bims-metlip 2025-06-01 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–06–01
34 papers selected by
Sofia Costa, Matterworks

Mass-spectrometry based metabolomics: an overview of workflows, strategies, data analysis and applications.
Standard Addition as a Method for Quantitative Mass Spectrometry Imaging.
Development and Validation of the LC-MS/MS Method and Its Application for Pharmacokinetic Studies for the Simultaneous Estimation of Motixafortide and Filgrastim in rat Plasma.
Enhanced Structure-guided Molecular Networking Annotation Method for Untargeted Metabolomics Data from Orbitrap Astral Mass Spectrometer.
Development and Validation of an Analytical Method for the Determination of Select 4-Position Ring-Substituted Tryptamines in Plasma by Liquid Chromatography-Tandem Mass Spectrometry.
A rapid and sensitive UHPLC-MS/MS method for epalrestat detection in micro-volumes of human plasma for the first time.
An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of 17β-estradiol in human serum and plasma.
Candidate reference measurement procedure based on isotope dilution-two dimensional-liquid chromatography-tandem mass spectrometry for the quantification of androstenedione in human serum and plasma.
Analysis of Methylated Quaternary Ammonium Compounds Using Hydrophilic Interaction Liquid Chromatography Combined With Mass Spectrometry.
Extension of an Ultra-High Performance Liquid Chromatography-MS/MS Method for the Determination of C3 Epimers of 25-Hydroxyl Derivatives of Vitamin D in Human Plasma.
Improvement of an ultra-high liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of eleven amino-polycyclic aromatic hydrocarbons in human urine.
Differential Protein Precipitation-Based GalNAc-siRNA Sample Preparation with LC/MS Method Development Workflow in Plasma.
A novel screening workflow for nitazene analogs using LC-MS/MS precursor ion scan acquisition.
Stable 13C-glutamine Tracing Resolved Metabolomics for Cancer Metabolism Study.
Histamine metabolite to basal serum tryptase ratios in systemic mastocytosis and hereditary alpha tryptasemia using a validated LC-MS/MS approach.
Enabling pan-repository reanalysis for big data science of public metabolomics data.
Comparison of electrospray ionization-lithium adduct formation and atmospheric pressure chemical ionization for lipid analysis by normal phase liquid chromatography.
Chemical characteristics vectors map the chemical space of natural biomes from untargeted mass spectrometry data.
Ultra-sensitive UHPLC-MS/MS method for simultaneous quantification of 31 neurochemicals in zebrafish larvae and brain.
Automatised online SPE-LC-MS/MS method for the enantioselective determination of chiral β-blockers and antidepressants in wastewater.
Comparison of per- and polyfluoroalkyl substance (PFAS) soil extractions and instrumental analysis: large-volume injection liquid chromatography-mass spectrometry, EPA Method 1633, and commercial lab results for 40 PFAS in various soils.
Equidistant regions of interest - multivariate curve resolution for clean spectra from plant-metabolomics by gas chromatography with high resolution mass spectrometry.
QuEChERS and UPLC-MS/MS-Based Quantification of Human Plasma of Eight Nucleoside Reverse Transcriptase Inhibitors and Platinum Anticancer Drugs for Hepatocellular Carcinoma.
Determination of Multiple Fluorescent Brighteners in Human Plasma Using Captiva EMR-Lipid Clean-Up and LC-MS/MS Analysis.
High-Throughput Determination of Multiclass Chemical Hazards in Poultry Muscles and Eggs Using UPLC-MS/MS.
Quantitative analysis of DNDI-6174 using UPLC-MS/MS: A preclinical target site pharmacokinetic study.
Liquid Chromatography Coupled to High-Resolution Mass Spectrometry for High-Throughput Pesticide Residue Detection: Methodological Advances and Insights for Herbal Medicine Analysis.
TD-ESI-MS/MS for High-Throughput Screening of 13 Common Drugs and 4 Etomidate Analogs in Hair: Method Validation and Forensic Applications.
TopLib: Building and Searching Top-Down Mass Spectral Libraries for Proteoform Identification.
Developing a Cell Quenching Method to Facilitate Single Cell Mass Spectrometry Metabolomics Studies.
Comprehensive profiling of folates across polyglutamylation and one-carbon states.
Metabolomics Biomarkers for Breast Cancer: An updated perspective.
Data-independent profiling of phenolic constituents in shea using comprehensive two-dimensional liquid chromatography (RPLC × HILIC) hyphenated to cyclic ion mobility-quadrupole-time-of-flight mass spectrometry.
Multiplex LC-MS/MS assay for simultaneous quantification of artesunate and its active metabolite dihydroartemisinin with pyronaridine, proguanil, cycloguanil, and clindamycin in pharmacokinetic studies.

Proteome Sci. 2025 May 26. 23(1): 5

Mass-spectrometry based metabolomics: an overview of workflows, strategies, data analysis and applications.

