bims-metlip 2025-02-09 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–02–09
29 papers selected by
Sofia Costa, Matterworks

Quantification of mycophenolic acid in plasma by isotope dilution liquid chromatography-tandem mass spectrometry candidate reference method.
An ultra-sensitive liquid chromatography tandem mass spectrometry method for the simultaneous quantification of 2H6-alectinib and alectinib in human plasma to support a microtracer food-effect trial.
A Validated Liquid Chromatography-Tandem Mass Spectrometry Assay for the Determination of NX-5948 in Beagle Dog Plasma: Application to a Pharmacokinetic Study.
Ion suppression correction and normalization for non-targeted metabolomics.
Liquid and gas-chromatography-mass spectrometry methods for exposome analysis.
Molecular Structure Discovery for Untargeted Metabolomics Using Biotransformation Rules and Global Molecular Networking.
Toward Machine Learning Electrospray Ionization Sensitivity Prediction for Semiquantitative Lipidomics in Stem Cells.
Preconcentration and purification of oligonucleotides from heat-treated human plasma by anion-exchange microextraction devices coupled to high performance liquid chromatography-mass spectrometry.
A Low-Cost, High-Resolution Thermoplastic Microfluidic Probe for Mass Spectrometry Imaging of Biological Tissue Samples.
Accelerating isotope dilution LC-MS-based desmosine quantification for estimating elastin turnover.
Bioanalytical Tandem Mass Spectrometry Method for Precise Measurement of Tranexamic Acid in Human Plasma With Unveiling Methodological Advancement: Green Assessment With Advanced Metrics.
Ultra-High-Performance Liquid Chromatography Combined With Mass Spectrometry Detection Analytical Method for the Determination of N-Nitrosoduloxetine in Duloxetine HCl.
Three-dimensional mass spectrometry imaging (3D MSI): incorporating top-hat IR-MALDESI and automatic z-axis correction.
A validated single-step saliva and serum sample extraction LC-MS/MS method for the analysis of nicotine, cotinine and 3'-hydroxycotinine for clinical vaping studies.
Method development and validation for determination of N-Nitrosamines in pharmaceutical preparations by LC-MS/MS: Application to extractables and leachables studies.
Dried blood spot microsampling: A semi-quantitative 4D-lipidomics approach using ultrahigh-performance liquid chromatography - high-resolution mass spectrometry (UHPLC - HRMS).
An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of cortisol in human serum and plasma.
Optimization of HPLC method for metanephrine and normetanephrine detection in urine: Enhancing diagnostic precision for pheochromocytoma.
Detection and application of 40 piperazine compounds in hair suitable for rapid separation of isomers.
Determination of veterinary drugs in foods of animal origin by QuEChERS coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).
Innovative determination of phytohormones in Aloe vera.
Pre-analytic assessment of dried blood and dried plasma spots: integration in mass spectrometry-based metabolomics and lipidomics workflow.
European Society of Toxicologic Pathology-Pathology 2.0 Mass Spectrometry Imaging Special Interest Group: Mass Spectrometry Imaging in Diagnostic and Toxicologic Pathology for Label-Free Detection of Molecules-From Basics to Practical Applications.
Matrix free laser desorption ionization coupled to trapped ion mobility mass spectrometry: an innovative approach for isomer differentiation and molecular network visualization.
A Protocol for Determination of Proteinogenic Amino Acids in Biological Fluids by the High-Speed UHPLC-MS Method: Application on Transgenic Spontaneously Hypertensive Rat-24 Plasma and Cerebrospinal Fluid Samples.
Pure Shift NMR with Solvent Suppression: A Robust and General Method for Determining Quantitative Metabolic Profiles in Biofluids.
Determination of Pesticide Residues in Ectoparasiticide-Impregnated Collars Using Ultrasound-Assisted Extraction and Liquid Chromatography-Mass Spectrometry.
Molecular mass spectrometric imaging of amino acids in dietary supplement tablets using laser ablation-atmospheric pressure chemical ionization-trapped ion mobility spectrometry-mass spectrometry.
Improved LC-MS Detection of Opioids, Amphetamines, and Psychedelics Using TrEnDi.

Anal Bioanal Chem. 2025 Feb 05.

Quantification of mycophenolic acid in plasma by isotope dilution liquid chromatography-tandem mass spectrometry candidate reference method.

Jing Lin, Min Shen, Ting Yu, Huimin Wang, Jingjue Pan, Gaipeng Huang, Quanle Li.

  Accurate measurements of plasma mycophenolic acid (MPA) are essential for therapeutic drug monitoring in transplant recipients and autoimmune diseases. The performance of plasma mycophenolic acid routine methods remains highly variable that calls for a candidate reference measurement procedure (cRMP) to improve the standardization of plasma mycophenolic acid measurements. In this study, sample preparation was based on protein precipitation with methanol followed by further dilution. The mycophenolic acid was quantified by the isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with electrospray ionization in positive ion mode. According to the Clinical and Laboratory Standards Institute (CLSI) documents C62-A and C50-A, the basic analytical performance of the candidate reference method was verified, such as linearity, limit of quantification, matrix effect, precision, accuracy, and uncertainty. Moreover, the candidate reference measurement procedure was compared with the routine liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a clinical laboratory. Based on the data, the mycophenolic acid in human plasma was well detected by ID-LC-MS/MS. No apparent interferences were found with the mycophenolic acid measurement. The calibration curve for the mycophenolic acid was linear in the concentration range of 0.1-50 μg/mL with a correlation coefficient of 0.9999 under the optimum experimental conditions. This method was sensitive because the low limit of quantitation (LOQ) was 0.05 μg/mL. The recoveries of MPA were 98.11-98.95%. The intra-day and inter-day coefficients of variations (CV) of our method were ≤ 1.53% and ≤ 0.51%, respectively. No obvious matrix effect was observed. There was a good correlation between this method and the clinical routine LC-MS/MS method. To sum up, we established and validated a reliable plasma MPA method using ID-LC/MS/MS. The desirable accuracy and precision of this method enable it to serve as a promising cRMP to improve the standardization for plasma MPA routine measurements.

Keywords:  Candidate reference method; Isotope dilution liquid chromatography-tandem mass spectrometry; Method validation; Mycophenolic acid

DOI:  https://doi.org/10.1007/s00216-025-05750-1
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jan 28. pii: S1570-0232(25)00040-6. [Epub ahead of print]1253 124488

An ultra-sensitive liquid chromatography tandem mass spectrometry method for the simultaneous quantification of 2H6-alectinib and alectinib in human plasma to support a microtracer food-effect trial.

L T van der Heijden, M M Tibben, M I Mohmaed Ali, L G J Aardenburg, N Steeghs, J H Beijnen, H Rosing, A D R Huitema.

