bims-metlip 2025-03-30 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–03–30
28 papers selected by
Sofia Costa, Matterworks

Novel Charge-Switch Derivatization Method Using 3-(Chlorosulfonyl)benzoic Acid for Sensitive RP-UHPLC/MS/MS Analysis of Acylglycerols, Sterols, and Prenols.
[Determination of four oxidative stress biomarkers in human urine using solid-phase extraction coupled with ultra performance liquid chromatography-tandem mass spectrometry].
Targeted analysis of seven selected tryptophan-melatonin metabolites: Simultaneous quantification of plasma analytes using fast and sensitive UHPLC-MS/MS.
Universal Photosensitizer for Isomer-Selective Lipid Imaging with High Molecular Coverage.
Iridium Isotope Tag-Assisted LC-MS Method for Global Profiling and Quantification of Nonvolatile Serum Fatty Acids in Nonalcoholic Fatty Liver Mice.
Robust UPLC-MS/MS Method With Acetonitrile for Precise Intracellular Quantification of Tacrolimus in PBMCs: A Step Toward Clinical Integration.
[Determination of 18 free amino acids in strawberries at different ripening stages by ultra performance liquid chromatography-triple quadrupole mass spectrometry based on hydrophilic interaction].
[Determination of eight organophosphate esters in animal-derived foods by ultra performance liquid chromatography-tandem mass spectrometry].
[Rapid and simultaneous determination of 11 ergot alkaloids in cereals and their products by ultra performance liquid chromatography-tandem mass spectrometry combined with Captiva EMR-Lipid column purification].
Protocol for spatial metabolomics and isotope tracing in the mouse brain.
Determination of carboxyatractyloside, the main toxic component of Xanthium strumarium L., and alkaloid toxins in soybean by liquid chromatography tandem mass spectrometry.
Dereplication of secondary metabolites from Sophora flavescens using an LC-MS/MS-based molecular networking strategy.
On-Tissue Chemical Derivatization for Mass Spectrometry Imaging of Fatty Acids with Enhanced Detection Sensitivity.
Direct 3D Mass Spectrometry Imaging Analysis of Environmental Microorganisms.
Direct Sampling Mass Spectrometry Analysis for the Assessment of Wounds: A Systematic Review.
Determination of skin-insect repellent icaridin and DEET in human urine using solid-phase extraction and liquid chromatography with tandem mass spectrometry and its application to a sample of Japanese adults.
Discovering Novel Short- and Medium-Chain Esters of Hydroxy Fatty Acids in Human Fecal Samples Using Untargeted Liquid Chromatography/Mass Spectrometry.
nmRanalysis: An Open-Source Web Application for Semi-automated NMR Metabolite Profiling.
Development and validation of a multiclass LC-MS/MS method for the analysis of cyanotoxins.
FluoroMatch IM: An Interactive Software for PFAS Analysis by Ion Mobility Spectrometry.
[Determination of four classes of 34 chlorinated persistent organic pollutants in seawater by solid-phase extraction and gas chromatography-electrostatic field orbitrap high resolution mass spectrometry].
Stability Indicating High Performance Liquid Chromatography Method Development, Validation, and Characterization of Forced Degradant Product by Liquid Chromatography-Mass Spectroscopy for Molnupiravir and Favipiravir.
Toxicological detection of the new psychoactive substances MDPHP and MDPHpP in human urine samples by elucidation of their urinary metabolites using gas chromatography-mass spectrometry.
Overcoming challenges in the analysis of the ubiquitous emerging contaminant trifluoroacetic acid employing trap column liquid chromatography tandem mass spectrometry.
[Effects of nicotine exposure on endogenous metabolites in mouse brain based on metabolomics and mass spectrometry imaging].
A 2D Microfluidic Paper-Based Analytical Device for Diagnosis of Canine Visceral Leishmaniasis via Mass Spectrometry-Based Immunoassay.
Development and validation of an isotope dilution-liquid chromatography (ID-LC-MS/MS)-based candidate reference measurement procedure for total 3,3',5-triiodothyronine in human serum.
A laboratory-friendly protocol for freeze-drying sample preparation in ToF-SIMS single-cell imaging.

Anal Chem. 2025 Mar 28.

Novel Charge-Switch Derivatization Method Using 3-(Chlorosulfonyl)benzoic Acid for Sensitive RP-UHPLC/MS/MS Analysis of Acylglycerols, Sterols, and Prenols.

Ondřej Peterka, Yasmin Kadyrbekova, Robert Jirásko, Zuzana Lásko, Bohuslav Melichar, Michal Holčapek.

Chemical derivatization involves the reaction of an analyte with a derivatization agent to modify its structure, improving the peak shape, chromatographic performance, structural analysis, ionization efficiency, and sensitivity. A novel derivatization method using 3-(chlorosulfonyl)benzoic acid is developed for the determination of monoacylglycerols, diacylglycerols, free sterols, and tocopherols using the reversed-phase ultra-high-performance liquid chromatography-tandem mass spectrometry (RP-UHPLC/MS/MS) method in the negative ion mode. The chromatographic and mass spectrometric properties of derivatized lipids are investigated by using 29 lipid standards spanning four lipid classes. The derivatization process is optimized using pooled plasma spiked by 9 internal standards, achieving an optimal yield with a reaction time of 40 min at 60 °C. The stability of the derivatives is confirmed, with short-term stability maintained for 10 h at 4 °C and long-term stability preserved for 5 days at -80 °C. The repeatability and reproducibility are verified by one/two operator(s), which underscores the simplicity and robustness of the method, and calibration curves with high linear regression coefficients illustrate the accuracy of the method. The derivatization approach, which combines RP-UHPLC/MS/MS and the use of specific fragmentation patterns, significantly reduces limits of detection, reaching 15-25 pmol/mL for free sterols in plasma. The optimized method is applied to the analysis of human plasma, leading to the identification of 92 lipid species in the targeted lipid classes. This represents a substantial improvement in sensitivity and detection capabilities compared to those of previously reported methods.

DOI: https://doi.org/10.1021/acs.analchem.4c06496
Se Pu. 2025 Apr 08. 43(4): 317-325

[Determination of four oxidative stress biomarkers in human urine using solid-phase extraction coupled with ultra performance liquid chromatography-tandem mass spectrometry].

Zhuang-Zhuang Feng, Xiao Lin, De-Jun Bao, Xiao-Jian Hu, Hai-Jing Zhang, Ying Zhu, Xu Zhang.

