bims-metlip 2025-03-16 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–03–16
23 papers selected by
Sofia Costa, Matterworks

Enhanced lipidomics workflows for plasma and extracellular vesicles through advanced liquid chromatography-tandem mass spectrometry integrated.
Pioneering Human Plasma Detection of Haloperidol With 5-pg/mL Sensitivity via Refined Sample Preparation and Advanced LC-MS/MS.
Profiling neuroactive steroids in pregnancy. A non-derivatised liquid chromatography tandem mass spectrometry method for the quantitation of allopregnanolone and four related isomers in maternal serum.
Automated Sequential Derivatization for Gas Chromatography-[Orbitrap] Mass Spectrometry-based Metabolite Profiling of Human Blood-based Samples.
imzML Writer: An Easy-to-Use Python Pipeline for Conversion of Continuously Acquired Raw Mass Spectrometry Imaging Data to imzML Format.
High-Throughput Solid Phase Extraction for Targeted and Nontargeted Exposomics.
A sensitive reverse-phase ultra performance liquid chromatography-tandem mass spectrometry method for the determination of tobramycin in Pseudomonas aeruginosa cell lysate.
Critical assessment of quenching and extraction/sample preparation methods for microorganisms in metabolomics.
Accuracy, linearity, and statistical differences in comparative quantification in untargeted plant metabolomics using LC-ESI-Orbitrap-MS.
A Comprehensive Review of Instrumentation and Applications in Post-Column and In-Source Derivatization for LC-MS.
Development and validation of a UPLC-MS/MS method for almonertinib with its active metabolite HAS-719 and anlotinib in human plasma.
The development of a bioanalytical method for the simultaneous analysis of gentamicin and tacrolimus in Rat whole blood using UHPLC-MS/MS.
Development and validation of a HPLC-MS/MS method the determination of genistein and equol in serum, urine and follicular fluid.
Tear Sampling and Biomarker Discovery: A Robust Workflow for Routine Clinical Applications Using UHPLC-MS/MS and Schirmer Strips.
Simultaneous quantification of four urinary biomarkers related to oxidative stress using UHPLC-QqQ-MS/MS.
Application of silica monoliths for improved storage stability of metabolites in human plasma.
Rapid Proteomic Amelogenin Sex Estimation of Human and Cattle Remains Using Untargeted Evosep-timsTOF Mass Spectrometry.
FIORA: Local neighborhood-based prediction of compound mass spectra from single fragmentation events.
Reference Library for Suspect Non-targeted Screening of Environmental Toxicants Using Ion Mobility Spectrometry-Mass Spectrometry.
Direct comparison of single peak and gradient chromatographic methods for routine analysis of surfactants in biopharmaceuticals.
Green liquid-liquid microextraction for quantification of ketamine and metabolites in human urine by gas chromatography-mass spectrometry.
Quantitative method for intestinal short chain fatty acids based on stable isotope labeling combined with liquid chromatography-mass spectrometry.
Advancing atherosclerosis research: The Power of lipid imaging with MALDI-MSI.

Talanta. 2025 Mar 01. pii: S0039-9140(25)00337-6. [Epub ahead of print]291 127847

Enhanced lipidomics workflows for plasma and extracellular vesicles through advanced liquid chromatography-tandem mass spectrometry integrated.

Adriana F L Vilela, Miguel R Patrício, Pedro Nobre-Azevedo, Jonatan C S de Carvalho, Thiago V Defelippo-Felippe, Nathan N H Pontes, Daniel L Rodrigues, Bianca T M Oliveira, Pedro V da Silva-Neto, Viviani Nardini, Ana P M Fernandes, Fausto Almeida, Lucia H Faccioli, Carlos A Sorgi.

  Lipidomics, a subfield of metabolomics, provides comprehensive analysis of lipids in biological systems and is essential for biomedical research, driven by advances in analytical technologies. Lipids are crucial biomolecules in cellular functions and have been increasingly recognized for their role in physiological and pathological processes. This study focuses on advanced strategies for the development, validation, and implementation of untargeted lipidomics methods in human plasma and extracellular vesicles (EVs) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Method validation demonstrated excellent accuracy (precision and trueness) (81-120 % of nominal value), precision with inter-day repeatability below 20 %, limits of quantification ranging from 0.25 to 25 μM, and recovery rates exceeding 80 % for most lipid classes, as well as matrix effects. Plasma samples were used as a proof-of-concept study, and the method was ultimately applied to human macrophage-derived EVs. Lipid extraction utilized four liquid-liquid extraction methods to ensure broad lipid class coverage, high recovery, and repeatability. Additionally, we demonstrated that a sonication-assisted homogenization step effectively facilitates lipid extraction from EVs. Through untargeted lipidomics, our study identifies and quantifies a diverse range of lipid species in human plasma (225 lipids analytes) and macrophage-derived EVs (124 lipids analytes) within different classes. Overall, we present sophisticated approaches that combine pre-analytical lipid extraction techniques with high-resolution LC-MS/MS to enhance lipidomics research. This approach enhances the characterization of lipid profiles and their biological implications, paving the way for applications in personalized medicine and the discovery of novel lipid biomarkers associated with EVs biogenesis.

Keywords:  Extracellular vesicles; LC-MS/MS; Lipid-extraction; Lipidomics

DOI:  https://doi.org/10.1016/j.talanta.2025.127847
Biomed Chromatogr. 2025 Apr;39(4): e70048

Pioneering Human Plasma Detection of Haloperidol With 5-pg/mL Sensitivity via Refined Sample Preparation and Advanced LC-MS/MS.

Santosh Tawari, Ujashkumar Shah.