Kosar Hajnajafi, Mohammad Askandar Iqbal.

   BACKGROUND: Metabolomics, a burgeoning field within systems biology, focuses on the comprehensive study of small molecules present in biological systems. Mass spectrometry (MS) has emerged as a powerful tool for metabolomic analysis due to its high sensitivity, resolution, and ability to characterize a wide range of metabolites thus offering deep insights into the metabolic profiles of living systems.
AIM OF REVIEW: This review provides an overview of the methodologies, workflows, strategies, data analysis techniques, and applications associated with mass spectrometry-based metabolomics.
KEY SCIENTIFIC CONCEPTS OF REVIEW: We discuss workflows, key strategies, experimental procedures, data analysis techniques, and diverse applications of metabolomics in various research domains. Nuances of sample preparation, metabolite extraction, separation using chromatographic techniques, mass spectrometry analysis, and data processing are elaborated. Moreover, standards, quality controls, metabolite annotation, software for statistical and pathway analysis are also covered. In conclusion, this review aims to facilitate the understanding and adoption of mass spectrometry-based metabolomics by newcomers and researchers alike by providing a foundational understanding and insights into the current state and future directions of this dynamic field.

Keywords:  Analytes; LC–MS; Mass spectrometry; Metabolic fingerprinting; Metabolites; Metabolomics

DOI:  https://doi.org/10.1186/s12953-025-00241-8
Anal Chem. 2025 May 27.

Standard Addition as a Method for Quantitative Mass Spectrometry Imaging.

Lucie Davidová, Ingela Lanekoff.

In mass spectrometry imaging (MSI), analytes are desorbed and ionized directly from a complex and unique chemical microenvironment in each pixel, which makes their quantification challenging. Matrix effects have been addressed by the use of isotopically labeled internal standards (IS), either included in the solvent or sprayed over the tissue section, for pixel-by-pixel relative quantification. However, in addition to requiring preselection, isotopically labeled IS may be costly or unavailable. Here, we introduce a novel approach for quantification in MSI, based on the standard addition method. We report a workflow for both acquiring and processing quantitative data. Furthermore, we compare the detected concentrations obtained by standard addition to the detected concentrations obtained using both IS quantification and external calibration. Finally, we show the applicability of using molecules extracted from tissue as an easily accessible standard mixture for standard addition quantification in MSI. The possibility of using analytical standards and readily available endogenous analytes as a source of calibration standards makes our standard addition-based quantitative approach cost-effective, accessible, and versatile.

DOI: https://doi.org/10.1021/acs.analchem.5c00549
Biomed Chromatogr. 2025 Jul;39(7): e70126

Development and Validation of the LC-MS/MS Method and Its Application for Pharmacokinetic Studies for the Simultaneous Estimation of Motixafortide and Filgrastim in rat Plasma.

Lurdhu Mary K, Naresh Podila, Afzal B Shaik, Jithendra Chimakurthy.

   INTRODUCTION: Motixafortide, a CXCR4 antagonist, and filgrastim, a granulocyte colony-stimulating factor, have shown significant potential in enhancing hematopoietic stem cell mobilization and improving cancer treatment outcomes when used in combination. However, critical challenges persist due to the absence of analytical methods and the necessity for reliable quantification of both drugs in biological matrices. This research aims to address these gaps by developing and validating a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of motixafortide and filgrastim in rat plasma.
EXPERIMENT: An isocratic mobile phase with acetonitrile and buffer was employed for separation, following protein precipitation with acetonitrile, using a C18 Waters X-Terra RP-18 column (150x4.6 mm,3.5 μm). Liquid chromatography-tandem mass spectrometry with an internal standard employs multiple reaction monitoring in electrospray ionization (positive ion mode) to monitor the transitions. The method was validated according to USFDA guidelines, assessing parameters such as selectivity, matrix effect, linearity, precision, accuracy, lower limit of quantification, and stability.
RESULTS: This method was successfully applied in pharmacokinetic studies, ensuring reliable and accurate quantification of co-administered motixafortide and filgrastim. These findings significantly contribute to optimizing therapeutic protocols and enhancing treatment outcomes for cancer patients.

Keywords:  Filgrastim; LC–MS/MS; Motixafortide; pharmacokinetics; rat plasma

DOI:  https://doi.org/10.1002/bmc.70126
Anal Chem. 2025 May 29.

Enhanced Structure-guided Molecular Networking Annotation Method for Untargeted Metabolomics Data from Orbitrap Astral Mass Spectrometer.

Xinxin Wang, Yao Chen, Zaifang Li, Ziquan Fan, Rui Zhong, Tian Liu, Xiangjun Li, Xin Lu, Guowang Xu.

