bims-metlip 2025-07-06 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–07–06
27 papers selected by
Sofia Costa, Matterworks

Comprehensive evaluation of dual-mode LC-MS conditions for enhanced metabolomic profiling: Application to microplastic exposure studies.
[Determination of six psychotropic drug metabolites in human plasma by LC-MS/MS method].
Evaluation of separation performance and quantification accuracy in lipidomics methods.
Longitudinal Fragment Profiles Based on Multi-Collision Energy Tandem Mass Spectra Improve the Accuracy of Metabolite Identification in Untargeted Metabolomics.
Chemical Derivatization-Based Liquid Chromatography-Mass Spectrometry Method for Fatty Acid Profiling in Biological Samples.
MassCube improves accuracy for metabolomics data processing from raw files to phenotype classifiers.
Determination of vitamin D3 (cholecalciferol) in wheat flour using a solid-liquid extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS).
LC-MS/MS-based determination of 25-hydroxyvitamin D in dried blood spots: crucial importance of combining a robust extraction with knowledge of the hematocrit.
MS2MP: A Deep Learning Framework for Metabolic Pathway Prediction from MS/MS-Based Untargeted Metabolomics.
[Fast determination of per- and polyfluoroalkyl substances in human serum by cold-induced phase separation coupled with liquid chromatography-tandem mass spectrometry].
Simultaneous Determination of Seven Benzotriazole UV Stabilizers in Surface Waters by Using Solid-Phase Extraction Coupled With Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry With Electrospray Ionization.
Assessment of matrix effects in quantitative GC-MS by using isotopologs.
Multiplexed Targeted Spatial Mass Spectrometry Imaging Assays to monitor lipids and NAD+ metabolites in CD38 knockout mice exhibiting improved metabolism.
Development and validation of a LC-MS/MS method for the simultaneous determination of simnotrelvir and ritonavir in human serum and bronchoalveolar lavage fluid.
Monitoring the degree of in-source phospholipid fragmentation during MALDI mass spectrometry imaging.
Novel Data-Driven Mechanistic Modeling of Untargeted Metabolome Data Reveals Feed Component Effects in CHO Cell Bioprocess Using Column Generation-Based EFMs.
Protoporphyrin IX plasma and blood pharmacokinetics and brain tumor distribution determined by a validated LC-MS/MS method.
[Preparation and chromatographic performance evaluation of hydrophilic interaction chromatography stationary phase based on amino acids].
Untargeted metabolomic profiling of serum and urine in kidney cancer: a non-invasive approach for biomarker discovery.
Affordable 3D-printed prototype integrated with open-source electronic platform for low-cost polyamide noncoated adsorption-based microextraction.
A High-Performance Liquid Chromatography-Mass Spectrometry method for simultaneous determination of dolutegravir, nevirapine, efavirenz, rifampicin and rifapentine concentrations in human plasma.
Constant Distance Mode Mass Spectrometry Imaging with a Confocal Sensor.
Integrated plasma metabolomics and lipidomics profiling highlight distinctive signatures with hashimoto's thyroiditis.
Construction of a Fritillaria alkaloids database to develop a multi-dimensional matching strategy for comprehensive annotation of known and unknown components.
Strychnos peckii B.L. Rob.: secondary metabolite annotation by liquid chromatography tandem high-resolution mass spectrometry with insights from biogenesis.
[Determination of squalene and oxidized squalene in edible oil by gas chromatography-tandem mass spectrometry and evaluation of the thermal stability of squalene].
Ion source component development for ambient mass spectrometry-based mycotoxin detection in ready-to-eat corn.

Anal Chim Acta. 2025 Sep 15. pii: S0003-2670(25)00707-X. [Epub ahead of print]1367 344313

Comprehensive evaluation of dual-mode LC-MS conditions for enhanced metabolomic profiling: Application to microplastic exposure studies.

Kuan-Lu Wu, Pei-Chen Lin, Wei-Chen Lin, Sung-Fang Chen.

  Polystyrene microplastics (PS-MPs) are emerging contaminants of concern due to their potential health impacts and widespread presence in the environment. Metabolomics offers a powerful approach to investigate biological responses to such exposures. However, current LC-MS methods are often limited by the suitability of chromatographic conditions for metabolites with diverse physicochemical properties, leading to suboptimal coverage and analytical redundancy. This study addresses these limitations by establishing a robust, broadly applicable dual-mode LC-MS strategy to improve coverage and analytical efficiency in microplastic exposure studies. This study evaluated 18 chromatographic conditions using six commercial columns including amide, silica, Obelisc N, C18, pentafluoophenyl (F5), and cyanopropyl (CN), to optimize metabolite separation in both positive and negative electrospray ionization (ESI) modes. Mouse large intestine extracts exposed to PS-MPs showed broad metabolome coverage under optimized conditions. In positive mode, the amide column with ammonium acetate/acetic acid (AmAc/AcA) effectively captured diverse polar metabolites. In negative mode, the F5 column with ammonium formate/formic acid (AmF/FA) excelled in phospholipid detection and lipid separation. Combining these conditions enabled complementary profiling with minimal overlap. Additionally, 42 differential metabolites affected by PS-MPs were associated with key metabolic pathways, including amino acid, taurine, hypotaurine, and glutathione metabolism. This optimized, high-coverage LC-MS strategy provides a novel analytical framework that maximizes metabolome profiling efficiency and minimizes sample input. It improves detection of diverse metabolite classes and supports robust biological interpretation, offering broad applicability for future studies on environmental exposures and complex biological challenges.

Keywords:  Liquid chromatography; Mass spectrometry; Metabolomics

DOI:  https://doi.org/10.1016/j.aca.2025.344313
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2025 Jun 20. 43(6): 445-448

[Determination of six psychotropic drug metabolites in human plasma by LC-MS/MS method].

Y X Cui, H L Li, Y Yu, J Ma, B Zhou, F Dong.

  Objective: To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of N-desalkylquetiapine, hydroxybupropion, N-desmethyl clozapine, N-desmethyl clomipramine, O-desmethyvenlafaxine and dehydro aripiprazole in human plasma. Methods: In June 2023, plasma samples were treated with methanol-acetonitrile (volume ratio of 1∶1) for protein precipitation and then detected by LC-MS/MS. It was separated by C18 column and eluted with 5 mmol/L ammonium acetate water and 5 mmol/L ammonium acetate methanol solution as mobile phase gradient. The metabolites of six psychotropic drugs were qualitatively and quantitatively by using electrospray positive ion multi-reactive ion monitoring scanning mode (MRM) . Results: The linear relationships of six psychotropic drug metabolites were good in the concentration range of 2-100 μg/L. The linear correlation coefficients were 0.9971-0.9999, the limits of quantification of the method were 0.10-6.00 μg/L, and the inter-and intra-day precision were 4.6%-9.8% and 1.5%-8.6%, with the recoveries of the spiked standards ranged from 90.0% to 106.1%. Conclusion: The LC-MS/MS method for the determination of metabolites of six psychotropic drugs in human plasma is simple, rapid and sensitive, and can be used for the qualitative and quantitative determination of metabolites in plasma samples of patients suspected of psychotropic drug poisoning.

Keywords:  Blood drug concentration; Chromatography, liquid; Drug poisoning; Psychotropic drug metabolites; Tandem mass spectrometry

DOI:  https://doi.org/10.3760/cma.j.cn121094-20240207-00053
J Chromatogr A. 2025 Jun 22. pii: S0021-9673(25)00511-4. [Epub ahead of print]1758 466165

Evaluation of separation performance and quantification accuracy in lipidomics methods.

Noriyuki Tomiyasu, Yoshihiro Izumi, Omidreza Heraviadeh, Masatomo Takahashi, Takeshi Bamba.

