bims-metlip 2025-03-23 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–03–23
twenty-one papers selected by
Sofia Costa, Matterworks

Bioanalytical method validation to quantify ketorolac in human vitreous and aqueous via surrogate matrix of human plasma.
Simultaneous Multiclass Analysis of Cyanotoxins in Cyanobacterial Samples Using Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry.
Determination of Free Amino Acid Concentrations in Bovine Plasma Using High-Pressure Liquid Chromatography With Electrospray Ionization Mass Spectrometry Detection.
Development and validation of a highly-sensitive, quantitative LC-MS/MS assay to evaluate plasma oxytocin.
Untargeted pixel-by-pixel metabolite ratio imaging as a novel tool for biomedical discovery in mass spectrometry imaging.
Novel Isotope-Coded Photochemical Derivatization Coupled with LC-MS and MS Imaging Platform Enables Sensitive Quantification and Accurate Localization of Amine Submetabolome in Pancreatic Disease.
Topographic imaging with automatic z-axis correction of Brassica oleracea var. viridis leaves by IR-MALDESI mass spectrometry imaging.
Multidimensional Separations for Characterization of Isomeric PFAS Using SLIM High-Resolution Ion Mobility and Tandem Mass Spectrometry.
Beyond Targeted Newborn Screening: A Nontargeted Metabolomics Workflow to Investigate Birthweight-Metabolome Correlations.
The metabolomic profile features of some bio logical fluids in serous ovarian adenocarcinoma patients.
Simultaneous Monitoring of 3 Antiviral Drugs in Serum Using Liquid Chromatography-Tandem Mass Spectrometry: Full Validation and Clinical Application.
Biomonitoring of azole fungicides in free-living blackbird plasma using on-line solid-phase extraction coupled to liquid chromatography-tandem mass spectrometry (SPE HPLC-MS/MS).
Analysis of N,N-dimethyltryptamine (DMT) and its metabolites using LC-MS/MS for forensic purposes.
Clinical Lipidomics Reveals High Individuality and Sex Specificity of Circulatory Lipid Signatures: A Prospective Healthy Population Study.
Combining per-aqueous and chiral reversed phase separation modes towards an enhanced comprehensive 2-dimensional liquid chromatographic based chiral screening platform.
Frontiers in Mass Spectrometry-Based Spatial Metabolomics: Current Applications and Challenges in the Context of Biomedical Research.
Pneumatically Assisted Microfluidic Probe for Enhanced Mass Spectrometry Imaging Performance.
LC-ESI-MS/MS method for the accurate quantification of eight nitrosamines in pharmaceutical products.
Application of rapid evaporative ionization mass spectrometry in preclinical and clinical analyses of steatotic liver tissues and cells.
A multiplex assay of leptin, resistin, and adiponectin by immunoaffinity enrichment and targeted mass spectrometry.
Enhancing pyrrolizidine alkaloid separation and detection: LC-MS/MS method development and integration of ion mobility spectrometry into the LC-HRMS workflow.

Future Sci OA. 2025 Dec;11(1): 2476866

Bioanalytical method validation to quantify ketorolac in human vitreous and aqueous via surrogate matrix of human plasma.

Prajita Pandey, Brianna A White, Colin Goswell, Neelanjan Bose, Sara Butterworth Connell, Nicolee Schulze, Jim Nevelos, Ana Najafi, Ramin Najafi, Ryan K Cheu.

   PURPOSE: Intracameral phenylephrine 1.0%/ketorolac 0.3% (OMIDRIA®) is used during cataract surgery to prevent intraoperative miosis and reduce postoperative pain. Although studied in beagles, no human data exist showing the duration ketorolac remains in the eye postoperatively. A clinical trial measuring ketorolac concentrations in aqueous/vitreous samples necessitated the development of a validation process for acquiring these measurements. Due to limited human aqueous/vitreous humor sample availability, a bioanalytical method was developed and validated to quantify ketorolac levels using human plasma as a surrogate matrix.
METHODS: The developed process involves extracting ketorolac and its internal standard (ketorolac-d5) from plasma as a surrogate for aqueous and vitreous humor using a protein precipitation sample preparation technique, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
RESULTS: The validated method can be successfully applied for quantitation of ketorolac over a concentration range of 2.5 ng/mL to 5000 ng/mL. The method met the acceptance criteria with respect to selectivity, specificity, precision, accuracy, linearity, dilution integrity, and stability.
CONCLUSIONS: The validated method can use plasma as a surrogate matrix for quantitation of ketorolac in aqueous and vitreous humor, thereby eliminating the need to procure human vitreous and aqueous samples for validation prior to initiation of a clinical trial.

Keywords:  OMIDRIA; Surrogate matrix; cataract surgery; ketorolac; liquid chromatography-tandem mass spectrometry; phenylephrine/ketorolac; validation study

DOI:  https://doi.org/10.1080/20565623.2025.2476866
J Sep Sci. 2025 Mar;48(3): e70121

Simultaneous Multiclass Analysis of Cyanotoxins in Cyanobacterial Samples Using Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry.

Rosemary Bergin, Siobhan Peters, Simon Mitrovic, David P Bishop.