The rapid, efficient, and accurate annotation of compounds in complex samples remains a significant challenge in metabolomics. The recently developed Orbitrap Astral mass spectrometer (MS) integrates a traditional quadrupole Orbitrap with a novel Astral mass analyzer, providing fast MS/MS scanning speed and high sensitivity. However, existing metabolomics annotation methods have not fully exploited the advanced capabilities of Astral MS. In this study, an enhanced structure-guided molecular networking (E-SGMN) method was developed, which is specifically tailored for the Orbitrap Astral mass spectrometer (MS). Unlike previous network annotation methods, E-SGMN extracted both previously detected metabolites and those potentially detected by Astral from the metabolome database, enabling more efficient and accurate network construction through structural similarity. E-SGMN expands annotation coverage by accurately improving network size, while minimizing the inclusion of irrelevant compounds, achieving a balance between annotation scale and accuracy. Validation results revealed that Astral-E-SGMN achieved an annotation coverage and accuracy of 76.84% and 78.08%, respectively, for a spiked plasma, significantly outperforming E-SGMN-Q Exactive HF (E-SGMN-QE HF). Notably, 5440 metabolite features from NIST SRM 1950 human plasma were annotated by Astral-E-SGMN, a 3.6-fold increase over QE HF-SGMN. Comparative analyses for six types of typical biological samples demonstrate that E-SGMN-Astral enhanced metabolite annotations by 3.7-44.2 times compared to conventional annotation methods, highlighting E-SGMN's wider metabolite annotation coverage. This method not only enhances annotation coverage, but also provides a transformative tool for understanding complex biological systems, holding significant potential for life science and clinical medicine.

DOI: https://doi.org/10.1021/acs.analchem.5c00314
J Anal Toxicol. 2025 May 26. pii: bkaf045. [Epub ahead of print]

Development and Validation of an Analytical Method for the Determination of Select 4-Position Ring-Substituted Tryptamines in Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

Ana Miguel Fonseca Pego, Malik Schoffner, Vamshikrishna Reddy Sammeta, Marilyn Naeem, David R Manke, Andrew Chadeayne, Grant C Glatfelter, Michael H Baumann, Marta Concheiro.

  4-Phosporyloxy-N, N-dimethyltryptamine (psilocybin) is a psychedelic tryptamine found in certain mushroom species that has shown efficacy in the treatment of various psychiatric disorders. In conjunction with the renewed interest in therapeutic effects of psychedelics, there has been an increase in psilocybin-like designer tryptamines appearing in non-medical drug markets. The present study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detecting and quantifying 4-position ring-substituted tryptamines and their 4-hydroxy metabolites in plasma. Specifically, we investigated 4-phosphoryloxy-N, N-dimethyltryptamine (psilocybin), 4-acetoxy-N, N-dimethyltryptamine (psilacetin), 4-propionoxy-N, N-dimethyltryptamine (4-Pro-DMT) and their shared metabolite 4-hydroxy-N, N-dimethyltryptamine (psilocin), along with 4-methyl carbonato-N, N-di-n-propyltryptamine (4-MeCO3-DPT) and its metabolite 4-hydroxy-N, N-di-n-propyltryptamine (4-HO-DPT). Mass spectrometry analysis employed electrospray ionization (ESI) in positive mode, with two multiple reaction monitoring (MRM) transitions per analyte. Plasma samples were acidified with ascorbic acid, followed by protein precipitation with acetonitrile. Linearity was achieved across a concentration range of 0.5-100 ng/mL for all analytes, except psilocybin, which displayed linearity from 5-100 ng/mL. Validation results demonstrated acceptable bias (±20%) and imprecision (<20%) for all analytes. Matrix effects, evaluated in 10 samples (CV < 18.3%), indicated minimal interference, although ion enhancement was observed for psilocin (31.9%) and psilocybin (45.7%). Extraction efficiency across all tryptamines was approximately 50%. The assay method was used to quantitate plasma samples from male rats treated with 1.0 mg/kg s.c. of the prodrug psilacetin, and collected before and 5, 30, 60, 120 and 240 min after injection. No psilacetin was detected, and psilocin concentrations ranged from non-detected up to 32.7 ng/mL. Overall, we successfully developed a sensitive and specific method for the detection and quantification of six tryptamines in plasma, providing a robust tool for future research and clinical applications.

Keywords:  4-MeCO3-DPT; 4-OH-DPT; 4-PrO-DMT; LC-MS/MS; Tryptamines; plasma; psilacetin; psilocin; psychedelics; therapeutic

DOI:  https://doi.org/10.1093/jat/bkaf045
Bioanalysis. 2025 May 26. 1-11

A rapid and sensitive UHPLC-MS/MS method for epalrestat detection in micro-volumes of human plasma for the first time.

Hong Chen, Kewei Liu, Jiawen Zhang, Xihong Zhao, Yatian Wang, Xiumei Lu, Feng Qin.