  The present study develops, refines, and validates an LC-MS/MS technique to detect haloperidol in human plasma with outstanding sensitivity (5 pg/mL), selectivity, specificity, fast analysis time, and low sample volume to enhance mental treatment regimens. Haloperidol and haloperidol D4 were tested on a Kromasil C18 stationary phase with a 35:65 ratio of 10-mM ammonium trifluoroacetate buffer to acetonitrile. A Strata-X PRO cartridge extracted the analyte and IS with improved sample extraction. ESI with multiple reaction monitoring measured haloperidol (Q1/Q3: 376.1/165.0) and D4 (Q1/Q3: 380.1/169.0). This method has high sensitivity (5.03 pg/mL), extraction efficiency (> 95%), rapid analysis (3 min), and a small sample volume (100 μL). The correction coefficient (r2) > 0.980 linearized the process from 5.03 to 6020.75 pg/mL. Nominal accuracy was 95.40%-102.52%, while intraday precision (% CV) was 0.92%-5.33%. Additionally, interday accuracy ranged from 95.40% to 102.66% and precision from 2.05% to 5.73%. This bioanalytical method for haloperidol detection in human plasma shows great sensitivity, selectivity, and linearity with an LLOQ of 5.03 pg/mL. It improves pharmacokinetics, bioequivalence, and scale-up bioavailability. Being precise, stable, and adaptable are all advantages in clinical and therapeutic monitoring.

Keywords:  LC–MS/MS; haloperidol; haloperidol D4; human plasma; method validation

DOI:  https://doi.org/10.1002/bmc.70048
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Feb 27. pii: S1570-0232(25)00093-5. [Epub ahead of print]1256 124541

Profiling neuroactive steroids in pregnancy. A non-derivatised liquid chromatography tandem mass spectrometry method for the quantitation of allopregnanolone and four related isomers in maternal serum.

Edward Hinchliffe, Alexander Heazell.

  Neuroactive steroids are metabolites of progesterone, synthesised during pregnancy by the placenta. Here, we describe development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantitation of allopregnanolone, pregnanolone, isopregnanolone, epipregnanolone and allopregnan-20α-ol-3-one in maternal serum. Following addition of deuterated internal standards, 200 μL of serum was subjected to solid phase extraction. Chromatography was performed using a pentafluorophenyl column, and LC-MS/MS on a Sciex 6500+. Sample injection volume was 20 μL, and injection-to-injection time 10.0 min. The assay was validated according to published guidelines; assay linearity and lower limit of quantification were suitable for analysis of each steroid in maternal serum, for all analytes mean recoveries were 100 % ± 15 %, intra- and inter-assay imprecision <15 %, and matrix effects negligible, and specificity experiments confirmed nil interference from a wide range of endogenous metabolites of progesterone. The method was applied to human serum samples obtained from a large cohort of third trimester pregnancies which were subsequently characterised by normal fetal and maternal outcomes, and relationships between maternal neuroactive steroid concentrations and fetal gestational age assessed. Positive correlations between maternal serum concentration and fetal gestational age were observed for isopregnanolone, allopregnanolone and allopregnan-20α-ol-3-one. The LC-MS/MS method offers significant advantages over previously published approaches for quantitation of neuroactive steroids in human maternal serum, notably obviating the need for derivatisation, whilst achieving exceptional specificity. Characterisation of normal maternal neuroactive steroid concentrations will aid future research as dysregulated placental progesterone metabolism is observed in pregnancies with poor outcomes.

Keywords:  Allopregnanolone; Epipregnanolone; Isopregnanolone; Liquid chromatography tandem mass spectrometry; Neuroactive steroids; Pregnanolone

DOI:  https://doi.org/10.1016/j.jchromb.2025.124541
Bio Protoc. 2025 Mar 05. 15(5): e5196

Automated Sequential Derivatization for Gas Chromatography-[Orbitrap] Mass Spectrometry-based Metabolite Profiling of Human Blood-based Samples.

Akrem Jbebli, Kateřina Coufalíková, Moira Zanaboni, Manuela Bergna, Renzo Picenoni, Jana Klánová, Elliott J Price.

  Many small molecules require derivatization to increase their volatility and to be amenable to gas chromatographic (GC) separation. Derivatization is usually time-consuming, and typical batch-wise procedures increase sample variability. Sequential automation of derivatization via robotic liquid handling enables the overlapping of sample preparation and analysis, maximizing time efficiency and minimizing variability. Herein, a protocol for the fully automated, two-stage derivatization of human blood-based samples in line with GC-[Orbitrap] mass spectrometry (MS)-based metabolomics is described. The protocol delivers a sample-to-sample runtime of 31 min, being suitable for better throughput routine metabolomic analysis. Key features • Direct and rapid methoximation on vial followed by silylation of metabolites in various blood matrices. • Measure ~40 samples per 24 h, identifying > 70 metabolites. • Quantitative reproducibility of routinely measured metabolites with coefficients of variation (CVs) < 30%. • Requires a Thermo ScientificTM TriPlusTM RSH (or comparable) autosampler equipped with incubator/agitator, cooled drawer, and automatic tool change (ATC) station equipped with liquid handling tools. Graphical overview Workflow for profiling metabolites in human blood using automated derivatization.

Keywords:  Automation; Derivatization; Gas chromatography–mass spectrometry (GC–MS); Metabolite profiling; Thermo ScientificTM TriPlusTM RSH

DOI:  https://doi.org/10.21769/BioProtoc.5196
Anal Chem. 2025 Mar 14.

imzML Writer: An Easy-to-Use Python Pipeline for Conversion of Continuously Acquired Raw Mass Spectrometry Imaging Data to imzML Format.

Joseph Monaghan, Kiera Nguyen, Nicholas Woytowich, Alora Keyowski, Kyle D Duncan.