  Oxidative stress biomarkers are measurable biological indicators that reflect the balance between the production of reactive oxygen species (ROS) and the body's ability to neutralize them using antioxidants. Elevated oxidative stress is associated with a number of health effects. Herein, we report the development of a comprehensive and sensitive method for quantifying four typical oxidative stress biomarkers in human urine using solid-phase extraction (SPE) in conjunction with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The quantified biomarkers include L,L-dityrosine (diY), 8-hydroxy-2'-deoxyguanosine (8-OHdG), 8-hydroxyguanosine (8-OHG), and 4-hydroxynonenal mercapturic acid (HNEMA), which are markers of oxidative-stress-related damage in proteins, DNA, RNA, and lipids, respectively. To that end, we systematically optimized the MS parameters, SPE cartridge, and elution conditions of the method. Briefly, 0.2 mL of a urine sample was mixed with 0.8 mL of pure water, after which an internal-standard mixture was added. The four target analytes were enriched and purified using an HLB SPE cartridge. The diY and the other three compounds were eluted with 2% (volume fraction) methanol aqueous solution and methanol, respectively. The two groups of eluates containing different target analytes were separately injected onto an Acquity UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm) and gradient eluted using 0.05% (v/v) acetic acid aqueous solution and methanol. The target analytes were identified using both negative and positive electrospray ionization (ESI- and ESI+) and multiple reaction monitoring (MRM) modes, and quantified using stable-isotope-labeled internal standards. The four typical oxidative-stress biomarkers exhibited good linearities within the mass concentration range of 0.01-100 μg/L, with correlation coefficients ≥0.9998, and limits of detection (LODs) and limits of quantification (LOQs) of 7-18 and 22-60 ng/L, respectively. The spiked recoveries of the target analytes at three levels (5, 10 and 50 μg/L) were 103.0%-105.6%(8-OHdG), 100.8%-104.2%(8-OHG), 97.2%-100.2%(diY) and 96.9%-106.0%(HNEMA), with intra-day precisions of between 1.6% and 5.2%. Moderate-to-strong matrix effects of between 42% and 137% were observed for each target analyte. The target compounds exhibited weak matrix effects of 99%-102% (8-OHdG), 97%-98% (8-OHG), 97%-106% (diY), and 94%-110% (HNEMA) after adjustment using the stable-isotope-labeled internal-standard method. The developed method was used to determine the abovementioned four typical oxidative stress biomarkers in 40 urine samples. All target compounds were detected in human urine at rates of 100%, with mass concentrations of 0.52-14.40 μg/L, 2.75-38.15 μg/L, 8.92-82.28 μg/L, and 1.74-575.29 μg/L recorded for 8-OHdG, 8-OHG, diY, and HNEMA, respectively, along with median values of 2.89, 12.36, 37.66, and 96.92 μg/L, respectively. The developed method is simple to operate, highly sensitive, and is very precise and accurate; consequently, it is suitable for determining the abovementioned four typical oxidative stress biomarkers in human urine.

Keywords:  human biomonitoring; oxidative stress biomarkers; solid-phase extraction (SPE); ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS); urine

DOI:  https://doi.org/10.3724/SP.J.1123.2024.10003
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Feb 13. pii: S1570-0232(25)00072-8. [Epub ahead of print]1256 124520

Targeted analysis of seven selected tryptophan-melatonin metabolites: Simultaneous quantification of plasma analytes using fast and sensitive UHPLC-MS/MS.

Michal Kaleta, Bhagya S Kolitha, Ondřej Novák, Farshid Mashayekhy Rad, Jonas Bergquist, S J Kumari A Ubhayasekera.

  Tryptophan-derived metabolites, a group of neurotransmitters essential for various brain functions, play key roles in regulating mood, movement, sleep, and cognition. However, the comprehensive characterisation of tryptophan-melatonin pathway metabolites is challenging due to factors such as their structural diversity, chemical complexity, low concentrations, and instability of these metabolites. In this study, we developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) methodology with electrospray ionisation for the simultaneous separation and quantification of tryptophan metabolites in human plasma. The analytical calibration ranges in plasma were 0.50-200 ng/mL for serotonin, 0.01-5 ng/mL for N-acetylserotonin, 0.01-20 ng/mL for tryptamine, 0.01-20 ng/mL for 6-sulfatoxymelatonin, 0.01-20 ng/mL for 6-hydroxymelatonin, 0.01-100 ng/mL for melatonin, and 0.10-20 ng/mL for N-acetyltryptamine, with correlation coefficients ranging from 0.954 for N-acetyltryptamine to 0.997 for tryptamine. The intraday and interday precision remained consistently below 15 % for all analytes. Most analytes met the accuracy criteria, except for N-acetyltryptamine at the lowest quality control level (0.2 ng/mL), where the intraday and interday accuracy were 22.4 % and 17.4 %, respectively. In conclusion, this novel method allows for rapid identification of tryptophan-melatonin pathway intermediates in less than ten minutes, including seven distinct melatonin-related analytes. This suggests that it may find use in everyday clinical and scientific endeavours.

Keywords:  Liquid chromatography; Mass spectrometry; Melatonin; Plasma; Tryptophan metabolites

DOI:  https://doi.org/10.1016/j.jchromb.2025.124520
Anal Chem. 2025 Mar 25.

Universal Photosensitizer for Isomer-Selective Lipid Imaging with High Molecular Coverage.

Sara Amer, Daisy Unsihuay, Manxi Yang, Julia Laskin.

Spatial lipidomics is a powerful technique for understanding the complexity of the lipidome in biological systems through mass spectrometry imaging (MSI). Recent advancements have enabled isomer-selected MSI (iMSI) of lipids in biological samples using both online and off-line derivatization strategies. Despite these impressive developments, most iMSI techniques are limited to either positive or negative ion mode analysis, restricting the molecular coverage achievable in a single experiment. Additionally, derivatization efficiency often varies across lipid classes, presenting challenges for comprehensive lipid analysis. In this study, we introduce tetrakis(4-carboxyphenyl)porphyrin (TCPP) as a universal photosensitizer that facilitates online lipid derivatization in both positive and negative ionization modes via singlet oxygen (1O2) reaction. This method enables the identification and localization of both acyl chain compositions and lipid carbon-carbon (C═C) isomers in liquid extraction-based ambient ionization techniques. We have also employed sodium fluoride (NaF) as a solvent dopant to enhance the analysis of low-abundance and poorly ionizable biomolecules. By integrating these online derivatization and signal enhancement strategies with nanospray desorption electrospray ionization (nano-DESI), we achieved dual polarity iMSI within the same sample. We demonstrate imaging of low-abundance isomeric lipids, which were otherwise below the noise level. Notably, TCPP significantly enhances the efficiency of the online derivatization of unsaturated fatty acids, for which other photosensitizers are inefficient. This novel approach allows for the imaging of isomeric fatty acids and phospholipids from multiple classes in the same experiment, revealing their distinct spatial localization within biological tissues.

DOI: https://doi.org/10.1021/acs.analchem.4c05538
Anal Chem. 2025 Mar 27.

Iridium Isotope Tag-Assisted LC-MS Method for Global Profiling and Quantification of Nonvolatile Serum Fatty Acids in Nonalcoholic Fatty Liver Mice.

Yongcheng Dai, Beicheng Zhu, Xueting Yan, Xiaobo Xie, Zixuan Zhan, Yi Lv.

Highly accurate and sensitive measurements of fatty acids (FAs) in biological samples are essential for advancing our understanding of their diverse biofunctions. In this work, based on the characteristic isotope pattern of iridium (191/193Ir), we employed an iridium-encoded amine (Ir-NH2) as the derivatization reagent to establish a selective and sensitive liquid chromatography-mass spectrometry (LC-MS) method for rapid identification and accurate quantification of FAs in biological samples. Upon derivatization, nonvolatile FAs were transformed into amide derivatives tagged with a charged iridium tag, exhibiting improved sensitivity and selectivity in the electrospray ionization (ESI) positive ion mode. By leveraging the unique 2.002 Da mass shift and the 3:5 peak intensity ratio from the natural 191Ir and 193Ir isotopes, we can rapidly and efficiently screen the potential carboxyl-containing metabolites from biological samples. Compared to other existing methods, our technique offers higher sensitivity, better signal-to-noise ratio, lower detection limit (1.2-8.4 pg/mL), and easier quantification due to the clear identification of iridium-tagged derivatives. With this method, a total of 58 FAs, including both saturated and unsaturated types, were detected in mice serum lipid extracts, with carbon chain lengths varying from C9 to C24. More importantly, this method was successfully employed for global profiling of nonvolatile serum FAs from mice with nonalcoholic fatty liver disease (NAFLD), providing a novel means for detecting them and offering new avenues for exploring their functional roles and disease associations.

DOI: https://doi.org/10.1021/acs.analchem.4c05310
Clin Transl Sci. 2025 Apr;18(4): e70210

Robust UPLC-MS/MS Method With Acetonitrile for Precise Intracellular Quantification of Tacrolimus in PBMCs: A Step Toward Clinical Integration.

Napatsanan Tanathitiphuwarat, Asada Leelahavanichkul, Pajaree Chariyavilaskul, Suwasin Udomkarnjananun.