  In mass-spectrometry-based lipidomics, diverse analytical methods are employed in different institutions, resulting in variations in isomer identification, the number of detected compounds, and quantitation accuracy. The primary analytical methods include flow injection (FI), reversed-phase liquid chromatography (RP-LC), and hydrophilic interaction liquid chromatography (HILIC). Recently, supercritical fluid chromatography (SFC) has gained attention for its enhanced separation of hydrophobic and structural isomers. However, the most suitable analytical method for lipidomics remains in debate because of the scarcity of comparative performance studies. In this study, we evaluated the quantitative performance of four methods (FI, RP-LC, HILIC, and SFC) each connected to a triple quadrupole mass spectrometer (MS/MS) using the NIST SRM1950 plasma reference under identical conditions, focusing purely on methodological differences. Quantification was performed for 355 lipid species across 14 lipid classes using one deuterated standard per class as an internal standard. The results revealed no significant differences in quantification across six lipid classes, whereas other classes showed notable method-specific variations. Additionally, chromatographic performance, including analysis time, pressure drop, theoretical plate height, and isomer separation, was compared between HILIC-MS/MS and SFC-MS/MS. SFC-MS/MS outperformed HILIC-MS/MS in all parameters, including height equivalent to a theoretical plate, resolution, peak height, and structural isomer separation performance. We conclude that although all methods are applicable in lipidomics, method selection and optimization should be consistent with specific analytical requirements, including target lipid class, sample consumption, and analysis time.

Keywords:  Flow injection; Hydrophilic interaction liquid chromatography; Lipidomics; Mass spectrometry; Quantification accuracy; Reverse-phase liquid chromatography; Supercritical fluid chromatography

DOI:  https://doi.org/10.1016/j.chroma.2025.466165
Anal Chem. 2025 Jul 01.

Longitudinal Fragment Profiles Based on Multi-Collision Energy Tandem Mass Spectra Improve the Accuracy of Metabolite Identification in Untargeted Metabolomics.

Xian Fu, Qiang Li, Hou-Hua Yin, Ya-Nan Liu, Wenlin Wu, Bruce D Hammock, Jianbo Pan, Jun-Yan Liu.

Metabolite identification in untargeted metabolomics via tandem mass spectrometry (MS/MS) spectral matching is commonly performed by comparing experimental and reference MS/MS spectra acquired at one or a few collision energies, which generates a similarity score based on the relative intensities of fragment ions within each spectrum, referred to as cross-sectional profiling. Here, we introduced a novel method that significantly improved identification accuracy by comparing longitudinal fragment profiles, which consisted of the intensities of individual MS/MS fragments across multiple collision energies. This approach, termed longitudinal profiling, highlighted low-abundance fragments that were often overlooked by conventional cross-sectional methods, emphasizing predominant ions. We optimized the Jaccard similarity algorithm for longitudinal profiling and established identification criteria using an in-house spectral database comprising approximately 1,80,000 MS/MS spectra. The robustness of the method was validated using inter-instrument datasets, spiked standards, and human plasma samples. Compared with cross-sectional profiling using the optimal entropy algorithm, the longitudinal profiling method improved annotation accuracy by 8.7-25.9% and reduced the false discovery rate by 28.6-41.7%, resulting in a fair increase in the number of confidently annotated metabolites. This method enhances the probability of discovering true diagnostic markers while reducing the likelihood of false diagnostic markers. Our results demonstrate that longitudinal profiling provides a promising new avenue for more accurate metabolite identification in untargeted metabolomics.

DOI: https://doi.org/10.1021/acs.analchem.5c01414
J Sep Sci. 2025 Jul;48(7): e70211

Chemical Derivatization-Based Liquid Chromatography-Mass Spectrometry Method for Fatty Acid Profiling in Biological Samples.

Jingxuan Yang, Yi Wu, Liang Zhao, Meng Li, Wenjun Guo, Yajuan Xu, Yang Wang.

  Fatty acids (FAs) are important metabolites for various biochemical functions in living organisms, including energy storage, cell structure, and cell signaling. Changes in FAs composition were significantly related to abnormal metabolic levels and body status. In this study, the derivatization-based liquid chromatography-quadrupole-orbitrap mass spectrometry method was established for FA analysis under parallel reaction monitoring mode. The 4-amino-1-benzylpiperidine (4A1BP) was used as a derivatization reagent to amide with FAs. The results showed that protonated molecules of FA+4A1BP-H2O were observed, and characteristic fragment ions at m/z 174.1280 and 91.0548 were detected in the tandem mass spectrum. The derivatization method was optimized in terms of sample preparation, chromatographic separation, and mass spectrometric conditions. The linearity, stability, and repeatability of the method were also investigated with good results. Finally, we applied the established method to analyze the serum and liver tissue samples, successfully evaluating the efficacy of Qizha Shuangye granules in the treatment of hyperlipidemia rats. The established method has broad utility and great potential in the detection of FAs, which can provide technical support for disease mechanism research, biomarker discovery, as well as food quality assurance, and nutritional value evaluation.

Keywords:  LC‐MS; biological sample; chemical derivatization; fatty acid; parallel reaction monitoring

DOI:  https://doi.org/10.1002/jssc.70211
Nat Commun. 2025 Jul 01. 16(1): 5487

MassCube improves accuracy for metabolomics data processing from raw files to phenotype classifiers.

Huaxu Yu, Jun Ding, Tong Shen, Min Liu, Yuanyue Li, Oliver Fiehn.

Nontargeted peak detection in LC-MS-based metabolomics must become robust and benchmarked. We present MassCube, a Python-based open-source framework for MS data processing that we systematically benchmark against other algorithms and different types of input data. From raw data, peaks are detected by constructing mass traces through signal clustering and Gaussian-filter assisted edge detection. Peaks are then grouped for adduct and in-source fragment detection, and compounds are annotated by both identity- and fuzzy searches. Final data tables undergo quality controls and can be used for metabolome-informed phenotype prediction. Peak detection in MassCube achieves 100% signal coverage with comprehensive reporting of chromatographic metadata for quality assurance. MassCube outperforms MS-DIAL, MZmine3 or XCMS for speed, isomer detection, and accuracy. It supports diverse numerical routines for MS data analysis while maintaining efficiency, capable for handling 105 GB of Astral MS data on a laptop within 64 min, while other programs took 8-24 times longer. MassCube automatically detected age, sex and regional differences when applied to the Metabolome Atlas of the Aging Mouse Brain data despite batch effects. MassCube is available at https://github.com/huaxuyu/masscube for direct use or implementation into larger applications in omics or biomedical research.

DOI: https://doi.org/10.1038/s41467-025-60640-5
Food Res Int. 2025 Oct;pii: S0963-9969(25)01120-2. [Epub ahead of print]217 116782

Determination of vitamin D3 (cholecalciferol) in wheat flour using a solid-liquid extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Marcia Becerra, Bárbara Riveros, Danahe Allende, Luis Honda, Edwar Fuentes.

  Vitamin D helps absorb calcium and preserve healthy bones. In several countries, vitamin D intake comes mainly from fortified foods. Fortification of wheat flour is a way to improve the low intake of vitamin D in low- and middle-income countries. At the same time, reliable analytical methods are required to perform quality controls to certify the vitamin D content in this matrix. This work presents an analytical method to determine vitamin D3 (cholecalciferol) in white wheat flour, based on a very simple solid-liquid extraction (SLE) and quantification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). SLE involves 5 g of sample and 15 mL of extractant solution (16 % ethyl acetate in ethanol), shaking for 12 min (in a vortex) and repeating the process. Evaporation of the extract at 1.5 mL complete the sample treatment. The LC-MS/MS determination use atmospheric pressure chemical ionization (APCI) source in positive mode and monitoring the m/z 385.4 to 259.1 transition. The average recovery was 84 % at the limit of quantification of the method (0.9 μg/100 g of vitamin D3). The coefficients of variation for repeatability and intermediate precision were 11 and 9 %, respectively. The proposed method compares favorably with those few reported in the literature. Due to the delay in the application of the Food Sanitary Regulations that indicate the fortification of flours with vitamin D3 at the national level in Chile, real samples were not available for the application of the method. However, this will allow adequate control when mandatory fortification comes into force.

