histidine) were determined as potential biomarkers for oral squamous cell carcinoma and hepatocellular carcinoma, respectively. Based on the above potential amino acid markers, discriminant equations for oral squamous cell carcinoma and hepatocellular carcinoma were established, respectively. Two biomarker combinations were successfully screened: lysine and 3-iodo-L-tyrosine (AUC = 0.998, sensitivity = 0.967, specificity = 1.0) for oral squamous cell carcinoma, and histidine and arginine (AUC = 0.944, sensitivity = 0.833, specificity = 0.933) for hepatocellular carcinoma. Our findings will provide new directions for cancer diagnosis.

Keywords:  biomarkers; hepatocellular carcinoma; metabolomics; oral squamous cell carcinoma

DOI:  https://doi.org/10.1002/jssc.70217
Anal Chim Acta. 2025 Sep 22. pii: S0003-2670(25)00720-2. [Epub ahead of print]1368 344326

An innovative method for simultaneous separation and determination of monosaccharide and sugar alcohol isomers associated with glycometabolism in beagle plasma using gas chromatography-mass spectrometry.

Haihui Ren, Wen Xiao, Jiameng Han, Qiaorong Liu, Zunjian Zhang, Yuan Tian.

   BACKGROUND: Tracking changes in plasma concentrations of monosaccharides and sugar alcohols related to glycometabolism, which plays a role as the main source of energy, is of great significance for the treatment of metabolic diseases. However, analysis of monosaccharides and sugar alcohols has always been a difficult task due to the high polarity of the polyhydroxy structure and the presence of a large number of isomers. So far, no suitable method has been developed for simultaneous determination of monosaccharides and sugar alcohols in beagle plasma.
RESULTS: This work performed an innovative gas chromatography-mass spectrometry method for the simultaneous chromatographic separation and absolute quantification of 11 monosaccharides and sugar alcohols associated with glycometabolism. After derivatization, monosaccharides converted to be oxime acetylated derivatives and sugar alcohols transformed into acetylated derivatives. All analytes achieved good separation after chromatographic conditions were optimized, especially the selection of mid-polarity capillary column(6 % cyanopropyl phenyl and 94 % dimethyl polysiloxane). The improvement of derivatization efficiency through single factor and response surface optimization ensured a high sensitivity, with LOQ of each analyte in the range of 0.2-50 ng/mL. The established method was fully validated in terms of linearity and range, sensitivity, accuracy and precision, recovery, matrix effect, stability, dilution integrity and proved to be reliable, which was applied to the comparative study on hypoglycemic effect of two antidiabetic agents with different pharmacological mechanisms, dorzagliatin and metformin, as well as possible metabolic pathways affected by them.
SIGNIFICANCE: This method solves the problems of multiple peaks after derivatization and frequent co-elution of analytes in previous analytical methods, significantly enhances derivatization efficiency of ketoses, which provides a promising approach for simultaneous analysis of aldose, ketose, and sugar alcohol. It offers new insights into a more comprehensive research on glycometabolism rather than merely focusing on glucose.

Keywords:  Derivatization; GC-MS; Glycometabolism; Isomer; Monosaccharide; Sugar alcohol

DOI:  https://doi.org/10.1016/j.aca.2025.344326
Toxicol Rep. 2025 Dec;15 102076

Determination of nitrofuran metabolites in sausage casings and crawfish using LC-Q-Orbitrap HRMS: Method development and validation.

Omar Khaled, Lamia Ryad, Nermine Gad, Fawzy Eissa.

  This study developed and validated an analytical method for determining four nitrofuran (NF) metabolites in sausage casings and crawfish matrices. The method utilizes liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry (LC-Q-Orbitrap HRMS) to analyze these metabolites. The validation of the developed method was carried out in accordance with the Commission Implementing Regulation (CIR) EU 2021/808, using three concentration levels ranging from 0.25 to 0.75 μg/kg. The recoveries ranged from 77 % to 109 %, while both repeatability and reproducibility remained consistently below 15 %. The calibration curves exhibited good linearity, with correlation coefficients (R) exceeding 0.9978. The limits of detection (LOD) ranged from 0.0218 to 0.0596 μg/kg, while the limits of quantification (LOQ) ranged from 0.0719 to 0.1966 μg/kg. The decision limit (CCα) and detection capability (CCβ) values ranged from 0.30 to 0.39 μg/kg and 0.29-0.35 μg/kg, respectively. Out of 30 crawfish samples collected from local markets in Egypt, 16.66 % contained NF metabolites residues, whereas sausage casings showed no NF metabolite residues. The reliability of the method was further demonstrated through successful participation in two proficiency testing (PT) rounds.