   AIM: A rapid and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for epalrestat detection in human plasma.
MATERIALS AND METHODS: A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source was used to quantify epalrestat and the internal standard epalrestat-d5 in the negative ion mode using multiple reaction monitoring (MRM). After acetonitrile-mediated protein precipitation, chromatographic separation was achieved using a reversed-phase C18 column (ACQUITY UPLC BEH, 2.1 × 50 mm, 1.7 μm; Waters Corp) with acetonitrile and 2 mM ammonium acetate in water as the mobile phase through gradient elution.
RESULTS AND CONCLUSION: The retention time of epalrestat was 0.92 min and the entire run time was only 2.00 min. The calibration curve was linear in the range 10.0-8.00 × 103 ng/mL (r ≥ 0.99). The within-run and between-run relative standard deviations (RSDs) were < 9.3%. The within-run and between-run relative errors (REs) ranged -8.4-4.2%. No significant matrix effects were observed and the recovery rate was high. The method was fully validated, including reinjection reproducibility in human plasma, and was successfully applied in a pharmacokinetic study, in which 100% incurred sample reanalysis met the criteria.

Keywords:  Epalrestat; UHPLC-MS/MS; detection; microsample; plasma

DOI:  https://doi.org/10.1080/17576180.2025.2509480
Clin Chem Lab Med. 2025 May 26.

An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of 17β-estradiol in human serum and plasma.

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

   OBJECTIVES: A new candidate isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based reference measurement procedure (RMP) has been developed for the accurate and precise quantification of 17β-estradiol (E2) in human serum and plasma covering a measurement range from 0.400 to 5,000 pg/mL (1.47-18,357 pmol/L). To address this broad range, two separate methods were created: a high sensitivity (HS) method for concentrations between 0.400 and 5.00 pg/mL (1.47-18.4 pmol/L) and a standard range (SR) method for concentrations between 5.00 and 5,000 pg/mL (18.4-18,357 pmol/L).
METHODS: As the primary reference material, E2 (CRM 6004-a) from the National Metrology Institute of Japan was used to ensure traceability to the international system (SI). A two-dimensional heart-cut LC approach was utilized for LC-MS/MS analysis, employing a supported liquid extraction sample preparation protocol for the SR method and a liquid-liquid extraction protocol followed by derivatization for the HS method. Assay validation was conducted following current guidelines. Selectivity and specificity were assessed using spiked serum samples, while potential matrix effects were evaluated through a post-column infusion experiment and comparison of standard line slopes. Precision, accuracy, and trueness were determined using an extensive 5-day protocol. Standard measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM), with three individual sample preparations performed on at least two different days. Equivalence with higher-order RMPs was demonstrated through participation in the CDC Steroid Hormones Standardization (HoSt) program.
RESULTS: The RMP enabled the quantification of E2 within the range of 0.400-5,000 pg/mL (1.47-18,357 pmol/L), demonstrating no interference from structurally related compounds and no evidence of matrix effects. The relative mean bias of the SR method ranged from -2.4 to 1.9 % across all levels, including secondary reference materials and spiked samples, whereas the HS method exhibited a mean bias ranging from -3.0 to 2.9 %. Expanded measurement uncertainties (k=2) for target value assignment ranged from 1.7 to 4.4 % for the SR method and were found to be ≤6.1 % for the HS method. The method's transferability was demonstrated in a comparison study at a second laboratory. Additionally, the candidate RMP exhibited excellent correlation and equivalence to JCTLM-listed RMPs through the CDC HoSt program.
CONCLUSIONS: In summary, the ID-LC-MS/MS-based RMP accurately quantifies E2. Its robust performance makes it suitable for standardizing routine assays and measuring individual patient samples, ensuring traceability.

Keywords:  17β-estradiol; SI units; isotope dilution-liquid chromatography-tandem mass spectrometry; qNMR characterization; reference measurement procedure; traceability

DOI:  https://doi.org/10.1515/cclm-2024-1255
Clin Chem Lab Med. 2025 May 23.

Candidate reference measurement procedure based on isotope dilution-two dimensional-liquid chromatography-tandem mass spectrometry for the quantification of androstenedione in human serum and plasma.

Myriam Ott, Friederike Bauland, Vanessa Fischer, Daniel Köppl, Alexander Gaudl, Andrea Geistanger, Uta Ceglarek, Manfred Rauh, Judith Taibon.