  The proliferation of cyanobacteria can result in algal blooms, which may cause environmental and biological harm due to the production and release of secondary metabolites, or cyanotoxins, into the affected waterway. Cyanobacteria can produce multiple classes of cyanotoxins; therefore, to understand the full toxic load of algal blooms, it is necessary to perform analyses that quantify each class. These classes are generally monitored individually due to the challenges associated with the differing physicochemical properties of the cyanotoxins. Hydrophilic interaction liquid chromatography (HILIC) is a form of chromatography capable of retaining multiple classes of cyanotoxins that differ in physicochemical properties. Here an HILIC-MS/MS method was developed and validated to detect 3 microcystins, 11 saxitoxins, and 2 anatoxins. The chromatographic conditions were optimized to allow for the separation of multiple pairs of saxitoxin epimers, and in-source fragmentation in the MS interface was used to develop unique MRMs between the pairs. The method was validated and had low limits of detection (LODs, between 0.00770 and 9.75 µg L-1) and limits of quantification (LOQs, between 0.0257 and 32.5 µg L-1) for all compounds. All analytes exhibited good linearity (R2 values ≥ 0.991) and low percentage relative standard deviations for retention time (0%-1.74%) and peak area (4.54%-27.6%), with spiked recoveries ranging from 75.6% to 117% for all compounds. A multiclass sample preparation method to extract the three classes of analytes from cyanobacterial samples was developed and validated, with 80:20 acetonitrile:water and 0.1% formic acid as the optimal extraction solvent. The newly developed sample preparation and analysis methods were applied to cultured cyanobacteria and field samples, with microcystins and saxitoxins detected. The multiclass sample preparation and analysis methods developed here improve on individual methods as they reduce the complexity and time of sample preparation and analysis and will assist ecotoxicologists in assessing the full toxic risk of cyanobacterial blooms.

Keywords:  algal blooms; cyanobacteria; cyanotoxins; hydrophilic interaction liquid chromatography; multiclass analysis

DOI:  https://doi.org/10.1002/jssc.70121
Rapid Commun Mass Spectrom. 2025 Apr 15. 39(11): e10027

Determination of Free Amino Acid Concentrations in Bovine Plasma Using High-Pressure Liquid Chromatography With Electrospray Ionization Mass Spectrometry Detection.

Laurie A Reinhardt, Levi Svaren, Ritvik Marathe, Geoffrey I Zanton, Michael L Sullivan.

   RATIONALE: Quantification of free amino acid concentrations in plasma has become an important tool in monitoring the health of dairy cows and health of their offspring under various management regimes, especially diet. Consequently, it was desirable to develop a robust, accurate, medium-throughput method to quantitate free amino acid concentrations in bovine plasma.
METHODS: Bovine plasma was deproteinated with methanol and amino acids partially purified using cation exchange resin. Samples were then subjected to precolumn derivatization with phenyl isothiocyanate, followed by high-pressure liquid chromatography with positive electrospray ionization single quadrupole mass spectrometry detection for analysis. The corresponding 13C and 15N labeled amino acids (mass unit difference > 3) were used as internal standards, while deuterium labeled standards were used for other metabolites.
RESULTS: All 20 amino acids showed linear fits to their individual calibration curves (correlation coefficients > 0.99) with concentration range of amino acids measured from 5 to 600 μM. Coefficient of variation (CV) values for the concentrations measured for all amino acids ranged from 2.0 to 6.7 for intraday aliquots and from 1.0 to 4.6 for interday aliquots with the exception of aspartic acid (11.1 and 12.6 for intraday and interday, respectively).
CONCLUSIONS: The use of a stable isotope labeled version of each amino acid analyte as internal standard added to plasma samples at the beginning of the procedure corrected for any losses, instrument variability, and chemistry of derivatization. Use of this method to quantify bovine plasma amino acids will allow better understanding of physiological processes underlying nutritional interventions in dairy production systems and may be more broadly applicable to ruminant and other animal production systems.

Keywords:  amino acid analysis; dairy cow; plasma

DOI:  https://doi.org/10.1002/rcm.10027
J Mass Spectrom Adv Clin Lab. 2025 Apr;36 19-28

Development and validation of a highly-sensitive, quantitative LC-MS/MS assay to evaluate plasma oxytocin.

E Grifnée, A Mackowiak, J Demeuse, M Schoumacher, L Huyghebaert, W Determe, T Dubrowski, P Massonnet, S Peeters, G Scantamburlo, E Cavalier, C Le Goff.

   Introduction: Oxytocin is a 9-amino acid peptide that serves as neuromodulator in the human central nervous system. This peptide is implicated in the regulation of diverse behaviors and plays a significant role in positive social interaction. Currently, oxytocin levels are measured using immunoassays. However, these methods have several limitations that can lead to false results and erroneous interpretation. Given the remarkably low endogenous level of oxytocin in human plasma (low ng/L levels), we developed and rigorously validated a novel and highly sensitive LC-MS/MS method for oxytocin quantification in plasma.
Methods: Oxytocin was initially extracted using solid-phase extraction with an Oasis HLB 30 mg plate and then subjected to LC-MS/MS analysis. PBS-0.1 % BSA served as surrogate matrix for the preparation of validation samples and the calibration curve, ensuring no endogenous interference. The validation design followed the Clinical Laboratory Standards Institute guidelines. Precision, accuracy, and measurement uncertainty were determined using single-nested analysis of variance and e.noval software.
Results: A lower limit of quantification of 1 ng/L was achieved. The method was validated for oxytocin concentrations ranging from 1 ng/L to 75 ng/L, with precision (coefficient of variation) below 10 %, accuracy ranging from 94 % to 108 %, and measurement uncertainty below 15 %.
Conclusion: In this work, we developed and validated a highly sensitive LC-MS/MS method for the quantification of oxytocin in plasma. Our novel methodology is well-suited for clinical applications.

Keywords:  CLSI guidelines; Liquid chromatography; Oxytocin; Psychiatric disorders; Solid phase extraction; Tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.jmsacl.2025.02.002
Elife. 2025 Mar 18. pii: RP96892. [Epub ahead of print]13

Untargeted pixel-by-pixel metabolite ratio imaging as a novel tool for biomedical discovery in mass spectrometry imaging.