  A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of 2H6-alectinib and alectinib was developed and validated for the support of a pilot microtracer food-effect trial. The aim of the bioanalytical method was the simultaneous quantification of low 2H6-alectinib concentrations and high alectinib concentrations that are present in study samples, using a single sample pre-treatment and analysis method. Sample preparation consisted of liquid-liquid extraction with tert-butyl methyl ether (TBME). The final extract was injected on a C18 column (1.7 μm particles, 50 × 2.1 mm ID) with gradient elution. A triple quadruple mass spectrometer operating in positive method was used for detection and quantification. The validated concentration ranges were from 5 to 400 pg/mL for 2H6-alectinib and from 25 to 2000 ng/mL for alectinib. The bias was within ±3.5 % and ± 5.1 % and precisions ≤5.7 % and ≤ 1.9 % for 2H6-alectinib and alectinib, respectively. By correcting for the interference of natural abundant isotopes of alectinib, 2H6-alectinib plasma concentrations between 1 and 5 pg/mL could be quantified, with bias was within ±15.9 % and precision ≤12.5 % in the presence of 400 ng/mL or 800 ng/mL alectinib. The clinical application was successfully applied to quantify 2H6-alectinib and alectinib in plasma samples from a participant enrolled in a microtracer food-effect study.

Keywords:  Alectinib; Food-effect; LC-MS/MS; Microtracer; Natural isotope interference correction; Pharmacokinetics

DOI:  https://doi.org/10.1016/j.jchromb.2025.124488
Biomed Chromatogr. 2025 Mar;39(3): e6090

A Validated Liquid Chromatography-Tandem Mass Spectrometry Assay for the Determination of NX-5948 in Beagle Dog Plasma: Application to a Pharmacokinetic Study.

Fang Wang, Wenfei Sun, Zheheng Ma, Furui Chu.

  Proteolysis targeting chimera (PROTAC) has been developed and currently enjoys widespread interest among the field of pharmaceutical manufacturing in recent decades. NX-5948 is an orally active PROTAC Bruton tyrosine kinase degrader, which shows anti-inflammatory and antitumor activities. In this study, a simple and fast bioanalytical method for the quantification of NX-5948 in beagle dog plasma was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A full method validation was performed according to regulatory guidelines. For the quantification, [M + H]+ was formed using an electrospray ionization (ESI) source in the positive ion mode, and selective reaction monitoring (SRM) was employed using a triple quadrupole mass spectrometry. The monitored precursor-to-product transitions were m/z 807.5 > 790.5 for NX-5948 and m/z 812.4 > 452.1 for internal standard. A linear response was obtained at the concentration range of 0.5-200 ng/mL, with correlation coefficient > 0.99. The interday accuracy ranged from -9.03% to 5.99% (RSD < 7.84%), and the intraday accuracy from -5.15% to 8.56% (RSD < 6.90%). NX-5948 was demonstrated to be stable under the present storage conditions. After validation, the method was successfully applied for the quantification of NX-5948 in beagle dog plasma after oral and intravenous administration of the NX-5948.

Keywords:  Bruton's tyrosine kinase; NX‐5948; method validation; pharmacokinetics; proteolysis targeting chimera

DOI:  https://doi.org/10.1002/bmc.6090
Nat Commun. 2025 Feb 04. 16(1): 1347

Ion suppression correction and normalization for non-targeted metabolomics.

Iqbal Mahmud, Bo Wei, Lucas Veillon, Lin Tan, Sara Martinez, Bao Tran, Alexander Raskind, Felice de Jong, Yiwei Liu, Jibin Ding, Yun Xiong, Wai-Kin Chan, Rehan Akbani, John N Weinstein, Chris Beecher, Philip L Lorenzi.

Ion suppression is a major problem in mass spectrometry (MS)-based metabolomics; it can dramatically decrease measurement accuracy, precision, and sensitivity. Here we report a method, the IROA TruQuant Workflow, that uses a stable isotope-labeled internal standard (IROA-IS) library plus companion algorithms to: 1) measure and correct for ion suppression, and 2) perform Dual MSTUS normalization of MS metabolomic data. We evaluate the method across ion chromatography (IC), hydrophilic interaction liquid chromatography (HILIC), and reversed-phase liquid chromatography (RPLC)-MS systems in both positive and negative ionization modes, with clean and unclean ion sources, and across different biological matrices. Across the broad range of conditions tested, all detected metabolites exhibit ion suppression ranging from 1% to >90% and coefficients of variation ranging from 1% to 20%, but the Workflow and companion algorithms are highly effective at nulling out that suppression and error. To demonstrate a routine application of the Workflow, we employ the Workflow to study ovarian cancer cell response to the enzyme-drug L-asparaginase (ASNase). The IROA-normalized data reveal significant alterations in peptide metabolism, which have not been reported previously. Overall, the Workflow corrects ion suppression across diverse analytical conditions and produces robust normalization of non-targeted metabolomic data.

DOI: https://doi.org/10.1038/s41467-025-56646-8
J Chromatogr A. 2025 Jan 25. pii: S0021-9673(25)00077-9. [Epub ahead of print]1744 465728

Liquid and gas-chromatography-mass spectrometry methods for exposome analysis.

Victor Castro-Alves, Anh Hoang Nguyen, João Marcos G Barbosa, Matej Orešič, Tuulia Hyötyläinen.

  Mass spectrometry-based methods have become fundamental to exposome research, providing the capability to explore a broad spectrum of chemical exposures. Liquid and gas chromatography coupled with low/high-resolution mass spectrometry (MS) are among the most frequently employed platforms due to their sensitivity and accuracy. However, these approaches present challenges, such as the inherent complexity of MS data and the expertise of biologists, chemists, clinicians, and data analysts to integrate and interpret MS data with other datasets effectively. The "omics" era advances rapidly, driven by developments of AI-based algorithms and an increase in accessible data; nevertheless, further efforts are necessary to ensure that exposomics outputs are comparable and reproducible, thus enhancing research findings. This review outlines the principles of MS-based methods for the exposome analytical pipeline, from sample collection to data analysis. We summarize and review both standard and cutting-edge strategies in exposome research, covering sample preparation, focusing on MS-based platforms, data acquisition strategies, and data annotation. The ultimate goal of this review is to highlight applications that enable the simultaneous analysis of endogenous metabolites and xenobiotics, which can help enhance our understanding of the impact of human exposure on health and disease and support personalized healthcare.

Keywords:  Chromatography; Environmental pollutants; Exposome; Mass spectrometry; Metabolomics

DOI:  https://doi.org/10.1016/j.chroma.2025.465728
Anal Chem. 2025 Feb 04.

Molecular Structure Discovery for Untargeted Metabolomics Using Biotransformation Rules and Global Molecular Networking.

Margaret R Martin, Wout Bittremieux, Soha Hassoun.

Although untargeted mass spectrometry-based metabolomics is crucial for understanding life's molecular underpinnings, its effectiveness is hampered by low annotation rates of the generated tandem mass spectra. To address this issue, we introduce a novel data-driven approach, Biotransformation-based Annotation Method (BAM), that leverages molecular structural similarities inherent in biochemical reactions. BAM operates by applying biotransformation rules to known "anchor" molecules, which exhibit high spectral similarity to unknown spectra, thereby hypothesizing and ranking potential structures for the corresponding "suspect" molecule. BAM's effectiveness is demonstrated by its success in annotating query spectra in a global molecular network comprising hundreds of millions of spectra. BAM was able to assign correct molecular structures to 24.2% of examined anchor-suspect cases, thereby demonstrating remarkable advancement in metabolite annotation.

DOI: https://doi.org/10.1021/acs.analchem.4c01565
J Chem Inf Model. 2025 Feb 05.

Toward Machine Learning Electrospray Ionization Sensitivity Prediction for Semiquantitative Lipidomics in Stem Cells.