   OBJECTIVES: Androstenedione (ASD) is a biomarker used in the diagnosis of hyperandrogenism and adrenal hyperplasia. Therefore, accurate measurement of serum ASD is pivotal in clinical settings. We aimed to develop a novel highly selective reference measurement procedure (RMP) based on isotope dilution-two dimensional-liquid chromatography-tandem mass spectrometry (ID-2D-LC-MS/MS) for the quantification of ASD in human serum/plasma.
METHODS: To achieve high selectivity and sensitivity, a two-dimensional heart-cut LC approach for LC-MS/MS and a supported liquid extraction sample preparation protocol were employed. Matrix effects were evaluated through a post-column infusion experiment and comparison of standard line slopes. Precision and accuracy were determined via a multi-day validation experiment. Reproducibility was assessed by comparing results from two independent laboratories, and measurement uncertainty (MU) was evaluated in compliance with current guidelines.
RESULTS: Our novel RMP proved effective for measuring ASD concentrations ranging from 0.00800 ng/mL to 12.0 ng/mL (0.0279 nmol/L to 41.9 nmol/L) and demonstrated matrix-independence. The relative mean bias varied between 0.6 and 2.2 % across different matrices and concentration levels. Intermediate precision was observed to be between 1.2 and 1.9 %. The expanded measurement uncertainty for ASD target value assignment ranged from 1.7 to 2.5 %, irrespective of sample concentration. Equivalence to the JCTLM-listed RMP was evaluated through a method comparison study, revealing a high degree of agreement (r≥0.997). Additionally, by comparing results from two independent laboratories, the transferability and reproducibility of the RMP were confirmed.
CONCLUSIONS: This isotope dilution two-dimensional LC-MS/MS method can be used for the evaluation and standardization of routine assays and for measuring individual patient samples, ensuring traceability.

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

DOI:  https://doi.org/10.1515/cclm-2024-1135
Mass Spectrom Rev. 2025 May 29.

Analysis of Methylated Quaternary Ammonium Compounds Using Hydrophilic Interaction Liquid Chromatography Combined With Mass Spectrometry.

Iman Zarei, Ambrin Farizah Babu, Ville M Koistinen, Marjo Tuomainen, Anna Kårlund, Retu Haikonen, Marko Lehtonen, Kati Hanhineva.

  Liquid chromatography-mass spectrometry (LC-MS) is a powerful technique for the detection and quantification of methylated quaternary ammonium compounds (mQACs), such as acylcarnitines and methylated amino-acid-derived (betainized) compounds, in biological matrices. Due to their high polarity and permanent charge, mQACs present analytical challenges, particularly in achieving efficient chromatographic retention and resolution. Here, we focus on the application of hydrophilic interaction liquid chromatography combined with mass spectrometric (HILIC-MS), for the analysis of these compound classes in biological samples. We highlight practical considerations in their analysis, including their MS/MS fragmentation patterns and identification in positive electrospray mode (ESI)+, to support researchers working with mQACs in targeted or untargeted metabolomics studies.

Keywords:  acylcarnitines; betainized compounds; electrospray ionization (ESI); hydrophilic interaction liquid chromatography (HILIC); liquid chromatography‐mass spectrometry (LC–MS); metabolomics; methylated quaternary ammonium compound (mQAC)

DOI:  https://doi.org/10.1002/mas.21942
J Chromatogr Sci. 2025 May 07. pii: bmaf031. [Epub ahead of print]63(5):

Extension of an Ultra-High Performance Liquid Chromatography-MS/MS Method for the Determination of C3 Epimers of 25-Hydroxyl Derivatives of Vitamin D in Human Plasma.

Mohamed Abouzid, Julia Kerner, Aniceta Mikulska-Sauermann, Dorota Filipowicz, Matylda Resztak, Franciszek Główka, Leonid Kagan, Marta Karaźniewicz-Łada.

Current vitamin D quantification methods do not account for 25-hydroxyl epimers, which can falsely increase concentrations and mask actual deficiencies. Previously, we developed an ultra-high performance liquid chromatography-tandem mass spectrometry method to measure 25(OH)D3, 3-epi-25(OH)D3 and 25(OH)D2; here, we extended this method to include 3-epi-25(OH)D2. Analytes were separated using a Shimadzu UPLC with a Kinetex F5 column (100 × 2.1 mm, 2.6 μm). The mobile phase contained 0.1% formic acid in methanol and water (70:30, v/v). The internal standard, deuterated 25(OH)D3 and analytes were extracted with hexane. Detection was performed by a mass spectrometer equipped with a triple quadrupole after prior electrospray ionization. It demonstrated sufficient precision and spike recovery within and between days, with a coefficient of variation ≤15% and an error of determination ≤18%. The method exhibited linearity in the 2-100-ng/mL concentration range. The limits of quantification and limits of detection were 2 and 1 ng/mL, respectively. Extraction recoveries ranged from 70.05% to 97.13%. The matrix effect, carryover and dilution integrity were evaluated and met the FDA acceptance criteria. The stability of all metabolites in plasma was confirmed after 3 h of storage at room temperature and after three cycles of freezing at -80°C and thawing. Applying the method to clinical samples showed a high 25-hydroxyl epimer derived from vitamin D.