Huiyong Cheng, Dawson Miller, Nneka Southwell, Paola Porcari, Joshua L Fischer, Isobel Taylor, J Michael Salbaum, Claudia Kappen, Fenghua Hu, Cha Yang, Kayvan R Keshari, Steven S Gross, Marilena D'Aurelio, Qiuying Chen.

  Mass spectrometry imaging (MSI) is a powerful technology used to define the spatial distribution and relative abundance of metabolites across tissue cryosections. While software packages exist for pixel-by-pixel individual metabolite and limited target pairs of ratio imaging, the research community lacks an easy computing and application tool that images any metabolite abundance ratio pairs. Importantly, recognition of correlated metabolite pairs may contribute to the discovery of unanticipated molecules in shared metabolic pathways. Here, we describe the development and implementation of an untargeted R package workflow for pixel-by-pixel ratio imaging of all metabolites detected in an MSI experiment. Considering untargeted MSI studies of murine brain and embryogenesis, we demonstrate that ratio imaging minimizes systematic data variation introduced by sample handling, markedly enhances spatial image contrast, and reveals previously unrecognized metabotype-distinct tissue regions. Furthermore, ratio imaging facilitates identification of novel regional biomarkers and provides anatomical information regarding spatial distribution of metabolite-linked biochemical pathways. The algorithm described herein is applicable to any MSI dataset containing spatial information for metabolites, peptides or proteins, offering a potent hypothesis generation tool to enhance knowledge obtained from current spatial metabolite profiling technologies.

Keywords:  adipose; brain; computational biology; embryo; mouse; systems biology

DOI:  https://doi.org/10.7554/eLife.96892
Anal Chem. 2025 Mar 17.

Novel Isotope-Coded Photochemical Derivatization Coupled with LC-MS and MS Imaging Platform Enables Sensitive Quantification and Accurate Localization of Amine Submetabolome in Pancreatic Disease.

Yaling Wu, Manjiangcuo Wang, Rui Wang, Shan Yang, Wanmeng Li, Siwei Bi, Xia Li, Yangjuan Bai, Qing Xia, Huimin Lu, Chenggong Hu, Dan Du.

Alterations in amine metabolite levels are closely associated with the poor progression of pancreatic disease, including acute pancreatitis (AP) and pancreatic cancer (PC). However, effectively quantifying and visualizing these metabolites through mass spectrometry (MS) has proven to be challenging. Here, we have designed a novel and rapid strategy for analyzing the amine submetabolome within liquid chromatography-mass spectrometry (LC-MS) and air-flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) platforms by inducing a pair of isotope-labeling-based photochemical derivatization reagents. The simultaneous introduction of a 4-amino-1-methylpyridinium moiety renders a 160- to 1037-fold higher response in MS. Coupled with full MS-ddMS2 and precursor ion scan modes, this labeling strategy allows for straightforward detection of 423 peaks for indazolone derivatives and identification of 82 amine metabolites in biological samples. The semiquantitation of the 82 amines in plasma from AP patients and healthy controls resulted in the discovery of unreported aromatic amines and aminoaldehydes with significant changes in AP and employing ethanolamine for distinguishing the severities of AP in the early stage. In the MSI platform, the photochemical reagent can efficiently derivatize primary amine metabolites avoiding spatial deviation and significantly enhancing imaging sensitivity in rat brain and kidney. Further joint analysis of amine submetabolome in plasma and pancreas from PC patients by use of these two platforms allowed for identifying the significant metabolite, methylamine. These results together enhance the role of amine-driven biomarker discovery in the diagnosis of pancreatic disease and accelerate the application of on-tissue photochemical derivation in MSI.

DOI: https://doi.org/10.1021/acs.analchem.4c06388
Anal Bioanal Chem. 2025 Mar 15.

Topographic imaging with automatic z-axis correction of Brassica oleracea var. viridis leaves by IR-MALDESI mass spectrometry imaging.

Quinn Mills, Sarah M Ashbacher, Alexandria L Sohn, David C Muddiman.

  Mass spectrometry (MS) is a versatile technique for elucidating the chemical composition of biological samples. Beyond analysis of crude extracts, MS can be further applied to spatially resolve compounds across the area of a sample with a technique called mass spectrometry imaging (MSI). The infrared matrix-assisted laser desorption ionization (IR-MALDESI) platform combines elements of matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) to enable MSI of mammalian tissue using endogenous water in the sample as a matrix. For laser-based techniques such as IR-MALDESI, changes in topography across the sample surface cause inconsistent ablation as the sample surface moves above and below the focal plane of the laser. The localization of chemical species in plants reveals crucial information about metabolic processes as reported by Nemes and Vertes (Anal. Chem. 79 (21), 8098-8106, 2007) and biosynthetic pathways by Zou et al. (Trends in Plant Science, 2024) and can even inform selective breeding of crops as discussed by Sakurai (Breed Sci 72 (1), 56-65, 2022); however, leaf topography raises a unique challenge. Features such as veins and trichomes exhibit unique topography, but flattening risks delocalization of analytes and activation of unwanted signaling pathways, and transferring metabolites to a membrane for indirect analysis may incur delocalization and limit metabolomic coverage. To overcome these challenges, a chromatic confocal sensor probe (CA probe) was incorporated for IR-MALDESI-MSI of sections of a collard (Brassica oleracea var. viridis) leaf. The CA probe measures the height at all points of the sample, and automatic z-axis corrections (AzC) are generated from height differences to continuously raise and lower the stage. These stage height corrections keep the sample surface in focus of the laser for the duration of analysis. This method has been applied to relatively homogenous samples, but has not yet been characterized on heterogeneous leaf tissue with considerable topography. Herein, data quality is compared between MSI analyses with and without AzC applied, focusing on the localization of analytes known to be concentrated in different layers of collard leaves.

