bims-metlip 2025-10-19 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–10–19
twenty-six papers selected by
Sofia Costa, Matterworks

Design and Analysis of Untargeted Metabolomics Experiments.
Plasma-Free Metanephrine and Normetanephrine Quantification for Clinical Applications Validated by Combining Solid-Phase Extraction and HPLC-MS/MS.
Sample preparation optimization for metabolomics and lipid profiling from a single plasma and liver tissue based on NMR and UHPLC-MS.
Development and validation of an LC-MS/MS method for the quantification of novel therapeutic TT-478, a selective adenosine receptor 2B antagonist, for a phase I/II clinical trial.
An isotope dilution-liquid-chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of mycophenolic acid in human serum and plasma.
Trace Determination of 16 Phenolic Endocrine-Disrupting Chemicals in Environmental Waters: A Validated SPE-UPLC-MS/MS Approach.
Simultaneous analysis of stimulants, opioids, gabapentin, xylazine, benzodiazepines, cannabinoids, and novel stimulants/hallucinogens in human urine.
JuliaMSI: A high-performance graphical platform for mass spectrometry imaging data analysis.
Subcellular mass spectrometry imaging of lipids and nucleotides using transmission geometry ambient laser desorption and plasma ionisation.
Coated blade spray-mass spectrometry for rapid screening: A tutorial.
Examination of lipid distributions in hydrogel-expanded mouse brain tissue using imaging mass spectrometry.
Dynamic Spray Mass Spectrometry for Direct Analysis of Complex Mixtures.
Aird-MSI: A High Compression Rate and Decompression Speed Format for Mass Spectrometry Imaging Data.
Dried blood spots analysis for targeted and non-targeted exposomics.
Lysosomal Immunoprecipitation (LysoIP) from Cells and Tissues for Metabolomic and Proteomic Analyses.
Demonstrating the application of a liquid chromatography-mass spectrometry method for the determination of phthalate diesters in complex solid and liquid environmental samples.
Construction and Comparison of UPLC-QE-Orbitrap-MS and UPLC-MS/MS Methods for the Detection of Diazepam Residues in Complex Aquatic Matrices.
Time-controlled online SPE: strategic approach for N-acylethanolamines quantification in complex matrices.
Real-Time Cell Analysis Using Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy at Single and Bulk Cellular Levels.
Method validation for quantification of five antineoplastic agents in urine using UPLC-ESI-MS/MS.
Determination of several cationic pesticides in food of plant and animal origin using the QuPPe method and ion chromatography with tandem mass spectrometry.
In-Capillary Chemoselective Tagging-Facilitated Direct nESI-MS Profiling of Carboxyl- and Carbonyl-Containing Metabolites in Urine.
A fast GC-MS/MS method for the simultaneous measurement of key metabolites of peroxisomal beta-oxidation and ether lipid biosynthesis in human fibroblasts.
New salty extraction of polar analytes technique and oil-based cleanup (SExPA-Oc) for the determination of nereistoxins in vegetable samples with HILIC-MS/MS.
Liquid chromatography-tandem mass spectrometry-based recalibration reduces inter-platform variability in aldosterone detection across chemiluminescence immunoassay platforms.
Spatial biology using single-cell mass spectrometry imaging and integrated microscopy.

Curr Protoc. 2025 Oct;5(10): e70232

Design and Analysis of Untargeted Metabolomics Experiments.

Zane G Long, Rachel W Martin.

  Untargeted metabolomics is a powerful approach for identifying small molecules from highly complex mixtures, such as biological tissues or environmental samples. This technology enables the relatively fast and inexpensive identification of metabolites in situations where many or most of the chemical species are unknown before the experiment begins. This situation often arises in biomedical and environmental research, as well as in the case described here, the discovery of metabolites from plants. The objective of this paper is to provide practical and technical knowledge about untargeted metabolomics using mass spectrometry as the detection method. Specifically, we focus on liquid chromatography tandem mass spectrometry (LC-MS/MS). We provide a consolidated protocol for new users, serving as a starting point for experimental design, data collection, and data analysis. We explain the terminology and technical details in the context of real experiments and samples. In addition to general background information, step-by-step protocols are provided for sample preparation, liquid chromatography-tandem mass spectrometry data collection, and data analysis, utilizing readily available and widely used software. The chosen example data set is based on plant metabolites with varying chemical properties; however, the approach is applicable to essentially any complex biological sample. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for LC-MS/MS Support Protocol 1: Preparing a 'master mix' sample for assessment of liquid chromatography and sensitivity Basic Protocol 2: LC-MS/MS data collection Basic Protocol 3: Data analysis using the software MSConvert, MZMine, and SIRIUS Support Protocol 2: Using the MZMine batch file.

Keywords:  LC‐MS/MS; electrospray ionization; mass spectrometry; plant metabolites; untargeted metabolomics

DOI:  https://doi.org/10.1002/cpz1.70232
Molecules. 2025 Sep 23. pii: 3847. [Epub ahead of print]30(19):

Plasma-Free Metanephrine and Normetanephrine Quantification for Clinical Applications Validated by Combining Solid-Phase Extraction and HPLC-MS/MS.

Hyebin Choi, Jisook Yim, Jiwon Yun, Jong Kwon Lee, Keun Ju Kim, Minjeong Nam, Myung Hyun Nam, Yunjung Cho, Seung Gyu Yun.

  Plasma-free metanephrines are the most sensitive and specific biochemical markers for diagnosing catecholamine-secreting tumors, such as pheochromocytoma and paraganglioma. In this study, we developed and validated a liquid chromatography-tandem mass spectrometry method for quantifying metanephrine and normetanephrine in human plasma, using solid-phase extraction with a weak cation-exchange mechanism. Validation was performed according to the FDA Bioanalytical Method Validation Guidance and CLSI guideline C62-A. The method showed excellent linearity over concentration ranges of 0.11-13.92 nmol/L for metanephrine and 0.14-26.43 nmol/L for normetanephrine, with correlation coefficients exceeding 0.999. The accuracy, precision, and lower limit of quantification met the acceptance criteria of the study. Matrix effect evaluation revealed a process efficiency of 121% for metanephrine at the lowest concentration, slightly exceeding the acceptable range of 100 ± 15%. This was likely because of matrix-induced ion enhancement or variability in extraction efficiency. However, all other tested concentrations were within the acceptable limits. Overall, this method demonstrated high sensitivity, specificity, and reproducibility, making it suitable for routine clinical applications. Minor deviations at low concentrations do not compromise reliability; however, future optimizations, such as matrix-matched calibration, may further improve performance.

