bims-metlip 2025-05-11 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–05–11
34 papers selected by
Sofia Costa, Matterworks

High-Sensitivity LC-MS/MS Approach for Accurate Quantification of N-Nitroso Duloxetine in Duloxetine Pharmaceutical Formulations.
Development and Validation of an HPLC-MS/MS Method for the Simultaneous Quantification of Vitexin and Isovitexin in Rabbit Plasma: Pharmacokinetic Insights on a Microcapsule Formulation.
LC-MS/MS Determination of 27 Antipsychotics and Metabolites in Plasma for Medication Management Monitoring.
Local Asymmetric Gaussian Fitting Algorithm for Enhanced Peak Detection of Liquid Chromatography-High Resolution Mass Spectrometry Data.
A Simple, Sensitive, and Stable LC-MS/MS Method for the Simultaneous Determination and Pharmacokinetic Study of Dapagliflozin and Its Metabolite D3OG in Human Plasma.
Liquid chromatography-tandem mass spectrometry assay for simultaneous quantification of catecholamines and metabolites in human plasma and cerebrospinal fluid.
Quantitative analysis of nonylphenol ethoxylates in textiles using ultrasonic/microwave-assisted extraction coupled with ultra-high-performance supercritical fluid chromatography-photodiode array-tandem mass spectrometry.
Development and Validation of a Serum Total Testosterone LC-MS/MS assay Certified by the CDC Hormone Standardization Program.
Development of an LC-MS method for the determination of simvastatin and its hydroxy acid form in muscle tissue and method application.
MetCohort: Precise Feature Detection and Correspondence for Untargeted Metabolomics in Large-Scale Cohort Studies.
Label-free quantitative shotgun analysis of bis(monoacylglycero)phosphate lipids.
Development of an isotope dilution gas chromatography - mass spectrometry candidate reference measurement procedure for glucose in human serum.
Preserving Full Spectrum Information in Imaging Mass Spectrometry Data Reduction.
A Validated Screening and Confirmation Method for 946 Drugs and Metabolites Using LC-QTOF-MS with SWATH Acquisition.
Distinguishing Chiral Amino Acids Using Chiral Derivatization and Differential Ion Mobility Mass Spectrometry.
Quantitative Nuclear Magnetic Resonance for Small Biological Molecules in Complex Mixtures: Practical Guidelines and Key Considerations for Non-Specialists.
Absolute Quantitation of Neopterin as an Endogenous Pharmacodynamic Biomarker: The Successful Method Development, Validation, and Use of a Surrogate Matrix for Clinical Sample Analysis.
Mapping the Edges of Mass Spectral Prediction: Evaluation of Machine Learning EIMS Prediction for Xeno Amino Acids.
Emerging Mycotoxins in Cheese: Simultaneous Analysis of Aflatoxin M1, Aflatoxicol, and Sterigmatocystin by LC-MS/MS.
Multi-Plug Filtration Purification Combined With Gas Chromatography-Tandem Mass Spectrometry for the Detection of 107 Pesticides in Livestock and Poultry Meat.
A HILIC-IM-MS-Based Pharmacometabodynamic Study of the Effects of Orally Administered Gefitinib on the Polar Urinary Metabolic Phenotypes of C57Bl6 Mice.
Development and analysis of 49 perfluoroalkyl and polyfluoroalkyl substances (PFASs) in contact lenses using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Three-Layer Electrospray Constructing Charged Microdroplets for Online Derivatization and Position Identification of Lipid C═C Bonds.
Development and validation of a QuEChERS extraction-UPLC-MS/MS method for the simultaneous determination of chloramphenicol drugs in Laminaria japonica and Porphyra yezoensis.
LC-MS System for Collecting Time-Resolved Metabolomics Data of Cultured Cells.
Separation and Quantification of Cyclic di-AMP, -GMP, and Cyclic GAMP in Bacteria Using LC-MS/MS.
A High-Throughput RP-UPLC Assay for Simultaneous Determination of Telmisartan and Azelnidipine in Pharmaceutical Formulations.
Onsite Analysis of Complex Samples Using Miniature Mass Spectrometry With Direct Sampling and Ionization.
A Comprehensive Model for Separating Systematic Bias and Noise in Metabolomic Timecourse Data-A Nonlinear B-Spline Mixed-Effects Approach.
Rapid processing of liquid chromatography mass spectrometry data for compound screening and characterization in complex matrix based on python: with Meconopsis quintuplinervia Regel as an example.
Quantification of Epigenetic DNA and RNA Modifications by UHPLC-MS/MS Technologies: New Concepts and New Improvements for the Special Collections.
Simultaneous determination of Favipiravir and its active metabolite, Favipiravir Ribofuranosyl-5'-triphosphate, in plasma and buccal cells using HPLC.
Simultaneous profiling of mercapturic acids, glucuronic acids, and sulfates in human urine.
Biomonitoring Livestock Antiparasitic Residues: Development of Fast Mass Spectrometric Methods for the Quantification of Ivermectin, Albendazole, and their Metabolites in Sheep Plasma and Feces.

Biomed Chromatogr. 2025 Jun;39(6): e70101

High-Sensitivity LC-MS/MS Approach for Accurate Quantification of N-Nitroso Duloxetine in Duloxetine Pharmaceutical Formulations.

Syam Sundhar Sai Kumar Boppana, Kiran Kumar Chagarlamudi, Rajesh Damarapurapu, Venkata Siva Suryanarayana Gurram, Vinod Kumar Manglige.

  The high-sensitivity analytical method for the detection of N-nitroso duloxetine, which can be carcinogenic in duloxetine medication products, was successfully developed by applying liquid chromatography-tandem mass spectrometry. Chromatographic separation was achieved by using liquid chromatography-mass spectrometry was carried out by employing an Agilent Zorbax Eclipse Plus C18, 4.6 × 150 mm, 5-μm column. The gradient elution mode was used to operate the mobile phase, which consisted of Phase A, which was a solution of 0.1% ammonia and 0.1% formic acid in water, and Phase B, 100% methanol. This approach overcame duloxetine challenges. Tandem mass spectrometric detection with positive electro spray ionization in MRM mode then found N-nitroso duloxetine. Quality control involves verifying the method for precision, specificity, linearity, accuracy, and robustness, following ICH and USP criteria <1225> to ensure suitability and consistent results. On the other hand, the correlation coefficient (r) was more than 1.000, the mean impurity recovery ranged from 100.5% to 102.4%, and the relative standard deviation (RSD) values (n = 6) ranged from 1.54% to 2.6% over the ranges of LOQ-150%. This work seeks to simplify risk assessment for N-nitroso duloxetine in duloxetine pharmaceutical formulations by providing a fast and reliable quantitative LC-MS/MS analytical method.

Keywords:  European Medicines Agency; LC‐MS/MS; N‐nitroso duloxetine; duloxetine

DOI:  https://doi.org/10.1002/bmc.70101
Molecules. 2025 Apr 10. pii: 1690. [Epub ahead of print]30(8):

Development and Validation of an HPLC-MS/MS Method for the Simultaneous Quantification of Vitexin and Isovitexin in Rabbit Plasma: Pharmacokinetic Insights on a Microcapsule Formulation.

Duc Tuan Nguyen, Trung Nguyen Nguyen Le, Duc Kien Ngo, Hien Minh Khuu, Khang Thien Tran, Hoang Thanh Le, Hung Viet Tran, Truong-Thang Nguyen Phan, Vo Thi Kim Khuyen, Han Hoang Do, Nhu Huynh Mai, Quan Minh Le.