Keywords:   Brassica oleracea var. viridis ; IR-MALDESI; Mass spectrometry imaging; Plant biology; Plant imaging; Plant metabolomics

DOI:  https://doi.org/10.1007/s00216-025-05820-4
Anal Chem. 2025 Mar 17.

Multidimensional Separations for Characterization of Isomeric PFAS Using SLIM High-Resolution Ion Mobility and Tandem Mass Spectrometry.

Heidi M Sabatini, Terra Pettit-Bacovin, Ralph Aderorho, Christopher D Chouinard.

Per- and polyfluoroalkyl substances (PFAS) are synthetic organofluorine compounds that accumulate in the environment due to significant industrial use and resistance to degradation. PFAS are of global interest because of their environmental and health concerns. They exist in a variety of linear and nonlinear forms containing a variety of isomers, as well as differing functional headgroups for each class. That structural complexity requires advanced analytical techniques, beyond current high-resolution mass spectrometry (HRMS) methods, for their accurate identification and quantification in a wide range of samples. Herein, we demonstrate the power of Structures for Lossless Ion Manipulations (SLIM)-based high-resolution ion mobility (HRIM) for separation of complex PFAS branched isomers. SLIM is integrated into a multidimensional LC-SLIM IM-MS/MS workflow, developed for the extensive characterization of a wide range of PFAS compounds. As we surveyed sulfonate and carboxylic acid classes of PFAS, we observed unique arrival time vs m/z trend lines that were representative of each class; these trend lines are important for allowing identification of emerging species based on their placement in that two-dimensional space. Next, we used complementary tandem mass spectrometry (MS/MS) approaches with all ion fragmentation (AIF), as well as energy-resolved MS/MS, to further investigate the structure of mobility-separated species. This allowed both investigation of fragmentation mechanism and identification of unique fragment ions that could allow differentiation of isomers when ion mobility was insufficient. Overall, the combination of chromatography, high-resolution SLIM, and MS/MS provided a comprehensive workflow capable of identifying unknown emerging PFAS compounds in complex environmental samples.

DOI: https://doi.org/10.1021/acs.analchem.4c06985
Anal Chem. 2025 Mar 18.

Beyond Targeted Newborn Screening: A Nontargeted Metabolomics Workflow to Investigate Birthweight-Metabolome Correlations.

Carter K Asef, Samuel G Moore, Charles Austin Pickens, Carlos A Saavedra-Matiz, Joseph J Orsini, Konstantinos Petritis, David A Gaul, Facundo M Fernández.

Newborn screening (NBS) is one of the United States' largest, most successful preventative public health initiatives, improving outcomes for newborns with inborn errors of metabolism. Most disorders on the Recommended Uniform Screening Panel are screened using triple-quadrupole mass spectrometry and flow injection analysis. While these methods are sensitive and well suited for high-throughput quantitative applications, the breadth of measured analytes is limited to a relatively small number of biomarkers, which often have considerable overlaps between healthy and diseased populations. High-resolution liquid chromatography-mass spectrometry (LC-MS)-based metabolomics is now capable of profiling thousands of metabolites, making it well suited for exploratory and biomarker discovery studies. To this end, we developed a robust workflow for performing nontargeted LC-MS analysis on dried bloodspot (DBS) specimens with coverage across many metabolic pathways relevant to NBS. HILIC chromatography enabled quantitation of amino acid and acylcarnitine species while also retaining lipid species, such as lyso-phosphatidylcholines. We analyzed 810 newborn-derived DBS samples across a wide range of newborn birthweights, identifying correlations with metabolites that help to better account for the lower accuracy observed for some NBS markers (e.g., isovalerylcarnitine). Additionally, we leveraged this nontargeted workflow to capture new biomarkers and metabolic phenotypes in newborns associated with parenteral nutrition administration and maternal nicotine exposure. Two critical biomarkers were identified as useful additions to targeted screening panels: N-acetyltyrosine as a qualitative marker for parenteral nutrition administration and N-acetylputrescine as a quantitative marker for controlling birthweight variability.

DOI: https://doi.org/10.1021/acs.analchem.4c06061
Klin Onkol. 2025 ;38(1): 38-44

The metabolomic profile features of some bio logical fluids in serous ovarian adenocarcinoma patients.

D S Kutilin, F E Filippov, O N Guskova, I A Alliluev, Y S Enin, A Y Maksimov.

   BACKGROUND: The search for effective biomarkers for ovarian cancer (OC) early diagnosis is an urgent task of modern oncogynecology. Metabolic profiling by ultra-high performance liquid chromatography and mass spectrometry (UHPLC-MS) provides information on the totality of all low molecular weight metabolites of patient's biological fluids sample, reflecting the processes occurring in the body. The aim of the study was to research blood plasma and urine metabolomic profile of patients with serous ovarian adenocarcinoma by UHPLC-MS.
MATERIAL AND METHODS: To perform metabolomic analysis, 60 blood plasma samples and 60 urine samples of patients diagnosed with serous ovarian carcinoma and 20 samples of apparently healthy volunteers were taken. Chromatographic separation was performed on a Vanquish Flex UHPLC System chromatograph (Thermo Scientific, Germany). Mass spectrometric analysis was performed on an Orbitrap Exploris 480 (Thermo Scientific, Germany) equipped with an electrospray ionization source. Bioinformatic analysis was performed using Compound Discoverer Software (Thermo Fisher Scientific, USA), statistical data analysis was performed in the Python programming language using the SciPy library.
RESULTS: Using UHPLC-MS, 1,049 metabolites of various classes were identified in blood plasma. In patients with OC, 8 metabolites had a significantly lower concentration (P < 0.01) compared with conditionally healthy donors, while the content of 19 compounds, on the contrary, increased (P < 0.01). During the metabolomic profiling of urine samples, 417 metabolites were identified: 12 compounds had a significantly lower concentration compared to apparently healthy individuals, the content of 14 compounds increased (P < 0.01). In patients with ovary serous adenocarcinoma, a significant change in the metabolome of blood plasma and urine was found, expressed in abnormal concentrations of lipids and their derivatives, fatty acids and their derivatives, acylcarnitines, phospholipids, amino acids and their derivatives, derivatives of nitrogenous bases and steroids. At the same time, kynurenine, myristic acid, lysophosphatidylcholine and L-octanoylcarnitine are the most promising markers of this disease.
CONCLUSION: The revealed changes in the metabolome can become the basis for improving approaches to the diagnosis of serous ovarian adenocarcinoma.