Keywords:  LC-MS/MS; bioanalytical method validation; metanephrine; normetanephrine; pheochromocytoma; plasma analysis; solid-phase extraction

DOI:  https://doi.org/10.3390/molecules30193847
J Pharm Biomed Anal. 2025 Sep 29. pii: S0731-7085(25)00514-X. [Epub ahead of print]268 117173

Sample preparation optimization for metabolomics and lipid profiling from a single plasma and liver tissue based on NMR and UHPLC-MS.

Chenxi Wang, Yan Gao, Ruibing Chen, Yuefen Lou.

  Currently, the majorty of metabolomics studies rely on the use of a single analytical platform, either NMR or MS, which hampers the ability to conduct an unbiased and comprehensive analysis. However, the use of multiple platforms poses an additional challenge: the limited sample material. Consequently, it is highly desirable to develop comprehensive, effective and sample-conserving procedures for metabolite extraction. In this study, we presented pretreatment strategies enabling sequential NMR and multi-LC-MS (UHPLC-Q-Orbitrap MS, and UHPLC-QqQ MS) platform analysis using a single plasma and liver tissue sample. For the plasma sample, the biphasic CHCl3/MeOH/H2O method was suitable for polar and lipid extraction after NMR-based metabolomics analysis, in terms of the number of annotated metabolites, reproducibility, and the amount of sample needed. While for the liver sample, the two-step extraction involving CHCl3/MeOH followed by MeOH/H2O was recommended. In this method, resuspension of dried lipid extracts was used for lipidomics, and polar extracts were transferred for further untargeted metabolic profiles by UHPLC-Q-Orbitrap MS following an NMR-based metabolomics study. Finally, the proposed preparation protocols were evaluated for robustness, and the identification data were used to generate a comprehensive metabolic map for plasma and liver tissue. In summary, this study provides a unique sample preparation procedure for two biological specimens, allowing for multi-platform analysis using a single sample. By adopting this approach, comprehensive metabolic profiling can be conducted to detect metabolic alterations under different physiological or pathological conditions.

Keywords:  (1)H NMR; A single sample; Lipidomics; Metabolomics; Sample preparation optimization; UHPLC-MS

DOI:  https://doi.org/10.1016/j.jpba.2025.117173
Bioanalysis. 2025 Sep;17(17): 1105-1112

Development and validation of an LC-MS/MS method for the quantification of novel therapeutic TT-478, a selective adenosine receptor 2B antagonist, for a phase I/II clinical trial.

Daniel Whitaker, Laura Francis, Sami Karaborni, Steven Smith, Jenny L Craigen, Svatopluk Svetlik, Shelby Barnett, Gareth J Veal.

   BACKGROUND: TT-478 is a novel adenosine receptor 2B antagonist, administered orally as a prodrug (TT-702) for the treatment of advanced metastatic prostate cancer in a Phase I/II clinical trial setting. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was required to quantify TT-478 in plasma samples obtained from patients recruited to the ongoing early-phase trial.
METHODS AND RESULTS: An LC-MS/MS method has been developed and fully validated that allows the quantification of TT-478 in patient plasma samples following a simple extraction procedure using acetonitrile. The assay was shown to be sensitive and selective for TT-478, with an analytical range of 75-25,000 ng/mL, and exhibited excellent precision (coefficient of variation < 12%) and accuracy in the range of 96-107%. Consistently high recovery was achieved, and no matrix effect observed. Analysis of patient samples confirmed that TT-702 rapidly and completely hydrolyzes to TT-478 following administration.
CONCLUSION: A novel robust method to quantify TT-478 in human plasma has been fully validated and is currently being utilized in an ongoing clinical trial. Analysis of TT-478 levels in plasma samples from these patients will provide first-in-human pharmacokinetic data for this novel compound.Clinical trial registration: https://clinicaltrials.gov/study/NCT05272709?term=AREA%5BConditionSearch%5D(Advanced%20Prostate%20Cancer)%20AND%20AREA%5BBasicSearch%5D(phase%203%20drug)%20AND%20AREA%5BOverallStatus%5D(NOT_YET_RECRUITING%20OR%20RECRUITING%20OR%20ENROLLING_BY_INVITATION%20OR%20ACTIVE_NOT_RECRUITING)&rank=10 identifier is NCT05272709.

Keywords:  A2BR; LC-MS/MS; TT-478; TT-702; cancer; pharmacokinetics; validation

DOI:  https://doi.org/10.1080/17576180.2025.2554564
Clin Chem Lab Med. 2025 Oct 20.

An isotope dilution-liquid-chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of mycophenolic acid in human serum and plasma.

Tobias Schierscher, Linda Salzmann, Neeraj Singh, Carina Schäfer, Luc Sungauer, Friederike Bauland, Andrea Geistanger, Lorenz Risch, Christian Geletneky, Christoph Seger, Judith Taibon.

   OBJECTIVES: A candidate reference measurement procedure (RMP) based on isotope dilution (ID) liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated to accurately measure serum and plasma concentrations of mycophenolic acid (MPA).
METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was utilized for determining the absolute content of the reference material, guaranteeing its traceability to SI units. Separation of MPA from potential interferences, whether known or unknown, was accomplished by using a C18 column. For sample preparation, a protocol for protein precipitation followed by a high dilution step was established. Assay validation and measurement uncertainty determination were conducted following the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement guidelines.
RESULTS: The RMP demonstrated high selectivity and specificity without any indication of a matrix effect, enabling the quantification of MPA in the range between 0.240 and 18.0 mg/L. The intermediate precision and repeatability (n=60, measurements) were found to be <3.7 % and <3.0 %, respectively, across all concentration levels and independent from the matrix. The relative mean bias ranged from -1.9 to 2.1 % for serum and from -0.5 to 1.8 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were found to be <6.4 % and <3.0 % (k=2), respectively.
CONCLUSIONS: We are pleased to present a new LC-MS/MS-based candidate RMP for MPA in human serum and plasma which offers a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Keywords:  ID-LC-MS/MS; mycophenolic acid; qNMR; reference measurement procedure; standardization; traceability

DOI:  https://doi.org/10.1515/cclm-2024-0860
Rapid Commun Mass Spectrom. 2026 Jan;40(1): e10155

Trace Determination of 16 Phenolic Endocrine-Disrupting Chemicals in Environmental Waters: A Validated SPE-UPLC-MS/MS Approach.