  Monitoring whole blood tacrolimus concentrations is standard in clinical practice; however, it may not fully reflect its therapeutic effects, as tacrolimus primarily acts within lymphocytes. While various intracellular quantification methods have been developed, many involve complex procedures such as evaporation, reconstitution, or specialized tools (e.g., magnetic beads, online solid-phase extraction), limiting their accessibility. This study aimed to develop and validate a streamlined, sensitive method for measuring intracellular tacrolimus concentrations using 5×105 peripheral blood mononuclear cells (PBMCs). Tacrolimus concentrations were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). PBMCs were aliquoted into 50 μL volumes containing 5×105 cells and prepared via acetonitrile-based protein precipitation. Chromatographic separation was performed using a Luna C18 column with a gradient mobile phase consisting of water with 20 mM ammonium acetate, 0.1% formic acid, and methanol at a flow rate of 0.4 mL/min. The method demonstrated excellent linearity between 0.1 and 25 ng/mL, corresponding to intracellular concentrations of 1-250 pg/5×105 cells (r2 = 0.999). Intra- and interday accuracy ranged from 98.1% to 109.8%, with precision between 2.08% and 8.70% across validation runs. Extraction recovery was high (93.0%-97.2%), with minimal matrix effects (100.9% at low QC and 111.6% at high QC). This validated LC-MS/MS method provides a rapid, reliable, and sensitive approach for pharmacokinetic studies and clinical applications, facilitating intracellular tacrolimus monitoring in transplant patients.

Keywords:  LC–MS/MS; intracellular tacrolimus; peripheral blood mononuclear cells; therapeutic drug monitoring

DOI:  https://doi.org/10.1111/cts.70210
Se Pu. 2025 Apr 08. 43(4): 372-381

[Determination of 18 free amino acids in strawberries at different ripening stages by ultra performance liquid chromatography-triple quadrupole mass spectrometry based on hydrophilic interaction].

Min Ju, Yu-Ming Song, Jin-Feng Zhao, Yu-Ming Sun, Li-Na Zhou, Qing-Xin Yin, Chen Wang, Rui Cai, Qiang Xu, Hui-Hui Wan.

  The determination of free amino acids is important for quality evaluation and nutritional studies of strawberries. In this study, an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method using hydrophilic interaction chromatographic column was established for the simultaneous determination of 18 free amino acids in strawberries, and the pretreatment, chromatography, and mass spectrometry conditions were optimized. Specific pretreatment processes include grinding, extraction with water, centrifugation, and membrane filtration. The treated samples were then analyzed by hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry. The separation was performed on an ACQUITY UPLC Glycan BEH Amide column (150 mm×2.1 mm, 1.7 μm), with a water-acetonitrile system containing 5 mmol/L ammonium formate and 0.1% (v/v) formic acid as the mobile phase for gradient elution. A triple quadrupole mass spectrometer was used in positive-ion electrospray ionization (ESI) scanning mode, with target amino acids quantified using the matrix-matched standard-curve method. Eighteen free amino acids were determined with good linearities in the range of 0.5-40.0 μmol/L, along with r2 greater than 0.992. The intra-day and inter-day precisions of the method were 1.0%-14.8% and 3.6%-17.6%, respectively. The limits of detection (LODs) were in the range of 50-250 nmol/L. The recoveries of the 18 amino acids were 75.0%-114.6% with relative standard deviations (RSDs) of 0.3%-13.5%. The contents of free amino acids in strawberries at different ripening stages were statistically analyzed, and a total of seven differentiated free amino acids (phenylalanine, isoleucine, glutamine, 4-aminobutyric acid, arginine, glutamic acid, and aspartic acid) were statistically screened. The method is rapid, accurate, reproducible and stable, and can quantitatively determine the content of amino acids in strawberries.

Keywords:  free amino acids; hydrophilic interaction chromatography (HILIC); strawberry; tandem mass spectrometry (MS/MS); ultra performance liquid chromatography (UPLC)

DOI:  https://doi.org/10.3724/SP.J.1123.2024.04017
Se Pu. 2025 Apr 08. 43(4): 309-316

[Determination of eight organophosphate esters in animal-derived foods by ultra performance liquid chromatography-tandem mass spectrometry].

Yan Tang, Sheng Wen, Wen-Cheng Cao, Xiao Liu, Cheng-Lin Lei, Qing-Yun Cheng, Hai-Chuan Chen, Ling Liu, Xiao-Fang Liu, Yan Zhou.

  Organophosphate esters (OPEs) are widely used as flame retardants in most regions, they adversely affect ecosystems and threaten human health. OPEs have attracted significant public attention because they are toxic and ubiquitously present in the environment. While China is among the world's largest users and producers of OPEs, limited data on the exposure of animal-derived foods to OPEs exist; consequently, a method for quantifying OPEs in animal-derived food samples is needed. In this study, a method was developed for the determination of eight OPEs, including triethyl phosphate (TEP), tripropyl phosphate (TPrP), tri-n-butyl phosphate (TnBP), tris(2-chloroethyl) phosphate (TCEP), tris(2-chloroisopropyl) phosphate (TCIPP), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), triphenyl phosphate (TPHP), and 2,2-bis(chloromethyl) trimethylene bis[bis(2-chloroethyl) phosphate] (V6), from twelve types of typical animal-derived foods by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The samples were purified using an HMR-Lipid SPE column. The effects of mobile phase A (water, 5 mmoL/L ammonium acetate aqueous solution, and 0.1% formic acid aqueous solution) and mobile phase B (methanol and acetonitrile), as well as the methanol/acetonitrile ratio on the separation and extraction efficiencies for the eight OPEs were investigated using one-way analysis. The results showed that optimal response values and peak shapes were obtained for the various compounds using 0.1% formic acid aqueous solution-acetonitrile system as the mobile phase. The following pretreatment procedure was used: A 0.5 g sample was accurately weighed and ultrasonically extracted with 5 mL of acetonitrile. The supernatant was collected after freezing and centrifugation, and cleaned-up was performed using an HMR-Lipid SPE column. The target analytes were analyzed using a Waters Acquity BEH C18 column (100 mm×2.1 mm, 1.7 μm) and ESI+ MS conditions. Compound V6 was quantified by the external standard method, with the other seven compounds quantified using the internal standard method. The method exhibited linearity with r2≥0.9900, limits of detection (LODs) of 0.01-0.87 μg/kg, and limits of quantification (LOQs) of 0.02-2.62 μg/kg for the various substances. Spiked recoveries of the eight OPEs at three levels (2, 20, and 100 μg/kg) were in the range of 80.5%-117.8% and RSDs ≤ 14.8% (n=6). Twelve animal-derived foods (grass carp, bass, Procambarus clarkii, milk, milk powder, yogurt, pork, beef, chicken, duck meet, egg, and duck egg) were analyzed using the developed method. Compounds TnBP and TCIPP were detected at rates of 100%, and TEP, TCEP, TPHP, and TDCIPP at rates greater than 50%, while TPrP and V6 were not detected. The method has a simple-to-operate pre-treatment step, analyzes rapidly with good recoveries and precisions, and is suitable for rapidly analyzing and detecting eight OPEs in a variety of animal-derived foods.

Keywords:  animal-derived foods; organophosphate esters (OPEs); ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)

DOI:  https://doi.org/10.3724/SP.J.1123.2024.07010
Se Pu. 2025 Apr 08. 43(4): 326-334

[Rapid and simultaneous determination of 11 ergot alkaloids in cereals and their products by ultra performance liquid chromatography-tandem mass spectrometry combined with Captiva EMR-Lipid column purification].

Bolin Liu, Dan Zhang, Zi-Wei Zhao, Ji-An Xie, Zi-Yue Zhan, Qi Zhang, Wei-Dong Li.