  Vitexin and isovitexin are natural flavone C-glucosides that have numerous benefits for human health. However, their low oral bioavailability and poor gastrointestinal absorption dramatically restrict their potential medicinal uses. To overcome this challenge, chitosan-coated alginate microcapsules were prepared for intragastrical administration to rabbits. An LC-MS/MS method was developed and validated for the simultaneous determination of vitexin and isovitexin in the plasma of treated rabbits, using salicylic acid as the internal standard. Raw rabbit plasma samples were deproteinized using acetonitrile as a precipitation agent. Chromatographic separation was performed on a reversed-phase C18 column (100 mm × 4.6 mm, 3.5 µm), with an isocratic mobile solvent system comprising methanol and 0.1% acetic acid (40:60) as the mobile phase. All the analytes and the internal standard were ionized on a triple quadrupole mass spectrometer and electrospray ionization, operating in negative mode and multiple reaction monitoring. The analytical approach developed underwent validation in terms of system suitability, specificity, selectivity, LLOQ of 2 ng/mL, linearity (2.0-200 ng/mL, R2 > 0.99), accuracy (the intra- and inter-day from 94 to 110% with the relative standard deviations no more than 8.7%, precision with the recoveries from 97% to 102%, matrix effect (90-100%), carry-over, dilution integrity (2 times), and stability at room and frozen temperature for up to 1 month, and all the parameters met FDA and EMA requirements for bioanalytical methods. The validated procedure was applied to measure the absorption of vitexin and isovitexin from encapsulated extracts in a pilot pharmacokinetic study on rabbit plasma. Compared to the raw traditional extracts, the microcapsules enhanced the bioavailability of vi-texin and isovitexin regarding Cmax and AUC values.

Keywords:  LC-MS/MS; encapsulation; isovitexin; pharmacokinetic; rabbit plasma; vitexin

DOI:  https://doi.org/10.3390/molecules30081690
Xenobiotica. 2025 May 04. 1-26

LC-MS/MS Determination of 27 Antipsychotics and Metabolites in Plasma for Medication Management Monitoring.

Shanshan Chen, Donghan Wang, Yuanyuan Zhao, Yaqi Sun, Yueyao Luan, Qixuan Sun, Jiaqi Wang, Yuhang Yan, Jing Yu, Chunhua Zhou.

  1. With the increasing prevalence and escalating complexity of mental disorders, precise medication has become critically important. This necessitates an efficient, accurate, and convenient method for drug concentration monitoring to support laboratory personnel and clinicians. In this study, three liquid chromatography-tandem mass spectrometry methods were developed and validated for simultaneously determining and quantifying 27 antipsychotics and related metabolites in human plasma. The plasma samples were subjected to protein precipitation using methanol, with isotope-labeled internal standards, followed by separation via isocratic elution on a BEH C18 column. Mass spectrometric analysis was performed using electrospray ionization in positive ionization mode with multiple reaction monitoring for quantitative detection. The analytes demonstrated high separation efficiency, with a single sample run time of 3.0 min. The method exhibited a wide linear range with excellent linearity across the concentration range. The intra- and inter-batch precision were ≤10.00%, the accuracy was 88.67%∼113.29%. Accurate quantification of antipsychotics remained unaffected under various storage conditions: 72h at room temperature, 7d at 4 °C refrigeration, and 14d at -80 °C freezing.This validated methodology has been successfully applied to plasma samples from patients with psychiatric disorders, demonstrating its practical utility for accurate quantification of antipsychotics in large-scale and complex matrices containing multiple analytes.

Keywords:  Analysis methods; Antipsychotic drugs; LC-MS/MS; Therapeutic drug monitoring

DOI:  https://doi.org/10.1080/00498254.2025.2498702
Anal Chem. 2025 May 05.

Local Asymmetric Gaussian Fitting Algorithm for Enhanced Peak Detection of Liquid Chromatography-High Resolution Mass Spectrometry Data.

Shengsi Zou, Qingxiao Cui, Jinyue Liu, Qiong Wu, Lijia Zhu, Da Chen, Yiping Du, Ting Wu.

Feature detection is a crucial step in the data preprocessing workflow of liquid chromatography-mass spectrometry (LC-MS). However, many existing methods are hindered by intricate parameter adjustments and high false positive rates during extracted ion chromatogram (EIC) construction and peak detection, which challenges the identification of spurious and missing compounds. This study introduces a novel algorithm, local asymmetric Gaussian fitting (LAGF), for peak detection. LAGF integrates with the "data points bins" EIC extraction algorithm to enhance the feature detection efficiency. By using a 1 Da data points bin for EIC extraction, computational time is significantly reduced, making the method well-suited for batch metabolomics analysis. LAGF minimizes parameter numbers of generalized two-sided asymmetric Gaussian fitting by automatically determining the peak center (μ) and height (α) while accommodating two-sided standard deviations (σ1 and σ2) to self-adaptively model peak patterns. Features are filtered based on a goodness-of-fit threshold of 0.5. The performance of LAGF was validated using standard mixtures and serum samples at different concentrations in reversed-phase or hydrophilic interaction LC mode. In most cases, LAGF outperformed conventional tools in terms of determination coefficient (R2) and relative standard deviation for automatically detected peak areas. The LAGF algorithm is available as open-source Python code alongside an interactive interface, facilitating implementation in both nontargeted and targeted LC-MS analysis to enhance peak detection and compound identification.

DOI: https://doi.org/10.1021/acs.analchem.5c00060
Biomed Chromatogr. 2025 Jun;39(6): e70108

A Simple, Sensitive, and Stable LC-MS/MS Method for the Simultaneous Determination and Pharmacokinetic Study of Dapagliflozin and Its Metabolite D3OG in Human Plasma.

Ancheng Gu, Chuwen Zhang, Dan Li, Bohong Cen, Wanwen Cao, Zhongyuan Xu.

  Dapagliflozin is a widely used sodium-glucose cotransporter 2 inhibitor that has been approved for the treatment of Type 2 diabetes. In this study, we present a simple and sensitive high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous quantification of dapagliflozin and its metabolite, dapagliflozin 3-O-glucuronide (D3OG). The experiments were conducted using an Agilent G6495B triple quadrupole mass spectrometer coupled with an Agilent 1290 Infinity II HPLC system, featuring a Poroshell 120 EC-C18 column. Gradient elution was performed with ammonium formate (10 mM) and methanol as the mobile phase. The G6495B was operated in negative ion mode with electrospray ionization and multiple reaction monitoring. The quantitative method was validated according to FDA and EMA guidelines, assessing parameters such as selectivity, linearity, accuracy, precision, dilution integrity, stability, and recovery. Methanol was used as a protein precipitant during sample preparation, resulting in consistent extraction recoveries ranging from 91% to 96% for all analytes. The linear range for the analytes was established at 2-800 μg/L with a sample volume of 100 μL. This validated method is sufficient for the simultaneous quantification of dapagliflozin and D3OG in plasma and has been successfully applied in pharmacokinetic studies, bioequivalence assessments, and clinical therapeutic monitoring. Trial Registration: Chinese Clinical Trial identifier: ChiCTR2100044600.

Keywords:  D3OG; LC–MS/MS; dapagliflozin; human plasma; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.70108
Pract Lab Med. 2025 Jul;45 e00471

Liquid chromatography-tandem mass spectrometry assay for simultaneous quantification of catecholamines and metabolites in human plasma and cerebrospinal fluid.

Yuting Wang, Quan Li, Yuhang Deng, Wenqing Wu, Cuiping Zhang, Yichi Zheng, Ming Guan, Haoqin Jiang.

  Catecholamines (CAs) and their metabolites in human cerebrospinal fluid (CSF) and plasma are potential biomarkers of Alzheimer's disease (AD) and facilitate early diagnosis. Liquid chromatography-tandem mass spectrometry is the gold standard method for analyzing CAs. The objective of this study was to develop and validate a liquid chromatography-tandem mass spectrometry assay capable of simultaneously quantifying dopamine (DA), epinephrine (E), norepinephrine (NE), metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT) in both human CSF and plasma. Samples were processed by solid-phase extraction with a weak cation exchange adsorbent and then separated using an ultra-performance reversed-phase chromatography column. Analyte detection was performed using a triple quadrupole mass spectrometer operated in positive-ion multiple reaction monitoring mode. The developed assay was validated according to standard guidelines. The linearity, specificity, precision, accuracy, carryover and stability were assessed to ensure compliance with specified criteria. The lower limits of quantification for DA, E, NE, MN, NMN, and 3-MT were 4.5, 2.5, 4.5, 2.5, 2, and 0.3 pg mL-1, respectively. The total runtime for a single sample was 6.5 min. These results demonstrated that the method was sensitive, rapid, and reliable for the simultaneous quantification of DA, E, NE, MN, NMN, and 3-MT in clinical practice. We successfully detected CAs and their metabolites in plasma and CSF samples from patients with normal cognition and AD. This study demonstrates an efficient laboratory workflow for high-throughput analysis of CAs and their metabolites and lays a foundation for further studies on AD biomarkers.