Bao Song, Xinting Wang, Anchen Liu, Dongxue Lei, Yuelong Qin, Cui Pan, Yangzhao Sun, Shujun Zhong.

   RATIONALE: Phenolic endocrine-disrupting chemicals (EDCs), including alkylphenols (APs) and bisphenols (BPs), pose significant environmental and public health concerns due to their widespread presence in aquatic ecosystems and potential toxicity at trace concentrations. This study developed and validated a sensitive analytical method for the simultaneous determination of 16 phenolic EDCs in environmental waters.
METHODS: The method involves sample acidification to pH 2, hydrophilic-lipophilic balance (HLB), solid-phase extraction (SPE), and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis with negative electrospray ionization (ESI-). Systematic optimization revealed that an alkaline mobile phase (methanol/0.1% ammonia) significantly enhanced the ionization efficiency of phenolic compounds, improving sensitivity by approximately an order of magnitude compared to acidic conditions. Chromatographic separation was optimized using an ACQUITY UPLC BEH C18 column, which provided superior peak shapes and improved retention of hydrophobic compounds.
RESULTS: Acidification of samples prior to SPE was critical for maximizing analyte recovery (57.9%-94.2%). The validated method demonstrated outstanding analytical performance, achieving low detection limits (0.01-0.4 ng/L), excellent linearity (R = 0.991-0.999) across a broad concentration range (1.0-50.0 μg/L) and high precision (RSDs = 1.3%-13.6%). Application of the method to environmental samples collected from rivers, wastewater treatment plants, and landfill leachates identified eight target compounds, with alkylphenols detected at higher concentrations than BPs.
CONCLUSION: This reliable and robust method provides a valuable tool for monitoring trace levels of phenolic EDCs in diverse aquatic environments, supporting technically comprehensive environmental assessments and regulatory compliance.

Keywords:  endocrine‐disrupting chemicals (EDCs); solid‐phase extraction (SPE); ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS)

DOI:  https://doi.org/10.1002/rcm.10155
J Chromatogr A. 2025 Oct 11. pii: S0021-9673(25)00805-2. [Epub ahead of print]1763 466461

Simultaneous analysis of stimulants, opioids, gabapentin, xylazine, benzodiazepines, cannabinoids, and novel stimulants/hallucinogens in human urine.

Zhong-Min Li, Sunmi Lee, Kurunthachalam Kannan.

  A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of 67 drugs including stimulants, opioids, gabapentin, xylazine, benzodiazepines, cannabinoids, novel stimulants/hallucinogens, and their metabolites in urine. Urine samples were deconjugated, diluted, and fortified with isotopically labelled internal standards prior to analysis. The method was linear for all analytes with regression coefficients (R-values) > 0.99. The method limits of quantification were in the range of 0.1-15 ng/mL for all analytes. The recoveries of all drugs fortified in human urine at low (0.15-6 ng/mL), medium (200-400 ng/mL), and high (400-800 ng/mL) concentrations were in the range of 75-126%. Repeated analysis of similarly fortified urine yielded intra- and inter-day variations in the range of 0.3-14% and 0.45-17%, respectively. Enzymatic deconjugation using β-glucuronidase significantly improved sensitivity and enabled positive identification of several drugs that were predominantly excreted as glucuronides. The validated method was applied in the determination of drugs in 200 urine samples collected from non-fatal overdose patients. Fentanyl, norfentanyl, hydromorphone, morphine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, 4-anilino-N-phenethylpiperidine, methamphetamine, benzoylecgonine, and xylazine were detected in ≥ 50% of the 200 urine samples analyzed, with the sum concentration of all drugs ranging from 8.59 to 2840,000 ng/mL. Several drugs belonging to different chemical classes (e.g., xylazine and opioids) showed positive correlations, suggesting co-exposure to multiple drugs in overdose patients.

Keywords:  Gabapentin; LC-MS/MS; Opioids; Overdose; Stimulants; Urine; Xylazine

DOI:  https://doi.org/10.1016/j.chroma.2025.466461
Anal Chim Acta. 2025 Dec 01. pii: S0003-2670(25)01007-4. [Epub ahead of print]1377 344613

JuliaMSI: A high-performance graphical platform for mass spectrometry imaging data analysis.

José Julián Sierra-Álvarez, Martín Orlando Camargo-Escalante, Carlos Daniel Sierra-Álvarez, Carmelo Hernández-Caricio, Juan Francisco Moreno-Luna, Isabel Buendía-Corona, Robert Winkler.

  Mass Spectrometry Imaging (MSI) generates large datasets that require efficient computational solutions for data handling and visualization. While R and Python are commonly used for MSI analysis, their limited performance can hinder Big Data workflows. Julia is a high-performance programming language widely adopted in computationally demanding fields such as physics and economics. Here, we present JuliaMSI, a graphical user interface (GUI) developed in Julia for reading and analyzing MSI data in open formats (.imzML, .ibd, .mzML). JuliaMSI accelerates data loading, preprocessing, and visualization, with benchmarks showing up to 4.2-fold faster processing on Windows/macOS and 5.2-fold on Linux compared to R-based tools. Beyond speed, JuliaMSI enables interactive analysis through features such as contrast-enhancing filters (TrIQ, median filter), 3D topographic visualizations of ion intensity landscapes, and overlays of ion images with optical reference images. Users can inspect mass spectra linked to ion images, select regions of interest (ROI), and export results in publication-ready formats (.png, .jpg, .bmp). The platform supports seamless integration with downstream workflows via open data standards and provides a computationally efficient, user-friendly environment for large-scale MSI analysis. JuliaMSI is cross-platform (Linux, macOS, Windows) and available under the MIT license at https://codeberg.org/LabABI/JuliaMSI.

Keywords:  Julia; Mass spectrometry imaging (MSI); Topography map; Visualization

DOI:  https://doi.org/10.1016/j.aca.2025.344613
Nat Commun. 2025 Oct 15. 16(1): 9130

Subcellular mass spectrometry imaging of lipids and nucleotides using transmission geometry ambient laser desorption and plasma ionisation.

Reuben S E Young, Ann-Katrin Piper, Luke McAlary, Jayden C McKinnon, Jeremy S Lum, Jens Soltwisch, Marcel Niehaus, Shane R Ellis.