Mass spectrometry imaging (MSI) is a technique that uncovers the contextual distribution of biomolecules in tissue. This involves collecting large data sets with information-rich mass spectra in each pixel. To streamline image processing and interpretation, the MSI community has developed toolboxes for image preprocessing, segmentation, statistical analysis, and visualization. These generally require data to be input as imzML files, an Extensible Markup Language file with vocabulary for mass spectrometry and imaging-specific parameters. While commercial systems (e.g., MALDI) come with proprietary file converters, to our knowledge, no open-access user-friendly converters exist for continuously acquired imaging data (e.g., nano-DESI, DESI). Here, we present imzML Writer, an easy-to-use Python application with a graphical user interface to convert data from vendor format into pixel-aligned imzML files. We package this application with imzML Scout, allowing visualization of the resulting file(s) and batch export of ion images across a range of image and data formats (e.g., PNG, TIF, CSV). To demonstrate the utility of files generated by imzML Writer, we processed nano-DESI data with popular tools such as Cardinal MSI and METASPACE. Overall, this work provides a simple open-access tool for emerging MSI modality users to access advanced MSI processing tools reliant on imzML format. ImzML Writer is available as a distributable Python package via pip or as a standalone program for Mac and PC at https://github.com/VIU-Metabolomics/imzML_Writer.

DOI: https://doi.org/10.1021/acs.analchem.4c06520
Anal Chem. 2025 Mar 13.

High-Throughput Solid Phase Extraction for Targeted and Nontargeted Exposomics.

Yunyun Gu, Max Lennart Feuerstein, Benedikt Warth.

Characterizing the chemical exposome relies on advanced instrumentation including tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS), and nontargeted analysis (NTA) using high-resolution MS. However, proper sample pretreatment, balancing broad analyte coverage, method robustness, and throughput remain a major bottleneck in exposomics. Here, we developed a robust and scalable solid phase extraction (SPE) protocol for human urine and plasma and optimized it for a panel of 94 highly diverse environmental and food-related contaminants (LogP -0.7 to 6.8). Extraction recoveries (RE) and signal suppression and enhancement (SSE) were determined using targeted LC-MS/MS. Acceptable REs (60-140%) were achieved for >70% of analytes, and acceptable SSE values (60-140%) for 86% and 90% in urine and plasma, respectively. Subsequently, the method was transferred to 96-well plate format, significantly improving throughput to meet the capacity requirements needed for exposome-wide association studies (ExWAS). The established workflow is approximately 10× faster than routinely used metabolomics-based protein precipitation approaches when comparing the estimated total analysis time for 1000 samples. The method's applicability for NTA and suspect screening was tested and compared to a generic protein precipitation protocol using NIST standard reference materials for urine (SRM 3672) and plasma (SRM 1950). Favorable performance was shown for the protein precipitation workflow while the SPE protocol demonstrated promising results. The developed workflow is thus not only superior for future high-throughput targeted exposomics but also offers an option for NTA applications. The presented well-balanced approach is scalable and also applicable to research in the fields of pharmacology, food safety, and systems toxicology.

DOI: https://doi.org/10.1021/acs.analchem.4c06177
J Pharm Biomed Anal. 2025 Feb 26. pii: S0731-7085(25)00084-6. [Epub ahead of print]260 116743

A sensitive reverse-phase ultra performance liquid chromatography-tandem mass spectrometry method for the determination of tobramycin in Pseudomonas aeruginosa cell lysate.

Woravimol Krittaphol, Lois W Martin, Iain L Lamont, Greg F Walker.

  Measurement of antibiotic accumulation in bacteria is essential for full understanding of the mechanisms of antimicrobial resistance but requires a highly sensitive analytical assay. A suitable ultra performance reverse phase liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) method was developed in the electrospray negative ionisation mode for quantifying tobramycin in Pseudomonas aeruginosa cell extracts. P. aeruginosa cell lysate extracts were prepared and spiked with tobramycin and kanamycin (internal standard). Following a protein precipitation extraction procedure, the sample was applied to a reverse phase C18 column, equilibrated in 10 mM ammonium hydroxide at pH 11. Tobramycin and kanamycin were eluted using an acetonitrile gradient and detected in the electrospray negative ionisation mode. The retention times for kanamycin and tobramycin were 1.9 and 2.5 min, respectively and total run time of 10 min. The assay demonstrated linearity in the range of 0.02 - 1 µg mL-1 (R = 0.9999), with limits of detection and quantitation at 1.42 ng mL-1 and 10 ng mL-1, respectively. The precision, expressed as the coefficient of variation, ranged from 1.4 % to 6.5 %, and the accuracy, expressed as bias, ranged from 0.4 % to 17.1 % (ICH, 1996). Using a simple protein precipitation method, the recoveries (%) of tobramycin from cell lysate supernatant for quality controls were 99.7-105.2 % and this result shows that the assay is quantitative. This validated analytical protocol will facilitate future studies aimed at determining the cellular uptake kinetics of tobramycin by P. aeruginosa under various controlled conditions and it could be used for other applications.

Keywords:  Cell lysates supernatant; Kanamycin; Pseudomonas aeruginosa; Tobramycin; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jpba.2025.116743
Metabolomics. 2025 Mar 13. 21(2): 40

Critical assessment of quenching and extraction/sample preparation methods for microorganisms in metabolomics.

Hossein Sedighikamal, Shohreh Mashayekhan.

   BACKGROUND: Advancements in the research of intracellular metabolome have the potential to affect our understanding of biological processes. The applications and findings of intracellular metabolome analysis are useful in understanding cellular pathways, microbial interactions, and the detection of secreted metabolites and their functions.
AIM OF REVIEW: This work focuses on the analysis of intracellular metabolomes in microorganisms. The techniques used for analyzing the intracellular metabolomes including metabolomics approaches such as mass spectrometry, nuclear magnetic resonance, liquid chromatography, and gas chromatography are discussed.
KEY SCIENTIFIC CONCEPTS OF REVIEW: Challenges such as sample preparation, data analysis, metabolite extraction, sample storage and collection, and processing techniques were investigated, as they can highlight emerging technologies and advancements in metabolome analysis, future applications in drug discovery, personalized medicine, systems biology, and the limitations and challenges in studying the metabolome of microorganisms.