  Ergot alkaloids (EAs) are mycotoxins produced by Claviceps and are present in cereals and their products; their residues pose significant threats to human health through food consumption, resulting in ergotism and sickness. Herein, a sensitive and rapid method for the determination of 11 EAs in cereals and their products using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) were developed. Eleven EAs were extracted with 20 mL acetonitrile-200 mg/L ammonium acetate solution (80∶20, v/v) for 15 min using the vortex shock method followed by 15 min of ultrasonication. The mixture was subsequently centrifuged at 10000 r/min for 10 min, and the supernatant was purified by a Captiva EMR-Lipid column. Target analytes were separated on an ACQUITY UPLC HSS T3 chromatography column (100 mm×3 mm, 1.8 μm) at a column temperature of 40 ℃ and a flow rate of 0.4 mL/min using an injection volume of 5 μL. Gradient elution was performed using 1 mmol/L ammonium acetate solution and acetonitrile as mobile phases. Data were collected in electrospray positive-ion (ESI+) and multi-reaction monitoring (MRM) modes, and quantified using matrix-matched standard curves. The 11 EAs exhibited good linearities in their linear ranges, with correlation coefficients (r2) of 0.9933-0.9999, with limits of detection (LODs) and limits of quantification (LOQs) of 0.002-0.2 and 0.006-0.6 μg/kg, respectively. Recoveries and relative standard deviations (RSDs) of the 11 EAs in matrix samples of wheat flour, coix seed, wheat flour products, and corn flour at low, medium, and high spiked levels were 80.1%-118% and 0.2%-13.3%, respectively. The established method was used to determine EAs in 240 wheat flour, 80 corn flour, 30 rice, and 30 coix seed samples, as well as 146 wheat flour products, with the detection rates of the 11 EAs of 0.57%-20.3%. A maximum total content of EAs of 56.7 μg/kg was recorded for a single sample. The sample pretreatment process used in this method is simple and fast, and the detection method is highly sensitive, with accurate and reliable results obtained. This method is suitable for simultaneously determining various EAs in cereals and their products. The results of this study provide valuable information for future EA risk-assessment studies.

Keywords:  cereals and their products; ergot alkaloids (EAs); mycotoxins; ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)

DOI:  https://doi.org/10.3724/SP.J.1123.2024.02022
STAR Protoc. 2025 Mar 27. pii: S2666-1667(25)00122-4. [Epub ahead of print]6(2): 103716

Protocol for spatial metabolomics and isotope tracing in the mouse brain.

Watit Sontising, Francine Yanchik-Slade, Carlos Rodriguez-Navas, Md Amir Hossen, Manuel Gonzalez-Rodriguez, Jake Vaynshteyn, Shinichi Yamaguchi, Mohamed Nazim Boutaghou, Isaac Marin-Valencia.

  Mass spectrometry imaging enables high-resolution spatial chemical mapping, yet its application for dynamic analysis with tracers poses challenges. Here, we present a protocol for spatial metabolomics and isotope tracing in the mouse brain. We describe steps for tracer administration, tissue collection, and cryosectioning. We then detail procedures for matrix application, ion identification, and data analysis. This protocol delivers high-quality spatial metabolomics data and is well suited for region-specific tracing analysis in the brain.

Keywords:  mass spectrometry; metabolism; neuroscience

DOI:  https://doi.org/10.1016/j.xpro.2025.103716
J Chromatogr A. 2025 Mar 22. pii: S0021-9673(25)00245-6. [Epub ahead of print]1749 465897

Determination of carboxyatractyloside, the main toxic component of Xanthium strumarium L., and alkaloid toxins in soybean by liquid chromatography tandem mass spectrometry.

Ádám Tölgyesi, Attila Cseh, Andrea Simon.

  The presence of carboxyatractyloside (CAT) in Xanthium strumarium L. (cocklebur weed) presents a high risk for both human and animal health. In this paper, we report a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the determination of CAT in soybean intended for animal feed. The herein described approach involves a fast sample preparation utilising QuEChERS extraction followed by dSPE clean-up. The optimised mobile phase composition flowing through an HPLC column packed with C18 fused-core particles consisted of alkaline conditions (pH = 8.8) in the aqueous eluent and pure methanol as the organic modifier. In this manner, it was also suitable for the analysis of tropane and ergot alkaloids together with CAT, which was attempted for the first time and may improve the analysis efficiency and reduce costs. During the method validation the matrix effect was also investigated, which clearly showed that matrix-matched calibration or standard addition is needed for appropriate quantification, mainly at low levels. The recoveries were between 94 % and 115 %, and the RSD % varied from 2.0 % to 7.3 %. The limits of quantification for alkaloids and CAT were 1 µg/kg and 100 µg/kg, respectively. The method implementation in other LC-MS/MS instruments was also tested. Finally, the validated method was successfully applied on soybean samples naturally contaminated with cocklebur.

Keywords:  Carboxyatractyloside; Ergot alkaloids; LC-MS/MS; Soybean; Tropane alkaloids; Xanthium strumarium L.

DOI:  https://doi.org/10.1016/j.chroma.2025.465897
Sci Rep. 2025 Mar 24. 15(1): 10148

Dereplication of secondary metabolites from Sophora flavescens using an LC-MS/MS-based molecular networking strategy.

Hua Wang, Hui Ding.

  A dereplication strategy was developed for the screening of secondary metabolites from Sophora flavescens. The strategy consisted of 4 procedures. First, the extract of the Sophora flavescens root was subjected to LC-MS/MS analysis with both data-independent acquisition (DIA) mode and data-dependent acquisition (DDA) mode. Then the DIA results were used to construct a molecular networking (MN) according to the GNPS workflow and consequently obtain annotations. In parallel, the DDA results were projected to both MN analysis and direct databases matching to obtain annotations. Finally, the isomers were discriminated and annotated by their extracted ion chromatogram. Through the combination of these approaches, a total of 51 compounds were annotated and dereplicated in the Sophora flavescens samples. The annotation results showed DIA and DDA approach are complementary to each other. MN on GNPS can overcome the challenges of trace compound identification compared to direct DB matching. This strategy provides a powerful tool for the dereplication study in plant chemistry.

Keywords:   Sophora flavescens ; Alkaloids; Dereplication; Flavonoids; Metabolites; Molecular networking

DOI:  https://doi.org/10.1038/s41598-025-94958-3
Biomolecules. 2025 Mar 03. pii: 366. [Epub ahead of print]15(3):

On-Tissue Chemical Derivatization for Mass Spectrometry Imaging of Fatty Acids with Enhanced Detection Sensitivity.

Malik Ebbini, Zicong Wang, Hua Zhang, Kelly H Lu, Penghsuan Huang, Cameron J Kaminsky, Luigi Puglielli, Lingjun Li.

  The dysregulation of fatty acid (FA) metabolism is linked to various brain diseases, including Alzheimer's disease (AD). Mass spectrometry imaging (MSI) allows for the visualization of FA distribution in brain tissues but is often limited by low detection sensitivity and high background interference. In this work, we introduce a novel on-tissue chemical derivatization method for FAs using Girard's Reagent T (GT) as a derivatization reagent combined with 2-chloro-1-methylpyridinium iodide (CMPI) as a coupling reagent and triethylamine (TEA) to provide a basic environment for the reaction. This method significantly enhances the detection sensitivity of FAs, achieving a 1000-fold improvement over traditional negative ion mode analysis. Our method enabled us to observe a notable depletion of oleic acid in the corpus callosum of AD mouse model brain tissue sections compared to wild-type control brain tissue sections. The reliability of our method was validated using LC-MS/MS, which confirmed the presence of eight distinct GT-labeled FAs across various tissue locations. This approach not only improves FA detection in brain tissues but also has the potential to provide a deeper understanding of FA dynamics associated with AD pathogenesis.

Keywords:  Alzheimer’s disease mouse model; MALDI; fatty acids; mass spectrometry imaging; on-tissue chemical derivatization

DOI:  https://doi.org/10.3390/biom15030366
Molecules. 2025 Mar 14. pii: 1317. [Epub ahead of print]30(6):

Direct 3D Mass Spectrometry Imaging Analysis of Environmental Microorganisms.