Demand for mass spectrometry imaging (MSI) technologies offering subcellular resolution for tissues and cell imaging is rapidly increasing. To accomplish this, efficient analyte ionisation is essential, given the small amounts of sample material in each pixel. Herein, we describe an atmospheric pressure transmission-geometry matrix-assisted laser desorption source equipped with plasma ionisation. By utilising a pre-staining method for sample preparation, lipid signal intensities were enhanced by an order of magnitude compared to conventional matrix-only methods, while serendipitously enabling imaging of numerous nucleotides. The system enables detection of up to 200 lipids and nucleotides in tissues at 1 µm-pixel size while informative MSI data is still obtained down to 250 nm pixel size. The use of sub-micron pixels is shown to discern subcellular features through combinations with fluorescence microscopy. This method provides a powerful tool for cellular and sub-cellular imaging of small molecules from tissues and cells for spatial biology applications.

DOI: https://doi.org/10.1038/s41467-025-64604-7
Anal Chim Acta. 2025 Dec 01. pii: S0003-2670(25)00852-9. [Epub ahead of print]1377 344458

Coated blade spray-mass spectrometry for rapid screening: A tutorial.

Wei Zhou, Janusz Pawliszyn.

  Coated blade spray-mass spectrometry (CBS-MS) integrates solid-phase microextraction (SPME) with ambient mass spectrometry (AMS), providing a powerful approach for analyzing trace analytes in complex sample matrices. In CBS-MS, analytes are extracted and enriched into a thin layer of coating material on a stainless-steel blade. Afterwards, a small volume of solvent is applied directly to the coating surface, enabling rapid desorption and subsequent electrospray ionization (ESI) from the blade tip under high voltage. By eliminating chromatographic separation and utilizing high-throughput extraction formats, CBS-MS enables rapid screening with total analysis times as short as 10 s per sample. The use of matrix-compatible coating materials facilitates direct analysis of complex biological matrices-including plasma, urine, whole blood, and tissue-without additional cleanup steps. This tutorial highlights the key features of CBS-MS, provides detailed guidance for its implementation, and offers perspectives on future developments of this emerging technology.

Keywords:  Coated blade spray; Green analytical chemistry; High throughput analysis; Mass spectrometry; Solid-phase microextraction

DOI:  https://doi.org/10.1016/j.aca.2025.344458
Anal Chim Acta. 2025 Dec 01. pii: S0003-2670(25)01023-2. [Epub ahead of print]1377 344629

Examination of lipid distributions in hydrogel-expanded mouse brain tissue using imaging mass spectrometry.

Jacob M Samuel, Nana M Baby, Elijah D Mayo, Tingting Yan, Zhongling Liang, Boone M Prentice.

   BACKGROUND: Imaging is essential in biological research, and imaging mass spectrometry uniquely provides a label-free approach with high molecular specificity. However, imaging mass spectrometry is limited in spatial resolution, which in turn limits the biological structures that can be studied. Custom lens setups and altered optical paths can shrink the diameter of the incident laser beam probe in matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry to achieve high spatial resolutions (<5 μm). However, these research-grade instruments are complex and expensive, making high spatial resolution imaging experiments unrealistic for the broader community. There is a need for inexpensive and widely-accessible means for high spatial resolution imaging.
RESULTS: An alternative method for improving spatial resolution is through physical magnification of the substrate, which has been well established in the subfield of expansion microscopy (ExM). ExM leverages superabsorbent hydrogels for isotropic expansion of tissues and retention of fluorescently labeled analytes. While typical ExM involves covalently anchoring analytes to the hydrogel network, lipid retention without anchoring has been recently demonstrated for imaging mass spectrometry. Herein, we demonstrate expansion imaging mass spectrometry (ExIMS) of expanded brain tissue and examine lipid distributions in both positive and negative ion modes across multiple tissue structures. A linear expansion factor of 4.5-fold is achieved and used to obtain high spatial resolution images of mouse brain cerebellum. Approximately 75 % of lipids in both positive and negative ion modes are detected in expanded tissue compared to unexpanded tissue. Additionally, the majority of lipid distributions across the brain are maintained post-expansion, though lipid delocalization can occur for some lipids in the hippocampus and the granular layer of the cerebellum. Alterations to the hydrogel formulation can significantly affect the ability of ExIMS to maintain accurate lipid distributions in expanded tissue. For example, stronger fixation can better maintain lipid distributions, while gentler digests can better maintain lipid distributions at higher tissue expansion factors.
SIGNIFICANCE: This work provides a methodical analysis of the effect of hydrogel embedding and expansion on lipid distributions across multiple tissue structures, ion polarities, and lipid classes. Computational tools are used to assess the spatial fidelity of lipid distributions prior to and following tissue expansion, which begins to assess the interaction between lipid delocalization and expansion parameters.

Keywords:  Expansion; High resolution; Hydrogel; Imaging mass spectrometry; MALDI

DOI:  https://doi.org/10.1016/j.aca.2025.344629
Anal Chem. 2025 Oct 17.

Dynamic Spray Mass Spectrometry for Direct Analysis of Complex Mixtures.

Purva S Damale, Dmytro S Kulyk, Alexander P Zumock, Abraham K Badu-Tawiah.

The analysis of small volumes of complex mixtures is important for biological, medicinal, toxicological, and forensic applications. Herein, we report the development of a single-step method using nanoelectrospray ionization (nESI), capable of atmospheric pressure chemical ionization (APCI) for mass spectrometry (MS) analysis of polar and nonpolar compounds. This capability was enabled by creating a novel dynamic power supply, which allowed spray voltage to be applied in a linear sweep, thus generating a composite mass spectrum over the specified ramp period. At lower voltages, polar analytes are ionized via electrospray. Upon reaching a higher voltage (>4 kV) in the ramp process, APCI is activated from the same nESI emitter, allowing the ionization of nonpolar analytes, typically undetectable via nESI. Collectively, the advantages associated with the dynamic spray process include (i) increased matrix tolerance, (ii) autocorrection of spray voltage based on analyte type and emitter tip size/shape, and (iii) simultaneous quantification of different compounds from a single mixture. The broad applicability of this strategy was demonstrated via the analysis of proteins, nucleic acids, steroids, illicit drugs, lipids, and fatty acids. Importantly, the method was applied to diagnose malaria by analyzing whole blood, without any sample pretreatment. The method provided good sensitivity, accuracy, and precision. These results encourage future developments where the simple platform discussed here can be utilized as an alternative to conventional MS methods for analyzing biofluids.