Keywords:  Catecholamine; Cerebrospinal fluid; Liquid chromatography-tandem mass spectrometry; Metanephrine; Plasma

DOI:  https://doi.org/10.1016/j.plabm.2025.e00471
J Chromatogr A. 2025 Apr 23. pii: S0021-9673(25)00333-4. [Epub ahead of print]1753 465985

Quantitative analysis of nonylphenol ethoxylates in textiles using ultrasonic/microwave-assisted extraction coupled with ultra-high-performance supercritical fluid chromatography-photodiode array-tandem mass spectrometry.

Haiyan Gao, Yuncheng Ge, Lili Tong, Lei Yin, Qiang Ma.

  A sensitive and robust analytical method was developed for the simultaneous determination of nonylphenol ethoxylates (NPEOs) in textiles. The method utilizes ultra-high-performance supercritical fluid chromatography with photodiode array detection (UHPSFC-PDA) to assess monomer proportions across varying polymerization degrees, followed by quantitation via tandem mass spectrometry (UHPSFC-MS/MS) in multiple reaction monitoring (MRM) mode. Under optimized conditions, NPEOs (n = 2-39) were separated within 7 min on a BEH C18 column using compressed CO₂ and methanol as the mobile phase. Additionally, response surface methodology and Box-Behnken design were employed to optimize ultrasonic/microwave-assisted extraction (UMAE) parameters. The method demonstrated excellent linearity (R² > 0.99) for 13 NPEO homologues (n = 2-14), with limits of detection (LODs) and quantitation (LOQs) values ranging from 0.26 to 45.09 μg/kg and 2.28-188.06 μg/kg, respectively. Intra- and inter-day precision values ranged from 0.8 % to 4.23 % and from 1.34 % to 9.59 %, respectively. Recoveries at low, medium, and high spiking levels were between 71.1 % and 102.5 % (RSD ≤ 12.98 %). The method was validated using quality control standards and successfully applied to commercial textile samples, revealing NPEO concentrations up to 2626.63 μg/kg. The combination of UMAE with UHPSFC-PDA-MS/MS offers a rapid, sensitive, and environmentally friendly solution for comprehensive NPEO analysis in textiles.

Keywords:  Nonylphenol ethoxylates; Photodiode array; Supercritical fluid chromatography; Tandem mass spectrometry; Textiles

DOI:  https://doi.org/10.1016/j.chroma.2025.465985
Ann Clin Lab Sci. 2025 Mar;55(2): 192-202

Development and Validation of a Serum Total Testosterone LC-MS/MS assay Certified by the CDC Hormone Standardization Program.

Jianbo Yang, Kimberly Robyak, Christopher Hamilton, Yusheng Zhu.

OBJECTIVE: We aimed to develop and validate a serum testosterone liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay and sought certification by the CDC Hormone Standardization (HoSt) Program.
METHODS: Serum samples mixed with internal standards were extracted with hexane: ethyl acetate. An Agilent Poroshell 120 EC-C18 column and an Agilent 1260 Infinity HPLC system were used for liquid chromatography. An Agilent 6460C QQQ triple quadrupole mass spectrometer was used for multiple reaction monitoring. The assay was validated for matrix effect, lower limit of quantification, analytical measurement range, precision, accuracy, dilution, specificity, interference, and carryover.
RESULTS: The LC-MS/MS testosterone assay demonstrated an analytical measurement range (AMR) of 2.9-2330.4 ng/dl with a mean bias of 0.4% (95% CI: -2.8% to 3.6%) for the certified reference materials from the HoSt Program. The total CVs were 2.4-4.7%. The correlation between this LC-MS/MS (y) and a reference laboratory LC-MS/MS (x) was excellent: y=0.939x-8.3 (R=0.9978). The method was certified by the CDC HoSt Program.
CONCLUSION: An accurate and sensitive LC-MS/MS assay to measure serum testosterone is developed, validated, and certified by the HoSt Program. The assay can be used for serum testosterone testing in pediatric, female, and hypogonadal male populations.

Keywords: LC-MS/MS; electrochemiluminescent immunoassay; radioimmunoassay; testosterone
PLoS One. 2025 ;20(5): e0322808

Development of an LC-MS method for the determination of simvastatin and its hydroxy acid form in muscle tissue and method application.

Ewa Paszkowska, Karolina Pietrowska, Steen Larsen, Emilia Fornal, Michał Ciborowski.

PURPOSE: Statins are the most commonly used drugs worldwide. Besides a significant decrease in cardiovascular diseases (CVDs) risk, the use of statins is also connected with a broad beneficial pleiotropic effect. At the same time, it is burdened with different side effects. The most common ones are muscle issues (from mild myalgia to rhabdomyolysis). The mechanisms of many of them are still unclear. Therefore, an analytical method for the determination of simvastatin (SIM) and its main metabolite (the hydroxy acid form - SIMA) in muscle tissue was developed.
METHODS: Muscle samples were homogenized with ammonium acetate buffer using the bead mill homogenizer, and then statins were extracted with a mixture of methanol and ethanol. Prepared samples were analyzed with liquid chromatography (using a reverse-phase column with a gradient elution) combined with the mass spectrometer which was operated in a multiple reaction monitoring mode.
RESULTS: The assay was linear over a 0.1-5 ng/mL range for both statin forms. Inter- and intra-day precision and accuracy were characterized. The method was considered precise (with the following relative standard deviation values: 6.0-6.9% for SIM, and 8.1-12.9% for SIMA) and accurate (with the following mean accuracies: 91.4-100.1% for SIM, and 102.2-115.4% for SIMA). The extraction efficiency was evaluated by recovery determination (76% for SIM, and 99% for SIMA). Moreover, the matrix effect was calculated with the following results: 87% for SIM, and 139% for SIMA. The proposed method was applied for SIM and SIMA determination in skeletal muscle tissues obtained from statin-treated patients.
CONCLUSION: The obtained results proved that the method may be a useful tool for explaining muscle effects related to statin therapy.

DOI: https://doi.org/10.1371/journal.pone.0322808
Anal Chem. 2025 May 06.

MetCohort: Precise Feature Detection and Correspondence for Untargeted Metabolomics in Large-Scale Cohort Studies.

Jun Yang, Pengwei Guan, Di Yu, Qi Li, Xiaolin Wang, Guowang Xu, Xinyu Liu.

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics is becoming increasingly popular in large-scale cohort studies. However, its data processing is complex and challenging. We present MetCohort, a computational tool for performing metabolomics raw data alignment for large-scale sample analysis, and accurate feature detection and quantification. By combining chromatogram profile alignment and local anchor matching with an outlier removal algorithm, the retention times of the raw data were aligned. With aligned retention times across all the samples, regions of interest (ROIs) are detected and stacked among samples to form a two-dimensional (2D) ROI-matrix. This 2D ROI-matrix, resembling an image with rows representing samples and columns corresponding to the time, allows the application of image processing techniques. Since the peaks are already aligned in the alignment step, features can be accurately detected and quantified with automatic correspondence of all the samples. Based on the 2D image processing technique, holistic scale feature detection is performed, which not only significantly decreases the number of false-positives and improves the detection of low-intensity compounds, but also avoids tricky peak matching and quantification uncertainty. Overall, MetCohort has potential to enhance the accuracy and efficiency of data processing in large-scale LC-HRMS.

DOI: https://doi.org/10.1021/acs.analchem.4c04906
Anal Bioanal Chem. 2025 May 09.

Label-free quantitative shotgun analysis of bis(monoacylglycero)phosphate lipids.

Lian Y Wang, Rico J E Derks, Kevin A J Brewster, Danilo Prtvar, Sabina Tahirovic, Stefan A Berghoff, Martin Giera.