Justyna Szulc, Tomasz Grzyb, Joanna Nizioł, Sumi Krupa, Wiktoria Szuberla, Tomasz Ruman.

  Assessing the spatial distribution of microorganisms' metabolites in growth medium remains a challenge. Here, we present the first use of the newly developed LARAPPI/CI-MSI 3D (laser ablation remote atmospheric pressure photoionization/chemical ionization mass spectrometry imaging) method for direct three-dimensional (3D) mass spectrometry imaging of bacterial and fungal metabolites in solid culture media. Two-dimensional (2D) MSI was also performed, and it indicated the presence of metabolites belonging to, and including, amino acids and their derivatives, dipeptides, organic acids, fatty acids, sugars and sugar derivatives, benzene derivatives, and indoles. Distribution at a selected depth within the culture medium with the estimation of concentration across all dimensions of 16 metabolites was visualized using LARAPPI/CI-MSI 3D. The imaging results were correlated with the results of ultra-high-performance liquid chromatography-ultra-high-resolution mass spectrometry (UHPLC-UHRMS). A total of 351-393 chemical compounds, depending on the tested microorganism, were identified, while 242-262 were recognized in the HMDB database in MetaboAnalyst (v 6.0). The LARAPPI/CI-MSI 3D method enables the rapid screening of the biotechnological potential of environmental strains, facilitating the discovery of industrially valuable biomolecules.

Keywords:  2D/3D microbiological culture imaging; bioactive compounds; mass spectrometry imaging; metabolomic analysis; microbial interactions; phytopathogens

DOI:  https://doi.org/10.3390/molecules30061317
Int Wound J. 2025 Apr;22(4): e70158

Direct Sampling Mass Spectrometry Analysis for the Assessment of Wounds: A Systematic Review.

Kanin Pruekprasert, Matthew Tan, Lauren Ford, Alun Huw Davies, Zoltan Takats, Sarah Onida.

  Mass spectrometry is increasingly utilised in medicine to identify and quantify small biomarkers for diagnostic and prognostic purposes. Conventional mass spectrometry, however, requires time-consuming sample preparation, hindering its clinical application. Direct sampling mass spectrometry, which allows for direct analysis of patient samples with minimal preparation, offers potential for clinical use. This systematic review examines the utility of direct sampling mass spectrometry for the assessment of external wounds and explores its translational applications in wound care. Out of 2 930 screened abstracts, six studies were included employing various direct sampling mass spectrometry technologies. These studies focused on burn wounds (n = 3), pressure ulcers (n = 2), and acute surgical wounds (n = 1). Both targeted and untargeted molecular profiling methods were used to examine biomarkers related to inflammatory and healing processes, including various proteins, lipid species, and other metabolites. Direct sampling mass spectrometry was found to complement conventional methods such as histology, providing additional insights into the spatial localisation and accumulation of metabolites within wounds. Additionally, imaging techniques equipped with this technology can spatially map wound surfaces and reveal dynamic changes in wounds as they age or progress through different healing processes, with specific metabolite and protein accumulations potentially aiding in prognostication.

Keywords:  biomarker; mass spectrometry; metabolomics; ulcer; wound

DOI:  https://doi.org/10.1111/iwj.70158
Environ Health Prev Med. 2025 ;30 18

Determination of skin-insect repellent icaridin and DEET in human urine using solid-phase extraction and liquid chromatography with tandem mass spectrometry and its application to a sample of Japanese adults.

Nanami Nishihara, Tomohiko Isobe, Mai Takagi, Toshiki Tajima, Yugo Kitahara, Mai Hayashi, Isao Saito, Satoru Watanabe, Miyuki Iwai-Shimada, Jun Ueyama.

   BACKGROUND: Icaridin and DEET are common insect repellents widely used on human skin and clothing (skin-insect repellents [skin-IR]) to repel common pests, such as mosquitoes and biting flies. Novel analytical methods for urinary skin-IR exposure biomarkers that can be effectively applied in epidemiological studies and provide strong evidence related to risk assessment associated with daily exposure are required. In this study, we aimed to develop a method for analyzing the concentrations of icaridin, DEET, and two DEET metabolites N,N-diethyl-3-(hydroxymethyl) benzamide and 3-(diethylcarbamoyl) benzoic acid in human urine.
METHODS: In this analysis, after formic acid-induced acidification of the urine sample, exposure biomarkers were extracted using solid-phase extraction composed of a modified polystyrenedivinylbenzene polymer for reversed phase (hydrophobic) retention. Subsequently, high-performance liquid chromatography-tandem mass spectrometry was performed within 10 min for a separation analysis. The present method was applied to five Japanese adults (aged 20-43 years) who used icaridin or DEET-containing products within a week.
RESULTS: Limits of detection were 0.06-0.11 µg/L. Extraction recoveries were 74%-88%. The intraday and interday variations were 1.5-17.5 and 0.9-15.8% relative standard deviation, respectively. All exposure biomarkers were successfully detected in all five adults. Urinary concentrations of exposure biomarkers reached their maximum values within 15 h after starting to use skin-IR.
CONCLUSIONS: This method was successful in measuring urinary exposure biomarkers of skin-IR, including icaridin and DEET. Moreover, this study presents the first application of biomonitoring of urinary icaridin concentrations after using a commercial product.

Keywords:  DEET; Human biomonitoring; Icaridin; Insect repellent; LC-MS/MS

DOI:  https://doi.org/10.1265/ehpm.24-00220
Rapid Commun Mass Spectrom. 2025 Apr 30. 39(12): e10032

Discovering Novel Short- and Medium-Chain Esters of Hydroxy Fatty Acids in Human Fecal Samples Using Untargeted Liquid Chromatography/Mass Spectrometry.

Jayashankar Jayaprakash, Divyavani Gowda, Rachana M Gangadhara, Shalini Jain, Hariom Yadav, Siddabasave Gowda B Gowda, Shu-Ping Hui.

   RATIONALE: Exploring novel metabolites produced by host gut microbiome communication is crucial for understanding their roles in various disease pathologies. We previously uncovered a novel class of lipids, short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs), in mouse fecal samples and demonstrated their promising physiological functions in mammals. However, the discovery of SFAHFAs in human samples remains unexplored.
METHODS: This study aimed to analyze the SFAHFAs and their structural analogs in human fecal samples using liquid chromatography/mass spectrometry.
RESULTS: We identified 26 isomeric lipid species, including SFAHFAs and novel medium-chain fatty acid esters of hydroxy fatty acids (MFAHFAs). The detected SFAHFAs and MFAHFAs were characterized by accurate mass measurements using MSn analysis. The results were validated by matching the mass spectral fragmentation and retention time with authentic standards. Two new MFAHFAs, enanthic acid and caprylic acid esters of long-chain hydroxy fatty acids (C24 and C26), were detected and characterized for the first time in human fecal samples. Among the 26 isomeric lipid species, SFAHFA 2:0/24:0 or 4:0/22:0 and SFAHFA 2:0/24:1 were most abundant among the saturated and unsaturated SFAHFAs, respectively.
CONCLUSIONS: This study offers the first insights into detecting and characterizing novel gut microbial lipids in human fecal samples. Further investigations are essential to recognize the metabolism and function of these lipids in the human gut.

Keywords:  LC/MS; MFAHFAs; MSn analysis; SFAHFAs; human fecal

DOI:  https://doi.org/10.1002/rcm.10032
Anal Chem. 2025 Mar 25.

nmRanalysis: An Open-Source Web Application for Semi-automated NMR Metabolite Profiling.

Javier E Flores, Anastasiya V Prymolenna, Logan A Lewis, Natalie M Winans, Elizabeth K Eder, William Kew, Robert P Young, Lisa M Bramer.