Keywords:  Cholecalciferol; Food analysis; LC-MS/MS determination; Solid-liquid extraction; Wheat flour

DOI:  https://doi.org/10.1016/j.foodres.2025.116782
Anal Chim Acta. 2025 Sep 15. pii: S0003-2670(25)00617-8. [Epub ahead of print]1367 344223

LC-MS/MS-based determination of 25-hydroxyvitamin D in dried blood spots: crucial importance of combining a robust extraction with knowledge of the hematocrit.

Liesl Heughebaert, Christophe P Stove.

   BACKGROUND: While many liquid chromatography - tandem mass spectrometry (LC-MS/MS)-based methods exist for the determination of 25-hydroxyvitamin D (25OHD) using dried blood spots (DBS), none of these comprehensively evaluated the robustness of the employed extraction method. The latter is critical as the hematocrit (Hct) as well as DBS ageing may affect analyte recovery and, thus, the accuracy of the obtained DBS-based 25OHD result. Moreover, as the application potential of DBS for the determination of 25OHD mainly lies in the analysis of DBS collected and archived in the context of newborn screening or epidemiological studies, a large variation in Hct and ageing is to be expected.
RESULTS: A Hct- and ageing-independent extraction of 25OHD from a single 6 mm DBS subpunch was obtained in the Hct range of 0.23-0.53 L/L, using thermoshaking in 50/50 acetonitrile/water for 1 h at 60 °C. The optimized LC-MS/MS-based method allowed fast (<2 min) separation of 3-epi-25OHD3 from 25OHD3 using an inverse gradient without the need for a dedicated column. The method was successfully validated for the determination of 25OHD3 and 25OHD2 in DBS, whole blood and plasma, with lower limits of quantification of 1.97 and 2.53 ng/mL in DBS and whole blood, and 3.94 and 5.06 ng/mL in plasma for 25OHD3 and 25OHD2, respectively. For both 25OHD3 and 25OHD2, accuracy and imprecision were respectively within -14.7 %-2.3 % and within 1.1 %-10.0 % for all matrices. Stability studies (up until three months of storage at room temperature) as well as the evaluation of spotted blood volume did not reveal any relevant impact on the quantification of 25OHD in DBS. Finally, a proof-of-concept study indicated that, when the Hct is taken into account, comparable results can be obtained in DBS and whole blood samples and plasma concentrations can be derived from DBS, respectively.
SIGNIFICANCE AND NOVELTY: The presented manuscript is the first to address in-depth one of the major remaining research gaps in the determination of 25OHD from DBS, namely the evaluation of the robustness of the employed extraction procedure. Using thermoshaking at elevated temperature, we demonstrated Hct- and ageing-independent recovery of 25OHD3 and 25OHD2, confirming the method's suitability for large-scale applications such as newborn screening and epidemiological studies.

Keywords:  25-Hydroxyvitamin D; Dried blood spot (DBS); Extractability; Hematocrit; LC-MS/MS

DOI:  https://doi.org/10.1016/j.aca.2025.344223
Anal Chem. 2025 Jun 30.

MS2MP: A Deep Learning Framework for Metabolic Pathway Prediction from MS/MS-Based Untargeted Metabolomics.

Han Bao, Xiuqiong Zhang, Xinxin Wang, Jinhui Zhao, Xinjie Zhao, Chunxia Zhao, Xin Lu, Guowang Xu.

MS/MS-based untargeted metabolomics generates complex data, but pathway enrichment analysis is constrained by the low annotation rates of metabolic features. Here, we propose MS2MP, a novel deep learning-based framework for KEGG pathway prediction directly from untargeted tandem mass spectrometry (MS2), eliminating the need for prior metabolite annotation. MS2MP utilizes a graph neural network architecture to learn the complex relationships between spectral features and metabolic pathways, representing MS2 spectra as fragmentation tree graphs. Trained on 33,221 experimental MS2 spectra, MS2MP achieves robust predictive performance with a balanced accuracy of 94.1% in cross-validation and 87.8%-91.2% on three independent test sets. Notably, MS2MP achieves an "exact match" for 97-98 out of 161 tested metabolite standards across diverse experimental conditions, underscoring its reliability and adaptability. Subsequently, a novel MS2-based pathway enrichment method was developed. The established methods were applied to identify significantly perturbed pathways in transgenic maize. The results uncovered disruptions in phenylpropanoid biosynthesis and related downstream pathways, including those involved in amino acid and secondary metabolite metabolism, which were overlooked by the conventional annotation-based enrichment analysis method. To the best of our knowledge, MS2MP is the first computational tool capable of directly predicting metabolic pathways from MS2 spectra. By linking MS2-based untargeted metabolomics data to metabolic pathways, MS2MP enables more efficient pathway enrichment analysis, thereby accelerating biological discoveries and enhancing our understanding of complex metabolic networks.

DOI: https://doi.org/10.1021/acs.analchem.4c06875
Se Pu. 2025 Jul;43(7): 756-766

[Fast determination of per- and polyfluoroalkyl substances in human serum by cold-induced phase separation coupled with liquid chromatography-tandem mass spectrometry].

Jian-di Wang, Yi-Wei Wang, Jia-Xin Wu, Zhi-Xiong Shi.

  Per- and polyfluoroalkyl substances （PFASs） are a large group of synthetic chemicals that have been widely used in various industrial and commercial products owing to their unique physicochemical properties. However， accumulating evidence suggests that PFASs are persistent， transmissive over long distances， bioaccumulative， and toxic； consequently， their adverse effects on ecosystems and humans is of widespread concern. Serum is the most commonly used human matrix for assessing internal exposure to environmental pollutants， and several analytical methods have been developed to measure PFASs in sera. Current methods are generally fast， convenient， and robust； however， their pretreatment steps require large amounts of organic solvents and materials， such as solid-phase extraction cartridges and/or sorbents. In this study， a novel and low-cost analytical method based on cold-induced phase separation （CIPS） strategy was developed for the simultaneous determination of 31 legacy and emerging PFASs in serum. The core mechanism and distinctive feature of CIPS involves cooling an acetonitrile-water （ACN-water） mixture at a low temperature to produce two clear-cut layers： one with a high ACN proportion （the ACN layer） and an aqueous layer （water layer）. Certain chemicals are significantly enriched in the ACN layer during cooling； at the same time， impurities， especially water-soluble impurities， remain in the aqueous layer. CIPS only requires the temperature to be varied， and no external impurities are introduced during pretreatment， which dramatically reduces material costs and avoids new impurities from intervening. Our method involves the following procedure： serum was drawn accurately （0.2 mL） into a 1.5 mL Eppendorf （EP） tube， 2 ng of each isotopically labeled internal standard was added， the mixture is vortexed， and 350 µL of ACN was added， followed by vortexing and ultrasonic extraction. Subsequently， 450 µL of water is added to adjust the volume proportion of ACN to 35% （the volume percentage of ACN in the total solution）. The protein at the bottom of the tube was collected following centrifugation at 15 000 r/min for 10 min， and the supernatant was transferred to a 1 mL syringe. The syringe was frozen in a -20 ℃ refrigerator for 1 h to obtain the two layers， after which the upper layer （approximately 80-100 μL） containing ACN and the target compounds was finally transferred to a glass vial for instrumental analysis. Liquid chromatography coupled with triple quadrupole mass spectrometry augmented with electrospray ionization （LC-ESI-MS/MS） was used to quantify the PFASs. The analytes were separated using a C18 column， with methanol and 2 mmol/L of ammonium formate-H2O used as mobile phases. Linearities， limits of detection （LODs） and， limits of quantification （LOQs）， recoveries， precisions， and matrix effects were determined under the optimal conditions. The LODs and LOQs of PFASs in serum were 0.01-25 and 0.03-83 pg/mL， respectively. Under two spiked levels， namely 5 ng/mL and 25 ng/mL， average recoveries ranged between 60.5% and 129.6%， with relative standard deviations （RSDs） of less than 22.8%. Under 5 pg/mL as LOD spiked level， average recoveries ranged between 61.6% and 199.1%，with RSDs<29.4%. While matrix-effect testing revealed slightly enhanced signals， the use of isotopically labeled internal standards compensated for these effects. Real samples were subsequently analyzed， with 50 human serum samples collected in first trimester of pregnancy women living in the Shunyi District， Beijing. Nine PFASs exhibited high detection frequencies （>80%）， which suggests that PFASs are ubiquitous in the population. The median and mean levels of Σ31PFASs （sum of 31 PFASs） in serum were 21.8 and 22.9 ng/mL， respectively， and the range was 0.456-73.9 ng/mL. Both legacy and emerging PFASs were detected at high frequencies and contamination levels， which suggests that they are widely used. In summary， the method developed in this study is fast， sensitive， and solvent- and material-efficient； it is also very linear and highly accurate， and exhibits satisfactory extraction recovery and enrichment factors； hence， it is suitable for surveying large populations as well as for use in environmental epidemiology.