DOI: https://doi.org/10.1021/acs.analchem.5c04327
J Proteome Res. 2025 Oct 17.

Aird-MSI: A High Compression Rate and Decompression Speed Format for Mass Spectrometry Imaging Data.

Shuochao Li, Hongping Sheng, Pengyuan Du, Jingying Chen, Xixi Wang, Junjie Tong, Jiahua Hong, Xiaohan Jing, Miaoshan Lu, Changbin Yu.

  Mass spectrometry imaging has emerged as a pivotal tool in spatial metabolomics, yet its reliance on the imzML format poses critical challenges in data storage, transmission, and computational efficiency. While imzML ensures cross-platform compatibility, its lower compressed binary architecture results in large file sizes and high parsing overhead, hindering cloud-based analysis and real-time visualization. This study introduces an enhanced Aird compression format optimized for spatial metabolomics through two innovations: (1) a dynamic combinatorial compression algorithm for integer-based encoding of m/z and intensity data; (2) a coordinate-separation storage strategy for rapid spatial indexing. Experimental validation on 47 public data sets demonstrated significant performance gains. Compared to imzML, Aird achieved a 70% reduction in storage footprint (mean compression ratio: 30.89%) while maintaining near-lossless data precision (F1-score = 99.75% at 0.1 ppm m/z tolerance). For high-precision-controlled data sets, Aird accelerated loading speeds by 13-fold in MZmine. The Aird format overcomes crucial bottlenecks in spatial metabolomics by harmonizing storage efficiency, computational speed, and analytical precision, reducing I/O latency for large cohorts. By achieving near-native feature detection accuracy, Aird establishes a robust infrastructure for translational applications, including disease biomarker discovery and pharmacokinetic imaging.

Keywords:  Aird; data compression; mass spectrometry imaging; spatial metabolomics

DOI:  https://doi.org/10.1021/acs.jproteome.5c00423
Environ Int. 2025 Oct 10. pii: S0160-4120(25)00565-3. [Epub ahead of print]205 109814

Dried blood spots analysis for targeted and non-targeted exposomics.

Vinicius Verri Hernandes, Maximilian Zeyda, Lukas Wisgrill, Benedikt Warth.

  Dried blood spots (DBS) are an established sample type, widely used in newborn screening programs for monitoring metabolic diseases. Their minimally invasive nature offers great promise for assessing chemical exposures, particularly during early life stages and in large-scale epidemiological studies. However, comprehensive evaluations of key analytical parameters such as extraction efficiency and matrix effects across multiple chemical classes remain limited. Moreover, the promising approach of broadly combining targeted and non-targeted mass spectrometric data evaluation remains unexplored in DBS small-molecule omics. Here, we present an optimized LC-HRMS workflow for combined exposomic and metabolomic analysis in DBS samples. Four extraction protocols were systematically compared, with analytical performance evaluated for >200 structurally diverse xenobiotics, including PFAS chemicals, personal care products, pesticides, mycotoxins and other food contaminants, flame retardants, polyphenols and additional pollutants/toxicants or their biomarkers of exposure. The optimized protocol demonstrated acceptable recoveries (60-140 %) and reproducibility (median RSD: 18 %) for a majority of compounds. Matrix effects showed a median value of 76 % (median RSD: 14 %). In a proof-of-principle study, eleven exposure compounds of the target panel with diverse physicochemical properties were identified in real-life samples, with several reported for the first time in DBS human biomonitoring. Complementary non-targeted analysis further expanded the detectable chemical space, enabling reliable annotation of additional exposures. Moreover, high-confidence identification of endogenous metabolites, including amino acids, biogenic amines, fatty acids and acylcarnitines demonstrated the integrated capacity to capture a broad snapshot of the human metabolome. These findings support the use of DBS for integrated exposomics and metabolomics applications, providing toxicological and biological insights from low-volume samples in both, prospective and retrospective studies.

Keywords:  Early-life prevention; Microsampling; Next-generation human biomonitoring; Public/environmental health; Untargeted metabolomics

DOI:  https://doi.org/10.1016/j.envint.2025.109814
Methods Mol Biol. 2026 ;2976 85-102

Lysosomal Immunoprecipitation (LysoIP) from Cells and Tissues for Metabolomic and Proteomic Analyses.

Nouf N Laqtom, Monther Abu-Remaileh.

  Lysosomes, known for degrading biomolecules and damaged cellular components, are now recognized as signaling hubs for nutrient sensing and metabolic adaptation, and their dysfunction is implicated in diseases including cancer and neurodegeneration. To understand the composition of the lysosome, the dynamic behavior of its contents, and its specific roles in health and disease, we describe a lysosomal immunoprecipitation method, termed "LysoIP," that enables the isolation of intact lysosomes from cultured cells and mouse tissues. This method utilizes a lysosome-localized 3xHA epitope tag (LysoTag) and a simple, yet robust organelle immunoprecipitation workflow. Isolated lysosomes are extracted with optimized buffers to allow the efficient retrieval of lysosomal proteins, polar metabolites, and lipids, maintaining compatibility with downstream liquid chromatography and mass spectrometry (LC-MS) analyses.

Keywords:  LC-MS analyses; LysoIP; LysoTag; LysoTag mouse; Lysosomes; Metabolomics; Proteomics; TMEM192

DOI:  https://doi.org/10.1007/978-1-0716-4844-5_8
Anal Methods. 2025 Oct 15.

Demonstrating the application of a liquid chromatography-mass spectrometry method for the determination of phthalate diesters in complex solid and liquid environmental samples.

Fiona Regan, Catherine Allen, Jenny Lawler.

Phthalate diesters are ubiquitous contaminants of concern, yet their reliable determination in complex environmental matrices remains analytically challenging due to low environmental concentrations, matrix interferences, and widespread background contamination. Here, we present a robust LC-MS/MS workflow for the simultaneous quantification of eleven low- and high-molecular weight phthalates across four contrasting matrices: surface water, landfill leachate, soils, and municipal solid wastes. Sample preparation was streamlined using ultrasonication or filtration followed by solid-phase extraction (SPE), providing recoveries of 70-98% without the need for derivatisation. The method delivered sub-ng L-1 detection limits (as low as 0.2 ng L-1) and high reproducibility (RSD <5%), while integrated contamination-control strategies, including delay columns and phthalate-free materials, enabled reliable trace analysis. Systematic evaluation of matrix effects confirmed robustness across diverse environmental samples. This method combines sensitivity, selectivity, and broad applicability, offering a practical platform for routine environmental monitoring of phthalates at trace levels.