Though data acquisition and initial signal preprocessing of nuclear magnetic resonance (NMR) spectra have achieved high degrees of automation, downstream processing─specifically the profiling of spectra─has bottlenecked the overall NMR analysis workflow. Several efforts have been made to mitigate this bottleneck, but these solutions often trade an increase in automation for limitations elsewhere. In this work, we introduce nmRanalysis, a user-friendly web application that integrates the strengths of existing profiling tools for a more automated profiling workflow. nmRanalysis additionally incorporates novel features, including a machine-learning-driven recommender system for metabolite identification, further increasing the utility of nmRanalysis over the individual tools that it incorporates.

DOI: https://doi.org/10.1021/acs.analchem.4c05104
Anal Bioanal Chem. 2025 Mar 27.

Development and validation of a multiclass LC-MS/MS method for the analysis of cyanotoxins.

Lydia Zamlynny, Hannah M Morris, Sabrina D Giddings, Johannes Kollatz, Timo H J Niedermeyer, Rob C Jamieson, Daniel G Beach.

  Cyanobacteria are prokaryotic organisms that can form large monospecific blooms, which pose a risk to human and animal health as some species produce toxic secondary metabolites called cyanotoxins. Multiclass cyanotoxin analysis is challenging due to varying chemical and physical properties between classes, as well as potentially large numbers of analogues within each class. Incorporating anatoxins (ATXs) into multiclass methods can be particularly challenging due to their small molecular size, potential interferences, polarity, and a lack of chemical standards for most analogues. Here, we present the development of a multiclass LC-MS/MS method and a quantitative calibration solution for aetokthonotoxin (AETX), an emerging cyanotoxin linked to mass mortalities of bald eagles in the Eastern United States. The developed method is capable of detecting 17 microcystins (MCs), nodularin-R, three cylindrospermopsins (CYNs), AETX, and 17 ATXs, including recently tentatively identified 10-hydroxy analogues. Analytes were identified by retention time and product ion ratio matching with available standards. The method was evaluated with respect to limits of detection (LODs), linear range, accuracy, and precision using neat and matrix matched standards. LODs in wet cyanobacterial biofilms ranged from 0.14 ng/g for CYN to 2.8 ng/g for [Dha7]MC-LR with accuracies ranging from 65% for [Leu1]MC-LY to 116% for CYN. Finally, the method's application was demonstrated through analysis of cyanobacterial field samples, a dietary supplement matrix reference material, and passive sampler extracts to assess versatility within different matrices.

Keywords:  Aetokthonotoxin; Anatoxins; Cyanobacteria; Cyanotoxins; Cylindrospermopsins; LC–MS/MS; Microcystins; Multiclass analysis

DOI:  https://doi.org/10.1007/s00216-025-05829-9
Environ Sci Technol. 2025 Mar 25.

FluoroMatch IM: An Interactive Software for PFAS Analysis by Ion Mobility Spectrometry.

Rachel Smolinski, Jeremy P Koelmel, Paul Stelben, David Weil, David Godri, David Schiessel, Michael Kummer, Sarah M Stow, Sheher Mohsin, Lauren Royer, Alan McKenzie-Coe, Thomas Lubinsky, Daniel DeBord, Olivier Chevallier, Emma E Rennie, Krystal J Godri Pollitt, Carrie McDonough.

  Per- and polyfluoroalkyl substances (PFASs) are often present in complex mixtures at trace levels in environmental samples, posing difficulties for analytical chemists. Ion mobility offers highly replicable identifiers, enabling the use of community-based libraries for PFAS annotation in nontargeted analysis. Currently, limited software exists to leverage the capabilities of liquid chromatography ion mobility high-resolution mass spectrometry (LC-IM-HRMS) for nontargeted analysis. FluoroMatch IM is a free vendor-neutral open-source tool for rapid annotation of PFASs in LC-IM-HRMS datasets. Annotation algorithms include collision cross-section (CCS) matching, formula prediction, homologous series detection, mass defect filtering, and accurate mass matching with a database of 194 PFAS ions that can be continuously expanded by the community. Results from FluoroMatch IM were compared to a targeted approach with a laboratory-prepared mixture of 63 PFASs and real wastewater samples. A nontarget workflow incorporating FluoroMatch IM revealed additional likely PFASs (n = 16) while confirming most targeted annotations (11/12) in wastewater samples. Validation of the standard mix showed a low false negative rate of 5% and a 5% false positive rate for features included in the CCS library, with a 0% false positive rate for features assigned confident scores. This study demonstrates the promise of FluoroMatch IM for streamlining PFAS analysis workflows.

Keywords:  HRMS; PFAS; ion mobility; nontarget analysis; software

DOI:  https://doi.org/10.1021/acs.est.4c13846
Se Pu. 2025 Apr 08. 43(4): 345-354

[Determination of four classes of 34 chlorinated persistent organic pollutants in seawater by solid-phase extraction and gas chromatography-electrostatic field orbitrap high resolution mass spectrometry].

Meng-Hao Gao, Xiao-Ying Li, Yuan Gao, Hai-Jun Zhang, Ji-Ping Chen.

  Ocean acts as a "sink" for pollutants in the natural environment. Consequently, issues focused on marine pollution from terrestrial origin is attracting increasing attention. Persistent organic pollutants (POPs), including organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), short-chain chlorinated paraffins (SCCPs), and dechlorane plus isomers (DPs), are serious hazards for both the environment and humans. These POPs have been widely detected in the marine environment and are typically present at trace levels; however, separating and determining individual contaminants require large amounts of sampling and time. Establishing an accurate analytical method for determining typical POPs is critical for studying their environmental behavior and associated ecological risks to the marine environment. In this study, we developed a method based on solid-phase extraction (SPE) combined with gas chromatography-electrostatic field orbitrap high resolution mass spectrometry (GC-Orbitrap-HRMS) for determining 34 chlorinated POPs in seawater, including 25 OCPs, six PCB congeners, SCCPs and two DPs. The chromatographic conditions and MS parameters were optimized, and the effects of the extraction solvent and purification method were systematically studied. Dichloromethane exhibited satisfactory extraction efficiencies during the liquid-liquid extraction (LLE) of seawater samples, with recoveries of 73.1%-120.5% for OCPs, 87.2%-101.7% for PCBs, 105.5% for SCCPs, and 74.9%-78.6% for DPs, respectively. Purification using a SPE column with 500 mg of Florisil was adopted, and 9∶1 (v/v) n-hexane/acetone was confirmed as the eluent with recoveries between 68.2% and 122.8% for all the 34 chlorinated POPs. A DB-5MS (15 m×0.25 mm×0.10 μm) capillary chromatographic column was used to separate the target compounds, with an electron ionization (EI) source used to detect OCPs and PCBs, whereas SCCPs and DPs were determined in negative chemical ionization (NCI) source. All target compounds were analyzed in full-scan mode. An internal standard quantification method was used for OCPs and SCCPs while isotope dilution quantification was used for PCBs and DPs. The severe interference observed during the detection of chlorinated POPs in the mixture of co-extracted substances was completely eliminated following the purification. The 34 target chlorinated POPs exhibited good linearities in their corresponding ranges, with correlation coefficients (R2) exceeding 0.9. The method demonstrated low detection limits under the optimized conditions, with values of 0.009-0.061 ng/L for the 25 OCPs, 0.006-0.016 ng/L for the six PCBs, 2.78 ng/L for the SCCPs, and 0.021-0.023 ng/L for the two DPs, with lower limits of determination of 0.06-0.24, 0.02-0.06, 11.12, and 0.08-0.09 ng/L, respectively. Accuracy and precision were validated by the recoveries of samples spiked at low, medium, and high levels, which ranged between 70.6% and 128.9%. Relative standard deviations (n=6) were determined to be 0.2%-19.2%. These results highlight the suitability of the developed method for analyzing trace amounts of chlorinated POPs in seawater. The method is characterized by simple sample pretreatment, high sensitivity, fast analytical throughput, cost-effectiveness, and good stability for trace-level detection; hence, it is suitable for the rapid and accurate analysis of typical chlorinated POPs in seawater. This method is expected to play a significant role in marine environmental monitoring and the emergency surveillance of seawater pollution. The developed method was applied to seawater samples collected from Bohai, which revealed that the highest detection frequency (90%) was recorded for the SCCPs, while α-hexachlorocyclohexane (α-HCH) was only detected in 30% of the samples. All other OCPs were below the detection limit. PCB-52 was the only PCB congener detected in the seawater samples. The SCCPs were detected in much higher concentrations than the other POPs, with the highest value of 130.6 ng/L recorded. Consequently, particular attention must be paid to SCCPs.