Keywords:  cold-induced; isotope dilution; liquid chromatography-tandem mass spectrometry （LC-MS/MS）; per- and polyfluoroalkyl substances （PFASs）; serum

DOI:  https://doi.org/10.3724/SP.J.1123.2024.11028
J Sep Sci. 2025 Jul;48(7): e70200

Simultaneous Determination of Seven Benzotriazole UV Stabilizers in Surface Waters by Using Solid-Phase Extraction Coupled With Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry With Electrospray Ionization.

Chen Huifan, Zhou Huimin, Wang Zhenguo, Hu Xialin, Yin Daqiang.

  Benzotriazole ultraviolet stabilizers (BUVSs) are emerging persistent pollutants, due to the weak polarity and wide log Kow values, there is no general optimal analytical method for BUVSs. In this study, a solid-phase extraction-ultra performance liquid chromatography tandem mass spectrometry (SPE-UHPLC/MS/MS) method was developed for the simultaneous determination of seven BUVSs in the aqueous environment, based on the generalized electrospray ionization (ESI+) mode. The conditions of ESI-UHPLC-MS/MS detection and SPE were systematically optimized. A 50 mm UHPLC column at a column temperature of 40°C gave the highest response for the target BUVSs using methanol-0.1% (v/v) formic acid solution as the mobile phase. The effects of the four packed SPE cartridges (HLB, HC-C18, LC-C18 and phenyl columns) and sample pH (3.0-9.0) on the SPE enrichment were comparatively analyzed, and the optimum condition was HLB cartridges at sample pH 3.0. The SPE elution solvent was optimized as 5 mL methanol + 5 mL dichloromethane (V:V = 1:1). The seven target BUVSs were quantified by isotope internal standard method, and the linear range of the standard curve was wide (0.1-50 ng∙L-1) with the correlation coefficient R2 > 0.998. The recoveries of the spiked standards for the purified water as well as the sample of Huangpu River were 80.6%-105.2% and 53.9%-103.7% (except UV-P: 141.6%-148.7%), respectively. The relative standard deviations (RSDs) of the intra-day were 4.1%-18.0%; and the RSDs of the inter-day were 3.4%-18.8%, respectively. The limits of detection of the method were in the range of 0.050-0.406 ng∙L-1. Compared with the reported studies, this method is more sensitive and stable, and the UHPLC-MS/MS instrument based on the ESI ionization source is more generalizable.

Keywords:  benzotriazole ultraviolet stabilizers; solid‐phase extraction; ultra‐high‐performance liquid chromatography tandem mass spectrometry; water samples

DOI:  https://doi.org/10.1002/jssc.70200
Anal Biochem. 2025 Jun 27. pii: S0003-2697(25)00167-8. [Epub ahead of print] 115928

Assessment of matrix effects in quantitative GC-MS by using isotopologs.

Dimitrios Tsikas.

  In quantitative analyses, thousands of compounds are extracted from a biological matrix in addition to the analytes of interest and can affect their quantification by many different effects. They are widely known as matrix effects (ME). A frequently used approach in LC-MS/MS to quantify ME is performing two series of analyses, that is 1) in the biological sample, and 2) in analyte solutions in water, organic solvents and/or in mixtures of them, and by comparing the slope values of the two standard curves. This article suggests a new approach for the quantification of ME in GC-MS using isotopologs, namely their specific peak area. The approach is exemplified for amino acids, representing an important group of physiological substances, for human serum and urine, two capital matrices in biological analysis.

Keywords:  Amino acids; Concentration; Derivatization; Deuterium; GC-MS; Peaks area; Stable isotopes

DOI:  https://doi.org/10.1016/j.ab.2025.115928
Res Sq. 2025 Jun 16. pii: rs.3.rs-6743284. [Epub ahead of print]

Multiplexed Targeted Spatial Mass Spectrometry Imaging Assays to monitor lipids and NAD+ metabolites in CD38 knockout mice exhibiting improved metabolism.

Birgit Schilling, Charles Schurman, Joanna Bons, Prasanna Kumaar, Jingji Fang, Andrea Roberts, Genesis Hormazabal, Rebeccah Riley, Nannan Tao, Eric Verdin.

Mass spectrometry imaging (MSI) is a rapidly advancing technology that provides mapping of the spatial molecular landscape of tissues for a variety of analytes. Matrix-assisted laser desorption/ionization (MALDI)-MSI is commonly employed, however, confident in situ identification and accurate quantification of analytes remain challenging. We present a novel imaging methodology combining trapped ion mobility spectrometry (TIMS)-based parallel accumulation-serial fragmentation (PASEF) with MALDI ionization for targeted imaging parallel reaction monitoring (iprm-PASEF). We investigated the spatial distribution of lipids and metabolites in liver tissues from wild-type and CD38 knockout mice (CD38-/-). CD38, an enzyme involved in nicotinamide adenine dinucleotide (NAD+) metabolism, significantly influences liver metabolic function and contributes to age-related NAD+ decline. Although CD38 deletion previously was linked to improved metabolic phenotypes, the underlying spatial metabolic mechanisms are poorly understood. The spatial iprm-PASEF workflow enabled confident identification and differentiation of lipid isomers at the MS2 fragment ion level and revealed increased NAD+ and decreased adenosine diphosphate ribose (ADPR), a by-product of NAD+ hydrolysis, in CD38-/- livers. This approach provided confident, specific, and robust MS2-based identification and quantification of fragment ions in spatial MSI experiments. Additionally, the innovative iprm-PASEF opens unprecedented opportunities for spatial metabolomics and lipidomics, offering spatially resolved insights into molecular mechanisms.

DOI: https://doi.org/10.21203/rs.3.rs-6743284/v1
BMC Chem. 2025 Jul 02. 19(1): 180

Development and validation of a LC-MS/MS method for the simultaneous determination of simnotrelvir and ritonavir in human serum and bronchoalveolar lavage fluid.

Xiaoyu Yang, Wanling Lin, Yushang Zhao, Chaoyue Zhao, Fei Zhou, Dong Wang, Zhihong Yue, Lin Pei, Mei Jia, Bin Cao, Lin-Lin Cao, Hui Wang.