Alexandria Van Grouw, Markace A Rainey, Olivia K Reid, Molly M Ogle, Samuel G Moore, Johnna S Temenoff, Facundo M Fernández.

Specificity, sensitivity, and high metabolite coverage make mass spectrometry (MS) one of the most valuable tools in metabolomics and lipidomics. However, translation of metabolomics MS methods to multiyear studies conducted across multiple batches is limited by variability in electrospray ionization response, making batch-to-batch comparisons challenging. This limitation creates an artificial divide between nontargeted discovery work that is broad in scope but limited in terms of absolute quantitation ability and targeted work that is highly accurate but limited in scope due to the need for matched isotopically labeled standards. These issues are often observed in stem cell studies using metabolomic and lipidomic MS approaches, where patient recruitment can be a years-long process and samples become available in discrete batches every few months. To bridge this gap, we developed a machine learning model that predicts electrospray ionization sensitivity for lipid classes that have shown correlation with stem cell potency. Molecular descriptors derived from these lipids' chemical structures are used as model input to predict electrospray response, enabling quantitation by MS with moderate accuracy (semiquantitation). Model performance was evaluated via internal and external validation using cultured cells from various stem cell donors, achieving global percent errors of 40% and 20% for positive and negative electrospray ion modes, respectively. Although this accuracy is typically insufficient for traditional targeted lipidomics experiments, it is sufficient for semiquantitative estimation of lipid marker concentrations across batches without the need for specific chemical standards that many times are unavailable. Furthermore, the precision for model-predicted concentrations was 16.9% for the positive mode and 7.5% for the negative mode, indicating promise for data harmonization across batches. The set of molecular descriptors used by the models described here was able to yield higher accuracy than those previously published in the literature, showing high promise toward semiquantitative lipidomics.

DOI: https://doi.org/10.1021/acs.jcim.4c02040
Anal Chim Acta. 2025 Mar 08. pii: S0003-2670(25)00018-2. [Epub ahead of print]1342 343624

Preconcentration and purification of oligonucleotides from heat-treated human plasma by anion-exchange microextraction devices coupled to high performance liquid chromatography-mass spectrometry.

Derek R Eitzmann, Jared L Anderson.

   BACKGROUND: Methodologies that preconcentrate high quality nucleic acids (NAs) for downstream assays are essential for their accurate and reproducible analysis by mass spectrometry (MS). Established methods rely on solid-phase extraction that involves numerous steps and user intervention or liquid-liquid extractions that are time-consuming and employ toxic organic solvents. A promising alternative methodology involves anion-exchange microextraction sorbents that selectively isolate NAs through electrostatic interactions with the negatively charged phosphodiester backbone. The microextraction devices recover and preconcentrate NAs using a salt-containing solution, which is generally incompatible with MS analysis.
RESULTS: Six anion-exchange microextraction sorbents featuring monomers derived from 2-aminoethyl methacrylate were synthesized and examined in this study to understand the interactions that take place between NAs and the ammonium cationic moiety. Sorbent affinities for bovine serum albumin, small single-stranded oligonucleotides, RNA, short double-stranded DNA), and 2000 bp dsDNA were determined. Recoveries of an oligonucleotide and 2000 bp dsDNA were measured and examined in salt solutions of varied concentration, anion species, and organic additives. High oligonucleotide preconcentration factors of 8.6 ± 0.2 were obtained for the sorbent featuring two cationic ammonium moieties on each monomer using 500.0 mM ammonium perchlorate. A separate sorbent composed of dimethyl ethyl ammonium moieties produced a preconcentration factor of 4.6 ± 0.2 using only 31.25 mM ammonium perchlorate. The sorbents were demonstrated in the complete workflow in which an analog antisense oligonucleotide was spiked into human plasma and purified, enabling successful molecular weight analysis.
SIGNIFICANCE: This study demonstrates the compatibility of microextraction recovery solutions comprised of ammonium perchlorate with reversed-phase and hydrophilic interaction chromatographic separations and time-of-flight MS for characterization of oligonucleotides. The combination of anion-exchange microextraction sorbents and HPLC-TOF-MS enables the purification, preconcentration, and identification of a oligonucleotides from heat-treated human plasma. The compatibility of the salt-containing recovery solutions with chromatographic separation modalities highlights that anion-exchange microextraction devices are a complete and compelling sample preparation methodology for oligonucleotides.

Keywords:  High performance liquid chromatography; Mass spectrometry; Nucleic acids; Oligonucleotides; Sample preparation

DOI:  https://doi.org/10.1016/j.aca.2025.343624
Anal Chem. 2025 Feb 04.

A Low-Cost, High-Resolution Thermoplastic Microfluidic Probe for Mass Spectrometry Imaging of Biological Tissue Samples.

Xiangtang Li, Hang Hu, Manxi Yang, Julia Laskin.

Mass spectrometry imaging (MSI) using nanospray desorption electrospray ionization (nano-DESI) has been extensively used for label-free mapping of hundreds of molecules in biological samples with minimal sample pretreatment. While both nano-DESI probes made of two fused silica capillaries and glass microfluidic probes (MFP) have been developed for imaging biological tissues with high spatial resolution, MFPs significantly enhance the robustness and throughput of nano-DESI MSI experiments. Despite their advantages, the fabrication of glass microfluidic devices is costly and requires specialized equipment or cleanroom facilities. Meanwhile, plastic microfluidic devices often suffer from limited solvent compatibility and low fabrication precision, restricting their achievable spatial resolution. To overcome these limitations, we have developed a low-cost microfluidic probe made from cyclic olefin copolymer (COC), a widely used thermoplastic material known for its excellent chemical resistance. The probe is fabricated using wire imprinting and thermal bonding in a standard laboratory setting. We estimate the achievable spatial resolution of the COC-MFP of 5-7 μm and demonstrate its robustness by imaging a large (20.0 mm × 9.5 mm) human kidney tissue section with high sensitivity. This affordable thermoplastic probe makes high spatial resolution nano-DESI MSI more accessible, broadening its applications in the scientific community.

DOI: https://doi.org/10.1021/acs.analchem.4c06087
Bioanalysis. 2025 Jan;17(2): 99-104

Accelerating isotope dilution LC-MS-based desmosine quantification for estimating elastin turnover.

Elena Kuzmanova, Angela McIntyre, Maaz Syed, Zaid Iskandar, David E Newby, Matt J Bown, Anna-Maria Choy, Jeffrey Tj Huang.

   AIMS: Circulating total desmosine, representing endogenous systemic elastin degradation activity, is an emerging biomarker for mortality risk in several diseases and aging. However, the existing analytical method takes more than 23 hours to complete, limiting its potential applications. The objective of this study was to shorten the turnover time of a stable isotope dilution liquid chromatogram mass spectrometry-based desmosine assay.
MATERIALS & METHODS: Plasma samples were analyzed using acid hydrolysis followed by solid-phase extraction and LC-MS. Two approaches to reduce assay time were tested: microwave-assisted acid hydrolysis and direct injection following solid-phase extraction.
RESULTS: The combination of acid hydrolysis at 180°C for 8 minutes and a low-volume elution design for solid-phase extraction reduced the overall assay time to ~ 30 minutes. The assay was validated with intra-day precision and accuracy ranging from 4% to 14%, and -7% to 9%, respectively, while inter-day precision and accuracy were 0% to 9% and 1% to 3%, respectively. The assay was tested in a cohort of patients with acute aortic dissection and control subjects, where desmosine concentrations were approximately three-fold higher in patients.
CONCLUSIONS: These results demonstrated that rapid desmosine analysis can be achieved with the use of both microwave-assisted hydrolysis and streamlined solid-phase extraction.