Keywords:  chlorinated persistent organic pollutants (POPs); gas chromatography-electrostatic field orbitrap high resolution mass spectrometry (GC-Orbitrap-HRMS); seawater; solid-phase extraction (SPE)

DOI:  https://doi.org/10.3724/SP.J.1123.2024.07017
J Chromatogr Sci. 2025 Mar 27. pii: bmaf015. [Epub ahead of print]63(4):

Stability Indicating High Performance Liquid Chromatography Method Development, Validation, and Characterization of Forced Degradant Product by Liquid Chromatography-Mass Spectroscopy for Molnupiravir and Favipiravir.

Trupesh M Pethani, Monika Sangani, Param S Santoki.

Coronavirus disease 2019 (COVID-19) is a disease caused by a virus named SARS-CoV-2. It is very contagious and has quickly spread around the world. COVID-19 most often causes respiratory symptoms that can feel much like a cold, a flu, or pneumonia. Molnupiravir (MLP) and Favipiravir (FAV) are two recently approved drugs for the ongoing COVID-19 pandemic drug combination help to reduce antiviral load with less side effect. MLP and Favipiravir standards subjected to degradation under different stress condition like acidic, basic, oxidative, thermal, and photostability. The current study endeavors to identify and characterize the degradation products of MLP and Favipiravir using Liquid Chromatography-Mass Spectrometry (LC-MS). Method developed and validated as per International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Guideline (ICH) Q2(R1). The high performance liquid chromatography separation was achieved using Phenomenex Gemini 5 μ C18 (250 mm × 4.6 mm, 5 μm) column using mobile phase A and mobile phase B (15: 85% V/V) and the composition of mobile phase A: 10 mM ammonium acetate and mobile phase B: Acetonitrile (ACN) and methanol 70:30% V/V. Injection volume of 10 μL with 1.0 mL/min flow rate. The detection wavelength was 275 nm and the study was performed at 40°C column temperature. Excellent linear relationship between peak area of MLP and Favipiravir concentration in the range of 5-500 μg/mL for both drugs has been observed R2 = 0.9995 and R2 = 0.9996, respectively. Precision, accuracy, and robustness were found to be < 2% in terms of Relative Standard Deviation (RSD) and all other parameter were found within the specified criteria as per ICH guidelines. Proposed method has been successfully applied for quantification of MLP and Favipiravir in the presence of its impurities.

DOI: https://doi.org/10.1093/chromsci/bmaf015
J Anal Toxicol. 2025 Mar 25. pii: bkaf026. [Epub ahead of print]

Toxicological detection of the new psychoactive substances MDPHP and MDPHpP in human urine samples by elucidation of their urinary metabolites using gas chromatography-mass spectrometry.

I Brueckner, J Welter-Luedeke, C Gutjahr-Ruhland, M Graw, L D Paul.

  The continuous emergence of new psychoactive substances on the illicit drug market provides challenges for forensic and clinical analytics. Reliable detection of a previous ingestion of these drugs in human urine samples requires elucidation of target metabolites and, in the case of gas chromatography-mass spectrometry (GC-MS), the knowledge of their derivatized mass spectra. The study presented here focused on the two pyrrolidinophenones 3,4-methylenedioxy-α-pyrrolidinohexanophenone (MDPHP) and 3,4-methylenedioxy-α-pyrrolidinoheptanophenone (MDPHpP), which could be identified in 25 and 3 authentic cases, respectively. Using a standard analytical procedure by means of full-scan GC-MS after acid hydrolysis and acetylation, phase I metabolites of both substances were identified in authentic urine samples by elucidation of their mass spectral fragmentation patterns. The postulated phase I metabolic steps of MDPHP and MDPHpP comprised demethylenation followed by methylation of the methylenedioxy moiety, oxidation of the pyrrolidine ring, N,N-bisdealkylation of the pyrrolidine ring to its primary amine, hydroxylation of the aliphatic side chain. Various combinations were detected. Acetylated mass spectra of the metabolites were provided for both substances. The analogy in mass fragmentation of the proposed metabolites for the homologous parent compounds indicated a high plausibility. Based on the frequency of occurrence and abundances of the metabolites in the urine samples, target analytes for both substances and base peak fragment ions for specific mass search could be recommended for the mentioned procedure: m/z 140, 154 and 86 for MDPHP and m/z 154, 168 and 100 for MDPHpP. The study could support the detection of these new substances in forensic and clinical cases.

Keywords:  MDPHP; MDPHpP; fragmentation pattern; mass spectra; metabolic pathway; metabolites in urine; new psychoactive substances; target analytes; α-pyrrolidinophenones

DOI:  https://doi.org/10.1093/jat/bkaf026
J Hazard Mater. 2025 Mar 19. pii: S0304-3894(25)00892-1. [Epub ahead of print]491 137976

Overcoming challenges in the analysis of the ubiquitous emerging contaminant trifluoroacetic acid employing trap column liquid chromatography tandem mass spectrometry.

Ramadan Sayed, Ahmed A Omran, Rabie S Farag, Hend A Mahmoud, Mostafa Soliman.

  Trifluoroacetic acid (TFA) is a ubiquitous ultra-short chain emerging contaminant and a widely observant pollutant in the environment. Its analytical determination usually encounters two main challenges, difficulties of chromatographic separation on reversed-phase C18 columns and the false positive results in blank samples due to TFA ubiquitous nature. This study presents the successful separation of TFA on Poroshell 120 EC-C18, 3.0 × 50 mm, 2.7 μm column, enabling the determination of TFA along with other long-chain contaminants. The application of trap column technique resulted in delaying the elution of TFA contamination arising from mobile phase later than the retention time of the target analyte. For direct samples injection, acetonitrile and methanol (MeOH) with acid and alkali modifications were evaluated as dilution solvents. 6 mL of 0.1 % NH4-modified MeOH and 4 mL of water sample provided the optimum TFA peak shape. The method was successfully validated and satisfied the requirements of SANTE/11312/2021 (V2) guidelines. The evaluated criteria were specificity and selectivity, trueness, precision, calibration linearity, limit of quantification (LOQ), and matrix effect. The validated method was applied to monitor the concentration levels of TFA in 30 real samples. The detected concentrations were <LOQ of the method in all the positively tested samples.

Keywords:  Analytical method validation; Emerging contaminants; LC-MS/MS; Trap column technique; Trifluoroacetic acid; Ultra-short chain pollutants; Water

DOI:  https://doi.org/10.1016/j.jhazmat.2025.137976
Se Pu. 2025 Apr 08. 43(4): 363-371

[Effects of nicotine exposure on endogenous metabolites in mouse brain based on metabolomics and mass spectrometry imaging].

Lu-Lu Guo, Chen Zhang, Yan-Jun Huang, Xing-Yu Liu, De-Shui Liu, Teng Long, Jin-Hao Sun, Shao-Feng Liu, Zhong-Hao Li, Jia-Zhong Wang, Jian Mao.