  "Xiannuoxin" (simnotrelvir/ritonavir) is a novel anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drug developed in China, which plays an antiviral role by inhibiting 3C-like protease (3CLpro). At present, it has been put into clinical use, while a simple, accurate and sensitive detection method is urgently needed for the quantification of simnotrelvir/ritonavir in human serum and bronchoalveolar lavage fluid (BALF) to ensure safe and efficacious antiviral therapeutics. In this study, we developed a liquid chromatography tandem mass spectrophotometry (LC-MS/MS) method for the simultaneous determination of simnotrelvir, ritonavir and urea concentrations in human serum and BALF samples. Prior to LC-MS/MS analysis, a user-friendly, one-step pre-analytical process was conducted, followed by a rapid chromatographic run lasting 3 min. This was then succeeded by positive and negative electrospray ionization and detection using a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode. Subsequently, the LC-MS/MS method underwent a comprehensive validation in aspects such as sensitivity (LoQs of 2.5 ng/mL, 0.1 ng/mL, and 1 μg/mL for simnotrelvir, ritonavir and urea), linearity, carryover, precision, trueness (recovery rates of simnotrelvir, ritonavir and urea were between 85 and 115%), matrix effect (within 85-115%) and stability (stable for 72 h at room temperature). The validation results demonstrated that this LC-MS/MS method was robust and reliable. Notably, we can use the urea dilution correction method to calculate the concentrations of simnotrelvir and ritonavir in epithelial lining fluid (ELF), which is of great significance for evaluating the effectiveness and safety of antiviral drug treatment.

Keywords:  Bronchoalveolar lavage fluid; Epithelial lining fluid; LC-MS/MS; Ritonavir; Serum; Simnotrelvir; Urea

DOI:  https://doi.org/10.1186/s13065-025-01534-x
Anal Chim Acta. 2025 Sep 15. pii: S0003-2670(25)00691-9. [Epub ahead of print]1367 344297

Monitoring the degree of in-source phospholipid fragmentation during MALDI mass spectrometry imaging.

Samantha L Cousineau, Mohammed H Sarikahya, Kristina Jurcic, Steven R Laviolette, Daniel B Hardy, Ken K-C Yeung.

  Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful tool for tissue lipid analysis. Yet, the validity of fatty acid (FA) signals is often questioned due to the potential of in-source lipid fragmentation. This study investigates the extent of in-source phospholipid fragmentation by examining different phospholipid headgroups, specifically phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS) and phosphatidylcholine (PC), containing a mix of saturated and unsaturated tails, comparing in-source fragmentation of FA tails. These standards evaluated phospholipid fragmentation during laser intensity optimization for MALDI-MS and MSI. Fragmentation was assessed in negative ion mode using four MALDI matrices: norharmane (NRM), 9-aminoacridine (9AA), 1,5-diaminonaphthalene (DAN), and 1,6-diphenyl-1,3,5-hexatriene (DPH). By examining the extent of in-source fragmentation during MALDI MSI, an exogenous standard can be chosen to monitor in-source fragmentation during experiments. Various techniques for standard deposition were evaluated, including manual spotting versus automated spraying and pre-depositing standards beneath tissue sections compared to applying them directly onto tissue surfaces. The results demonstrate the ability of these phospholipid standards to detect in-source fragmentation using different matrices, as demonstrated by the detection of the exogenous FA 17:0 tail signal. Furthermore, the study highlights the importance of selecting a standard that closely matches the lipid of interest to optimize laser energy for MSI, enhancing endogenous FA signals while minimizing in-source fragmentation. This work provides a workflow for mitigating in-source fragmentation in MALDI-MSI experiments, enabling more reliable lipid analysis in complex biological tissues.

Keywords:  Brain tissue; Fatty acids; Fragmentation; Lipids; MALDI; Mass spectrometry imaging

DOI:  https://doi.org/10.1016/j.aca.2025.344297
Biotechnol J. 2025 Jul;20(7): e70008

Novel Data-Driven Mechanistic Modeling of Untargeted Metabolome Data Reveals Feed Component Effects in CHO Cell Bioprocess Using Column Generation-Based EFMs.

Meeri E-L Mäkinen, Markella Zacharouli, Sigrid Särnlund, Yun Jiang, Veronique Chotteau.

  This study presents a novel approach for applying mechanistic metabolic modeling to untargeted metabolomics data. The approach was applied to the production process of a difficult-to-express enzyme by CHO cells, to identify key feed medium component candidates responsible for improved productivity through feed modification. The exploitation of untargeted metabolomics implies no prior decision of the metabolites or pathways and thus allows screening of metabolic phenomena and bringing an objective perspective. However, such exploitation is challenging due to the high-dimensionality, complexity, relative quantitative information, and high analysis cost of the data, leading to data scarcity. A combination of untargeted metabolomics data exploration and mechanistic modeling was developed to leverage metabolomics data. The study analyzed LC/MS/MS metabolomics data (563 cellular and 386 supernatant metabolites) to determine the key metabolites involved in the productivity increase associated with a feeding modification. The metabolome data was utilized to expand the original stoichiometric reaction network of 127 reactions to 370 reactions. Mechanistic modeling using elementary flux modes-based column generation identified and simulated the underlying metabolic pathways. Twenty-one key metabolites significant for productivity improvement were revealed. This included several unexpected metabolites, such as citraconate and 5-aminovaleric acid, in addition to well-known components, as well as their underlying metabolic pathways. This study offers a novel approach for investigating nutrient supplementation in terms of metabolic fluxes and process performance, paving the way for rational process optimization supported by mechanistic understanding.

Keywords:  Chinese hamster ovary cells; bioprocessing; column generation; elementary flux mode; mechanistic metabolic model; metabolomics

DOI:  https://doi.org/10.1002/biot.70008
Sci Rep. 2025 Jul 01. 15(1): 21521

Protoporphyrin IX plasma and blood pharmacokinetics and brain tumor distribution determined by a validated LC-MS/MS method.

William Knight, Tigran Margaryan, Kamal Shaik, Nader Sanai, Artak Tovmasyan.

  Protoporphyrin IX (PPIX) is a fluorescent metabolite in the heme biosynthesis pathway, and cancer cells accumulate it when 5-aminolevulinic acid (5-ALA), a precursor, is administered. In the U.S., 5-ALA is approved for visualizing high-grade gliomas (HGG) during fluorescence-guided surgery. PPIX is also central to experimental photodynamic and sonodynamic therapies for HGG. Additionally, PPIX measurement is critical for diagnosing and monitoring porphyrias. Despite the need for a sensitive and rapid bioanalytical method for accurate quantification of PPIX in biospecimens, no reliable validation of an LC-MS/MS method is available due to challenges related to its chemical instability, poor solubility, and tendency to aggregate. This work is the first to present a fully validated, sensitive, and rapid LC-MS/MS method for determining PPIX levels in human plasma, blood, and brain tumors. The method overcomes stability concerns, achieving a 3.5-min total run-time with a concentration range of 1-2000 nmol/L for plasma and tumors, and 10-2000 nmol/L for blood. Application of the method in a clinical trial, which assesses sonodynamic therapy for HGG patients, shows significant PPIX production, peaking in plasma and blood six hours post-5-ALA administration. In recurrent HGG patients, PPIX levels were notably higher in gadolinium-enhancing tumor regions compared to non-enhancing areas, indicating preferential accumulation in tumors.

Keywords:  5-aminolevulinic acid (5-ALA); Brain tumor; Liquid chromatography tandem mass spectrometry (LC–MS/MS); Photodynamic therapy; Protoporphyrin IX (PPIX); Sonodynamic therapy

DOI:  https://doi.org/10.1038/s41598-025-05780-w
Se Pu. 2025 Jul;43(7): 734-743

[Preparation and chromatographic performance evaluation of hydrophilic interaction chromatography stationary phase based on amino acids].

Gai-Gai Xu, Yang Yi, Ping-Ping Liu, Wen-Fen Zhang.