Keywords:  Extraction; Intracellular; Metabolites; Microorganisms; Quenching

DOI:  https://doi.org/10.1007/s11306-025-02228-0
Anal Bioanal Chem. 2025 Mar 10.

Accuracy, linearity, and statistical differences in comparative quantification in untargeted plant metabolomics using LC-ESI-Orbitrap-MS.

Christina Maisl, Rainer Schuhmacher, Christoph Bueschl.

  High-resolution mass spectrometers, particularly when paired with liquid chromatography, are the instrument of choice for untargeted metabolomics approaches. Instruments, such as the Orbitrap, offer high sensitivity, selectivity, and exceptional mass accuracy, though they pose certain technical challenges, complicating absolute and comparative quantification. Consequently, method validation is crucial to ensure reliable results, as untargeted metabolomics approaches require the detection and quantification of a large number of metabolites in a broad dynamic range. Methods can be assessed using performance characteristics like accuracy and linearity to ensure analytical reliability. This study evaluates the suitability of untargeted metabolomics methods for discovery-based investigations. A stable isotope-assisted strategy was used with wheat extracts analyzed by a Q Exactive HF Orbitrap. Results showed that 70% of all detected 1327 metabolites displayed non-linear effects in at least one of the nine dilution levels employed. However, when considering fewer levels, 47% of all metabolites demonstrated linear behavior in at least four levels (i.e., a difference factor of 8). Moreover, the analysis further suggests that the observed abundances in less concentrated samples and those outside the linear range were mostly overestimated compared to expected abundances, but hardly ever underestimated. Consequently, during statistical analysis, which is an important step in prioritizing detected metabolites and correlating them with the biological hypothesis, the number of false-positives was not inflated, but the number of false-negatives might be increased. Generally, (non-)linear behavior did not correlate with specific compound classes or polarity, suggesting non-linearity is not easily predictable based on chemical structures.

Keywords:  Accuracy; Comparative quantification; Linearity; Orbitrap; Plant metabolomics; Untargeted metabolomics

DOI:  https://doi.org/10.1007/s00216-025-05818-y
Mass Spectrom Rev. 2025 Mar 12.

A Comprehensive Review of Instrumentation and Applications in Post-Column and In-Source Derivatization for LC-MS.

Nadeem Muhammad, Irshad Hussain, Xiao-An Fu, Amjad Ali, Dandan Guo, Laila Noureen, Qamar Subhani, Naushad Ahmad, Quan-Fei Zhu, Hairong Cui, Yu-Qi Feng.

  Liquid chromatography-mass spectrometry (LC-MS) has become an indispensable tool for elucidating molecular structures and quantifying diverse compounds within complex mixtures. Despite its versatility, it faces various challenges such as ion suppression, low sensitivity, analyte instability, and matrix effects, which are being overcome by different kinds of offline and online derivatization techniques to improve specificity and reduce potential interferences. In this context, considerable advancements have been made in reviewing and critically evaluating a wide range of developed methods and techniques; however, little attention has been given to post-column derivatization (PCD) in LC-MS. Therefore, this comprehensive review highlights state-of-the-art advancements in LC-MS with a specific focus on various types of chemical and physical PCD, and in-source derivatization. It also examines the latest instrumentation developments, highlights methods and influencing factors, and explores applications in food, proteomics, biology, pharmaceuticals, and environmental analysis from the past four decades. Besides, this review critically examines the role of PCD in LC-MS along with outlining its advantages and disadvantages. Furthermore, special emphasis is also made on prospects and insights for developing more versatile LC-PCD-MS techniques and in-source methodologies, to address ongoing challenges and aim to open new research avenues for analysts.

Keywords:  derivatization; fragmentation; insource; liquid chromatography; mass spectrometry; post‐column

DOI:  https://doi.org/10.1002/mas.21930
J Pharm Biomed Anal. 2025 Feb 24. pii: S0731-7085(25)00107-4. [Epub ahead of print]260 116766

Development and validation of a UPLC-MS/MS method for almonertinib with its active metabolite HAS-719 and anlotinib in human plasma.

Yi Qin, Xiaojun Guan, Shichao Zhang, Zijie Zhang, Chenrong Huang, Liyan Miao.

  Almonertinib and anlotinib are tyrosine kinase inhibitors used to treat malignant tumors, with their combination currently applied to non-small cell lung cancer (NSCLC) patients. This study aimed to develop a simple and rapid UPLC-MS/MS method for simultaneously detecting almonertinib, its active metabolite HAS-719, and anlotinib in human plasma. The analytes were separated on a Waters HSST3-C18 column following protein precipitation with acetonitrile. Mass detection was performed on a Waters TQS triple quadrupole mass spectrometer under positive electrospray ionization mode. The MRM ions were m/z 526.5→71.96 for almonertinib, m/z 512.4 → 455.41 for HAS-719, m/z 408.16→ 339.03 for anlotinib and m/z 518.5→372.26 for d6-HAS-000719 (internal standard). Method validation followed FDA guidelines and Chinese Pharmacopoeia regulations, demonstrating acceptable accuracy, precision, matrix effects, recovery, and stability. The method showed excellent linearity over 0.5-500 ng/mL for all three analytes, with correlation coefficients (r²)≥ 0.99. The validated UPLC-MS/MS method successfully monitored almonertinib and anlotinib concentrations in clinical, particularly for NSCLC patients.

Keywords:  Almonertinib; Anlotinib; LC-MS/MS; TKI

DOI:  https://doi.org/10.1016/j.jpba.2025.116766
Sci Rep. 2025 Mar 13. 15(1): 8761

The development of a bioanalytical method for the simultaneous analysis of gentamicin and tacrolimus in Rat whole blood using UHPLC-MS/MS.