  Interest in the role of bis(monoacylglycero)phosphate (BMP) lipids in lysosomal function has significantly grown in recent years. Emerging evidence highlights BMPs as critical players not only in Niemann-Pick disease type C (NPC) but also in other pathologies such as neurodegeneration, cardiovascular diseases, and cancers. However, the selective analysis of BMPs is significantly hindered by isomeric phosphatidylglycerol (PG) lipids. While this can be addressed by chromatographic separation, it poses a significant challenge for shotgun lipidomics approaches. Here, we present a shotgun lipidomics strategy to detect and separate BMPs from PGs using differential fragmentation of sodiated ions. This approach, including isotope correction, is integrated into an existing quantitative shotgun lipidomics workflow (Lipidyzer combined with Shotgun Lipidomics Assistant software) that simultaneously quantifies >1400 lipids. Validation using K-562 cell extracts demonstrated acceptable linearity, trueness, repeatability, and a limit of quantification of 0.12 µM, confirming robust analytical performance. Finally, characteristic accumulation of BMP lipids is shown in bone marrow-derived macrophages from NPC mice, demonstrating its applicability. Our method presents a quantitative, selective, rapid, and robust solution for shotgun-based BMP analysis without the need for extensive chromatographic separation or derivatization. The integration of BMP lipid detection into the Lipidyzer platform, alongside the recently launched iSODA data visualization tool, empowers chemists and biologists to gain deeper insights into BMP lipid biology.

Keywords:  BMP; Flow injection; Label free; Mass spectrometry; Shotgun lipidomics

DOI:  https://doi.org/10.1007/s00216-025-05890-4
J Mass Spectrom Adv Clin Lab. 2025 Apr;36 63-72

Development of an isotope dilution gas chromatography - mass spectrometry candidate reference measurement procedure for glucose in human serum.

Komal Dahya, Heather C Kuiper, Sarah W Kingsley, Uliana Danilenko, Hubert W Vesper.

   Introduction: Diabetes is the seventh leading cause of death in the United States, impacting over 37 million people. Accurate glucose measurements are critical for effective diabetes management. A reliable candidate reference measurement procedure (cRMP) for assessing the analytical performance of glucose tests performed in patient care is essential for ensuring measurement accuracy.
Methods: We have developed a gas chromatography-mass spectrometry (GC-MS)-based cRMP for glucose in human serum. In this procedure, glucose is measured as the aldononitrile acetate derivative and quantitated using a 13C6-glucose internal standard.
Results: Analytical selectivity was achieved through chromatographic separation and monitoring the quantitation ion/confirmation ion ratios in samples. With bias ranging from -0.79 % to 0.67 % for eight levels of serum-based certified reference materials from the National Institute of Standards and Technology (NIST) and Laboratoire national de métrologie et d'essais (LNE) and total CVs of 1.11 %, 0.68 % and 0.74 % at the low, medium, and high glucose concentration levels, respectively, the cRMP provided excellent accuracy and precision. The calibration curve was linear throughout the 13.51-378.21 mg/dL [0.75-21 mmol/L] measurement range (R2 = 0.9999), with a mean slope of 270.73 (95 % CI, 270.19 to 271.27) and an intercept of 0.021 (95 % CI, -0.157 to 0.199). The limit of detection was 0.25 mg/dL (0.014 mmol/L) and the limit of quantitation was 0.83 mg/dL (0.046 mmol/L).
Conclusion: The described GC-MS method, with metrological traceability to the International System of Units (SI), provides highly accurate and precise measurements of glucose in human serum.

Keywords:  Candidate Reference Measurement Procedure; Gas Chromatography; Glucose; Mass Spectrometry; Point of Care Testing

DOI:  https://doi.org/10.1016/j.jmsacl.2025.04.005
Bioinformatics. 2025 May 08. pii: btaf247. [Epub ahead of print]

Preserving Full Spectrum Information in Imaging Mass Spectrometry Data Reduction.

Roger A R Moens, Lukasz G Migas, Jacqueline M Van Ardenne, Eric P Skaar, Jeffrey M Spraggins, Raf Van de Plas.

MOTIVATION: Imaging mass spectrometry (IMS) has become an important tool for molecular characterization of biological tissue. However, IMS experiments tend to yield large datasets, routinely recording over 200,000 ion intensity values per mass spectrum and more than 100,000 pixels, i.e., spectra, per dataset. Traditionally, IMS data size challenges have been addressed by feature selection or extraction, such as by peak picking and peak integration. Selective data reduction techniques such as peak picking only retain certain parts of a mass spectrum, and often these describe only medium-to-high-abundance species. Since lower-intensity peaks and, for example, near-isobar species are sometimes missed, selective methods can potentially bias downstream analysis towards a subset of species in the data rather than considering all species measured.
RESULTS: We present an alternative to selective data reduction of IMS data that achieves similar data size reduction while better conserving the ion intensity profiles across all recorded m/z-bins, thereby preserving full spectrum information. Our method utilizes a low-rank matrix completion model combined with a randomized sparse-format-aware algorithm to approximate IMS datasets. This representation offers reduced dimensionality and a data footprint comparable to peak picking, but also captures complete spectral profiles, enabling comprehensive analysis and compression. We demonstrate improved preservation of lower signal-to-noise-ratio signals and near-isobars, mitigation of selection bias, and reduced information loss compared to current state-of-the art data reduction methods in IMS.
AVAILABILITY: The source code is available at https://github.com/vandeplaslab/full_profile and data is available at https://doi.org/10.4121/a6efd47a-b4ec-493e-a742-70e8a369f788.
SUPPLEMENTARY INFORMATION: Supplementary materials are available at Bioinformatics online.
CONTACT: raf.vandeplas@tudelft.nl.

DOI: https://doi.org/10.1093/bioinformatics/btaf247
J Anal Toxicol. 2025 May 05. pii: bkaf037. [Epub ahead of print]

A Validated Screening and Confirmation Method for 946 Drugs and Metabolites Using LC-QTOF-MS with SWATH Acquisition.

Maria Sarkisian, Luke N Rodda.

  A streamlined liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) method utilizing protein precipitation and filtration extraction was developed to achieve rapid and reliable screening and confirmation for blood and urine matrices. This method targets 946 drugs and metabolites across 35 drug classes via sequential window acquisition of all theoretical mass spectra (SWATH) acquisition with variable customized windows to enhance spectral clarity, and was validated per established guidelines to ensure high accuracy and reproducibility. Combined with complementary in-house methods, this approach meets and exceeds the testing requirements outlined in ANSI/ASB standards and recommendations for postmortem, drug-facilitated crime (DFC) and Tier I and II driving under the influence of drug (DUID) analyses. The method demonstrated efficient and sensitive performance, achieving limits of detection as low as 0.1 ng/mL. It accurately identified expected detections across 67 proficiency test samples and 224 authentic case samples, with high accuracy and reliability in the detection of both traditional drugs and novel psychoactive substances (NPS). The method employs an in-house built library and incorporates in-batch standards analyzed alongside case samples to ensure contemporaneous identification criteria, making it suitable for confirmation and reporting purposes. By expanding the analytical capabilities to include a vast range of analytes, this method improves the likelihood of identifying substances that may otherwise go undetected, and reduces the need for multiple separate tests, thereby enhancing the overall effectiveness of toxicological investigations.

Keywords:  Forensic Toxicology; General Unknown Screening; HRMS; Method Validation; NPS; Q-TOF; SWATH; Testing Regimes

DOI:  https://doi.org/10.1093/jat/bkaf037
J Am Soc Mass Spectrom. 2025 May 07.

Distinguishing Chiral Amino Acids Using Chiral Derivatization and Differential Ion Mobility Mass Spectrometry.

Simin Zhang, Xiangfeng Chen, T-Y Lui, Danna Hu, T-W Dominic Chan.

The precise differentiation of chiral amino acids (AAs) holds significant potential for elucidating health and disease status. Herein, we developed an analytical strategy integrating chiral derivatization with differential ion mobility spectrometry (DMS)-mass spectrometry (MS) to achieve the enantiomeric separation of AAs. Utilizing N-(4-nitrophenoxycarbonyl)-l-phenylalanine 2-methoxyethyl ester (S-NIFE) as a chiral derivatization reagent, sodium-adducted S-NIFE-AA ions were generated and resolved via DMS-MS. By using the optimized carrier gas with 0.2% 1-propanol as a modifier, baseline separation of 11 enantiomeric AA pairs was successfully achieved. Compared with existing reports on DMS-based chiral AAs separation, the method proposed in this work exhibits a better separation efficiency for chiral AAs pairs. This further demonstrates the potential of DMS for chiral AAs and other metabolite analyses in the future.