Keywords:  Urine; blood plasma; metabolomic profile; serous ovarian adenocarcinoma; ultra-high performance liquid chromatography and mass spectrometry; urine

DOI:  https://doi.org/10.48095/ccko202538
Ther Drug Monit. 2025 Mar 18.

Simultaneous Monitoring of 3 Antiviral Drugs in Serum Using Liquid Chromatography-Tandem Mass Spectrometry: Full Validation and Clinical Application.

Laura Conesa, Gonzalo Gonzalez-Silva, Lydia Peris-Serra, Sarai Garriga-Edo, Laura Castellote, Roser Ferrer, Yolanda Villena.

BACKGROUND: Patients undergoing solid organ and hematopoietic stem cell transplantation are at risk of opportunistic pathogenic infections that increase morbidity and mortality. Universal antiviral prophylaxis improves the outcomes in this context. Therapeutic drug monitoring of antiviral drugs is not universally recommended but may be necessary in certain complex or polymorbid patients. The authors aimed to develop and validate a high-performance liquid chromatography-tandem mass spectrometry method to simultaneously quantify ganciclovir, acyclovir, and letermovir in human serum.
METHODS: A stable isotopically labeled internal standard was used for each antiviral drug. Compounds were extracted by protein precipitation, evaporation, and reconstitution in an aqueous mobile phase. Samples were analyzed using reverse-phase chromatography with subsequent detection by electrospray ionization in the positive ion mode on a triple quadrupole mass spectrometer (run time: 6.5 minutes).
RESULTS: Analytical curves for ganciclovir and acyclovir exhibited linearity within 0.1-25 mg/L (R2 > 0.993), whereas for letermovir, the linear range was 0.01-2 mg/L (R2 = 0.999). Matrix effects were not observed. Intraday and interday precision and accuracy were within ±15%. A therapeutic drug monitoring-guided strategy was explored to optimize preemptive antiviral drug therapy in 3 cohorts of transplant recipients. Seventy-nine samples from 35 patients were quantified, revealing median trough concentrations of 0.2 mg/L for ganciclovir (n = 21), 0.28 mg/L for acyclovir (n = 26), and 0.29 mg/L for letermovir (n = 32).
CONCLUSIONS: This method has been successfully applied in clinical settings and allows reliable and accurate drug-level measurements.

DOI: https://doi.org/10.1097/FTD.0000000000001321
J Chromatogr A. 2025 Feb 22. pii: S0021-9673(25)00074-3. [Epub ahead of print]1748 465725

Biomonitoring of azole fungicides in free-living blackbird plasma using on-line solid-phase extraction coupled to liquid chromatography-tandem mass spectrometry (SPE HPLC-MS/MS).

Louise Prouteau, Marie-Hélène Dévier, Frédéric Angelier, Olivier Chastel, François Brischoux, Patrick Pardon, Karyn Le Menach, Hélène Budzinski.

  In this study, a rapid and sensitive method using on-line solid-phase extraction (SPE) coupled to liquid chromatography - tandem mass spectrometry (SPE HPLC-MS/MS) was developed to analyse 15 azole fungicides currently used in vineyards in blackbird plasma samples. The monitored fungicides included 13 triazoles (cyproconazole, difenoconazole, epoxiconazole, fenbuconazole, flusilazole, flutriafol, metconazole, penconazole, propiconazole, tebuconazole, tetraconazole, triadimefon, triadimenol) and 2 imidazoles (imazalil and prochloraz). After a rapid preparation step by protein precipitation with acetonitrile on 25 µL of plasma samples, final extracts diluted with Milli-Q water were analyzed by on-line SPE-LC-MS/MS in positive electrospray mode (ESI+) using the dynamic multi-reaction monitoring mode (dMRM). Following optimization, method validation was achieved through studies of linearity, sensitivity, accuracy, precision, and sample extract conservation. The limits of quantification (LOQs) obtained for a low volume of plasma (25 µL) ranged from 0.01 to 0.43 ng g-1 plasma, except for triadimenol (1.37 ng g-1). Finally, the validated method was successfully applied to 34 Eurasian blackbird plasma samples, with blackbirds from different habitats (city, forest, vineyards) submitted to contrasted azole pressures. Five of them were detected, tebuconazole and tetraconazole being the predominant ones. As expected, azoles concentrations were more elevated in blackbirds sampled in vineyards where most of these fungicides are used.

Keywords:  Azoles; Bird plasma; LC-MS/MS; On-line SPE; Pesticides

DOI:  https://doi.org/10.1016/j.chroma.2025.465725
J Forensic Sci. 2025 Mar 16.

Analysis of N,N-dimethyltryptamine (DMT) and its metabolites using LC-MS/MS for forensic purposes.

Munchelou M Gomonit, Madeleine J Swortwood, Michael T Truver, Britni N Skillman.