Keywords:  Desmosine; aortic aneurysm; aortic dissection; micro solid phase extraction; microwave; stable isotope dilution LC-MS/MS

DOI:  https://doi.org/10.1080/17576180.2025.2452723
Biomed Chromatogr. 2025 Mar;39(3): e6086

Bioanalytical Tandem Mass Spectrometry Method for Precise Measurement of Tranexamic Acid in Human Plasma With Unveiling Methodological Advancement: Green Assessment With Advanced Metrics.

Anil Kollapareddy, Kousrali Sayyad, Leela Prasad Kowtharapu, Naresh Konduru, Tanmoy Mondal, Mohan Varkolu, Sreedhar Gundekari.

  For the control of excessive blood loss occurring from major trauma, postpartum bleeding, surgery, or hereditary angioedema, antifibrinolytic compound tranexamic acid (TXM) is highly efficient in controlling blood loss by reversibly binding with on lysine receptor sites. A selective and sensitive method has been developed and validated for the quantitation of TXM in human plasma using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Tranexamic acid-D2 (TXM-D2) was used as internal standard (ISTD) to minimize the errors in TXM quantification. TXM quantification was performed with positive polarity mode using a Shimadzu high performance liquid chromatography coupled with AB-SCIEX API-4000 tandem mass spectrometer. A Zorbax Eclipse C18 (150 × 4.6 mm, 5 μ) column was used for the TXM quantification, 8-mM ammonium formate buffer with 0.1% formic acid ionization enhancer was used as a mobile phase-A, and acetonitrile was used as a mobile phase-B. (38:62) v/v portion of mobile phases A and B selected to elute the TXM and TXM-D2 with the flow rate of 0.8 mL/min. An electrospray ionization (ESI) technique was selected for detection of TXM in the human plasma. Linearity was assessed in the concentration range from 75 to 15,000 ng/mL by using least squares of weighting factor linear regression (1/X2).

Keywords:  ICH M10 guidelines; bioequivalence; high‐performance liquid chromatography–tandem mass spectrometer; solid‐phase extraction; tranexamic acid

DOI:  https://doi.org/10.1002/bmc.6086
Biomed Chromatogr. 2025 Mar;39(3): e6084

Ultra-High-Performance Liquid Chromatography Combined With Mass Spectrometry Detection Analytical Method for the Determination of N-Nitrosoduloxetine in Duloxetine HCl.

Anna Marlés-Torres, Sergio Bessa-Jambrina, Cristóbal Galán-Rodríguez.

  In the present study, one simple, fast, feasible, and sensitive ultra-high-performance liquid chromatography combined with mass spectrometry detection method, using electrospray ionization, is proposed. This method was developed and validated for the determination of N-nitrosoduloxetine content in Duloxetine HCl active pharmaceutical ingredient in 11 min employing a simple preparation method for both reference and sample solutions. The proposed method was validated as per regulatory guidelines. Acquity HSS T3 (3.0 × 100 mm, 1.8 μm) column and formic acid 0.1% in water combined with acetonitrile were used for chromatographic separation. The limit of detection and the limit of quantification-reporting threshold-were found to be 0.7 and 70 ppb, respectively. The trueness and precision of the method have been demonstrated in the working range, giving values of recovery within the range of 100%-110%, and the regression coefficients (R) were found to be in the range of 0.9990-0.9991. This method could be used for controlling N-nitrosoduloxetine content in duloxetine HCl drug substance batches manufactured at Moehs group.

Keywords:  N‐nitrosoduloxetine; UHPLC–MS/MS; duloxetine; mutagenic impurities; nitrosamines

DOI:  https://doi.org/10.1002/bmc.6084
Anal Bioanal Chem. 2025 Feb 03.

Three-dimensional mass spectrometry imaging (3D MSI): incorporating top-hat IR-MALDESI and automatic z-axis correction.

Alexandria L Sohn, John G Witherspoon, Robert C Smart, David C Muddiman.

  Leveraging a depth profiling approach expands the chemical elucidation of mass spectrometry imaging techniques to another dimension. Three-dimensional MSI (3D MSI) reveals the distribution of analytes with greater anatomical detail to add another level of information in a biological study. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) has demonstrated utility for an ablation-based approach, enabling simplified sample preparation workflows and streamlined data processing pipelines compared to a serial-sectioning strategy. To improve 3D MSI on the IR-MALDESI platform, two technologies have been characterized in tandem for the intention of minimizing sampling bias: (1) a top-hat optical train and (2) a chromatic confocal probe (CA probe). While the modified optical train creates a square spot size to avoid a Gaussian ablation crater after the analysis of subsequent layers, the CA probe enables automatic z-axis correction (AzC) to maintain the laser's focus on the surface of the sample. The work herein demonstrates the integration and optimization of these technologies on mouse skin, motivated by the clear biological skin layers that result in differential lipid expression and subsequent detection. Results support that a laser energy of 1.3 mJ/burst with the top-hat optical train and a 120 µm step size in the X and Y dimensions presented a comparable depth resolution to previous studies at under 7 µm. Further, the optimized parameters were utilized on two biological replicates to evaluate method reproducibility where lipid annotations and their abundance were considered.

Keywords:  3D; Automatic z-axis correction; Lipids; Mass spectrometry imaging; Top-hat

DOI:  https://doi.org/10.1007/s00216-025-05755-w
J Pharm Biomed Anal. 2025 Jan 27. pii: S0731-7085(25)00044-5. [Epub ahead of print]258 116703

A validated single-step saliva and serum sample extraction LC-MS/MS method for the analysis of nicotine, cotinine and 3'-hydroxycotinine for clinical vaping studies.

Vera van der Velpen, Evangelia Liakoni, Mats B Hirt, Celina M Vonwyl, Samuel E Christen, Urs Duthaler, Peyton Jacob, Manuel Haschke.

   INTRODUCTION: Quantifying low nicotine and metabolite concentrations in biofluids is challenging due environmental nicotine contamination. However, accurate quantification of low concentrations is crucial for studies on electronic nicotine delivery systems (ENDS) using e-liquids with varying nicotine content.
METHODS: We developed an LC-MS/MS method to quantify nicotine, cotinine, and 3'-hydroxycotinine (3-OH-cotinine) in serum and saliva for pharmacokinetic (PK) analyses and large studies.
RESULTS: For reliable chromatography and to limit bench work, C18 chromatography was used with single-step extraction using methanol and 0.1 M ZnSO4 (4:1, v/v) in serum and 80 % methanol in saliva. Environmental nicotine contamination was addressed through implementation of a C18 delay column, which separated the environmentally abundant nicotine present in the mobile phases from sample nicotine peaks. Total run-time was 6 min and lower limits of quantification were 0.5, 0.25 and 0.5 ng/ml for nicotine, cotinine and 3-OH-cotinine, respectively, in serum and 3, 1 and 2 ng/ml in saliva. The standard curves in both biofluids ranged up to 1000 ng/ml with R-values > 0.995. The within- and between-run accuracy ranged from 97.1 % to 106.9 % with a precision of ≤ 10.8 %. Cross-validation of serum samples with another laboratory showed good agreement with a bias of 0.56, -3.0 and -6.5 ng/ml for nicotine, cotinine and 3-OH-cotinine, respectively.
CONCLUSIONS: The integration of a delay column into the LC-MS/MS method mitigated the interference from environmental nicotine and facilitated the quantification of very low nicotine concentrations and two of its major metabolites in saliva and serum. C18 chromatography and single-step sample extraction make the method stable and suitable for large sample loads.