DOI: https://doi.org/10.1093/chromsci/bmaf031
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 May 19. pii: S1570-0232(25)00203-X. [Epub ahead of print]1262 124649

Improvement of an ultra-high liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of eleven amino-polycyclic aromatic hydrocarbons in human urine.

Haibin Li, Shiwei Cui, Xiaoying Zhou, Qian Zhou, Yanhao Zhang, Shusheng Zhang, Xue Tao, Xin Sun.

  Urinary amino-polycyclic aromatic hydrocarbons (amino-PAHs) are metabolites of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) and serve as biomarkers for assessing occupational exposure to nitro-PAHs, particularly in populations exposed to diesel exhaust. Current methods for detecting urinary amino-PAHs mainly rely on liquid-liquid extraction, which has limitations in covering a wide range of nitro-PAHs and often struggles to resolve interference from isomeric compounds. This study aimed to develop a reliable UPLC-MS/MS method incorporating solid-phase extraction (SPE) pretreatment for detecting 11 amino-PAHs in urine samples from workers exposed to nitro-PAHs. Briefly, urine samples were enzymatically hydrolyzed, purified using SPE extraction, and analyzed with an optimized UPLC-MS/MS method. Effective separation of the 11 amino-PAHs was achieved using reversed-phase HSS PFP columns with a gradient elution of 0.1 % formic acid in water and acetonitrile. Improved MS/MS conditions were established to achieve sufficient sensitivity using positive ion electrospray ionization (ESI), with limits of quantification (LOQs) ranging from 0.009 to 0.378 μg L-1. The method was optimized for linearity, accuracy, precision, and matrix effects, utilizing two isotopically labeled internal standards (1-Aminonaphthalene-d7 and 1-Aminopyrene-d9). This method enabled precise and accurate quantification of the 11 amino-PAHs, with a coefficient of variation (CV) of less than 11 % and recovery rates between 82.0 % and 106.9 %. The improved method provides a unique analytical approach that can be used for biomonitoring workers exposed to diesel exhaust.

Keywords:  Amino-polycyclic aromatic hydrocarbons; Diesel exhaust; Nitro-polycyclic aromatic hydrocarbons; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2025.124649
Anal Chem. 2025 May 26.

Differential Protein Precipitation-Based GalNAc-siRNA Sample Preparation with LC/MS Method Development Workflow in Plasma.

Youngjae Kim, Ting Ting Zhang, Hiroshi Sugimoto, Mark G Qian, Linlin Dong.

Liquid chromatography-mass spectrometry (LC/MS) plays a crucial role in the quantification of small interfering RNAs (siRNAs) in biological matrices. However, the recovery of siRNA from complex biological matrices remains a significant challenge. Focusing on liver-targeted N-acetylgalactosamine (GalNAc)-siRNA conjugates, the primary extraction methods currently used are solid-phase extraction (SPE) and hybridization. While both methods have advantages, SPE recovery can vary depending on the analyte and is costly, whereas the hybridization method requires specific reagents, limiting its applicability. To address these challenges, we developed a novel extraction method for LC/MS bioanalysis of GalNAc-siRNA. Our innovative approach uses a differential protein precipitation method with an optimized organic solvent mix to remove large, high-abundance plasma proteins as precipitates while preserving the GalNAc-siRNAs in the liquid phase. The workflow was optimized to identify the most intense MS/MS transitions, and LC-MS/MS parameters were fine-tuned using high-resolution Orbitrap and QTRAP hybrid mass spectrometers for the highly sensitive detection of targeted siRNA molecules. This approach achieved a lower limit of quantification in the single-digit ng/mL range for four FDA-approved GalNAc-siRNAs (Givosiran, Lumasiran, Inclisiran, and Vutrisiran) and a major Givosiran metabolite, AS(N-1)3'. The applicability of this approach was successfully demonstrated by analyzing plasma samples from an in vivo rat study involving three molecules (Givosiran, Givosiran AS(N-1)3', and Inclisiran). This method is straightforward, robust, highly sensitive, and cost-effective and should be readily adaptable for the bioanalysis of diverse GalNAc-siRNAs and, potentially, for late-stage sample analyses.

DOI: https://doi.org/10.1021/acs.analchem.5c01587
J Anal Toxicol. 2025 May 26. pii: bkaf046. [Epub ahead of print]

A novel screening workflow for nitazene analogs using LC-MS/MS precursor ion scan acquisition.

Amanda L Pacana, Britni N Skillman.