DOI: https://doi.org/10.1021/jasms.5c00054
Molecules. 2025 Apr 19. pii: 1838. [Epub ahead of print]30(8):

Quantitative Nuclear Magnetic Resonance for Small Biological Molecules in Complex Mixtures: Practical Guidelines and Key Considerations for Non-Specialists.

Eva Drevet Mulard, Véronique Gilard, Stéphane Balayssac, Gilles J P Rautureau.

  Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical approach that enables both the structural determination and precise quantification of small molecules, such as metabolites. However, achieving precise quantification with NMR involves more than simply comparing integrals derived from NMR peaks to a concentration reference; quantitative NMR (qNMR) is a distinct and specialized application within the field. To obtain absolute quantitative results, spectra must be acquired under strict experimental conditions. Unfortunately, these acquisition parameters can be challenging to implement experimentally and often require trade-offs that compromise high throughput or practicality. In such situations, alternative strategies based on relative quantification and advanced software tools offer valuable solutions. This review aims to provide non-specialists with the key concepts and methodologies required for accurate NMR-based quantification in biomedical research, focusing on practical guidelines and experimental considerations. Unlike prior reviews, it prioritizes accessibility and practical implementation for researchers outside the field, emphasizing key experimental workflows and applications in biological and clinical studies. It clarifies the distinctions between absolute and relative concentration determinations and emphasizes the critical importance of sample preparation, pulse sequence selection, and rigorous control of experimental parameters. Recent technological advancements, such as high-field spectrometers and cryoprobes, have significantly enhanced the sensitivity and accuracy of NMR, enabling the reliable detection of low-concentration metabolites. Quantitative NMR thus offers critical potential in elucidating metabolic processes, supporting drug development, and aiding disease diagnosis.

Keywords:  biochemistry; biomolecules; metabolites; metabolomics; quantitative NMR

DOI:  https://doi.org/10.3390/molecules30081838
Anal Biochem. 2025 May 07. pii: S0003-2697(25)00131-9. [Epub ahead of print] 115893

Absolute Quantitation of Neopterin as an Endogenous Pharmacodynamic Biomarker: The Successful Method Development, Validation, and Use of a Surrogate Matrix for Clinical Sample Analysis.

Ryan De Palma, Murali Matta, Jeffry Florian, Vikram Patel, Rodney Rouse.

  Biomarkers are playing an increasing role in the drug discovery and drug development process. Molecular biomarkers pose a bioanalytical challenge due to their low concentrations and endogenous presence. Inaccurate quantitation could lead to biased study results. Surrogate analyte and surrogate matrix approaches can be used to overcome the lack of a blank matrix and provide accurate quantitation. However, the suitability of the surrogate analyte or matrix must be established during method validation. Here we describe the development and validation of a surrogate matrix approach for the absolute quantitation of neopterin as a PD biomarker for use in an FDA-sponsored clinical study (NCT04183491). Matrix suitability was established through parallelism, precision and accuracy, and internal standard response. Parallelism experiments showed FBS, and serum had identical slopes 0.0145. Additionally, the difference in the X-intercepts was able to accurately predict the amount of endogenous neopterin (1.0 ng/mL). Inter-day accuracy across four surrogate QC levels ranged from 92.08 - 109.06% while precision ranged from 3.36 - 16.00%. Inter-day accuracy for the QCs in study matrix ranged from 96.81 - 108.86% and precision ranged from 4.13 - 6.01%. The internal standard response in FBS was only 6.9% different from serum. Additionally, there was no matrix effect, injection carryover, or cross-analyte interference observed. The method was then qualified for automated sample processing.

Keywords:  2018 FDA Bioanalytical Guidance; Surrogate matrix; endogenous biomarker; method validation; parallelism

DOI:  https://doi.org/10.1016/j.ab.2025.115893
Anal Chem. 2025 May 07.

Mapping the Edges of Mass Spectral Prediction: Evaluation of Machine Learning EIMS Prediction for Xeno Amino Acids.

Sean M Brown, Evan Allgair, Robin Kryštůfek.

Mass spectrometry is one of the most effective analytical methods for unknown compound identification. By comparing observed m/z spectra with a database of experimentally determined spectra, this process identifies compound(s) in any given sample. Unknown sample identification is thus limited to whatever has been experimentally determined. To address the reliance on experimentally determined signatures, multiple state-of-the-art MS spectra prediction algorithms have been developed within the past half decade. Here we evaluate the accuracy of the NEIMS spectral prediction algorithm. We focus our analyses on monosubstituted α-amino acids given their significance as important targets for astrobiology, synthetic biology, and diverse biomedical applications. Our general intent is to inform those using generated spectra for detection of unknown biomolecules. We find predicted spectra are inaccurate for amino acids beyond the algorithms training data. Interestingly, these inaccuracies are not explained by physicochemical differences or the derivatization state of the amino acids measured. We thus highlight the need to improve both current machine learning based approaches and further optimization of ab initio spectral prediction algorithms so as to expand databases for structures beyond what is currently experimentally possible, even including theoretical molecules.

DOI: https://doi.org/10.1021/acs.analchem.5c00286
Molecules. 2025 Apr 15. pii: 1774. [Epub ahead of print]30(8):

Emerging Mycotoxins in Cheese: Simultaneous Analysis of Aflatoxin M1, Aflatoxicol, and Sterigmatocystin by LC-MS/MS.

Maurizio Cossu, Andrea Sanna, Giuseppe Mangano, Giuseppe Ledda, Giannina Chessa, Pasquale Gallo, Antonio Vella, Ivan Pecorelli, Stefano Sdogati, Marilena Gili, Carlo Boselli.

  The presence of mycotoxins in cheese is a significant concern due to their potential health risks. Mycotoxins can contaminate cheese through two main routes: indirectly via contaminated animal feed, and/or directly, because of mold growth on dairy products. It has been reported that cheese may contain metabolites of aflatoxin B1 such as aflatoxin M1 (AFM1), aflatoxicol (AFL), and, its precursor, sterigmatocystin (STC). This study presents a reliable method for the simultaneous determination of AFM1, AFL, and STC in cheeses made from ovine, goat, or buffalo milk. The method was developed using single liquid extraction, clean-up by an immunoaffinity column (IAC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination. The method was subjected to initial validation according to EU regulations, which outline the required performance parameters and criteria of analytical methods for official food control. The limits of quantification (LOQs) of the method for AFM1, AFL, and STC are 2.0 ng/kg, 5.0 ng/kg, and 1.0 ng/kg, respectively. The method was applied in a study for the assessment of mycotoxin transfer from milk to cheeses and also their growth.

Keywords:  LC-MS/MS; aflatoxicol; aflatoxin M1; cheese; emerging mycotoxins; sterigmatocystin

DOI:  https://doi.org/10.3390/molecules30081774
Rapid Commun Mass Spectrom. 2025 Aug 30. 39(16): e10062

Multi-Plug Filtration Purification Combined With Gas Chromatography-Tandem Mass Spectrometry for the Detection of 107 Pesticides in Livestock and Poultry Meat.

Xuwei Guo, Youzhi Su, Jun Liu, Hongqin Lei, Fang Li, Yanmei Li.

   RATIONALE: With the global rise in foodborne diseases, excessive pesticide residues in livestock and poultry meat have emerged as a critical food safety issue. China is a large consumer of livestock and poultry meat, and the traditional national method standard can only detect a few or one type of pesticide in these meats. Establishing a method for the simultaneous detection of multiple types of pesticides in these meats can effectively protect people's health and safety.
METHODS: The extraction process was done using acetonitrile direct extraction, followed by clean-up through multi-plug filtration. Separation was achieved using gas chromatography on an HP-5MS capillary column with a programmed temperature gradient, and detection was performed via mass spectrometry in multiple reaction monitoring modes.
RESULTS: The linear ranges of 107 analytes exhibited strong correlation coefficients, all exceeding 0.9901, with limits of quantification spanning from 1.0 to 15.2 μg/kg. The average recoveries, inter-day relative standard deviations (inter-day RSDs), and intra-day relative standard deviations (intra-day RSDs) of the 94 analytes ranged from 70.2% to 120.0%, 4.0% to 16.0%, and 4.5% to 16.8%, respectively, at the three different spiked levels.
CONCLUSION: A multi-plug filtration purification combined with gas chromatography-tandem mass spectrometry method was successfully applied to the detection of 107 pesticide residues in livestock and poultry meat, which is simple, reliable, and reproducible. It provides a comprehensive solution for the detection of pesticide residues in livestock and poultry meat.