DOI: https://doi.org/10.1039/d5ay01057h
Foods. 2025 Sep 23. pii: 3296. [Epub ahead of print]14(19):

Construction and Comparison of UPLC-QE-Orbitrap-MS and UPLC-MS/MS Methods for the Detection of Diazepam Residues in Complex Aquatic Matrices.

Yuanhao Yang, Yi Li, Qiangbing Tian, Juanning Yang, Yiming Xu, Wen Su, Fei Wang, Siyao Qu, Sien Wen, Wei Cao.

  A standardized pretreatment protocol was established for simultaneous determination of diazepam and its metabolites-nordazepam, oxazepam, and temazepam-in aquatic products using liquid chromatography-mass spectrometry. Samples were extracted with 0.2% formic acid in acetonitrile (solid-liquid ratio 1:5, m/v), purified via MCX solid-phase extraction, eluted with 5% ammoniated methanol, and concentrated under reduced pressure. The residue was reconstituted in 0.2% formic acid-50% acetonitrile aqueous solution. Chromatographic and mass spectrometric conditions were optimized on two platforms: UPLC-QE-Orbitrap-MS and UPLC-MS/MS, with quantification based on internal standards. Both platforms showed excellent linearity across 0.2-200 ng/mL (R2 > 0.997), with detection and quantification limits as low as 0.1 μg/kg and 0.2 μg/kg, respectively. Following Codex Alimentarius guidelines (CAC/GL-71), 330 matrix samples (intra-batch n = 6, inter-batch n = 5) were validated, showing strong inter-platform agreement (Pearson r > 0.990, p < 0.001). Intra-batch RSDs ranged from 1.86% to 14.64%, and inter-batch RSDs from 1.10% to 11.41%. Recoveries ranged from 73.8% to 117.9% (p > 0.05). The dual-platform detection system developed herein demonstrates high sensitivity, strong matrix interference resistance, and excellent reproducibility, enabling accurate trace quantification of diazepam and its metabolites in heterogeneous aquatic samples.

Keywords:  complex aquatic matrices; diazepam metabolites; high-resolution MS; tandem triple quadrupole MS; ultra-performance liquid chromatography-orbitrap MS

DOI:  https://doi.org/10.3390/foods14193296
J Chromatogr A. 2025 Oct 08. pii: S0021-9673(25)00793-9. [Epub ahead of print]1763 466449

Time-controlled online SPE: strategic approach for N-acylethanolamines quantification in complex matrices.

Nicoletta Gatti, Valentina Greco, Salvatore Cuzzocrea, Rosalia Crupi, Enrico Gugliandolo, Alessandro Giuffrida.

  Quantifying and monitoring the level of N-acylethanolamines (NAEs) in biological fluids is becoming increasingly important to better understand their role in health and disease. The complexity of biological matrices, however, poses significant challenges for accurate quantification, with traditional pretreatment methods often proving insufficient in certain cases. This study introduces a novel approach utilizing Online Solid Phase Extraction (SPE) to quantify NAEs in equine plasma, specifically Palmitoyl Ethanolamide (PEA) and Oleoyl Ethanolamide (OEA). In the original analytical method here developed and validated, established toluene liquid-liquid extraction was used to isolate the lipid-like fraction, followed by an innovative Time-Controlled Online SPE coupled to HPLC-MS/MS. This strategic temporal approach allows target analytes to rapidly elute to the analytical column while retaining lipidic interferents, preventing matrix contamination and ensuring selective analysis. The method validation demonstrated excellent linearity, while providing high recovery and suitable matrix effects. The limits of detection (LOD) and quantification (LOQ) were determined as 0.27 ng/mL and 0.83 ng/mL for PEA; 0.04 ng/mL and 0.11 ng/mL for OEA. The innovative approach here presented allowed for reliable NAE quantification in equine plasma and provided unprecedented data on the endogenous levels of PEA and OEA in this species. This method has potential applications for the analysis of other complex biological matrices with high levels of interferents that share chemical similarities with the target compounds. This novel approach significantly advances the understanding of the endocannabinoid system in equines and serves as a valuable tool for future research in this domain.

Keywords:  Equine plasma; HPLC ESI-MS/MS; N-acylethanolamines; Online SPE; Palmitoylethanolamide; Quantification

DOI:  https://doi.org/10.1016/j.chroma.2025.466449
Crit Rev Anal Chem. 2025 Oct 17. 1-31

Real-Time Cell Analysis Using Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy at Single and Bulk Cellular Levels.

Swati Ramesh Pawar, Deepak Kumar, Rushikesh Biradar, Gayathri Loganathan, Pirangi Srikanth, Khaja Moinuddin Shaik, Sukhendu Nandi.

  Outlining biological processes and disease mechanisms requires a real-time understanding of cellular metabolism. Mass spectrometry (MS) and Nuclear magnetic resonance (NMR) serve as potent analytical methods for metabolomics, leveraging their advantages in the identification and quantification of metabolites. In this review we have discussed MS and NMR based techniques, such as matrix-assisted laser desorption/ionization (MALDI), secondary ionization mass spectrometry (SIMS), desorption electrospray ionization (DESI), direct analysis in real time (DART), and nano electrospray ionization (nano ESI), to develop and implement a MS based technique in its live state for single cell and NMR strategies for thorough, real-time analysis of cell metabolism at bulk cellular level. Single-cell metabolomic investigations using mass spectrometry is a valuable tool for understanding cellular heterogeneity and cell-to-cell variation. However, they can reveal hidden processes and heterogeneity across cells that are often missed by bulk cell analysis. Overcoming the inherently low sensitivity of NMR is crucial for omics studies. This review examines hyperpolarization techniques, including dynamic nuclear polarization (DNP), parahydrogen-induced polarization, sample amplification by reversible exchange (SABER), high-resolution magic angle spinning (HRMAS) NMR, and in vivo magnetic resonance spectroscopy (MRS) for the analysis of live bulk cells. Our discussion encompasses the technological platforms and recent applications of these techniques, utilizing both NMR and MS, along with an overview of metabolomics data analysis tools, such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA).

Keywords:  Analytical techniques; live cell metabolomics; mass spectrometry; nuclear magnetic spectroscopy

DOI:  https://doi.org/10.1080/10408347.2025.2572754
Arch Toxicol. 2025 Oct 16.

Method validation for quantification of five antineoplastic agents in urine using UPLC-ESI-MS/MS.