A persistent problem in the detection of novel psychoactive substances (NPS) is the inability of traditional screening methodologies to rapidly adapt to evolving drug trends. As such, high-resolution mass spectrometry (HRMS) screening methods have gained popularity in recent years for the ability to use non-targeted acquisition to detect a wide variety of compounds without necessarily returning to method development. However, these instruments may be unattainable for some forensic laboratories due to the associated high capital costs. The described method provides an alternative screening method using precursor ion scan (PIS) acquisition on a liquid chromatography tandem mass spectrometry (LC-MS/MS) platform to screen for nitazene analogs. Four ions were evaluated (m/z 72.1, 98.0, 100.1, and 112.1) for D0 analytes and one ion (m/z 104.1) for the metodesnitazene-D4 internal standard. Using a liquid-liquid extraction in whole blood, the method was validated with a 0.5 ng/mL limit of detection and 1.0 ng/mL administrative cutoff. Observed matrix effects did not affect limit of detection and there was no demonstration of carryover or interferences. As a proof-of-concept study, authentic (n = 3) and blind fortified (n = 20) samples were evaluated using this method, which was able to identify all nitazenes with no false negatives or positives. Several nitazenes not initially included in the scope of method development or validation were also presumptively identified. To accommodate this novel instrumental analysis, a workflow is also proposed to assist in the identification of known and emerging nitazene analogs. LC-MS/MS is widely available among forensic laboratories and presents a viable alternative to HRMS screening for nitazene analogs when operated in PIS acquisition, in such cases that HRMS is unavailable for assessing emerging NPS threats.

DOI: https://doi.org/10.1093/jat/bkaf046
Bio Protoc. 2025 May 20. 15(10): e5322

Stable 13C-glutamine Tracing Resolved Metabolomics for Cancer Metabolism Study.

Yaogang Zhong, Liqing He, Xinmin Yin, Logan Mazik, Xiang Zhang, Deliang Guo.

  Stable isotopes have frequently been used to study metabolic processes in live cells both in vitro and in vivo. Glutamine, the most abundant amino acid in human blood, plays multiple roles in cellular metabolism by contributing to the production of nucleotides, lipids, glutathione, and other amino acids. It also supports energy production via anaplerosis of tricarboxylic acid cycle intermediates. While 13C-glutamine has been extensively employed to study glutamine metabolism in various cell types, detailed analyses of specific lipids derived from 13C-glutamine via the reductive carboxylation pathway are limited. In this protocol, we present a detailed procedure to investigate glutamine metabolism in human glioblastoma (GBM) cells by conducting 13C-glutamine tracing coupled with untargeted metabolomics analysis using liquid chromatography-mass spectrometry (LC-MS/MS). The method includes step-by-step instructions for the extraction and detection of polar metabolites and long-chain fatty acids (LCFAs) derived from 13C-glutamine in GBM cells. Notably, this approach enables the distinction between isomers of two monounsaturated FAs with identical masses: palmitoleic acid (16:1n-7) (cis-9-hexadecenoic acid) and palmitelaidic acid (16:1n-7) (trans-9-hexadecenoic acid) derived from 13C-glutamine through the reductive carboxylation process. In addition, using this protocol, we also unveil previously unknown metabolic alterations in GBM cells following lysosome inhibition by the antipsychotic drug pimozide. Key features • Methods for analyzing the flux of the stable isotope 13C-glutamine in cancer cells and identifying its derived polar metabolites and long-chain fatty acids (LCFAs). • Distinguishes isomers of long-chain fatty acids, such as palmitoleic acid (16:1n-7) (cis-9-Hexadecenoic acid) and palmitelaidic acid (16:1n-7) (trans-9-Hexadecenoic acid), which share the exact same mass. • The method is utilized to investigate glutamine metabolism reprogramming in cancer cells following lysosome inhibition.

Keywords:  13C-glutamine; GBM cells; LC–MS/MS; Long-chain fatty acids; Lysosome; Pimozide; Polar metabolites

DOI:  https://doi.org/10.21769/BioProtoc.5322
Clin Chem Lab Med. 2025 May 22.

Histamine metabolite to basal serum tryptase ratios in systemic mastocytosis and hereditary alpha tryptasemia using a validated LC-MS/MS approach.

Abdulrazzaq Alheraky, Claude P Van Der Ley, Martijn Van Faassen, Saskia K Klein, Hanneke N G Oude Elberink, Caroline Roozendaal, Michel J Vos, André B Mulder, Ido P Kema.