Keywords:  C18 chromatography; LC-MS/MS; Nicotine; delay column; electronic nicotine delivery systems; pharmacokinetic studies

DOI:  https://doi.org/10.1016/j.jpba.2025.116703
J Chromatogr A. 2025 Jan 29. pii: S0021-9673(25)00090-1. [Epub ahead of print]1745 465741

Method development and validation for determination of N-Nitrosamines in pharmaceutical preparations by LC-MS/MS: Application to extractables and leachables studies.

Oğuzhan Dalkılıç, İbrahim Hakkı Demircioğlu, Saffet Çelik, Hasan Can, Tugrul Cagri Akman, Alptuğ Atila, Hamdullah Kılıç, Levent Kandemir.

  The use of highly sensitive and reliable analytical methods is essential for Extractables & Leachables studies. Especially the determination of N-Nitrosamines in drugs, which have carcinogenic properties and may contaminate drugs at trace levels, is quite important. In this study, a new, sensitive, short-time and reliable liquid chromatography with tandem mass spectrometry method was developed for the analysis of 15 N-Nitrosamines defined in the European Pharmacopoeia within the scope of Extractables & Leachables studies and validated according to the International Council for Harmonization (ICH Q2 (R2)). The analysis of N-Nitrosamines was carried out in positive mode using an Atmospheric Pressure Chemical Ionization source in the dynamic multiple reaction monitoring scanning mode. In the chromatographic separation, gradient elution was applied using a reverse phase Phenyl column and the mobile phase (A: 0.1 % formic acid in ultrapure water, B: 0.1 % formic acid in methanol); total analysis time was 16 mins and the flow rate was optimized as 0.6 mL/min. N-Nitroso-dimethylamine-d6 was used as an internal standard. The developed method was used in extractables studies to control the potential presence of N-Nitrosamines that may be caused by interactions between the product and primary packaging materials (e.g. polypropylene bag, LDPE container, disposable eye drop packaging and bromobutyl stopper). It was also successfully applied to pharmaceutical preparations containing sugammadex, metformin, gliclazide and paracetamol in the leachables studies.

Keywords:  Extractables & Leachables; LC-MS/MS; N-nitrosamines; Packaging materials; Validation

DOI:  https://doi.org/10.1016/j.chroma.2025.465741
Talanta. 2025 Feb 01. pii: S0039-9140(25)00163-8. [Epub ahead of print]287 127677

Dried blood spot microsampling: A semi-quantitative 4D-lipidomics approach using ultrahigh-performance liquid chromatography - high-resolution mass spectrometry (UHPLC - HRMS).

Jayden Lee Roberts, Monique J Ryan, Luke Whiley, Melvin Gay, Vimalnath Nambiar, Elaine Holmes, Jeremy K Nicholson, Julien Wist, Nicola Gray, Nathan G Lawler.

  Dried blood spot (DBS) sample collections can offer a minimally invasive, cost-effective alternative to traditional venepuncture for remote sampling and high-frequency metabolic profiling. We present an optimized protocol for DBS-based extraction and comprehensive untargeted 4D lipid profiling using ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry (trapped ion mobility - mass spectrometry), designed to support large-scale applications in population-wide lipidomics research. Inclusion of stable isotopically labelled internal standards allowed for semi-quantitative subclass-level correction for 10 μL DBS samples, enhancing the number of reproducible lipids within our curated target list (focussed on 432 unique rule-based lipid annotations out of 6845 features) across positive and negative heated electrospray ionization modes. The reproducibility of unique lipid features detected in replicate DBS (n = 6) was assessed on both peak areas (351 lipids <25 % CV) and calculated concentrations relative to internal standards (432 lipids <25 % CV), underscoring the benefit of internal standard addition. Storage conditions for DBS were also evaluated to determine short-term lipid stability at different temperatures (-20 °C, 4 °C, room temperature, and 45 °C). The majority of lipid subclasses, excluding a minority of glycerophospholipids and oxylipins, were stable up to 1 week at -20 °C and 4 °C (log2-fold change <30 % difference), which supports the short-term storage capacity for DBS in field and clinical settings. Similar stability was observed within a week at room temperature, excluding phosphatidylethanolamines and phosphatidylglycerols (log2-fold change >30 % difference). Application of the optimized workflow to a microsampling device (n = 6) identified 432 unique lipid features (CV < 25 %) with three repeated samplings over an hour showing minimal impact on lipid profiles by principal component analysis, showing promise for high-frequency, longitudinal DBS monitoring in population health. This work represents a significant advance, highlighting the potential for reliable lipid analysis from DBS samples with short-term stability under various storage conditions, an important logistical benefit for remote or resource-limited settings.

Keywords:  4D lipidomics; Dried blood spots (DBS); Lipids; Mass spectrometry; Microsampling; Patient-centric sampling; Storage stability

DOI:  https://doi.org/10.1016/j.talanta.2025.127677
Clin Chem Lab Med. 2025 Feb 10.

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

Myriam Ott, Neeraj Singh, Friederike Bauland, Daniel Köppl, Kerstin Kandler, Alexander Gaudl, Andrea Geistanger, Uta Ceglarek, Manfred Rauh, Christian Geletneky, Judith Taibon.

   OBJECTIVES: Accurate measurement of serum cortisol is crucial for the diagnosis and management of adrenal disorders. Thus, we have developed a novel isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) to quantify cortisol in human serum/plasma, offering higher sensitivity and reliability compared to existing RMPs.
METHODS: Quantitative Nuclear Magnetic Resonance spectroscopic (qNMR) methodology has been utilized to assign the absolute content (g/g) and SI-traceability to the reference materials. A novel two-dimensional heart-cut liquid chromatography (LC) approach was implemented for the LC-MS/MS, combined with a supported liquid extraction (SLE) sample preparation protocol. A multi-day validation experiment assessed precision and accuracy. Reproducibility was assessed by comparing procedure results between two independent laboratories, and measurement uncertainty (MU) was evaluated in compliance with current guidelines.
RESULTS: The established RMP exhibited high sensitivity, with a quantification range of 0.800-600 ng/mL (2.21-1,655 nmol/L), exceeding the ranges of existing JCTLM-listed RMPs. Intermediate precision was ≤2.6 %, and repeatability ranged from 0.9 to 1.9 % across all concentration levels. The relative mean bias ranged from -1.3 to 1.4 % for all matrices and concentration levels. Measurement uncertainties (MU) for cortisol in single measurements were ≤2.8 % regardless of the concentration level and sample type. Using the certified International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference panel, the equivalence between the candidate RMP and the Joint Committee on Traceability in Laboratory Medicine (JCTLM) listed RMPs (NRMeth 57 and NRMeth 8) was assessed, revealing excellent agreement.
CONCLUSIONS: This RMP allows for highly sensitive and reproducible determination of cortisol. The performance of the RMP facilitates the standardization of routine assays and ensures traceability in the measurement of individual patient samples.