Shrooq Altaweel, Ann Van Schepdael, Erwin Adams, Aliyah Almomen.

  Tacrolimus (TAC) is commonly administered to patients who have undergone organ transplantation to prevent the immune system from rejecting the transplanted organ. Multidrug-resistant bacterial infections are the most frequent complications during the first-month post-transplantation. Old antimicrobial agents such as gentamicin (GEN) are widely used to treat opportunistic nosocomial infections in immunosuppressed TAC patients. Nephrotoxicity is a significant side effect of GEN and TAC, but some studies indicated their concurrent administration. However, there is no information on whether the combination of the two drugs may result in a more significant impairment of kidney function than either drug used separately. To investigate this, both drugs should be monitored in blood. Sample preparation was carried out using protein precipitation, requiring only 50 µL of WB sample with an extraction recovery of not less than 95.2% (GEN) and 93.2% (TAC). Analytes and internal standard (IS) were monitored using mass spectrometry (MS) in positive ion mode by multiple reaction monitoring (MRM). Chromatographic analysis was performed on an Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm), kept at 50 °C and using gradient elution. Mobile phase A contained 2 mmol/L ammonium formate acidified with 0.1% formic acid in water, and mobile phase B was a mixture of 2 mmol/L ammonium formate and 0.1% formic acid in methanol, pumped at a flow rate of 0.25 mL/min. The analysis time was only 6 min. The method was verified according to the European Medicines Agency (EMA) guidelines over a concentration range of 19.5-2500 ng/mL for GEN and 1.95-250 ng/mL for TAC. Determination coefficients for the calibration curves were found to be ≥ 0.999. Within- and between-run precision and accuracy were evaluated for both drugs with relative standard deviations (RSD) ≤ 6.5% and inaccuracy ≤ 6.6%. The proposed method was successfully applied to analyze the WB samples at different time points after the co-administration of GEN and TAC to Wistar rats. In this work, a new bioanalytical UHPLC-MS/MS method was developed and validated for simultaneous quantification of total GEN congeners (C1, C1a, and C2/C2a) and TAC in Wistar rats whole blood (WB). The protein precipitation method has been chosen to extract the drug from the WB sample. The assay method has been successfully used to estimate the concentration of TAC and GEN after co-administration in rats.

Keywords:  Gentamicin; Ion pairing reagent; Rats whole blood; Tacrolimus; UHPLC–MS/MS

DOI:  https://doi.org/10.1038/s41598-025-92418-6
J Pharm Biomed Anal. 2025 Mar 05. pii: S0731-7085(25)00141-4. [Epub ahead of print]260 116800

Development and validation of a HPLC-MS/MS method the determination of genistein and equol in serum, urine and follicular fluid.

Xia Zheng, Yue-Jin Wu, Li-Mei Wu, Ling Zhang, Lin Zhang, Zhen Jin, Fang Gao, Qing-Qing Li, Yin Wang, Yi-Dan Wu.

  Soy isoflavones exert estrogen-like synergistic or antagonistic effects by binding to estrogen receptors, and potentially impact the function of female reproductive system, but their distribution profile in human remains little clarified. To determination of genistein (GEN) and equol (EQ) in human urine, serum and follicular fluid (FF), an analytical method based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed and validated. The enrichment and clean-up are performed on a solid-phase extraction (SPE) column; the elution is a gradient one, with the mobile phase (A) of 0.1 % (v/v) formic acid aqueous solution and the mobile phase (B) of 0.1 % (v/v) formic acid in acetonitrile; the column temperature is 40 °C. Mass spectrometry is performed using negative ion mode electrospray ionization (ESI -) in multiple reaction monitoring (MRM) mode. The method was validated over the linear ranges of 7.8-1000.0 ng/mL and 39.1-5000.0 ng/mL, for serum and urine, with correlation coefficients (r) of 0.9948-0.9984. The precision, accuracy and stability meet the U.S. Food and Drug Administration guidance. This method has been used to detect genistein (GEN) and equol (EQ) in serum, follicular fluid, and urine, to report equol in follicular fluid for the first time, and to study the correlation between genistein and equol in three body fluids. The study showed that the average concentration of EQ in follicular fluid was 18.5 ng/mL and there was a significant positive Spearman's correlation between concentrations of GEN in serum and FF (r = 0.44, p ≤ 0.05).

Keywords:  Equol; Genistein; HPLC-MS/MS; Soy isoflavone

DOI:  https://doi.org/10.1016/j.jpba.2025.116800
Int J Mol Sci. 2025 Feb 26. pii: 2041. [Epub ahead of print]26(5):

Tear Sampling and Biomarker Discovery: A Robust Workflow for Routine Clinical Applications Using UHPLC-MS/MS and Schirmer Strips.

Rossana Comito, Carmen Ciavarella, Gloria Astolfi, Matteo Conti, Emanuele Porru, Francesco Saverio Violante, Piera Versura.

  Human tear analysis is gaining increasing attention as a non-invasive tool for several applications such as proteomics and biomarker identification in various diseases, including cancer. The choice of the correct sampling method determines the result of the analysis. In this study, we developed and validated a robust method for tear protein quantification using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Tear samples were collected with Schirmer strips, a low-cost and practical tool for tear collection. It is the first time that internal standards have been used to enhance the analytical performance of a method based on Schirmer strips for tear sampling, overcoming the issues widely reported in the literature regarding protein extraction and data reproducibility. Non-human proteins were used for method development, ensuring improved accuracy and analytical precision. The method demonstrated excellent recovery, high sensitivity, and reproducibility. The use of Schirmer strips, combined with this advanced analytical method, highlights their potential as a reliable support for tear protein quantification and biomarker discovery. This study provides a cost-effective and reliable workflow for tear proteome analysis and contributes to the growing field of tear-based diagnostics, making it suitable for routine clinical and research applications in precision medicine.