Keywords:  GC–MS/MS; livestock and poultry meat; multi‐plug filtration purification; pesticide residues

DOI:  https://doi.org/10.1002/rcm.10062
J Sep Sci. 2025 May;48(5): e70163

A HILIC-IM-MS-Based Pharmacometabodynamic Study of the Effects of Orally Administered Gefitinib on the Polar Urinary Metabolic Phenotypes of C57Bl6 Mice.

Adam King, Lee A Gethings, Robert S Plumb, Ian D Wilson.

  The effects of the anilinoquinazoline tyrosine kinase inhibitor (TKI) gefitinib on the polar urinary metabolome of mice following oral administration (50 mg/kg) were studied over the 24 h period post dose. Analysis was performed using a hydrophilic interaction liquid chromatographic separation (HILIC) hyphenated to cyclic ion mobility (cIM) and mass spectrometry (MS). This investigation revealed numerous time-dependent changes in the polar urinary metabolic phenotype of gefitinib-dosed mice that mirrored the plasma concentrations and urinary excretion of the drug and its metabolites. These changes showed both relative increases and decreases in the amounts of various endogenous metabolites, including 8-hydroxydeoxyguanosine, thymidine, acetylcarnitine, isobutyrylcarnitine, myristoylglycine, 3-phenylpropionylglycine, cyclic AMP, 19-oxotestosterone, and 3'-asparagine-AMP. These changes were generally seen to be greatest at the time of the highest plasma and urinary concentrations of gefitinib. The concordance of these effects on the urinary metabolome with plasma/urine drug concentrations strongly implies a range of pharmacometabodynamic effects pointing to mechanism-based regulation of a number of endogenous metabolic pathways by gefitinib.

Keywords:  metabolic pathways; metabolite identification; polar metabolome; urinary metabotypes

DOI:  https://doi.org/10.1002/jssc.70163
Anal Bioanal Chem. 2025 May 05.

Development and analysis of 49 perfluoroalkyl and polyfluoroalkyl substances (PFASs) in contact lenses using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Yi-Chun Chiang, Chuan-Kai Yang, Sheng-Wei Wang, Shou-Chieh Huang, Min-Hsiung Pan, Su-Hsiang Tseng.

  Perfluoroalkyl and polyfluoroalkyl substances (PFASs) possess water-repellent, oil-repellent, and heat-resistant properties, making them widely used in consumer goods and industrial products. However, PFASs have been increasingly associated with health concerns, including reproductive toxicity, endocrine disruption, and thyroid disease. In 2023, the US consumer platform "Mamavation" reported organic fluorine levels ranging from 105 to 20,700 ppm in 18 contact lenses, raising concerns about the potential presence of PFASs in these products. Given the absence of established international testing methods and regulatory limits for PFASs in contact lenses, this study evaluated nine standard international methods, ultimately selecting 49 PFASs as target analytes after eliminating overlaps. To facilitate detection, novel extraction methods were developed for contact lenses and their lens care solutions. Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed for analysis, utilizing an analytical column (Atlantis Premier BEH C18 AX, 1.7 µm, 2.1 × 100 mm) and an isolator column (Atlantis Premier BEH C18 AX, 5 µm, 2.1 × 50 mm) to minimize background interference. Electrospray ionization in combination with multiple reaction monitoring mode enabled rapid analysis within 24 minutes. For contact lenses, the limit of quantification (LOQ) ranged from 1 to 400 ng/g, while for lens care solutions, the LOQ ranged from 0.04 to 0.8 ng/mL. When applied to 12 contact lens samples, the method detected 6:2 FTS (1H,1H,2H,2H-perfluorooctane sulfonic acid) in only one lens care solution at a concentration of 0.087 ng/mL. This study highlights the importance of further investigation into PFAS contamination in contact lenses and the need for international standardization in testing methodologies.

Keywords:  Contact lenses; Liquid chromatography-tandem mass spectrometry (LC–MS/MS); Perfluoroalkyl and polyfluoroalkyl substances (PFASs)

DOI:  https://doi.org/10.1007/s00216-025-05886-0
Anal Chem. 2025 May 07.

Three-Layer Electrospray Constructing Charged Microdroplets for Online Derivatization and Position Identification of Lipid C═C Bonds.

Yuan Cao, Chunlin Yue, Jinqi Yang, Lingyu Zhao, Jinchao Wei, Shan Shu, Yuanyuan Zhang, Yangyang Zhang, Zhenwen Zhao.

Numerous studies have demonstrated that charged microdroplets can significantly accelerate chemical reactions. In this work, we developed a novel three-layer electrospray ionization source (TL-ESI). This source generated charged microdroplets capable of facilitating the rapid epoxidation of unsaturated lipid C═C bonds with the derivatization reagent 3-chloroperbenzoic acid (m-CPBA) in an online process, achieving a conversion rate of up to 92.8%─a performance not achievable with conventional electrospray ionization source (ESI). Compared to conventional ESI, the TL-ESI effectively compartmentalized reactants, enabled precise control over the reaction process, and supported rapid online derivatization of lipid C═C bonds with m-CPBA. When integrated with tandem mass spectrometry (MS/MS), this source further enabled the localization of lipid C═C bond positions. The TL-ESI is characterized by its simplicity in design, ease of operation, and seamless integration with mass spectrometry (MS). These advantages make it an efficient and practical tool for locating C═C bond positions in unsaturated lipids, even within complex sample matrices. Additionally, this work highlights the potential of the TL-ESI as an innovative platform for accelerating chemical reactions via charged microdroplets, offering a valuable addition to the toolbox of modern analytical chemistry.

DOI: https://doi.org/10.1021/acs.analchem.4c06691
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 May 02. pii: S1570-0232(25)00186-2. [Epub ahead of print]1260 124632

Development and validation of a QuEChERS extraction-UPLC-MS/MS method for the simultaneous determination of chloramphenicol drugs in Laminaria japonica and Porphyra yezoensis.

Kaili Gao, Kunming Zheng, Juan Yuan, Qian Huang, Ruiqi Liu, Jiamin Tian, Xiaoping Wu, Xingang Meng.

  The analysis of the content of chloramphenicol (CAPs) in Laminaria japonica and Porphyra yezoensis samples is crucial for quality control of seaweed products. In this study, the analytical method using QuEChERS combined with Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) was established to separate and detect residues of chloramphenicol, thiamphenicol, and florfenicol in Laminaria japonica and Porphyra yezoensis. The extraction solvent used was acetonitrile, and a 1:1 mixture of graphitized carbon black (GCB) and C18 was employed as the purifying agent. Electrospray ionization (ESI) in negative ion mode with multiple reaction monitoring (MRM) was employed, coupled with the isotope internal standard quantification method. The results demonstrated good linearity within a range of 0.0005-0.050 μg/mL for chloramphenicol, thiamphenicol, and florfenicol with correlation coefficient (r) of 0.999. The method detection limit was 0.4 μg/kg, while the quantitation limit was 1.0 μg/kg. Furthermore, during validation experiments where concentrations ranging from 1.0 μg/kg to 10.0 μg/kg were added to samples, recovery rates for chloramphenicol ranged from 87.4 % to 103.4 %, with relative standard deviations (RSDs) between 2.1 % and 5 %. This developed method was applied to test twenty batches of samples in which one batch of Laminaria japonica was found to contain a chloramphenicol concentration of approximately 2.0 μg/kg. The developed method, which uses simple and cost-effective sample pretreatment techniques, is suitable for qualitative determination. It satisfies the experimental requirements in terms of accuracy and sensitivity when monitoring large quantities of marine algae products containing CAPs.

Keywords:  Chloramphenicol residues; Laminaria japonica; Porphyra yezoensis; QuEChERS; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2025.124632
Anal Chem. 2025 May 08.

LC-MS System for Collecting Time-Resolved Metabolomics Data of Cultured Cells.

Carly C Y Chan, Ryan A Groves, Raied Aburashed, Thomas Rydzak, Ian A Lewis.

Temporal metabolic dynamics are difficult to capture but are critical to understanding biology. We developed an automated liquid chromatography-mass spectrometry system that collects time-resolved metabolomics data from cultured cells, enabling sub-minute sequential sampling, broad metabolite coverage, robust metabolite identification, and parallel monitoring of up to 72 experimental conditions. Using this system, we identified temporal metabolic phenotypes of Escherichia coli and Proteus mirabilis that could not be captured from single time points.