Keywords:  SI units; cortisol; isotope dilution-liquid chromatography-tandem mass spectrometry; qNMR characterization; reference measurement procedure; traceability

DOI:  https://doi.org/10.1515/cclm-2024-0879
Toxicol Rep. 2025 Jun;14 101903

Optimization of HPLC method for metanephrine and normetanephrine detection in urine: Enhancing diagnostic precision for pheochromocytoma.

Hafiza Monaza Batool, Madeeha Batool.

  Catecholamines and their metabolites play critical physiological roles in the human body. Paragangliomas and pheochromocytomas are rare adrenal tumors that significantly alter catecholamine metabolism, particularly the concentrations of metanephrine (MN) and normetanephrine (NMN). This study presents the development and validation of a rapid and straightforward analytical method using reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with a photodiode array (PDA) detector for quantifying MN and NMN in 24-h urine samples. Sample preparation involved adding 1 mL of urine to a tube containing the internal standard 3-methoxy-4-hydroxy benzylamine hydrochloride (MHBA) and a 2 g/L solution of 2-aminoethyl-diphenylborinate. After vortex mixing and centrifugation, ethyl acetate was used for extraction, and the organic layer was dried under nitrogen at 50-60 °C before reconstitution in the mobile phase. Chromatographic separation was achieved on an RP C-18 column with an isocratic flow of the mobile phase (sodium dihydrogen phosphate, citric acid monohydrate, acetonitrile, and sodium octyl sulfate). Detection was performed at 347 nm, with peak identification based on standard retention times. The method was validated for linearity (10-2000 ng/mL), recovery, sensitivity, precision, accuracy, selectivity, carryover, stability, and dilution effects. It showed a strong correlation coefficient (>0.99) and accuracy within ± 15 %. Inter- and intra-day precision confirmed the method reliability. This validated technique is suitable for clinical and research applications involving catecholamine metabolite screening.

Keywords:  High-Performance Liquid Chromatography; Metanephrine; Normetanephrine; Pheochromocytoma

DOI:  https://doi.org/10.1016/j.toxrep.2025.101903
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jan 28. pii: S1570-0232(25)00025-X. [Epub ahead of print]1253 124473

Detection and application of 40 piperazine compounds in hair suitable for rapid separation of isomers.

Jinting Liu, Xin Wang, Wanting Xie, Liying Zhou, Lina Pei, Shuo Yang, Guoqing Gao, Keming Yun, Yan Shi.

  A simple and sensitive procedure, using benzylpiperazine-D7 (BZP-D7) as an internal standard, has been developed and validated for the qualitative and quantitative analysis of 40 piperazines in hair. Drugs were extracted from 20 mg of hair with 0.5 mL of methanol containing 1 ng/mL BZP-D7. After ultrasonication, centrifugation and filtration, the supernatant was analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) operating in the multiple reaction monitoring mode. Piperazine-type substances were separated in 10 min on a T3 column using a mobile phase gradient composed of A (water, formic acid 0.1 %, acetonitrile 5 %, and 20 mmol/L ammonium acetate) and B (acetonitrile). The developed and validated method showed good selectivity, sensitivity (limit of detection: 0.5-20 pg/mg and lower limit of quantitation: 5-20 pg/mg), linearity (R2 > 0.99), accuracy, precision, and dilution integrity. The method also showed good recovery and acceptable matrix effects for most of the targeted compounds. This analytical approach was successfully applied for the identification and quantification of piperazine-type substances in hair from rat and guinea pig.

Keywords:  Animal experiment; Human hair; Isomer; Piperazine; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2025.124473
J Chromatogr A. 2025 Jan 26. pii: S0021-9673(25)00075-5. [Epub ahead of print]1744 465726

Determination of veterinary drugs in foods of animal origin by QuEChERS coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

Qianran Sun, Jun Liu, Yuan Gou, Tieyuan Chen, Xiaofang Shen, Tao Wang, Yongli Li, Huizhen He, Huidan Deng, Yi Hua.

  A method using QuEChERS coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of the residues of 19 veterinary drugs in ten animal-derived matrices, including beef, pork, sheep, horse, chicken, prawn, fish, liver, milk, and fat. This method was based on the enactment of veterinary drug compounds by Korea, Canada, the United States, and the European Union in recent years. The samples were extracted using 85% acetonitrile and separated on an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) with a gradient elution of methanol-0.2% formic acid water as the mobile phase. The detection of the analytes was achieved through the use of positive ion electrospray ionization (ESI) and multiple reaction monitoring (MRM) modes, while the quantification was conducted via the matrix-matched external standard method. Following optimization, the linearity of the target veterinary residues in the ten matrices was observed to be satisfactory, having a range of 0.5-50.0 ng/mL (R2 > 0.991). The limits of detection (LOD) were in the range of 0.01-1.29 μg/kg, while the limits of quantification (LOQ) were in the range of 0.02-4.31 μg/kg. The recoveries were observed to be in the range of 60.6-117.7 %, with relative standard deviations (RSDs) of ≤20.6 %. The method is straightforward and highly sensitive, and it satisfies the maximum limits set by the relevant standards of Korea, Canada, the USA, and the EU. It is well-suited for the rapid screening, qualitative, and quantitative analyses of metomidate, acetanilide, dl-methylephedrine, and other substances in foods of animal origin, providing technical assistance for cross-border food safety and testing.

Keywords:  Foods of animal origin; QuEChERS; UPLC-MS/MS; Veterinary drugs

DOI:  https://doi.org/10.1016/j.chroma.2025.465726
Front Chem. 2024 ;12 1490639

Innovative determination of phytohormones in Aloe vera.

Muhammad K Hakeem, Meera Maraqa, Sampath K Elangovan, Esam Eldin Saeed, Ajay Kumar Mishra, Khaled M Hazzouri, Iltaf Shah, Khaled M A Amiri.

   Introduction: Aloe vera is widely known for its therapeutic properties, but concerns regarding the levels of phytohormones and their potential impact on human health highlight the need for advanced analytical techniques. This study aims to develop and validate a sensitive method for the determination of six key phytohormones in Aloe vera using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
Methods: A validated LC-MS/MS method was optimized for the determination and quantification of six phytohormones in Aloe vera: Abscisic Acid (ABA), Salicylic Acid (SA), Indole-3-Acetic Acid (I3AA), Gibberellic Acid (GA), 6-Benzylaminopurine (6BAP), and Isopentenyladenine (ISA). The sample extraction process and mobile phase composition were optimized to enhance chromatographic separation and mass spectrometry sensitivity. A C-18 column was used for separation, and a triple quadrupole mass spectrometer was employed for quantification. The method's performance was assessed in terms of linearity, sensitivity, and limits of detection.
Results: The LC-MS/MS method exhibited excellent linearity (R 2 > 0.99) and low limits of detection for all six phytohormones. Four of the six analytes were identified as predominant in Aloe vera. Quantitative analysis showed that ABA was the most abundant phytohormone, with a median concentration of 8.39 ng/mL, followed by I3AA (4.32 ng/mL), SA (3.16 ng/mL), and GA (1.55 ng/mL).
Discussion: This study provides a comprehensive and validated LC-MS/MS method for profiling phytohormones in Aloe vera. The results underscore the significant role of ABA, I3AA, SA, and GA in the plant's hormonal profile, offering a valuable tool for the analysis of phytohormonal content in Aloe vera and other plant species. The method is particularly beneficial for addressing health-related concerns regarding the presence and concentration of phytohormones in Aloe vera.