Eline Verscheure, Dina Vandervoort, Emily Deruyck, Katrien Poels, Manosij Ghosh, Jeroen Vanoirbeek, Lode Godderis.

  Antineoplastic agents are hazardous compounds frequently used in cancer treatment. It is already known that the hospital environment poses a risk of occupational exposure to these agents. However, recent years, the rise of outpatient treatment and at-home treatment has introduced an additional risk including also cohabitants of patients. We identified a clear need for highly sensitive monitoring methods to assess exposure to high-risk compounds in a home setting. This study presents two validated methods for quantifying five high-risk antineoplastic agents in urine: one for cyclophosphamide, etoposide, mitomycin C and imatinib, and one for alpha-fluoro-beta-alanine. Liquid-liquid extraction with ethyl acetate was used for extraction of cyclophosphamide, etoposide, mitomycin C and imatinib from urine. Alpha-fluoro-beta-alanine was extracted using solid-phase extraction with Oasis HLB cartridges. All samples were analysed using ultra-performance liquid chromatography coupled to tandem mass spectrometry. During method validation, selectivity, extraction efficiency, matrix effect, process efficiency, linearity, sensitivity, precision and accuracy were established. The lower limits of quantification were determined to be 0.1 ng/mL (cyclophosphamide and mitomycin C), 0.7 ng/mL (etoposide), 1 ng/mL (alpha-fluoro-beta-alanine) and 10 ng/mL (imatinib). The methods were fully validated and are now ready for application in the field.

Keywords:  Anticancer drug; Biomonitoring; Chemical exposure; Liquid chromatography; Mass spectrometry; Trace analysis

DOI:  https://doi.org/10.1007/s00204-025-04220-y
J Chromatogr A. 2025 Oct 01. pii: S0021-9673(25)00774-5. [Epub ahead of print]1763 466430

Determination of several cationic pesticides in food of plant and animal origin using the QuPPe method and ion chromatography with tandem mass spectrometry.

Ann-Kathrin Schäfer, Walter Vetter, Michelangelo Anastassiades.

  Several highly hydrophilic pesticides and metabolites are either permanently cationic or become cationic at low pH value. This makes them particularly suited for the analysis by ion chromatography (IC), especially since the suppressor technique enables a safe and reliable connection of IC with tandem mass spectrometry (IC-MS/MS). Here, we present a multi-method for the determination of 16 cationic pesticides using the 'quick polar pesticides' (QuPPe) extraction procedure and IC-MS/MS. The method covers permanently cationic compounds like chlormequat, mepiquat, diquat, and paraquat, as well as ionizable compounds like nicotine. Two different cation exchange columns were evaluated. Also, the impact of infusing an organic make-up solvent (e.g. acetonitrile and methanol) to the LC eluate between column exit and ion source entrance was tested at different infusion ratios, with acetonitrile performing best overall in terms of signal enhancements. Moreover, matrix effects tested with different types of matrices proved to be less problematic (±20 % in most cases) compared to LC-MS/MS methods. The final method was successfully validated at low levels in food of plant and animal origin (raspberry, sesame, rice, milk) and its suitability was verified by analysing >100 samples with incurred residues.

Keywords:  Diquat; Food; IC-MS/MS; Mepiquat; Nicotine; Paraquat; Polar pesticide

DOI:  https://doi.org/10.1016/j.chroma.2025.466430
Anal Chem. 2025 Oct 12.

In-Capillary Chemoselective Tagging-Facilitated Direct nESI-MS Profiling of Carboxyl- and Carbonyl-Containing Metabolites in Urine.

Yifan Wei, Xiumei Zheng, Zili Huang, Chunxiao Mou, Xiaobo Xie, Yi Lv.

Carboxyl- and carbonyl-containing metabolites (CCMs) are essential for energy metabolism and signaling in living cells and can serve as diagnostic biomarkers for various diseases. However, their low ESI ionization efficiencies and structural diversities could bring difficulties for their direct detection by mass spectrometry. In this work, a chemoselective tagging and acid-trigged release (CTAR) strategy-based nanoelectrospray ionization-mass spectrometry (nESI-MS) platform was developed by immobilizing a cis-diol-containing amine probe (i.e., 3-((2-aminoethyl)amino)propane-1,2-diol, denoted as AEAP-diol) on a coated ESI capillary with boronate esters as acid cleavage site, which can directly and selectively "fish out" targeted CCMs from biological matrices. Following the removal of unwanted species, the AEAP-diol tagged CCMs derivatives can be online released and directly detected by nESI-MS within 1 min. MS/MS fragmentation profiles of these CCMs derivatives exhibited a neutral loss of 91 Da and diagnostic fragments at m/z 118 for carboxyl- and carbonyl-containing metabolites, respectively, thereby enabling unambiguous structural elucidation of unknown CCMs. With high specificity, superior salt tolerance, and outstanding robustness, our CTAR-nESI-MS platform enabled direct and sensitive analysis of CCMs from a single drop of biofluid (2.5 μL of urine), achieving a low detection limit of 0.1 ppb for butyric acid. Using this method, 26 carboxyl- and carbonyl-containing metabolites were identified and quantified in urine samples from esophageal cancer patients and healthy volunteers, revealing significant differences in several reported CCM biomarkers. These findings collectively underscore the potential of CTAR-nESI-MS as a sensitive, cost-effective, and versatile tool for comprehensive metabolite profiling and high-throughput screening applications.

DOI: https://doi.org/10.1021/acs.analchem.5c04351
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Oct 06. pii: S1570-0232(25)00360-5. [Epub ahead of print]1267 124806

A fast GC-MS/MS method for the simultaneous measurement of key metabolites of peroxisomal beta-oxidation and ether lipid biosynthesis in human fibroblasts.

Karin Preindl, Thomas Stimpfl, Gunda Koellensperger, Fabian Dorninger, Johannes Berger, Christoph Wiesinger.