  Ayahuasca contains N,N-dimethyltryptamine (DMT), the primary alkaloid responsible for its psychedelic effects. DMT oxidative deamination yields indole-3-acetic acid (IAA) as the predominant metabolite, while N-oxidation produces N,N-dimethyltryptamine-N-oxide (DMT-NO) as the second most abundant metabolite. An LC-MS/MS method was developed and validated to quantify DMT, IAA, and DMT-NO in human plasma, as well as DMT and DMT-NO in human urine. Protein precipitation using a 75:25 (v/v) acetonitrile:methanol yielded analyte recoveries ≥91% in both plasma and urine. Key parameters including matrix effects, linearity, bias, precision, stability, carryover, and dilution integrity met their respective acceptability criterion outlined by ANSI/ASB 036 recommendations. In plasma, the linear range was 0.5-500 ng/mL (DMT), 0.25-125 ng/mL (DMT-NO), and 240-6000 ng/mL (IAA), while the DMT and DMT-NO range in urine was 2.5-250 ng/mL. Bias was within ±17.5%, and precision was ≤6.4% in both plasma and urine. Analytes were free from exogenous/endogenous interferences, and carryover was negligible. Extracts were also stable in the autosampler compartment (4°C) for 48 hours. A proof-of-concept study was conducted using authentic paired peripheral blood and urine samples. Results showed higher concentrations of DMT and DMT-NO found in urine as compared to plasma, highlighting the rapid metabolism and clearance of DMT and its metabolites. This study proposes the utility of DMT and DMT-NO as direct and distinctive biomarkers for forensic determination of exogenous DMT consumption. While IAA is the predominant metabolite of DMT, IAA should not be relied upon as the sole biomarker due to its substantial endogenous presence in both plasma and urine.

Keywords:  LC–MS/MS; N,N‐dimethyltryptamine; N,N‐dimethyltryptamine‐N‐oxide; ayahuasca; indole‐3‐acetic acid

DOI:  https://doi.org/10.1111/1556-4029.70013
J Lipid Res. 2025 Mar 18. pii: S0022-2275(25)00040-9. [Epub ahead of print] 100780

Clinical Lipidomics Reveals High Individuality and Sex Specificity of Circulatory Lipid Signatures: A Prospective Healthy Population Study.

Jessica Medina, Nicolas Goss, Gonçalo Dos Santos Correia, Rebecca Borreggine, Tony Teav, Zoltan Kutalik, Pedro Marques Vidal, Hector Gallart-Ayala, Julijana Ivanisevic.

  Lipid metabolism and circulatory lipid levels are tightly associated with the (cardio)metabolic health. Consequently, mass spectrometry (MS)-based lipidomics has emerged as a powerful phenotyping tool in epidemiological, human population, and in clinical intervention studies. However, ensuring high throughput and reproducible measurement of a wide panel of circulatory lipid species in large-scale studies poses a significant challenge. Here, we applied a recently developed quantitative LC-MS/MS lipidomics approach to a subset of 1086 fasted plasma samples belonging to apparently healthy participants from prospective Lausanne population study. This high-coverage and high-throughput HILIC-based methodology allowed for the robust measurement of 782 circulatory lipid species spanning 22 lipid classes and six orders of magnitude-wide concentration range. This was achieved by combining semi-automated sample preparation using a stable isotope dilution approach and the alternate analysis of NIST plasma reference material, as a quality control. Based on NIST QC analysis, median between-batch reproducibility was 8.5%, over the course of analysis of 13 independent batches comprising 1086 samples collected from 364 individuals at three time points. Importantly, the biological variability, per lipid species, was significantly higher than the batch-to-batch analytical variability. Furthermore, the significantly lower between-subject (compared to within-subject) variability, and unsupervised sample clustering demonstrated the high individuality and sex-specificity of circulatory lipidome. The most prominent sex differences were reported for sphingomyelins and ether-linked phospholipids present in significantly higher concentrations in female plasma. The high individuality and sex specificity of circulatory lipidome constitute important advantages for the application of lipidomics in next-generation metabolic health monitoring.

Keywords:  Circulatory lipids; Clinical lipidomics; HILIC-MS/MS; Personalized signatures; Prospective healthy population; Sex differences

DOI:  https://doi.org/10.1016/j.jlr.2025.100780
J Chromatogr A. 2025 Mar 14. pii: S0021-9673(25)00223-7. [Epub ahead of print]1748 465875

Combining per-aqueous and chiral reversed phase separation modes towards an enhanced comprehensive 2-dimensional liquid chromatographic based chiral screening platform.

Turaj Rahmani, Elena Bandini, Stephanie A Schuster, Lander Iterbeke, Jean-François Focant, Frédéric Lynen.

  The differentiation of enantiomers in complex mixtures is crucial in various fields such as food science, pharmaceuticals, and environmental studies. While this is achievable in principle through comprehensive two-dimensional liquid chromatography (LC × LC), practical challenges emerge when the mobile phase used in the first dimension (1D) is too strong or incompatible with the one in the second dimension (2D), leading to peak broadening and reduced resolution. These drawbacks become particularly evident when analyzing a diverse range of chiral compounds. In order to obtain timely elution of such compounds, typically spanning a range of hydrophobicity, the use of gradients (in both dimensions) is inevitable. This in turn leads to changing mobile phase compositions transferred from 1D to 2D, and hence to variations in the effectiveness of the modulation process. Lowering of the eluotropic strength of the mobile phase used in 1D allows to mitigate such problems. In this study, a novel achiral × chiral platform for fully automated screening of chiral compounds was developed. In the 1D of this platform, reversed HILIC (or per-aqueous liquid chromatography (PALC)) was employed, utilizing a commercially available HILIC column, which ensures robust and reproducible results with a water-rich mobile phase. In 2D, chiral chromatography with a broad range of gradients was utilized. Moreover, the impact of varying concentrations of organic solvent transferred to the 2D on enantioseparation was investigated. The water-rich mobile phase in the 1D facilitates the complete refocusing of organic solutes before entering the second dimension, preventing the loss of resolution in the 2D. This also allows for longer sampling times, consequently longer 2D running times, reducing the need for ultra-fast columns in the 2D. Furthermore, it enables the application of various mobile phase compositions in the 2D. Finally, this tool was successfully used to analyze compounds in urine and plasma matrices.