Keywords:  Aloe vera; LC-MS/MS; liquid chromatography mass spectrometry; method validation; phytohormones; plant hormone

DOI:  https://doi.org/10.3389/fchem.2024.1490639
Anal Bioanal Chem. 2025 Feb 05.

Pre-analytic assessment of dried blood and dried plasma spots: integration in mass spectrometry-based metabolomics and lipidomics workflow.

Eleonora Bossi, Simone Serrao, Pierluigi Reveglia, Antonietta Ferrara, Marta Nobile, Elena Limo, Gaetano Corso, Giuseppe Paglia.

  Microsampling, especially dried blood spots (DBS), emerged in recent years as a viable alternative to conventional blood collection since it is rapid, simple, minimally invasive, and has user-friendly characteristics. Moreover, DBS are able to avoid analyte degradation thanks to their great stability. Due to their versatility, clinical applications with DBS have increased, including mass spectrometry-based metabolomics and lipidomics studies. In this work, we evaluated and optimized extraction protocols testing five different extraction solutions to perform metabolomics and lipidomics studies on the same spot considering three commercially available microsampling devices, Capitainer, Whatman, and Telimmune. Parallelly, we also evaluated the short-term stability of the three devices at room temperature for up to 5 days. Our results showed that pure methanol was the best compromise to simultaneously extract from the same spot both the lipidome and polar metabolome. However, we also propose a two-step protocol combining methanol and water extraction that improves polar metabolite extraction and shows improved reproducibility in Capitainer and Whatman. Short-term stability results highlighted that both polar metabolites and lipids were stable for up to 6 days using the Capitainer device, while with Whatman and Telimmune, some significant variations were observed after 3 days for some classes of metabolites/lipids, suggesting the need for cold-chain storage when working with these devices.

Keywords:  Dried blood spots; Lipidomics; Metabolomics; Method optimization; Microsampling

DOI:  https://doi.org/10.1007/s00216-025-05760-z
Toxicol Pathol. 2025 Feb 04. 1926233241311269

European Society of Toxicologic Pathology-Pathology 2.0 Mass Spectrometry Imaging Special Interest Group: Mass Spectrometry Imaging in Diagnostic and Toxicologic Pathology for Label-Free Detection of Molecules-From Basics to Practical Applications.

Enrico Vezzali, Michael Becker, Fernando Romero-Palomo, Marjolein van Heerden, Caroline Chipeaux, Gregory Hamm, Dinesh S Bangari, Thomas Lemarchand, Barbara Lenz, Bogdan Munteanu, Bhanu Singh, Celine Thuilliez, Seong-Wook Yun, Andrew Smith, Rob Vreeken.

  Mass Spectrometry Imaging (MSI) is a powerful tool to understand molecular pathophysiology and therapeutic and toxicity mechanisms, as well as for patient stratification and precision medicine. MSI, a label-free technique offering detailed spatial information on a large number of molecules in different tissues, encompasses various techniques including Matrix-Assisted Laser Desorption Ionization (MALDI), Desorption Electrospray Ionization (DESI), and Secondary Ion Mass Spectrometry (SIMS) that can be applied in diagnostic and toxicologic pathology. Given the utmost importance of high-quality samples, pathologists play a pivotal role in providing comprehensive pathobiology and histopathology knowledge, as well as information on tissue sampling, orientation, morphology, endogenous biomarkers, and pathogenesis, which are crucial for the correct interpretation of targeted experiments. This article introduces MSI and its fundamentals, and reports on case examples, determining the best suited technology to address research questions. High-level principles and characteristics of the most used modalities for spatial metabolomics, lipidomics and proteomics, sensitivity and specific requirements for sample procurement and preparation are discussed. MSI applications for projects focused on drug metabolism, nonclinical safety assessment, and pharmacokinetics/pharmacodynamics and various diagnostic pathology cases from nonclinical and clinical settings are showcased.

Keywords:  diagnostic pathology; drug development; label-free; mass spectrometry imaging; multiplex

DOI:  https://doi.org/10.1177/01926233241311269
Talanta. 2025 Jan 23. pii: S0039-9140(25)00112-2. [Epub ahead of print]287 127626

Matrix free laser desorption ionization coupled to trapped ion mobility mass spectrometry: an innovative approach for isomer differentiation and molecular network visualization.

Manon Meunier, Martina Haack, Dania Awad, Thomas Brück, Khalijah Awang, Marc Litaudon, Frédéric Saubion, Marc Legeay, Dimitri Bréard, David Guilet, Séverine Derbré, Andreas Schinkovitz.

  The chemical profiling of complex mixtures of natural products (NPs) is a major challenge in analytical chemistry and generally addressed by liquid chromatography coupled to mass spectrometry (LC-MS). In recent years also matrix free laser desorption ionization-mass spectrometry (LDI-MS) has become a versatile and time efficient complement to LC-MS. However, the absence of chromatographic separation in LDI-MS does not permit the differentiation of isomers. Providing a potential solution to this problem, the current work presents a combined LDI-Ion mobility spectrometry-tandem mass spectrometry (LDI-IMS-MS2) approach, which facilitated the successful differentiation of four constitutional xanthone isomers namely butyraxanthone D, cratoxylone, garcinone D and parvixanthone G. In addition, the experimental collision cross section (CCS) distribution values of nine unreported xanthones are described. Based on these results, a proof of concept for the so far unexplored concept of a LDI-IMS-MS2 based molecular network is being presented.

Keywords:  Ion mobility spectrometry; Isomer differentiation; Mass spectrometry; Matrix free laser desorption ionization; Molecular network; Xanthones

DOI:  https://doi.org/10.1016/j.talanta.2025.127626
J Sep Sci. 2025 Feb;48(2): e70089

A Protocol for Determination of Proteinogenic Amino Acids in Biological Fluids by the High-Speed UHPLC-MS Method: Application on Transgenic Spontaneously Hypertensive Rat-24 Plasma and Cerebrospinal Fluid Samples.

Juraj Piestansky, Dominika Olesova, Petra Majerova, Petra Chalova, Andrej Kovac.

  Recently, proteinogenic amino acids have become very interesting molecules, accompanied by a large variety of metabolic processes in humans and are associated with various diseases. In the era of system biology, including a broad spectrum of associated disciplines (e.g., metabolomics, lipidomics, proteomics, etc.), the possibility of identifying trustworthy biomarkers of diseases becomes much more likely. Changes in amino acid levels in plasma, serum, or cerebrospinal fluid reflect physiological or pathological conditions and, therefore, their regular monitoring can lead to early detection of the occurrence of a disease. Therefore, the exact determination of amino acids in biological fluids is of great importance. However, it is necessary to dispose with an effective, accurate, precise, selective, and robust analytical method. This protocol describes the complex procedure of amino acid analysis based on a combination of UHPLC with single quadrupole MS. The protocol presents a highly reproducible and robust methodology that has already been established in the quality control of biopharmaceuticals and determination of proteinogenic amino acids in urine in our laboratory. Here, the application potential is extended to the most frequently investigated biological fluid, that is, plasma and to the cerebrospinal fluid, which is investigated in many neurological conditions.