DOI: https://doi.org/10.1021/acs.analchem.4c06697
J Sep Sci. 2025 May;48(5): e70157

Separation and Quantification of Cyclic di-AMP, -GMP, and Cyclic GAMP in Bacteria Using LC-MS/MS.

Silvio Uhlig, Kristina Vevik, Joke Van De Vyver, Inger Sofie Dragland, Lene A Grutle, Maria Pain, Roger Simm.

  This study summarizes the optimization of a selective method for UHPLC-separation and subsequent tandem mass spectrometric detection of adenosyl- and guanosyl-containing cyclic dinucleotides in bacteria. Cyclic dinucleotides are a class of bacterial second messenger molecules, and their cellular concentrations are tightly regulated by biosynthesis and enzymatic breakdown. Although bacteria, according to present knowledge, only produce 3',3'-linked cyclic dinucleotides, other nucleotide variants also exist, including structural isomers, which may lead to misidentifications. Mixtures of the 2',2'-, 2',3'-, and 3',3'-linked isomers of cyclic di-AMP, cyclic di-GMP, and cyclic GAMP were separated using an octadecylsilane-amide column. Subsequent tandem mass spectrometric detection was based on monitoring the adenosyl- or guanosyl-product ions for quantification, and additional monitoring of up to three product ions for verification. We show the presence of an unidentified putative structural isomer of cyclic di-AMP in several bacterial species. Cyclic di-AMP was quantified using an isotope dilution approach and 15N10-cyclic di-AMP as an internal standard, whereas instrument calibration for other variants was performed using matrix-matched calibration. The combined measurement uncertainty u' for the quantification of the nine cyclic dinucleotide variants in anion-exchanged bacterial extracts was 10%-41%, determined at an extract concentration of 12 nM. Our study also demonstrated the application of total protein measurements in resuspended, nucleotide-extracted, bacterial pellets to normalize nucleotide concentrations.

Keywords:  LC–MS/MS; anion‐exchange; bacteria; cyclic dinucleotides; isotope dilution

DOI:  https://doi.org/10.1002/jssc.70157
Biotechnol Appl Biochem. 2025 May 04. e2765

A High-Throughput RP-UPLC Assay for Simultaneous Determination of Telmisartan and Azelnidipine in Pharmaceutical Formulations.

BhargavaKrishna Pogaku, Ajitha Makula, Mohandass G, Sheeba Santhosh, Harish Gnanasambanthan, Sambit Sarkar, Rama Krishna Reddy Guduru.

  Telmisartan, an angiotensin II receptor blocker, and azelnidipine, a dihydropyridine calcium channel blocker, are often co-prescribed for the effective management of hypertension. The development of accurate and efficient analytical methods is crucial for ensuring the quality control of these combination formulations. This study presents a rapid and reliable reversed-phase ultra-performance liquid chromatography (RP-UPLC) assay for the simultaneous determination of telmisartan and azelnidipine in pharmaceutical formulations. Efficient chromatographic separation was achieved on an ACQUITY BEH C18 column (100 mm × 2.1 mm, 3 µm) using an isocratic mobile phase of pH 4.0 ammonium acetate buffer and acetonitrile (75:25% v/v) at a flow rate of 0.5 mL/min. Detection was performed at 260 nm, with telmisartan and azelnidipine eluting at 1.833 and 3.583 min, respectively. The method demonstrated good efficiency and minimal tailing (<1.5). Validation parameters, including accuracy, precision, linearity, specificity, and sensitivity, were determined according to International Council for Harmonisation guidelines. Calibration curves for both analytes exhibited excellent linearity (correlation coefficients > 0.999) over a concentration range of 50-150%. Recoveries from tablet dosage forms ranged from 98.0% to 102.0%, with assay values falling within the prescribed range. This validated that reversed-phase ultra-performance liquid chromatography assay offers a high-throughput approach suitable for routine quality control analysis of telmisartan and azelnidipine in pharmaceutical formulations.

Keywords:  azelnidipine; hypertension; reversed‐phase ultra‐performance liquid chromatography; simultaneous determination; telmisartan

DOI:  https://doi.org/10.1002/bab.2765
Rapid Commun Mass Spectrom. 2025 ;39(16): e10056

Onsite Analysis of Complex Samples Using Miniature Mass Spectrometry With Direct Sampling and Ionization.

Yiming Zou, Xuan Liu, Linzhi Du, Jiafan Yang, Zhishuang Liu, Zicheng Yuan, Lina Liu, Bicheng Yang, Zhe Shao, Igor A Popov, Bin Hu.

   RATIONALE: Miniature mass spectrometry (mini-MS) is a powerful method for onsite analysis of complex. The onsite sampling/ionization approach is critical in rapid screening of complex samples by mini-MS in field environments. Therefore, new development of direct ionization/sampling method coupled with mini-MS is needed for direct sample analysis.
METHODS: A direct sampling/ionization kit using wooden tip or plant tissue was developed to couple with mini-MS for onsite analysis of complex samples. Targeted analytes were detected and identified by mini-MS/MS.
RESULTS: Various raw samples including powders, liquid, and viscous samples were directly sampled and ionized using wooden tip. Direct pesticide detection in juice was used to further evaluate the analytical performances of the newly developed wooden tip kit, obtaining excellent sensitivity, reproducibility, and quantitation capacity for direct sample analysis. Furthermore, direct tissue analysis was also successful demonstrated by direct ionization, showing a fast, simple, and efficient method for onsite analysis of biological tissue.
CONCLUSIONS: Overall, our data showed that this direct sampling/ionization method using wooden tip or plant tissue coupled with mini-MS approach is simple, fast, and sensitive method for onsite direct analysis of raw samples in field environments.

Keywords:  direct ionization; direct sample analysis; miniature mass spectrometry; onsite analysis; wooden‐tip ESI

DOI:  https://doi.org/10.1002/rcm.10056
Biotechnol Bioeng. 2025 May 08.

A Comprehensive Model for Separating Systematic Bias and Noise in Metabolomic Timecourse Data-A Nonlinear B-Spline Mixed-Effects Approach.

Kathy Sharon Isaac, Stanislav Sokolenko.

  The simultaneous detection of tens to hundreds of metabolites in a single metabolomic timecourse sample offers a unique but often unrealized opportunity for quantification validation. An individual timecourse fit for each metabolite fundamentally convolutes measurement noise with systematic sample bias (stemming from, for example, variable sample dilution, extraction, and normalization). However, since systematic bias, by its definition, influences all metabolites within a sample in a similar fashion, it can be identified and corrected through the simultaneous fit of all detected metabolites in a single timecourse model. This study presents a nonlinear B-spline mixed-effects model as a convenient formulation capable of estimating and correcting such bias. The proposed model was successfully applied to real cell culture data and validated using simulated timecourse data perturbed with varying degrees of random noise and systematic bias. The model was able to accurately correct systematic bias of 3%-10% to within 0.5% on average for typical data. An R package for the correction model has been developed to facilitate model adoption and use. The proposed nonlinear B-spline mixed-effects formulation is general enough for application to a broad range of research areas beyond just cell culture metabolomics.

Keywords:  B‐spline; cell culture; metabolomics; mixed‐effects; systematic bias

DOI:  https://doi.org/10.1002/bit.29008
J Chromatogr A. 2025 May 05. pii: S0021-9673(25)00370-X. [Epub ahead of print]1753 466022

Rapid processing of liquid chromatography mass spectrometry data for compound screening and characterization in complex matrix based on python: with Meconopsis quintuplinervia Regel as an example.

Jiahao Yang, Xialin Zhu, Le Li, Fan Yang, Zhixin Tang, Haiqiang Jiang.