  Peroxisomes are subcellular compartments that host a variety of metabolic pathways, including the chain shortening of fatty acids (FAs) by beta-oxidation and certain steps in the formation of ether lipids. Here, we describe the development of a GC-MS/MS-based method for the simultaneous and reproducible determination of key metabolites of these pathways, also including less common FA species related to peroxisomal metabolism that are typically not part of standard analytical methods. We for the first time utilize 1-chlorobutane for the extraction of FAs as an effective alternative to commonly used extraction solvents. 1-Chlorobutane offers a broader polarity range than hexane and lower toxicity relative to chloroform with solvent consumption of less than one mL per sample. Six saturated long to very long-chain FAs, nine polyunsaturated FAs (PUFAs), two dicarboxylic FAs and 1-O-octadecyl glycerol (ODG, batyl alcohol) were extracted simultaneously. The method was validated using fibroblasts and for the majority of FA species accuracies ranged from 80 to 110 % with precision values (CV %) from 6 to 20 %. The measurement of ODG is for the first time described as marker for the estimation of the cellular ether lipid synthesis rate. The suitability of the method was demonstrated by the analysis of primary human fibroblasts from controls and individuals with peroxisomal disorders. This cell type represents a widely used model system for the investigation of peroxisomal metabolism and disease, thus rendering our protocol a valuable addition to the toolkit for studying peroxisomal pathways.

Keywords:  1-O-octadecyl glycerol; Ether lipids; GC–MS/MS; PUFA; VLCFA; X-ALD; Zellweger spectrum disorder

DOI:  https://doi.org/10.1016/j.jchromb.2025.124806
Food Chem. 2025 Oct 07. pii: S0308-8146(25)03920-2. [Epub ahead of print]496(Pt 1): 146668

New salty extraction of polar analytes technique and oil-based cleanup (SExPA-Oc) for the determination of nereistoxins in vegetable samples with HILIC-MS/MS.

J L Pérez-Figueroa, P Fernández-Hernando, M A García Mayor, R M Garcinuño.

  Nereistoxins are polar, unstable insecticides that present significant analytical challenges in agricultural products due to rapid degradation, limiting reliable monitoring in food safety programs. This study was carried out to address these challenges by developing a stable, efficient, and cost-effective method for the extraction and quantification of nereistoxins (cartap, nereistoxin, thiocyclam, thiosultap) in tomatoes, avocados, and olive oil. The novel SExPA-Oc (salty extraction of polar analytes-oil cleanup) method uses a salt-assisted liquid-liquid partition combined with an olive oil cleanup step, eliminating the need for expensive sorbents while improving analyte stability. Analysis by Hydrophilic Interaction Liquid Chromatography coupled with tandem mass spectrometry (HILIC-MS) achieved a linear range of 3-800 μg/kg and recoveries exceeding 70 % in most matrices, with limits of quantification meeting regulatory standards. This strategy provides a robust, environmentally friendly solution for nereistoxin analysis, addressing gaps in food safety monitoring and broader applicability to other polar, unstable compounds.

Keywords:  Agrochemical residues; Food safety monitoring; Liquid-liquid extraction; Olive oil cleanup

DOI:  https://doi.org/10.1016/j.foodchem.2025.146668
Clin Chim Acta. 2025 Oct 14. pii: S0009-8981(25)00483-8. [Epub ahead of print] 120604

Liquid chromatography-tandem mass spectrometry-based recalibration reduces inter-platform variability in aldosterone detection across chemiluminescence immunoassay platforms.

Jing Zhang, Baobin Luo, Zifu Fan, Xiaoyu Ma, Yungang Pu, Xiangyi Liu.

   BACKGROUND: Accurate aldosterone (ALD) measurement is vital in managing diseases such as primary aldosteronism (PA). However, inter-platform inconsistency across chemiluminescence immunoassay (CLIA) platforms complicates clinical decisions. This study conducted the largest comparison of CLIA platforms (six CLIA platforms) and one liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for ALD detection to date, systematically evaluated inter-platform consistency and explored the role of recalibration strategy based on LC-MS/MS in improving consistency among CLIA platforms.
METHODS: Fifty pooled clinical plasma samples were used to evaluate ALD levels across six different CLIA platforms and one LC-MS/MS assay. Friedman's test, Spearman correlation, Passing-Bablok regression, and Bland-Altman analysis were used to evaluate the consistency among assays. In addition, CLIA platforms were recalibrated against LC-MS/MS using regression equations with five pooled clinical plasma samples as calibration materials, and consistency was evaluated before and after recalibration.
RESULTS: LC-MS/MS yielded significantly lower ALD levels than six CLIA platforms (median: 120.75 vs. 129.29 to 216.25 pg/mL, P < 0.05). All assays correlated strongly (R ≥ 0.955), yet regression parameters revealed most slopes deviated from 1 (0.909 to 1.444) and intercepts ranged from -31.424 to 52.272 pg/mL. Bland-Altman plots demonstrated large relative mean differences (-2.396 % to 55.876 %) between assays. The recalibration process significantly reduced relative mean differences, whereas it showed limited improvement in addressing both proportional and systematic biases.
CONCLUSIONS: These findings demonstrate that the consistency among CLIA platforms and LC-MS/MS assays is suboptimal. Actionable design strategies for developing recalibration coefficients, which reduce relative mean differences, are provided for CLIA platforms, emphasizing the necessity for standardized calibration to reduce inter-platform variability in clinical ALD measurement. The persistent proportional and systematic biases underscore the urgency for optimization in calibration strategy and detection methodology.

Keywords:  Aldosterone; Chemiluminescence immunoassay; Liquid chromatography-tandem mass spectrometry; Method comparison; Recalibration

DOI:  https://doi.org/10.1016/j.cca.2025.120604
Nat Commun. 2025 Oct 15. 16(1): 9129

Spatial biology using single-cell mass spectrometry imaging and integrated microscopy.

Alexander Potthoff, Jan Schwenzfeier, Marcel Niehaus, Sebastian Bessler, Emily Hoffmann, Oliver Soehnlein, Jens Höhndorf, Klaus Dreisewerd, Jens Soltwisch.

Spatial biology pursues the analysis of cells in their native microenvironment, often with regard to morphology and gene- or protein expression. The spatial analysis of lipid and metabolic profiles by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can add another layer of molecular information. A seamless integration of MSI data with other (multimodal) methods at a single-cell level is often hampered by insufficient co-registration and resolution. Here, we introduce a MALDI-MSI based method that integrates in-source bright-field and fluorescence microscopy, allowing for a coupled (sub-)cellular investigation of the same sample in both modalities. Presented examples from cell culture and tissue analysis include the visualization of intracellular lipid distributions in macrophages during phagocytosis, and the heterogeneity of lipid profiles of tumor infiltrating neutrophils correlated to their individual microenvironments. The achieved combination of lipid profiling with morphological features and protein expression on the single-cell level constitutes a powerful method for cell biology.

DOI: https://doi.org/10.1038/s41467-025-64603-8