Keywords:  Schirmer strip; cancer biomarkers; mass spectrometry; non-invasive matrices; proteomics; tear film

DOI:  https://doi.org/10.3390/ijms26052041
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Mar 06. pii: S1570-0232(25)00104-7. [Epub ahead of print]1256 124552

Simultaneous quantification of four urinary biomarkers related to oxidative stress using UHPLC-QqQ-MS/MS.

Yingfeng Gao, Ruiwei Xu, Huixia Liu, Shuyu Jia, Yi Zhang, Xin Meng, Jicheng Gong.

  Oxidative stress biomarkers have been associated with both acute and chronic health outcomes. However, traditional methods analyze different biomarkers separately, resulting in complex sample preparation, high sample consumption, lengthy processing time, and limited comparability. In this study, we presented a newly developed and validated method for the simultaneous determination of oxidative stress from multiple perspectives (DNA, lipids, and antioxidants). Using a one-step solid-phase extraction and ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS), we measured four oxidative stress related biomarkers in urine simultaneously, within a run time of only 12 min. These biomarkers included 8-hydroxy-2'-deoxyguanosine (8-OHdG), 6-sulfatoxymelatonin (aMT6s), 8-isoprostaglandin-F2α (8-isoPGF2α), and 11-dehydro thromboxane B2 (11-DH-TXB2). The calibration curves showed wide linear ranges (0.4-800 ng/mL for 8-OHdG, 0.2-600 ng/mL for aMT6s, 0.4-600 ng/mL for 8-isoPGF2α, and 0.4-600 ng/mL for 11-DH-TXB2), with r2 values above 0.9932 for all analytes. The method demonstrated excellent sensitivity, with detection limits below 0.12 ng/mL, and good precision, with intra- and inter-day coefficients of variation ranging from 1.2 % to 14.4 %. We applied this method to urine samples from two populations living at different altitudes and found significantly higher levels of both 8-OHdG and 11-DH-TXB2 in the high-altitude group, likely due to hypobaric hypoxia. In the future, this new method could be applied in large-scale epidemiological studies to investigate biological mechanisms of oxidative stress in health risks or for clinical diagnosis.

Keywords:  Oxidative stress; Simultaneous determination; Solid-phase extraction; UHPLC-QqQ-MS/MS; Urinary biomarkers

DOI:  https://doi.org/10.1016/j.jchromb.2025.124552
J Biosci Bioeng. 2025 Mar 13. pii: S1389-1723(25)00049-0. [Epub ahead of print]

Application of silica monoliths for improved storage stability of metabolites in human plasma.

Kazuhiro Kawamura, Eiichiro Fukusaki.

  In metabolomic studies, sample collection and analysis are typically performed at separate locations, necessitating the transport and storage of samples. However, sample transport and storage conditions are often constrained by the available facilities. Specifically, metabolite levels in biological and food samples can fluctuate due to the activity of endogenous enzymes, depending on the transport and storage conditions. Therefore, in this study, we aimed to achieve metabolite stabilization during storage by sampling human plasma on silica monoliths. Silica monoliths maintained the metabolite samples in a dry state, enabling their transport and storage at high temperatures. Plasmas stored at room temperature and refrigerated were measured using gas chromatography/mass spectrometry (GC/MS), and the fluctuations in metabolites between normal storage and storage on silica monoliths were compared. As a result, fluctuations in several metabolites such as glucose and amino acids were observed during normal storage. However, these were suppressed during storage on silica monoliths. Overall, our findings highlight the efficiency of silica monoliths for sample transport and storage at high temperatures.

Keywords:  Gas–solid phase derivatization; Metabolite stability; Metabolomics; MonoTrap; Plasma; Silica monolith

DOI:  https://doi.org/10.1016/j.jbiosc.2025.02.007
Rapid Commun Mass Spectrom. 2025 Apr 15. 39(11): e10022

Rapid Proteomic Amelogenin Sex Estimation of Human and Cattle Remains Using Untargeted Evosep-timsTOF Mass Spectrometry.

Charllotte Blacka, Adam Dowle, Mik Lisowski, Michelle Alexander, Jessica Hendy, Kirsty Penkman, Jackie Mosely.

   RATIONALE: Sex estimation by analysis of amelogenin peptides in archaeological and fossil material has recently been gaining great traction within the fields of archaeology and palaeontology. Current widely used proteomic amelogenin sex estimation methods are hindered by relatively long mass spectrometric run times, or targeting peptides specific to human amelogenin proteins. Untargeted, high-throughput amelogenin sexing would be invaluable for a range of applications, from sex estimation of remains at mass grave sites to broadening the application of rapid amelogenin sexing to non-hominin species for husbandry and evolutionary studies.
METHODS: A new acid etch protocol followed by Evosep-LC-TIMS-TOF mass spectrometry is presented for amelogenin analysis, providing global peptide data through rapid mass spectrometric methods in under 20 min per sample (including sample preparation, mass spectrometric acquisition and data processing). This sampling protocol was developed on modern cattle (Bos taurus) teeth, before Evosep-timsTOF partial validation with archaeological cattle and human (Homo sapiens) teeth, demonstrating the potential of straightforward application of this rapid amelogenin sexing method to a range of taxa.
RESULTS: The rapid Evosep-LC-TIMS-TOF mass spectrometry methods gave comparable peptide counts to conventional long untargeted methods, while maintaining similar (or faster) acquisition times to those reported in methods targeting specific human amelogenin peptides. Implementation of the novel acid etch sampling approach also streamlined sample preparation without compromising peptide counts.
CONCLUSIONS: Rapid, untargeted Evosep-LC-TIMS-TOF mass spectrometry was successfully implemented in sex estimation of modern and archaeological material from Bos taurus and Homo sapiens teeth. This demonstrates an advancement in low-cost, high-throughput amelogenin sex estimation, for both human and zooarchaeological applications.