Keywords:  Chiral separation; Per-aqueous liquid chromatography (PALC); Two-dimensional liquid chromatography

DOI:  https://doi.org/10.1016/j.chroma.2025.465875
Trends Analyt Chem. 2024 Jun;pii: 117713. [Epub ahead of print]175

Frontiers in Mass Spectrometry-Based Spatial Metabolomics: Current Applications and Challenges in the Context of Biomedical Research.

Kate Wheeler, Camil Gosmanov, Michael Jimenez Sandoval, Zhibo Yang, Laura-Isobel McCall.

  Metabolites are critical products and mediators of cellular and tissue function, and key signals in cell-to-cell, organ-to-organ and cross-organism communication. Many of these interactions are spatially segregated. Thus, spatial metabolomics can provide valuable insight into healthy tissue function and disease pathogenesis. Here, we review major mass spectrometry-based spatial metabolomics techniques and the biological insights they have enabled, with a focus on brain and microbiota function and on cancer, neurological diseases and infectious diseases. These techniques also present significant translational utility, for example in cancer diagnosis, and for drug development. However, spatial mass spectrometry techniques still encounter significant challenges, including artifactual features, metabolite annotation, open data, and ethical considerations. Addressing these issues represent the future challenges in this field.

Keywords:  DESI mass spectrometry imaging; MALDI mass spectrometry imaging; chemical cartography; mass spectrometry; metabolomics; secondary ion mass spectrometry; spatial analysis

DOI:  https://doi.org/10.1016/j.trac.2024.117713
J Am Soc Mass Spectrom. 2025 Mar 21.

Pneumatically Assisted Microfluidic Probe for Enhanced Mass Spectrometry Imaging Performance.

Li-Xue Jiang, Julia Laskin.

  A pneumatically assisted microfluidic probe (MFP) with two microfluidic channels has been developed for nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI) of biological samples. This design simplifies the experimental setup, making it independent of the vacuum suction at the mass spectrometer inlet. The implementation of pneumatically assisted solvent flow through the probe enables stable, high solvent flow rates required to maintain a consistent liquid bridge during high-throughput MSI experiments. This approach addresses challenges associated with using MFP nano-DESI probes on mass spectrometers that have limited vacuum suction and the operation of MFPs with small microfluidic channels. We demonstrate the robustness of the pneumatically assisted MFP with 30 μm channels, which cannot be used for high-throughput MSI experiments without pneumatic assistance, by successfully imaging five mouse brain tissue sections without interruptions.

Keywords:  High throughput; Mass spectrometry imaging (MSI); Nanospray desorption electrospray ionization (nano-DESI); Pneumatically assisted microfluidic probe

DOI:  https://doi.org/10.1021/jasms.5c00011
Eur J Mass Spectrom (Chichester). 2025 Mar 21. 14690667251328823

LC-ESI-MS/MS method for the accurate quantification of eight nitrosamines in pharmaceutical products.

Ibrahim Danis, Durisehvar Ozer Unal.

  In 2018, the Food and Drug Administration and the European Medicines Agency identified nitrosamine impurities in certain drugs, prompting detailed investigations by international regulatory authorities. According to ICH M7 (R1) guidelines, nitrosamines are classified as Class 1 substances, the most hazardous category, based on their carcinogenic and mutagenic properties. They are also recognized as probable human carcinogens by the International Agency for Research on Cancer. Since nitrosamine-induced DNA damage poses significant health risks, identifying potential nitrosamines in pharmaceutical products is crucial. In this study, a simple and efficient extraction method was designed to minimize matrix effects. These effects were evaluated using calibration curves prepared for each drug product in their respective matrices. The developed method was performed using an Agilent 1260 series HPLC system and an Agilent 6460 triple quadrupole tandem mass spectrometer. An Inertsil ODS-3 C18 (5 µm, 4.6 × 150 mm) column was employed for chromatographic separation. A triple quadrupole mass detector with electrospray ionization was used for detection, and multiple reaction monitoring was employed for quantification. The correlation coefficients (r²) were at least 0.999 for all eight nitrosamines. Limit of detection and limit of quantification values were determined as 0.05-0.8 ng/mL and 0.1-2.0 ng/mL, respectively. Validation results demonstrated satisfactory selectivity, accuracy, precision, and stability. The results demonstrated that the developed method is capable of reliably detecting potential nitrosamines present in pharmaceutical products at trace levels. This method contributes significantly to pharmaceutical safety and can serve as a valuable tool for future analyses.

Keywords:  Nitrosamine; chromatography; impurity; mass spectrometry; pharmaceutical products

DOI:  https://doi.org/10.1177/14690667251328823
Sci Rep. 2025 Mar 18. 15(1): 9226

Application of rapid evaporative ionization mass spectrometry in preclinical and clinical analyses of steatotic liver tissues and cells.

Julian Connor Eckel, Lena Seidemann, Mohamed Albadry, Gerda Schicht, Marija Skvoznikova, Sandra Nickel, René Hänsel, Daniel Seehofer, Grit Gesine Ruth Hiller, Hans-Michael Tautenhahn, Uta Dahmen, Georg Damm.