Keywords:  AHD; AMOZ; AOZ; Food safety; Nitrofuran metabolites; Residue determination; SEM

DOI:  https://doi.org/10.1016/j.toxrep.2025.102076
Foods. 2025 Jun 26. pii: 2268. [Epub ahead of print]14(13):

Lipidomic Profiling of Edible Japanese Sea Urchins by LC-MS.

Sahana Amai, Kisara Yuki, Siddabasave Gowda B Gowda, Divyavani Gowda, Shu-Ping Hui.

  Sea urchins (Echinoidea) are marine echinoderms commonly consumed as seafood in East Asia. To date, various metabolic components of sea urchins have been analyzed, and their health benefits for humans have also been attracting attention. Lipids are the major biomolecules present in sea urchins. However, the comprehensive lipid profiling of sea urchins is limited. In this study, we aimed to perform the comprehensive lipid profiling of six types of sea urchins using liquid chromatography-mass spectrometry (LC/MS). The application of untargeted lipidomics led to the identification of 281 lipid molecular species in six varieties of fresh sea urchin gonads. Each lipid metabolite was identified based on its retention time and MS/MS fragmentation pattern. The results of the analysis showed the highest abundance of lipid percentage in Kitamurasakiuni (14.3%), followed by Hokuyobafununi (12.4%). In all the analyzed sea urchins, glycerolipids such as triacylglycerols were found to be the most abundant lipid components. Multivariate analysis revealed that Murasakiuni showed a different lipid profile from the other types. Interestingly, the polyunsaturated fatty acid to saturated fatty acid ratios and health-related nutritional indices factors were found to be higher in Hokuyobafununi compared to other varieties. The ω-3 fatty acids, such as docosapentaenoic acid (FA 22:6) and eicosapentaenoic acid (FA 20:5), were also abundant in Hokuyobafununi. Lipids such as ether and N-acyl-type lysophosphatidylethanolamines were detected for the first time in sea urchins. This study highlights the nutritional significance of sea urchins and their potential use in the development of functional foods.

Keywords:  lipidomics; liquid chromatography; mass spectrometry; sea urchin

DOI:  https://doi.org/10.3390/foods14132268
BMC Plant Biol. 2025 Jul 17. 25(1): 920

Quantitative analysis of ethylene precursor ACC in plant samples by liquid chromatography-tandem mass spectrometry.

Jianhua Tong, Wenkui Zhao, Keming Wang, Feiying Zhu, Danyi Deng, Langtao Xiao.

   BACKGROUND: Ethylene (C2H4) is a gaseous phytohormone that regulates various plant physiological processes and mediates the responses of the plants to various environmental stresses. 1-Aminocyclopropane-1-carboxylic acid (ACC) is the direct precursor of the phytohormone ethylene, and is also considered as a plant growth regulator. Accurate quantification of ACC is critically important in investigating its function. However, it remains challenging to accurately quantify ACC in plant tissues because it is a small, electroneutral molecule with very low concentrations.
METHODS: An easy, cost-saving, and highly efficient quantitative method for ACC in plant tissues was set up by liquid-liquid micro-extraction (LLME) purification with green solvent ethyl-acetate, and the precise control of the mobile phase entering into the mass spectrometer combined with the ultra-high performance liquid chromatography electrospray ionization-triple quadrupole mass spectrometer (UHPLC-ESI-MS/MS).
RESULTS: The contents of ACC in 10 mg of different fresh fruits were detected. The established method had limit of detection (LOD) (2.5 pg), matrix effect (ME) (92.6%), good precision (3.54%), recovery rate (95.82%), and a good linear relationship within the range of 0.5 to 1500 ng.mL-1 (R2 = 0.9998).
CONCLUSION: We have developed an easy, sensitive, and cost-saving quantitative method for ACC in plant tissues without derivatization which is useful for the precise quantification of ACC in plant tissues.