Keywords:  LC–MS/MS; TIMS‐TOF; amelogenin; palaeoproteomics; sex estimation

DOI:  https://doi.org/10.1002/rcm.10022
Nat Commun. 2025 Mar 07. 16(1): 2298

FIORA: Local neighborhood-based prediction of compound mass spectra from single fragmentation events.

Yannek Nowatzky, Francesco Friedrich Russo, Jan Lisec, Alexander Kister, Knut Reinert, Thilo Muth, Philipp Benner.

Non-targeted metabolomics holds great promise for advancing precision medicine and biomarker discovery. However, identifying compounds from tandem mass spectra remains a challenging task due to the incomplete nature of spectral reference libraries. Augmenting these libraries with simulated mass spectra can provide the necessary references to resolve unmatched spectra, but generating high-quality data is difficult. In this study, we present FIORA, an open-source graph neural network designed to simulate tandem mass spectra. Our main contribution lies in utilizing the molecular neighborhood of bonds to learn breaking patterns and derive fragment ion probabilities. FIORA not only surpasses state-of-the-art fragmentation algorithms, ICEBERG and CFM-ID, in prediction quality, but also facilitates the prediction of additional features, such as retention time and collision cross section. Utilizing GPU acceleration, FIORA enables rapid validation of putative compound annotations and large-scale expansion of spectral reference libraries with high-quality predictions.

DOI: https://doi.org/10.1038/s41467-025-57422-4
bioRxiv. 2025 Feb 27. pii: 2025.02.22.639656. [Epub ahead of print]

Reference Library for Suspect Non-targeted Screening of Environmental Toxicants Using Ion Mobility Spectrometry-Mass Spectrometry.

Devin Teri, Noor A Aly, James N Dodds, Jian Zhang, Paul A Thiessen, Evan E Bolton, Kara M Joseph, Antony J Williams, Emma L Schymanski, Ivan Rusyn, Erin S Baker.

As our health is affected by the xenobiotic chemicals we are exposed to, it is important to rapidly assess these molecules both in the environment and our bodies. Targeted analytical methods coupling either gas or liquid chromatography with mass spectrometry (GC-MS or LC-MS) are commonly utilized in current exposure assessments. While these methods are accepted as the gold standard for exposure analyses, they often require multiple sample preparation steps and more than 30 minutes per sample. This throughput limitation is a critical gap for exposure assessments and has resulted in an evolving interest in using ion mobility spectrometry and MS (IMS-MS) for non-targeted studies. IMS-MS is a unique technique due to its rapid analytical capabilities (millisecond scanning) and detection of a wide range of chemicals based on unique collision cross section (CCS) and mass-to-charge ( m/z ) values. To increase the availability of IMS-MS information for exposure studies, here we utilized drift tube IMS-MS to evaluate 4,685 xenobiotic chemical standards from the Environmental Protection Agency Toxicity Forecaster (ToxCast) program including pesticides, industrial chemicals, pharmaceuticals, consumer products, and per- and polyfluoroalkyl substances (PFAS). In the analyses, 3,993 [M+H] + , [M+Na] + , [M-H] - and [M+] + ion types were observed with high confidence and reproducibility (≤1% error intra-laboratory and ≤2% inter-laboratory) from 2,140 unique chemicals. These values were then assembled into an openly available multidimensional database and uploaded to PubChem to enable rapid IMS-MS suspect screening for a wide range of environmental contaminants, faster response time in environmental exposure assessments, and assessments of xenobiotic-disease connections.

DOI: https://doi.org/10.1101/2025.02.22.639656
Eur J Pharm Sci. 2025 Mar 12. pii: S0928-0987(25)00064-8. [Epub ahead of print] 107065

Direct comparison of single peak and gradient chromatographic methods for routine analysis of surfactants in biopharmaceuticals.

Maksymilian M Zegota, Juliane Achenbach, Georg Schuster, Christian Schöneich, Tim Menzen, Andrea Hawe.

  The non-ionic surfactants polysorbate 20, polysorbate 80 and poloxamer 188 are prone to degradation, which necessitates their monitoring as part of the analytical strategy for surfactant- containing biopharmaceuticals. In this study, we discuss the challenges of analyzing partially degraded surfactant samples in the context of the most common quantification method - online solid-phase extraction using a mixed-mode column with analyte elution as a single peak. Additionally, we compare this single peak approach with gradient methods for surfactant quantification. To facilitate this comparison, we developed a simple gradient approach that allows for the rapid profiling of both polysorbates in 5.5 minutes and poloxamer 188 in 11 minutes, using liquid chromatography (LC) coupled with charged aerosol detection (CAD) or mass spectrometry (MS). We also included polyethylene glycol 15 hydroxystearate (HS15) as a possible alternative to the established surfactants. The gradient approach is a stability-indicating method that can detect compositional changes due to common degradation pathways, such as those induced by hydrolytic or oxidative stress, based on changes in the elution profile. The sensitivity of the single peak approach to degradation varies depending on the root cause. In conclusion, we present a workflow in which one chromatographic column employing fast gradients enables effective separation of the main surfactant components, facilitating both qualitative and quantitative analysis, as well as root cause analysis in case of observed degradation.

Keywords:  charged aerosol detection; degradation; mass spectrometry; poloxamer; polysorbate; surfactants

DOI:  https://doi.org/10.1016/j.ejps.2025.107065
J Anal Toxicol. 2025 Mar 11. pii: bkaf022. [Epub ahead of print]

Green liquid-liquid microextraction for quantification of ketamine and metabolites in human urine by gas chromatography-mass spectrometry.