  To overcome current limitations in polar compound separation and better understand hydrophilic interaction liquid chromatography （HILIC） retention mechanisms， we designed and synthesized two novel amino acid-functionalized stationary phases using highly hydrophilic L-hydroxyproline and L-proline as modifiers through a continuous solid-liquid reaction method. The synthesized stationary phases were thoroughly characterized using Fourier-transform infrared spectroscopy （FT-IR）， thermogravimetric analysis （TGA）， and elemental analysis. Comparative elemental analysis revealed a substantial increase in carbon （C）， hydrogen （H）， and nitrogen （N） contents in both L-hydroxyproline-functionalized （L-OH-PSil） and L-proline-functionalized （L-PSil）stationary phases relative to the cyanuric chloride-bonded aminopropyl silica gel （TCT-Sil） intermediate， confirming successful functionalization. Quantitative analysis demonstrated distinct ligand densities for each phase， with L-OH-PSil exhibiting a higher loading （0.193 mmol/g） compared to L-PSil （0.178 mmol/g）. Thermal stability assessments indicated both materials maintained excellent structural integrity across a wide temperature range （20-600 ℃）， as evidenced by TGA results. To explore the chromatographic separation performance of the prepared L-OH-PSil and L-PSil stationary phases， sulfonamides were selected as solutes， and preliminary chromatographic separation investigations were conducted. The sulfonamide compounds exhibited excellent separation efficiency on both stationary phases， with retention behavior following consistent elution orders strongly correlated with analyte polarity. This observed retention pattern strongly suggested hydrophilic interactions constituted the predominant retention mechanism between the amino acid-functionalized stationary phases and sulfonamide analytes. Further supporting this conclusion， a systematic decrease in retention factors （lg k values） with increasing aqueous content in the mobile phase was observed as a characteristic feature of HILIC. Under optimized HILIC conditions， we further systematically evaluated the separation performance of both stationary phases using heterocyclic amines and nucleosides as model analytes， with direct comparison to a commercial Hypersil NH₂ column. Both custom phases exhibited exceptional column efficiency， with L-OH-PSil achieving 11 582.87 theoretical plates for 2-amino-3-methyl-9H-pyrido［2，3-b］indole （MeAαC） compared to 8 661.45 for L-PSil， while maintaining excellent performance across diverse analyte classes including plant growth hormones， flavonoids， and amines. The L-OH-PSil phase demonstrated superior chromatographic performance relative to both its L-PSil counterpart and the commercial NH₂ column. This superiority is attributable to its unique bifunctional design incorporating two hydroxyl groups， which combine the advantageous features of amino acid and diol-based stationary phases. This structural characteristic enables multiple synergistic interaction mechanisms， including π-π stacking， enhanced ion-exchange capacity， and additional hydrogen bonding sites， collectively yielding improved selectivity for polar small molecules. To further evaluate the chromatographic performance of the amino acid-based stationary phases， we investigated the effects of flow rate and column temperature using nucleosides and heterocyclic amines as model analytes. Remarkably， baseline separation was maintained even at elevated flow rates， demonstrating the robustness of both phases under high-throughput conditions. Temperature-dependent studies revealed that retention times exhibited only minor decreases or remained stable with increasing column temperature， suggesting minimal thermal effects on retention behavior. Van't Hoff analysis yielded excellent linear correlations （r²=0.992 9-0.999 7） for all tested analytes， confirming that the retention mechanism remains unchanged across the studied temperature range while indicating an exothermic separation process. Method validation confirmed the reliability of the developed system， with chromatographic peaks maintaining excellent shape and retention time stability across varying analyte concentrations. The relative standard deviations （RSDs） of retention times for six nucleosides ranged from 0.29% to 0.59%， underscoring the outstanding operational stability and analytical reproducibility of the L-OH-PSil stationary phase. These results collectively demonstrate the robustness of the amino acid-functionalized stationary phases under varying chromatographic conditions， further supporting their potential for practical applications in polar compound analysis. These results indicate that the L-OH-PSil stationary phase has excellent potential for broad applications in pharmaceutical analysis， environmental monitoring， and bioanalytical separations.

Keywords:  L-hydroxyproline; L-proline; amino acid-functionalized stationary phase; hydrophilic interaction liquid chromatography （HILIC）

DOI:  https://doi.org/10.3724/SP.J.1123.2025.04015
Metabolomics. 2025 Jul 01. 21(4): 97

Untargeted metabolomic profiling of serum and urine in kidney cancer: a non-invasive approach for biomarker discovery.

Anna Ossolińska, Aneta Płaza-Altamer, Krzysztof Ossoliński, Tadeusz Ossoliński, Tomasz Ruman, Joanna Nizioł.

   INTRODUCTION: Kidney cancer (KC) is a significant global health burden. Early diagnosis remains challenging due to the limited sensitivity and specificity of existing biomarkers. Metabolomics enables the detection of disease-specific metabolic alterations, offering potential for improved non-invasive biomarker discovery.
OBJECTIVES: This study aims to characterize metabolic signatures distinguishing KC patients from non-cancer controls and evaluate the diagnostic potential of annotated metabolites in serum and urine.
METHODS: An untargeted metabolomic analysis was performed on serum and urine samples from 56 KC patients and 200 controls using ultra-high-resolution mass spectrometry coupled with ultra-high-performance liquid chromatography (UHPLC-UHRMS in both positive and negative ionization modes with vacuum insulated probe heated electrospray ionization (VIP-HESI)). Samples were collected from the same individuals, which helped minimize inter-individual variability and enabled cross-biofluid comparison of metabolic profiles. Multivariate statistical techniques were applied to detect metabolic differences, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). An external validation strategy using training and validation subsets was employed to assess the robustness of candidate metabolite biomarkers matched in the discovery dataset.
RESULTS: Distinct metabolic signatures were observed between KC patients and controls, with key metabolic pathways involving lipid metabolism, amino acid biosynthesis, and glycerophospholipid metabolism. 19 serum and 12 urine metabolites showed high diagnostic potential (AUC > 0.90), demonstrating strong sensitivity and specificity.
CONCLUSION: These findings support the application of metabolomics for RCC detection and highlight the metabolic alterations associated with kidney cancer. Further validation in larger cohorts is necessary to confirm the clinical utility of these potential biomarkers.

Keywords:  Biomarkers; Kidney cancer; Metabolomics; Serum; UHPLC-UHRMS; Urine

DOI:  https://doi.org/10.1007/s11306-025-02294-4
Anal Chim Acta. 2025 Sep 15. pii: S0003-2670(25)00711-1. [Epub ahead of print]1367 344317

Affordable 3D-printed prototype integrated with open-source electronic platform for low-cost polyamide noncoated adsorption-based microextraction.

Camila Scheid, Sofia Aquino Monteiro, Carolina Souza Machado, Monique Deon, Josias Merib.

   BACKGROUND: The development of automated sample preparation approaches is considered a significant challenge in analytical chemistry. In general, commercially available devices exhibit impressive performance; however, their high acquisition costs remain a limitation for many laboratories worldwide. Consequently, efforts have been directed toward developing cost-effective instruments that can offer efficient and more independent sample handling. In this study, we propose a novel 3D-printed device integrated with an open-source electronic platform for polyamide noncoated adsorption-based microextraction (PANDA). To date, literature lacks electronically controlled apparatus designed for this technique. The device was evaluated for the determination of twenty organic pollutants using a 48-deep well plate system, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
RESULTS: The best experimental conditions consisted of polyamide + 15 % carbon fiber (PA + 15 % C) as sorbent phase, 120 min of extraction using 3 mL of water sample under stirring. Additionally, liquid desorption was performed using 300 μL of methanol:acetonitrile (1:1 v/v) for 10 min under stirring. Notably, the methodology demonstrated high throughput, enabling up to 48 simultaneous extractions with z-axis movements permitting more automated extraction and desorption steps. The method was validated, providing coefficients of determination higher than 0.9841 for all analytes; limits of detection (LODs) and limits of quantification (LOQs) ranged from 0.03 to 3.0 μg L-1 and from 0.1 to 10.0 μg L-1, respectively. Intraday precision ranged from 1.5 to 16.3 %, and interday precision varied from 4.0 to 18.2 %. Accuracy was examined through relative recoveries and ranged from 83.2 to 113.4 %. This method was successfully applied to analyze sixteen environmental water samples. In addition, the apparatus did not require expert skills to be designed and assembled.
SIGNIFICANCE: This novel 3D-printed device was successfully manufactured and assembled with an open-source electronic platform for polyamide noncoated adsorption-based microextraction. This device offered a more automated alternative to the apparatus previously used in this technique, requiring less analyst intervention and allowing for customization of the 3D-printed components. Importantly, production cost was approximately 10 times lower than that of similar commercially available devices, offering a cost-effective alternative for laboratories with limited budgets. Additionally, this first electronically controlled PANDA opens new possibilities to exploit a higher degree of automation in further studies.