Keywords:  Alzheimer's disease; biological samples; proteinogenic amino acids; single quadrupole mass spectrometer; ultraperformance liquid chromatography

DOI:  https://doi.org/10.1002/jssc.70089
Anal Chem. 2025 Feb 04.

Pure Shift NMR with Solvent Suppression: A Robust and General Method for Determining Quantitative Metabolic Profiles in Biofluids.

Xi Chen, Cédric Caradeuc, Gildas Bertho, Covadonga Lucas-Torres, Nicolas Giraud.

Ultrahigh-resolution pure shift NMR has recently been shown as a promising approach for providing quantitative metabolic profiles that can be used to study the metabolic footprint left by cancer cells in their aqueous growth medium. In this approach, a library of reference 1H pure shift spectra with water suppression was implemented to determine metabolite concentrations from the NOESY-presat-PSYCHE-SAPPHIRE spectrum recorded on the extracellular medium. This achievement clearly called for a generalization of a quantification method relying on ultrahigh-resolution data to other biological samples of interest (urine, plasma, tissue extracts, etc.), which requires evaluating the robustness of the analytical workflow. We have first addressed the influence of sample preparation on the quality of metabolite quantification. The quantification performed on a model mixture of metabolites prepared under different conditions shows good linearity, trueness, and precision, which highlights the high reproducibility of the proposed analytical protocol regardless of the physicochemical conditions in the sample. Second, we have successfully implemented this quantification protocol to determine metabolite levels in real urine and plasma samples, thereby paving the way for the use of the library of pure shift reference spectra for accurate and quantitative metabolic profiling of a broad range of aqueous samples.

DOI: https://doi.org/10.1021/acs.analchem.4c05261
Biomed Chromatogr. 2025 Mar;39(3): e70010

Determination of Pesticide Residues in Ectoparasiticide-Impregnated Collars Using Ultrasound-Assisted Extraction and Liquid Chromatography-Mass Spectrometry.

Pricília Santos Pereira Gomes, Luís Fabrício Santana Santos, Sandro Navickiene.

  Ectoparasiticide-impregnated collars are used on dogs and cats to control zoonoses, acting as repellents and/or killing vectors. The contact of animals or people with pesticides can cause intoxication, so ectoparasiticide-impregnated collars may be a potentially important source of these compounds. The aim of this work was to develop a method for the determination of propoxur, dichlorvos, diazinon, chlorpyrifos, deltamethrin, and flumethrin in commercial ectoparasiticide-impregnated collars, using ultrasound-assisted extraction and liquid chromatography-mass spectrometry. The total ultrasound extraction time was 45 min, resulting in recovery values between 40 ± 6% and 103 ± 8%, in the concentration range from 0.005 to 1.0 mg L-1. Under optimal conditions, good linearity and sensitivity were obtained in the concentration range from 0.005 to 5.0 mg L-1 with coefficients of determination above 0.99. The relative standard deviations for triplicate determinations were lower than 24%, and the limits of detection and quantification were in the ranges 0.013-1.36 and 0.036-4.13 mg kg-1, respectively. The proposed method was applied in the analysis of a commercial collar, after its expiry date, and flumethrin residues were found.

Keywords:  liquid chromatography–mass spectrometry; multiresidue method; pet collar; residues

DOI:  https://doi.org/10.1002/bmc.70010
J Pharm Biomed Anal. 2025 Jan 29. pii: S0731-7085(25)00060-3. [Epub ahead of print]257 116719

Molecular mass spectrometric imaging of amino acids in dietary supplement tablets using laser ablation-atmospheric pressure chemical ionization-trapped ion mobility spectrometry-mass spectrometry.

Johannes Schmeinck, Uwe Karst.

  A UV laser ablation system coupled to atmospheric pressure chemical ionization-trapped ion mobility spectrometry-mass spectrometry (LA-APCI-TIMS-MS) is presented as a novel mass spectrometry imaging (MSI) tool to investigate the distribution of amino acids and vitamins in dietary supplement tablets. The setup employs a custom-built LA interface for commercially available APCI sources. Average single pulse response (SPR) durations of less than 0.2 s full peak width at 1 % maximum (FW0.01 M) were achieved. Imaging analyses resulted in the detection of 16 amino acids and 10 vitamins, all with unique and complementary distributions that are in most cases heterogeneously distributed over the sample surface. To differentiate between leucine and isoleucine, a second analysis was conducted on the same amino acid tablet with TIMS, showing distinct distributions for the two compounds. These results illustrate the potential of the presented setup to gain insight into the composition and manufacturing conditions of dietary supplements.

Keywords:  Ambient ionization; Amino acids; LA-APCI-MS; Mass spectrometry imaging; Trapped ion mobility spectrometry

DOI:  https://doi.org/10.1016/j.jpba.2025.116719
J Am Soc Mass Spectrom. 2025 Feb 03.

Improved LC-MS Detection of Opioids, Amphetamines, and Psychedelics Using TrEnDi.

Christian A Rosales, Noah A Lepinsky, Wondewossen Gebeyehu, Karl V Wasslen, Fraser Colquhoun, Benjamin B Warnes, Jasmine Chihabi, Jeffrey M Manthorpe, Jeffrey C Smith.

  Substances of misuse are becoming increasingly difficult to analyze as unique methods of smuggling are adopted and due to the rapid emergence of new psychoactive substances, increasing the pool of compounds to characterize and identify. Technologies such as gas chromatography and liquid chromatography coupled to mass spectrometry (MS) represent the gold standard for accurate and robust analysis, with on-site ambient- and portable-MS systems providing rapid methods of drug screening and testing. For many samples containing residual analyte quantities, methods to improve sensitivity through chemical derivatization are critical for accurate determination. Herein, we demonstrate for the first time the use of trimethylation enhancement using diazomethane (TrEnDi) to improve the MS-based sensitivity of 13 different drugs of misuse. All analytes were successfully permethylated, with 11 demonstrating improved analytical characteristics from TrEnDi with MS sensitivity enhancements ranging from 1.2-fold to as high as 24.2-fold in the case of psilocybin, as well as increases in reversed-phase chromatographic retention for most species. Derivatization using 13C-isotopically labeled TrEnDi reagents were used to successfully resolve isobaric interference issues between three pairs of controlled substances. By using an unconventional aprotic solvent system for electrospray ionization, the benefit of a fixed-permanent positive charge was highlighted as TrEnDi-modified amphetamine was easily measured while unmodified was not detected. Finally, TrEnDi was employed to boost the sensitivity of morphine in a real urine matrix. Our results demonstrate a percent recovery of 103.1% and a sensitivity enhancement of 2.4-fold, demonstrating the versatility and applicability of TrEnDi to pre-existing analytical workflows for trace analysis.

Keywords:  HPLC; TrEnDi; amphetamines; diazomethane; mass spectrometry; opioids; psychedelics

DOI:  https://doi.org/10.1021/jasms.4c00382