Hsueh-Hui Yang, Chia-Sui Kao, Ahai C Lua, Tsong-Yung Chou.

  This study aimed to develop and validate a green method for the detection of ketamine and its metabolites in human urine samples and subsequently determine which metabolite is more suitable for judgment of ketamine abuse. Ketamine and its metabolites were extracted from urine samples with 1-undecanol using liquid-liquid microextraction based on the solidification of floating organic droplet extraction. The extracts were directly analyzed using gas chromatography-mass spectrometry (GC-MS) without derivatization. The sample pretreatment procedure prior to GC-MS analysis was simple, fast, and required little solvent consumption. Parameters that affect the extraction efficiency, including pH of the urine sample and centrifugation speed, were optimized. Under the optimized conditions, the limits of detection for norketamine, dehydronorketamine, and ketamine were 1.5, 1.7 and 1.0 ng/mL, respectively. The method was used to analyze eight real urine samples from drug abusers and exhibited acceptable accuracy and precision. By analyzing real samples, this study revealed that detection of dehydronorketamine in urine samples for the judgment of ketamine abuse may be more suitable than detection of norketamine or ketamine. The method used in this study addresses the need for green analytical techniques in toxicology.

Keywords:  Green chemistry; Ketamine; Liquid-liquid microextraction; Solidification of floating organic droplet; gas chromatography mass spectrometry

DOI:  https://doi.org/10.1093/jat/bkaf022
J Pharm Biomed Anal. 2025 Mar 10. pii: S0731-7085(25)00139-6. [Epub ahead of print]260 116798

Quantitative method for intestinal short chain fatty acids based on stable isotope labeling combined with liquid chromatography-mass spectrometry.

Yang Zhang, Yan Qiao, Xin Ding, Ya Zhang, Xuemei Su, Lei Zhang, Qian Zhou, Guangguo Tan.

  Short chain fatty acids (SCFAs) are produced from the breakdown of dietary proteins and fiber by gut microbes, and they have a close relationship with the health and diseases of the host. However, due to the similar structures of SCFAs, the abundance of active molecules, the wide concentration range in biological samples, and the characteristics such as high polarity, poor chromatographic separation, and ionization performance, it is challenging to comprehensively and accurately quantify SCFAs. This study utilized a stable isotope-labeled carboxyl derivatization reagent d0-/d6-2,4-dimethoxy-6-piperazin-1-yl pyrimidine (d0-/d6-DMPP) to establish a new method for the wide-coverage quantification analysis of SCFAs using ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS), capable of detecting the content of 16 SCFAs. The method demonstrated low limits of detection (LODs) of 0.05-0.5 nmol/L and limits of quantification (LOQs) of 0.1-1.0 nmol/L, with excellent linearity (R² > 0.99), intra-day precision (RSD < 8.5 %), and inter-day precision (RSD < 7.8 %). Using this quantitative analysis method, we successfully quantified 16 SCFAs from the colonic contents of rats with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ulcerative colitis (UC) and Sini decoction (SND) intervention. It was found that after the interventional treatment with SND, the levels of 7 SCFAs in the colonic contents of rats with UC were significantly up regulated, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, 2-methylbutyric acid, and hexanoic acid, while 4 SCFAs were significantly down regulated, including 3-hydroxyisovaleric, 3-methyl-2-oxobutanoic acid, 3-methyl-2-oxovaleric acid, and 4-methyl-2-oxovaleric acid. These findings suggested that SND might exert its therapeutic effect on UC by regulating the metabolism of SCFAs. Overall, this study not only provides a new method for the analysis of SCFAs with high sensitivity and wide-coverage but also offers important scientific evidence for understanding the mechanism of SND against UC.

Keywords:  Derivatization; Quantification; Short chain fatty acids; Sini decoction; Stable isotope labeling; Ulcerative colitis

DOI:  https://doi.org/10.1016/j.jpba.2025.116798
Atherosclerosis. 2025 Feb 15. pii: S0021-9150(25)00027-9. [Epub ahead of print] 119130

Advancing atherosclerosis research: The Power of lipid imaging with MALDI-MSI.

Christoph H M Bookmeyer, F Xavier Correig, Luis Masana, Paolo Magni, Óscar Yanes, Maria Vinaixa.

  Atherosclerosis is a chronic inflammatory disease that is one of the leading causes of mortality globally. It is characterized by the formation of atheromatous plaques in the intima layer of larger arteries. The (fibro-)fatty plaques usually develop asymptomatically within the vessel until a serious event such as myocardial infarction or stroke occurs. Lipids play a pivotal role in disease progression, but while the causal role of cholesterol is beyond doubt, the distribution of numerous other lipids within the heterogeneous layers of atherosclerotic plaques, and their biological function remain unclear. A deeper understanding of the pathophysiological progression of the disease for prognostics, diagnostics, treatment, and prevention is of great need. Mass spectrometry imaging (MSI), in particular with matrix-assisted laser desorption/ionization (MALDI) offers an unprecedented untargeted characterization of the physiological microenvironment, unraveling the spatial distribution of numerous biochemical compounds. MALDI-MSI offers an advantageous balance of sample preparation, chemical sensitivity, and spatial resolution, and thus has been established as a key technology in modern biomedical analysis. This review focuses on the analysis of lipids in atherosclerotic lesions with MALDI-MSI, for which the past years showed major developments in the spatial characterization of lipids and their interaction within atherosclerotic plaques. We will cover main contributions with a focus on the recent decade, elaborate possibilities, limitations, main findings, and recent developments from sample handling to instrumentation, and estimate current challenges and potentials of MALDI-MSI with respect to a clinical application.

Keywords:  Atherosclerosis; Lipids; MALDI-MSI; Mass spectrometry imaging; Spatial lipidomics

DOI:  https://doi.org/10.1016/j.atherosclerosis.2025.119130