  Nicotine, the principal alkaloid in tobacco, exhibits significant central nervous system activity and induces a wide array of physiological effects. In addition to its well-documented role in tobacco dependence, previous studies have suggested that nicotine also has diverse pharmacological properties. These include alleviating symptoms associated with Parkinson's disease, potentially reducing the risk of Alzheimer's disease, mitigating oxidative stress, as well as anti-inflammatory and anxiolytic effects. Neuroscientists frequently use an array of molecular biology techniques to elucidate the mechanisms responsible for the effects of nicotine on the central nervous system. However, disease onset is invariably accompanied by metabolic dysfunction, and organisms often exhibit complex and unpredictable responses to pharmacological stimuli. As a bioactive alkaloid with potent pharmacological properties, nicotine is able to cross the blood-brain barrier and induce brain-compound changes, which serves as the basis for its effects on the central nervous system. Consequently, examining the extensive impact of nicotine exposure on endogenous metabolites and metabolic pathways in the brain is an indispensable step toward providing a more robust foundation for understanding the complex physiological effects of nicotine. In this study, an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) metabolomic-analysis method was established to systematically examine the effects of repeated nicotine exposure on endogenous metabolites in mouse brains. Two chromatographic systems fitted with Acquity UPLC BEH HILIC (150 mm×2.1 mm, 1.7 μm) and BEH C18 (150 mm×2.1 mm, 1.7 μm) columns were used to determine the nicotine present in samples. As a result, the established UHPLC-MS/MS method identified a total of 759 endogenous metabolites. Compared with the saline group, nicotine exposure resulted in 575 significantly different metabolites, with 434 metabolites down-regulated and 141 up-regulated. Further pathway-enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that nicotine exposure primarily affects essential-amino-acid, lipid, nucleotide, carbohydrate, cofactor, and vitamin metabolism, as well as other amino-acid metabolic pathways in the brain. Although non-targeted metabolomics can simultaneously detect and analyze all small-molecule metabolites in an unbiased manner, accurately capturing metabolite changes in specific brain regions is challenging when dealing with complex brain-tissue systems. Targeting the aggregation of material bases and the delivery of precision treatment to certain brain regions is expected to be significant for the targeted therapy of central nervous system diseases. Airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) was further used to directly visualize the nicotine-induced distributions and variations of differentially expressed metabolites in various brain regions, which revealed that nicotine exposure leads to the significant downregulation of choline, serine, aspartate, and malate levels throughout the brain. Specifically, taurine, acetylcholine, and adenosine levels were notably affected in the cortical, hippocampal, and striatal regions, respectively. Essential-amino-acid metabolism was most affected by nicotine, with lipid metabolism found to be the next-most affected pathway. These metabolic pathways predominantly affected the cortical region, whereas the striatum, hippocampus, thalamus, and cerebellum were affected to varying degrees. These findings provide novel experimental evidence that enhances our understanding of metabolic biomarkers associated with nicotine exposure.

Keywords:  mass spectrometry imaging; metabolomics; mouse brain; nicotine; ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)

DOI:  https://doi.org/10.3724/SP.J.1123.2024.10005
Anal Chem. 2025 Mar 24.

A 2D Microfluidic Paper-Based Analytical Device for Diagnosis of Canine Visceral Leishmaniasis via Mass Spectrometry-Based Immunoassay.

Hianka J C de Carvalho, Ayesha Seth, Ruth Speidel, Ana L Abreu-Silva, Hélida M de Andrade, Maria A Miglino, Abraham K Badu-Tawiah.

This work presents the first indirect immunoassay performed on a paper-based microfluidic platform for the diagnosis of canine visceral leishmaniasis (CVL). The IgG antibody biomarker, which signifies the presence of this infectious disease, was captured with a recombinant K39 antigen and detected with secondary antibodies that were conjugated with cleavable ionic probes. The use of ionic probes enabled direct analysis of the assay results through an on-chip paper spray mass spectrometry (MS) technology. This MS-based immunoassay was developed to allow for early detection of CVL in asymptomatic dogs. The sensitivity required for such a diagnostic method was demonstrated through internal standard calibration in which sample dilution as low as 1/4000 was achieved. Aside from high sensitivity, the ionic probes are stable, which allowed the paper device to be stored at room temperature and under ambient conditions for 30 days without affecting the diagnostic outcome. Our method was used to analyze 20 clinical canine serum samples, where we detected a 2 orders of magnitude higher signal for CVL-positive samples compared to negative samples. MS signal derived from the 10 CVL-positive serum clinical samples showed a strong correlation with antibody titers determined by immunofluorescence assay. This correlation was confirmed through Pearson's statistical analysis. Overall, the high sensitivity and positive results from stability tests observed for our platform are expected to enable large-scale CVL screening in asymptomatic dogs in remote areas, especially when combined with portable mass spectrometers.

DOI: https://doi.org/10.1021/acs.analchem.4c05962
Anal Bioanal Chem. 2025 Mar 22.

Development and validation of an isotope dilution-liquid chromatography (ID-LC-MS/MS)-based candidate reference measurement procedure for total 3,3',5-triiodothyronine in human serum.

Wenya Li, Bingling Gao, Tao Yang, Liping Tang, Dan Song, Hongmei Li, Keqi Sun, Peng Xiao.

  3,3',5-Triiodothyronine (T3) plays a crucial role in the diagnosis and evaluation of thyroid diseases. The current reference measurement procedure (RMP) has been in place for 20 years and is widely adopted by reference laboratories around the world. To enhance the performance of the RMP, we developed and validated a simplified, SI-traceable candidate RMP specifically targeting serum total T3 (TT3) using isotope dilution-liquid chromatography with tandem mass spectrometry (ID-LC-MS/MS). Specifically, T3 reference material was employed as the standard. Compared to the RMP, our method incorporates a simplified serum cleanup process involving protein precipitation and nitrogen drying. The method validation followed ISO guidelines for measurement uncertainty assessment. Our results indicated that there was no significant conversion of T4 into metabolites during the serum pretreatment. The lower limit of the measuring interval was determined to be 0.077 nmol/L, and the regression model was linear across a range of 0.016 to 13.086 nmol/L. The imprecision of intra-assays ranged from 0.1 to 1.9%, while inter-assay imprecision ranged from 0.5 to 1.2%. The bias observed when analyzing SRM971 was between - 0.34 and - 1.65%. This candidate RMP demonstrated excellent agreement with the RMPs developed by RELA activity, showing a mean bias falling within the educational mean ratio (6%). The measurement uncertainties were 0.022 nmol/L for RELA2023A and 0.120 nmol/L for RELA2023B. In conclusion, the developed candidate RMP is traceable, reliable, and easy to operate, showing promising potential for the development of serum reference materials and the standardization of routine assays.

Keywords:  ID-LC-MS/MS; Reference measurement procedure; SI-Traceable; Serum 3,3′,5 Triiodothyronine; Thyroid

DOI:  https://doi.org/10.1007/s00216-025-05834-y
Front Chem. 2025 ;13 1523712

A laboratory-friendly protocol for freeze-drying sample preparation in ToF-SIMS single-cell imaging.

Xiujuan Shi, Mingru Liu, Yue Qi, Hongzhe Ma, Zhaoying Wang, Yanhua Chen, Zeper Abliz.

  ToF-SIMS is a high spatial resolution imaging technique for cellular or subcellular analysis of biological samples. Accurate molecular data in single-cell studies depend on proper cell morphology and chemical integrity, highlighting the importance of sample preparation. In this work, we standardized a more efficient freeze-drying method using standard lab materials and improved the sample preparation process. Our comprehensive freeze-drying protocol for cellular samples, encompassing washing, fixation, and drying steps, facilitates the acquisition of enhanced cellular information and ensures high reproducibility. These improvements are poised to significantly advance single-cell mass spectrometry imaging research.

Keywords:  ToF-SIMS; freeze-drying; mass spectrometry imaging; sample preparation; single cell imaging

DOI:  https://doi.org/10.3389/fchem.2025.1523712