Keywords:  3D-printing; Adsorption-based microextraction; Automation; Environmental pollutants; Open-source platform

DOI:  https://doi.org/10.1016/j.aca.2025.344317
medRxiv. 2025 Jun 09. pii: 2025.06.08.25329229. [Epub ahead of print]

A High-Performance Liquid Chromatography-Mass Spectrometry method for simultaneous determination of dolutegravir, nevirapine, efavirenz, rifampicin and rifapentine concentrations in human plasma.

Denis Omali, Moses Ocan, Hindum Lanyero, Martin Kamilo Angwe, Barbara Castelnuovo, Allan Buzibye.

Variations in clinical pharmacokinetics and genetics, and polypharmacy affect attainment of target drug concentrations in HIV-TB co-infected patients. However, scarcity of analytical methods hinders drug concentration measurement for therapeutic drug monitoring in this doubly vulnerable population in low-and-middle income countries. This study aimed to develop and validate an LC-MS method for simultaneous determination of dolutegravir, nevirapine, efavirenz, rifampicin, isoniazid and rifapentine concentrations in plasma. ThermoScientific LCQ ion trap operated by Xcalibur™ software was used. Analyte extraction from plasma was achieved by protein precipitation using 100% acetonitrile. Injection volume was 30µl for dolutegravir, efavirenz and nevirapine and 10µl for rifampicin and rifapentine. Analyte separation was achieved on Waters Atlantis dC18 column by 400µl flow rate of mobile phase under gradient elution. Transitions used were; dolutegravir (419.13→277.00), efavirenz (315.03→316.00), nevirapine (266.16→267.10), rifampicin (822.41→790.20), rifapentine (876.45→845.50), dolutegravir-d4 (423.13→280.10) and rifampicin-d4 (826.41→795.20). Calibration curves were constructed using quadratic regression with dynamic weighting of x = y, 1/x and 1/x 2 and accepted at r 2 ≥ 0.95 for all validation parameters. Method linearity and recovery ranged from 0.25µg/ml to 10.00µg/ml and 86.12% to 109.89% respectively for all analytes. Intra-and inter-day accuracy ranged from 88.73% to 109.67% and 93.38% to 104.30% respectively for all analytes. Both intra-and inter-day precision ranged from 2.47 RSD to 12.39 RSD and 5.34 RSD to 16.83 RSD respectively for all analytes. The method was selective and specific with no significant ghost peak identified in blank samples at expected retention times of dolutegravir, efavirenz, nevirapine, rifampicin and rifapentine. An LC-MS method for simultaneous determination of dolutegravir, efavirenz, nevirapine, rifampicin and rifapentine in human plasma was developed and validated. The method could be useful to the Uganda Ministry of Health for therapeutic drug monitoring in HIV-TB patients in health facilities in the country.

DOI: https://doi.org/10.1101/2025.06.08.25329229
Anal Chem. 2025 Jun 28.

Constant Distance Mode Mass Spectrometry Imaging with a Confocal Sensor.

Tommy Zhang, Ryan T Hilger, Li-Xue Jiang, Julia Laskin.

Mass spectrometry imaging in constant distance mode is critical for analyzing samples with complex topography, achieving high spatial resolution, and providing a robust imaging platform. Regardless of the ionization technique employed, variations in sample height during an imaging experiment can cause signal instability. In this work, we present the use of a confocal distance sensor for noncontact, real-time control of the sample height in MSI experiments. The confocal sensor offers submicrometer distance resolution and compatibility with a wide variety of surface optical properties. Although we demonstrate the performance of the sensor for constant distance mode nanospray desorption electrospray ionization (nano-DESI) experiments, the sensor is expected to be compatible with other MSI techniques, making it a versatile solution for challenging MSI applications.

DOI: https://doi.org/10.1021/acs.analchem.5c00742
Sci Rep. 2025 Jul 01. 15(1): 21081

Integrated plasma metabolomics and lipidomics profiling highlight distinctive signatures with hashimoto's thyroiditis.

Guoqiang Wang, Xuan Ye, Ting Lu, Yunxi Yang, Bingwei Sun, Hui Xiao.

  Hashimoto's Thyroiditis (HT) is an autoimmune disorder characterized by metabolic disturbances. However, a comprehensive metabolic and lipid profile of HT has not been reported. The metabolic and lipid profile of HT can be comprehensively analyzed through metabolomics and lipidomics technologies, providing a detailed understanding of the disease's biochemical alterations. Plasma samples were obtained from 20 mice, comprising 10 from the control group and 10 from the HT group. Plasma metabolomics and lipidomics were analyzed via LC-MS/MS. PCA, PLS-DA, and OPLS-DA of the preprocessed data matrix were conducted using the ropls package (V.1.6.2) in R. The identification of significant differential metabolites was based on the VIP scores in the OPLS-DA model and p-values (Student's t-test). Metabolites (VIP exceeding 1 and p < 0.05) were classified as significantly different. Pathway annotation of these metabolites was carried out using the KEGG database to identify associated metabolic pathways. Pathway enrichment analysis was performed using the Python scipy. Stats package, with Fisher's exact test employed to identify biological pathways most relevant to the experimental conditions. Metabolomics identified 6384 metabolites (2943 in positive ion mode and 3441 in negative), with 195 differential metabolites, comprising 114 upregulated and 81 downregulated in the HT group. Lipidomics analysis revealed 1054 lipid metabolites (695 detected in positive ion mode and 359 in negative), and 247 differentially expressed lipids were identified, including 165 upregulated and 82 downregulated in the HT group. KEGG enrichment analysis indicated that metabolites upregulated in the HT group were primarily associated with pathways such as Autophagy, Choline metabolism, PPAR signaling, and Glycerophospholipid metabolism. In contrast, pathways involved in Apoptosis, Cholesterol metabolism, Sphingolipid metabolism, EGFR tyrosine kinase inhibitor resistance, Th1 and Th2 cell differentiation, viral infection, and Chemokine signaling were suppressed. Metabolic and lipidomic dysregulation was observed in HT animal models, with pronounced alterations in Phospholipids, Eicosanoids, and Carnitines. Choline, Glycerophospholipid, and Linoleic acid metabolism pathways exhibited significant enrichment in HT.

Keywords:  Carnitines; Eicosanoids; Hashimoto’s thyroiditis; LC-MS/MS; Lipidomics; Metabolomics; Phospholipids

DOI:  https://doi.org/10.1038/s41598-025-07905-7
Food Res Int. 2025 Oct;pii: S0963-9969(25)01165-2. [Epub ahead of print]217 116827

Construction of a Fritillaria alkaloids database to develop a multi-dimensional matching strategy for comprehensive annotation of known and unknown components.

Leixin Zhuang, Hao Wang, Yue Wang, Dongxv Lu, Yiqiang Zhang, Wei Guan, Qingshan Chen, Lili Zhang, Shu Liu, Xiaochi Ma, Xiaoqing Yu, Haixue Kuang, Bingyou Yang, Yan Liu.