  Rapid evaporative ionization mass spectrometry (REIMS) shows promise as a preparation-free tissue analysis tool with the prospect for real-time diagnostics. Given that hepatic steatosis is characterized by shifts in lipid species and abundance, we selected it as basis for method development, as REIMS specifically measures lipidomic profiles. However, further validation and protocol refinement are necessary to establish its clinical utility. In this study, we applied REIMS to steatotic human liver tissues, focusing on its ability to differentiate varying degrees of steatosis. We established standardized protocols for tissue handling and lipid analysis, which were essential for reliable data interpretation. Notably, our findings revealed that tissue size impacts REIMS sensitivity, with smaller samples yielding lower total ion counts and altered lipid profiles. Through principal component analysis, we identified key lipid classes, namely triacylglycerides, fatty acids, and glycerophospholipids. Despite a missing link between triacylglyceride abundance and degree of steatosis, we successfully identified condition-specific lipid patterns, with ceramides emerging as markers of advanced steatosis. Our study provides a protocol for the measurements of lipid standards showing the detailed degradation of specific lipids using iKnife-coupled REIMS. It highlights the pitfalls and limitations and provides critical recommendations for REIMS use. It also emphasizes the need for standardized biobanking and tissue preparation to ensure accurate lipid profiling, laying the groundwork for future protocol adjustments required for clinical application.

Keywords:  Lipidomics; Lipids; MASLD; Rapid evaporative ionization mass spectrometry; Steatosis

DOI:  https://doi.org/10.1038/s41598-025-93305-w
J Mass Spectrom Adv Clin Lab. 2025 Apr;36 11-18

A multiplex assay of leptin, resistin, and adiponectin by immunoaffinity enrichment and targeted mass spectrometry.

Jie Pu, Xinxin Yang, Tai-Tu Lin, Thomas L Fillmore, Marina A Gritsenko, Shane S Kelly, Adam C Swensen, Tujin Shi, Stephen R Master, James P DeLany, Bret H Goodpaster, Wei-Jun Qian, Jun Qu.

   Background: Leptin, resistin, and adiponectin are critical adipokines involved in the pathophysiology of obesity and its related disorders, including type 2 diabetes. Although these biomarkers have historically been quantified using immunoassays, the specificity of antibody-based methods has frequently been questioned. As a result, there is an increasing interest in developing reliable, multiplexed clinical assays that utilize mass spectrometry for improved accuracy. In this study, we present a multiplexed immunoaffinity liquid chromatography-tandem mass spectrometry (multi-IA-LC-MS/MS) assay designed for the sensitive and selective measurement of leptin, resistin, and adiponectin in human plasma.
Methods: Leptin, resistin, and adiponectin were selectively enriched from plasma samples using an antibody cocktail composed of monoclonal antibodies targeting each respective adipokine. The enriched adipokines underwent enzymatic digestion, and the resulting tryptic peptides were quantified using LC-MS/MS. The validated assay was subsequently applied to plasma samples collected from a cohort of subjects representing various weight categories, including normal weight, overweight, and obesity.
Results: The lower limits of quantification for the assay were determined to be 0.5 ng/mL for both leptin and resistin, and 50 ng/mL for adiponectin. Intra- day, inter- day, and total imprecision measurements were all < 15 %, while spike recovery consistently exceeded 83 %. Comparative analysis with individual immunoassays demonstrated strong correlation, with all correlation coefficients (r) being equal to or greater than 0.869. Notably, when comparing subjects with obesity to those with normal weight, there was an approximately nine-fold increase in circulating leptin levels and a ∼1.6-fold decrease in circulating adiponectin levels.
Conclusions: A multi-IA-LC-MS/MS assay was developed for the simultaneous and sensitive measurement of leptin, resistin, and adiponectin in clinical samples. This quantitative method shows significant potential for applications related to obesity and could facilitate improved clinical management and understanding of obesity-related conditions.

Keywords:  Adipokines; Immunoaffinity enrichment; LC-MS; Mass Spectrometry; Obesity

DOI:  https://doi.org/10.1016/j.jmsacl.2025.01.003
J Chromatogr A. 2025 Mar 10. pii: S0021-9673(25)00211-0. [Epub ahead of print]1748 465863

Enhancing pyrrolizidine alkaloid separation and detection: LC-MS/MS method development and integration of ion mobility spectrometry into the LC-HRMS workflow.

Laura Carbonell-Rozas, Nicola Dreolin, Henry Foddy, Chiara Dall'Asta.

  Pyrrolizidine alkaloids (PAs) are plant toxins occurring in different foodstuffs, including teas, herbal infusions and species. Additionally, PAs may be transferred to honey and pollen when honeybees come into contact with contaminated plants. Due to their adverse effect, PAs occurrence in food must be controlled to ensure public health. Nevertheless, the presence of numeours PA epimers complicates their chromatographic separation and detection. In this regard, a method using liquid chromatography coupled with tandem mass spectrosmetry (LC-MS/MS) has been developed allowing the separation of 31 out of the 35 regulated PAs, which was sucessfully validated in different food matrices such as herbal infusions, spices and honey. Afterwards, travelling wave ion mobility spectrometry hyphenated with quadrupole time-of-flight mass spectrometry (TWIMS-QTOF) was evaluated to improve the analytical performance of PAs determination. Thus, collision cross section (CCS) values of PAs have been therefore obtained for the first time. The CCS library for PAs was also compared with predicted values by machine learning and with those meassured in real food matrices (bias <2 %). In addition, an in-house library was used in the suspect screening of PAs to complement the targeted analysis of the studied samples, all of which tested positive for several PAs.

Keywords:  CCS; Herbal infusions; Honey; Ion mobility mass spectrometry; Pyrrolizidine alkaloids; Spices

DOI:  https://doi.org/10.1016/j.chroma.2025.465863