  Herbal extracts are time-consuming and laborious to analyse by traditional methods due to the complexity of their composition. Therefore, it is necessary to develop a rapid identification tool. In this study, a mass spectrometry data processing workstation was designed based on python language, which can rapidly screen and comprehensively characterize the components of herbal medicines. The main steps are as follows: (1) data acquisition by ultra-high-performance liquid chromatography coupled with Q-Exactive MS/MS (UPLC-QE-MS/MS); (2) conversion of mass spectrometry raw data file format and collection of key information; (3) enumeration of permutations and combinations of core structures and substituents based on the structural features of common flavonoids, and establishment of a database of precursor ions. (4) matching precursor ions from mass spectrometry data to a database and labeling mass spectrometry fragment ions based on structural information provided by the database;(5) establishment of a rating system for evaluating compound combinations based on mass spectrometry fragmentation information; (6) information on combinations of highly rated compounds was collected, analyzed for retention times and ionic behavior, and compared with online databases for further structural confirmation. Taking Meconopsis quintuplinervia Regel as an example, 140 flavonoids were finally preliminarily characterized. The results showed that the collection and identification of mass spectrometry information with the help of the mass spectrometry data processing workstation is fast and effective, and can be used for the rapid screening and characterization of compounds in traditional Chinese medicine or other complex matrix.

Keywords:  Data processing workstation; Flavonoids; Meconopsis quintuplinervia Regel; Python; UPLC-QE-MS/MS

DOI:  https://doi.org/10.1016/j.chroma.2025.466022
J Sep Sci. 2025 May;48(5): e70159

Quantification of Epigenetic DNA and RNA Modifications by UHPLC-MS/MS Technologies: New Concepts and New Improvements for the Special Collections.

Rui Zhang, Hailong Liu, Biao Bai, Hailin Wang.

Dynamic and reversible DNA and RNA modifications are essential for cell differentiation and development. Aberrant epigenetic modifications are closely associated with the occurrence and progression of diseases, serving as potential markers for cancer diagnosis and prognosis. Ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) offers distinct advantages in the qualitative and quantitative analysis of various modifications due to its sensitivity, specificity, and accuracy. This review provides a comprehensive overview of the current knowledge regarding the liquid chromatography-mass spectrometry (LC-MS) analysis of DNA and RNA modifications, including analytical procedures, advancements, and biological applications, with a focus on tracing the source of (N6-2'-deoxy-adenosine) 6mdA in eukaryotes. Additionally, we examine the integration of UHPLC-MS/MS with other separation techniques to achieve accurate quantification of modifications in specific regions, certain fragments, and free nucleosides.

DOI: https://doi.org/10.1002/jssc.70159
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Apr 29. pii: S1570-0232(25)00169-2. [Epub ahead of print]1259 124615

Simultaneous determination of Favipiravir and its active metabolite, Favipiravir Ribofuranosyl-5'-triphosphate, in plasma and buccal cells using HPLC.

Yuriko Kondou, Moeka Obataya, Takeshi Uchikura, Kenji Momo, Masaru Kato.

  Favipiravir, recently approved for the treatment of tickborne severe fever with thrombocytopenia syndrome, is a prodrug that can be metabolized in vivo into its active phosphorylated form. Given that the accurate pharmacokinetic analysis of favipiravir and its active metabolite is crucial for dosage adjustment and, hence, therapeutic efficacy optimization, we herein developed an HPLC-based method for the simultaneous quantification of favipiravir and its active metabolite in plasma and buccal cells. Separation within 17 min was achieved using a mixed-mode C18 column with an anion-exchange functionality as the stationary phase, a phosphate buffer (pH 6.38) as the mobile phase, and gentisic acid as the internal standard. Fluorescence-based detection (excitation/emission = 370/440 nm) enabled the quantification of both compounds within the therapeutic range of 10-500 μM without interference from endogenous biological substances. The developed method, which allows for the rapid and reliable simultaneous determination of the prodrug and its active metabolite, is expected to be useful for improving the therapeutic outcome of favipiravir while minimizing side effects.

Keywords:  Active metabolite; Buccal cell; Favipiravir; Plasma; Simultaneous analysis

DOI:  https://doi.org/10.1016/j.jchromb.2025.124615
Environ Int. 2025 May 04. pii: S0160-4120(25)00267-3. [Epub ahead of print]199 109516

Simultaneous profiling of mercapturic acids, glucuronic acids, and sulfates in human urine.

Jin Y Chen, Saurin R Sutaria, Zhengzhi Xie, Manjiri Kulkarni, Rachel J Keith, Aruni Bhatnagar, Clara G Sears, Pawel Lorkiewicz, Sanjay Srivastava.

  Humans are constantly exposed to both naturally-occurring and anthropogenic chemicals. Targeted mass spectrometry approaches are frequently used to measure a small panel of chemicals and their metabolites in environmental or biological matrices, but methods for comprehensive individual-level exposure assessment are limited. In this study, we applied an integrated library-guided analysis (ILGA) with ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) to profile phase II metabolites, specifically mercapturic acids (MAs), glucuronic acids (GAs), and sulfates (SAs) in human urine samples (n = 844). We annotated 424 metabolites (146 MAs, 171 GAs, 107 SAs) by querying chromatographic features with in-house structural libraries, filtering against fragmentation patterns (common neutral loss and ion fragment), and comparing mass spectra with in-silico fragmentations and external spectral libraries. These metabolites were derived from over 200 putative parent compounds of exogenous and endogenous sources, such as dietary compounds, benzene/monocyclic substituted aromatics, pharmaceuticals, polycyclic aromatic hydrocarbons, bile acids/bile salts, and 4-hydroxyalkenals associated with lipid peroxidation process. Further, we performed statistical analyses on 214 metabolites found in more than 75% of samples to examine the association between metabolites and demographic characteristics using integrated network analysis, principal component analysis (PCA), and multivariable linear regression models. The network analysis revealed four distinct communities of 37 positively correlated metabolites, and the PCA (using the 37 metabolites) presented 4 principal components that meaningfully explained at least 80% of the variance in the data. The multivariable linear regression models showed some positive and negative associations between metabolite profiles and certain demographic variables (e.g., age, sex, race, education, income, and tobacco use).

Keywords:  Endobiotics; Glucuronic acids; Integrated Library-Guided Analysis; LC-HRMS; Mercapturic acids; Sulfates; Xenobiotics

DOI:  https://doi.org/10.1016/j.envint.2025.109516
J Sep Sci. 2025 May;48(5): e70156

Biomonitoring Livestock Antiparasitic Residues: Development of Fast Mass Spectrometric Methods for the Quantification of Ivermectin, Albendazole, and their Metabolites in Sheep Plasma and Feces.

Theodora E Barmpouni, Athanasios I Gelasakis, Eirini Tsimpouri, Serkos A Haroutounian, Kyriaki Machera, Konstantinos M Kasiotis.

  Antiparasitic substances are extensively used in livestock farming against common parasitic infections. Improper utilization of these medicines often leads to the development of antiparasitic resistance and the detection of residues across the food chain, posing significant challenges to animal health, food safety, and public health. The objective of this study was to monitor antiparasitics' pharmacokinetics after the administration of ivermectin and albendazole in farm sheep. To achieve this goal, a fast LC-ESI-MS/MS method was developed and validated for the simultaneous quantification of ivermectin, albendazole, and their metabolites in sheep plasma. Concurrently, a probe electrospray tandem mass spectrometry ultrafast method was also developed for the real-time monitoring of albendazole and its metabolites in plasma. In addition, the liquid chromatography methodology was extended and validated for the detection of ivermectin in sheep feces, offering a non-invasive alternative to tissue and biofluids analysis. The detection capability values estimated for the techniques developed herein for the studied compounds and their metabolites in plasma ranged from 0.1 to 50.2 ng/mL, while the respective values for ivermectin in feces ranged between 5.0 and 26.9 ng/g. Recoveries fluctuated from 64% to 119%, proving the competence of the methods. The application of these methods in sheep samples, after the administration of the antiparasitic substances, effectively indicated the distribution of their concentrations, although in some cases, the individual animal effect seemed to interplay with the expected results. Also, these methods can contribute to the adjustment of the farm animal antiparasitic protocols, promoting the most effective utilization of the respective drugs while minimizing the risk of developing antiparasitic resistance. In a broader context, they may aid the advancement of veterinary medicine, offering practical solutions for monitoring the drug residues in farm animals and the derived products, and mitigating the risks associated with the extensive and uncontrolled utilization of antiparasitic drugs.

Keywords:  albendazole; antiparasitic drugs; feces; ivermectin; mass spectrometry; plasma; probe electrospray; sheep

DOI:  https://doi.org/10.1002/jssc.70156