  Fritillaria, a dual-purpose medicinal and edible herb of the Liliaceae family, contains bioactive alkaloids as its key therapeutic components. Precise characterization of these alkaloids is critical for quality control and pharmacological mechanism studies. However, conventional analytical approaches face significant challenges due to the inherent complexity of plant metabolite matrices and the ubiquitous presence of structural isomers, resulting in low identification accuracy and difficulties in detecting unknown compounds. First, a novel dimension-enhanced approach was developed by integrating solid-phase extraction (SPE) with liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (LC/IM-QTOF-MS) to acquire four-dimensional structural information (tR, MS, MS/MS, and CCS). Subsequently, collision cross-section (CCS) values were predicted using machine learning, and a multidimensional database for Fritillaria alkaloids, namely FasMID, was established. In the analysis of prediction tools, ALLCCS/CCSbase demonstrated superior accuracy and applicability. Leveraging FasMID, which contained information on 248 Fritillaria alkaloids (including m/z, CCS, and MS/MS data), an integrated multi-dimensional matching strategy was developed for annotating both known and unknown chemical structures. Automated identification of known Fritillaria alkaloids was achieved by processing data with UNIFI and searching against FasMID, elevating the identification accuracy from a baseline of 12 % with traditional methods to 70 % with optimized approach. Furthermore, feature-based molecular networking (FBMN) combined with multidimensional characteristics of Fritillaria alkaloids was utilized to annotate unknown components, facilitating the exploration of novel chemical structures. In future research, prediction accuracy can be improved by expanding sample sources and optimizing deep learning algorithms, while extending this analytical strategy to other complex systems.

Keywords:  Collision cross section; Fritillaria alkaloids; Molecular networking; Multidimensional information database

DOI:  https://doi.org/10.1016/j.foodres.2025.116827
Anal Bioanal Chem. 2025 Jul 01.

Strychnos peckii B.L. Rob.: secondary metabolite annotation by liquid chromatography tandem high-resolution mass spectrometry with insights from biogenesis.

Fernando Cassas, Carla L G Santos, Felipe M A da Silva, Quezia B Cass.

  Alkaloids from the Strychnos genus exhibit remarkable chemical diversity and are considered promising candidates for drug development. However, many endemic species remain unexplored. In this study, we report the use of a scouting liquid chromatographic system for method development and a liquid chromatography system hyphenated to high-resolution mass spectrometry (LC-HRMS) to efficiently profile and annotate the specialized metabolites of Strychnos peckii B.L. Rob. A structural database was employed to curate the data through MS2 fragmentation experiments and the propagation of the molecular network using in silico tools. Manual data curation, based on biosynthetic pathways, comparisons with public data, and the creation of an in-house database, led to the annotation of 46 metabolites. The annotated metabolites were classified into alkaloids (33), benzoic acids (6), and flavonoids (5). Based on fragmentation patterns and their biogenesis, the alkaloids were further categorized into β-carbonyl compounds (1-5), monoterpene-indole alkaloid aglycones (6-11), monoterpene-indole alkaloids (12-20), monoterpene-indole alkaloid lactones (21-25), quinolinic compounds (26), and tryptophan-derived alkaloids (27-30), among others. The annotated alkaloids are well-known for their pharmacological properties. Overall, this study enhances the chemical understanding of Strychnos peckii.

Keywords:   β-carboline alkaloids; Biogenesis; Orthogonal columns; Specialized metabolites; Strictosidine derivates

DOI:  https://doi.org/10.1007/s00216-025-05967-0
Se Pu. 2025 Jul;43(7): 815-822

[Determination of squalene and oxidized squalene in edible oil by gas chromatography-tandem mass spectrometry and evaluation of the thermal stability of squalene].

Geng-Peng Xiao, Dan-Dan Liao, Gui-Sheng Li, Xiang Luo, Lu Yuan.

  The quality and safety of edible oils have frequently been compromised in recent years， seriously threatening consumer's legitimate rights and health. Hence， establishing methods for determining the quality of edible oils based on their endogenous components is greatly significant. Squalene is widely present in various oils； hence， studying the thermal stability of squalene in edible oils is expected to provide a new storage-management model and a method for rapidly determining oil quality. A method for determining squalene and oxidized squalene in edible oils was established based on QuEChERS-gas chromatography-tandem mass spectrometry. Edible oil samples were extracted by n-hexane and purified using a mixed adsorbent consisting of ethylenediamine-n-propyl silanized silica gel （PSA） and silica gel （CNW BOND Si）. Separation was used a TG-5ms column （30 m×0.25 mm×0.25 μm） and the squalene was used as an internal standard for quantitative analysis in selective reaction monitoring （SRM） mode. The chromatography column and adsorbent were judiciously optimized. Methodological verification revealed good linear relationships for squalene and oxidized squalene in the ranges of 0.03-0.4 and 0.29-3.80 mg/L， respectively， with correlation coefficients （r）≥0.992 under the optimized experimental conditions. Limits of detection （LODs， S/N=3） of 0.4 and 4.0 mg/kg were obtained for squalene and oxidized squalene， respectively， with corresponding limits of quantification （LOQs， S/N=10） of 1.2 and 12 mg/kg， respectively. Spiked recovery experiments were conducted at low， medium， and high spiked levels using three different oils， the average recoveries of squalene and oxidized squalene were 81.9%‒102.5% and 89.4%‒116.1%， respectively， with relative standard deviations （RSDs， n=6） of 3.5%‒6.8% and 3.2%‒7.4%， respectively. The developed method has the advantages of operational simplicity， stability， reliability， low LODs， and is suitable for detecting squalene and oxidized squalene in edible oils. The developed method was used to evaluate the thermal stability of squalene in peanut oil. The results showed that when the temperature was lower than 120 ℃， squalene did not undergo the phenomenon of conversion to oxidized squalene. However， when the temperature exceeds 120 ℃， the peak area of squalene shows a cliff-like decline， and the formation of oxidized squalene can be clearly detected. This method was used to test rapeseed oil， peanut oil， soybean oil and corn oil. The results showed that squalene was detected in all samples， while oxidized squalene was not detected in any samples. This method aims to provide new ideas for the storage management and rapid quality identification of edible oil.

Keywords:  QuEChERS; gas chromatography-tandem mass spectrometry （GC-MS/MS）; oxidized squalene; squalene

DOI:  https://doi.org/10.3724/SP.J.1123.2024.05031
Mikrochim Acta. 2025 Jul 04. 192(8): 478

Ion source component development for ambient mass spectrometry-based mycotoxin detection in ready-to-eat corn.

Hui-Chao Wang, Xiu-Li Xu, Xiu-Juan Wang, Jie Lian, Feng Zhang.

  A rapid hydrophilic covalent organic frameworks coated steel substrate (HCOFCS) was developed for the simultaneous determination of 9 mycotoxins in ready-to-eat corn using ambient mass spectrometry (AMS). The HCOFCS platform efficiently combines solid-phase extraction with electrospray ionization mass spectrometry. The established HCOFCS-ESI-MS method was employed to detect a variety of mycotoxins in ready-to-eat corn, including aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), Zearalenone (ZEN), Zearalanone (ZAN), Zearalenol (ZEL), Zearalanol (ZAL) and ochratoxin A (OTA). After the extraction process, HCOFCS was subjected to high voltage for ESI-MS, with a detection time of approximately 1 min. The method demonstrated a coefficient of determination (R2) exceeding 0.99 over a concentration range 0.1-100 μg/L. Limits of detection (LODs) and limits of quantitation (LOQs) ranged from 0.01-0.12 µg/kg to 0.1-0.7 μg/kg, respectively. The results indicated that the developed HCOFCS-ESI-MS method enables rapid and sensitive detection of multiple mycotoxins in ready-to-eat corn samples. Recoveries for the mycotoxins ranged from 83.62% to 96.97%, with relative standard deviations (RSDs) below 11.53%. Moreover, HCOFCS exhibited excellent inter-day and intra-day RSDs ranges, and the material could be reused at least six times without significant loss of adsorption efficiency.

Keywords:  Ambient mass spectrometry; Covalent organic framework materials; HCOFCS-ESI–MS; Mycotoxins; Solid-phase extraction

DOI:  https://doi.org/10.1007/s00604-025-07316-5