Keywords:  1-Aminocyclopropane-1-carboxylic acid; Ethylene; Liquid-liquid micro-extraction; Phytohormone; Precise control of the mobile phase; Tandem mass spectrometry

DOI:  https://doi.org/10.1186/s12870-025-06943-7
J Sep Sci. 2025 Jul;48(7): e70223

Sustainable Microextraction Using Switchable Solubility Solvent for the Liquid Chromatographic Determination of Three Profenoid Drugs in Urine Samples.

Christina Patakidou, Marianna Ntorkou, Constantinos K Zacharis.

  Herein, a switchable solubility solvent (sodium salicylate) was employed for the microextraction of "profenoid" drugs (i.e., ketoprofen, fenoprofen, and flurbiprofen) from human urine samples. This approach utilizes the phase transition of salicylic acid (0.75 mol/L) by altering the sample's pH (addition of 10 µL of H3PO4 10 M), facilitating efficient dispersion and phase separation in a single step. The solidified salicylic acid was easily collected using a syringe filter (nylon). In addition, the extensive contact area between the solidified solvent and the sample solution ensured effective extraction of the target analyte. The critical experimental parameters affecting the extraction performance of the analytes were examined. The separation of the drugs was carried out on a C18 analytical column using gradient elution. Excellent linearity was observed in the dynamic range of 50-3000 ng/mL while the precision (%RSD) was less than 14.3% in all cases. The intraday and interday trueness were satisfactory, being in the range of 82.3%-110.1%. The green potential of the proposed analytical scheme was examined based on AGREEprep, ComplexMoGAPI, CACI, and AGREE metric tools. The developed method was utilized for the analysis of authentic urine samples after oral administration of a ketoprofen-containing formulation.

Keywords:  HPLC; microextraction; non‐steroidal anti‐inflammatory drug; switchable solubility solvent; urine

DOI:  https://doi.org/10.1002/jssc.70223
J Chem Inf Model. 2025 Jul 16.

Machine Learning-Based Retention Time Prediction Tool for Routine LC-MS Data Analysis.

Sofiia A Dymura, Oleksandr O Viniichuk, Kostiantyn P Melnykov, Dmytro S Radchenko, Oleksandr O Grygorenko.

Accurate retention time (RT) prediction models can significantly improve liquid chromatography-mass spectrometry (LC-MS) data analysis widely used in chemical synthesis. As hundreds of thousands of syntheses are performed annually at Enamine, a large amount of experimental data has been generated internally. In this paper, we present the development of an RT prediction model based on the GATv2Conv + DL graph neural network (NN) architecture, trained on the internal data and further evaluated using the METLIN SMRT data set. The final model achieved a mean absolute error (MAE) of 2.48 s for the 120 s LC-MS method. We also conducted a detailed analysis of RT prediction errors and determined that the interval between RT - 7.12 s and RT + 9.58 s contained over 95% of the data. The developed model has been successfully integrated into the existing in-house LC-MS analysis toolkit, enhancing its predictive and analytical capabilities. Additionally, we have published a curated subset of 20,000 data points from our internal data set to support community benchmarking and further research.

DOI: https://doi.org/10.1021/acs.jcim.5c00514
bioRxiv. 2025 May 01. pii: 2025.04.28.650893. [Epub ahead of print]

LipidCruncher: An open-source web application for processing, visualizing, and analyzing lipidomic data.

Abdi Hamed, Yohannes A Ambaw, Chandramohan Chitraju, Shubham Singh, Zon Weng Lai, Robert V Farese, Tobias C Walther.

Advances in mass spectrometry (MS)-based lipidomics have led to a surge in data volume, underscoring a need for robust tools to evaluate and visualize these data comprehensively. Current workflows are often hampered by manual spreadsheet handling and insufficient assessment of data quality prior to analysis. Here, we introduce LipidCruncher , an open-source, web-based platform designed to process, visualize, and analyze lipidomic data with high efficiency and rigor. LipidCruncher consolidates key steps of the workflow, including data standardization, normalization, and stringent quality control to identify anomalies. The platform also provides advanced visualization and analysis tools, such as volcano plots, lipid saturation profiles, pathway mapping, and lipid heatmaps, that enable detailed and holistic data exploration. To demonstrate LipidCruncher 's utility, we analyzed lipidomic data from adipose tissue of mice lacking the triacylglycerol synthesis enzymes DGAT1 and DGAT2. We anticipate that LipidCruncher will be a valuable and user-friendly tool for standardizing and analyzing lipidomics data.

DOI: https://doi.org/10.1101/2025.04.28.650893