   OBJECTIVES: Histamine, mainly produced in mast cells (MC), plays a key role in allergy and inflammation. Measuring its urinary metabolites, N-methylhistamine (NMH) and 1-methyl-4-imidazoleacetic acid (MIMA), is essential in assessing histamine-related pathologies. Patients with concurrent systemic mastocytosis (SM) and hereditary alpha tryptasemia (HαT) may show increased MC mediator-related symptom severity. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify histamine, NMH, and MIMA, and explored their correlation with basal serum tryptase (BST) levels.
METHODS: Using an in-matrix double derivatization, enhancing extraction, we analyzed urinary histamine, NMH, and MIMA with an online solid-phase extraction LC-MS/MS system. Analytical method validation assessed recovery, imprecision, and detection limits. For clinical validation, correlation analysis between BST levels, NMH, and MIMA in SM and HαT patients was performed.
RESULTS: The assay demonstrated recoveries>98 %, imprecision<3 %, and limits of quantification at 2.0 nmol/L for histamine, 0.53 nmol/L for NMH, and 0.011 μmol/L for MIMA. Patients with a combination of SM and HαT showed a 2.6-3.6 fold increase in BST compared to those with SM alone. A BST/NMH ratio>0.129 predicted HαT with 91.3 % sensitivity and 85.6 % specificity, and a BST/MIMA ratio>7.46 predicted HαT with 89.9 % sensitivity and 86.0 % specificity, independent of SM status.
CONCLUSIONS: Our LC-MS/MS method provides highly accurate and efficient quantification of histamine, NMH, and MIMA. Integrating BST/NMH and BST/MIMA ratios in diagnostic protocols enhances detection of HαT in MC-related disorders, supporting improved diagnostics and tailored patient management.

Keywords:  N-methylhistamine 1-methyl-4-imidazoleacetic acid; basal serum tryptase; hereditary alpha tryptasemia; histamine; systemic mastocytosis

DOI:  https://doi.org/10.1515/cclm-2025-0189
Nat Commun. 2025 May 24. 16(1): 4838

Enabling pan-repository reanalysis for big data science of public metabolomics data.

Yasin El Abiead, Michael Strobel, Thomas Payne, Eoin Fahy, Claire O'Donovan, Shankar Subramamiam, Juan Antonio Vizcaíno, Ozgur Yurekten, Victoria Deleray, Simone Zuffa, Shipei Xing, Helena Mannochio-Russo, Ipsita Mohanty, Haoqi Nina Zhao, Andres M Caraballo-Rodriguez, Paulo Wender P Gomes, Nicole E Avalon, Trent R Northen, Benjamin P Bowen, Katherine B Louie, Pieter C Dorrestein, Mingxun Wang.

Public untargeted metabolomics data is a growing resource for metabolite and phenotype discovery; however, accessing and utilizing these data across repositories pose significant challenges. Therefore, here we develop pan-repository universal identifiers and harmonized cross-repository metadata. This ecosystem facilitates discovery by integrating diverse data sources from public repositories including MetaboLights, Metabolomics Workbench, and GNPS/MassIVE. Our approach simplified data handling and unlocks previously inaccessible reanalysis workflows, fostering unmatched research opportunities.

DOI: https://doi.org/10.1038/s41467-025-60067-y
J Chromatogr A. 2025 May 21. pii: S0021-9673(25)00406-6. [Epub ahead of print]1756 466058

Comparison of electrospray ionization-lithium adduct formation and atmospheric pressure chemical ionization for lipid analysis by normal phase liquid chromatography.

Sonia Abreu, Bastien Prost, Audrey Solgadi, Pierre Chaminade.

  We previously published a normal-phase liquid chromatography (NPLC) method for separating thirty classes of lipids. The non-polar solvents used required coupling with APCI-MS or APPI-MS ionization sources, which cause significant fragmentation and do not always allow the observation of pseudo-molecular ions. This is particularly true for molecular species such as sterol esters (SE), triacylglycerols (TG), and acylsterol glucosides (ASG). Additionally, the ionization of monoacylglycerols (MG) and lysophosphatidylcholines (LPC) is low, which can create challenges in analysis. In this study, we enabled the hyphenation of NPLC with an ESI ionization source via a post-column addition of lithium chloride (LiCl) in water-isopropanol. Optimization was performed using a mixture of standards comprising 21 lipid classes distributed over the entire chromatogram and then applied to wheat and soya lipid extracts. Data from NPLC-ESI+-MS-lithium adduct formation were compared with NPLC-APCI+-MS. As a result, the intensities of free fatty acids, MG, and LPC ESI+-MS were significantly improved. For low- and medium-polarity lipids, the formation of lithium adducts provided access to molecular species. MS² and MS³ fragmentation provide structural information to identify the nature of fatty acids and the mass of sterol nuclei. However, using ESI+-MS, squalene (SQ), cholesterol (Chol), and acyl-monogalactosyldiacylglycerol (acyl-MGDG) were not observed, and some phospholipids (PL) show the coexistence of several adducts, making data processing complex. This work evidences the complementarity of the two approaches.

Keywords:  Atmospheric pressure chemical ionization; Corona CAD®; Electrospray ionization; LC-HRMS; Lipids; Lithium adducts; Normal-phase liquid chromatography

DOI:  https://doi.org/10.1016/j.chroma.2025.466058
J Cheminform. 2025 May 26. 17(1): 82

Chemical characteristics vectors map the chemical space of natural biomes from untargeted mass spectrometry data.

Pilleriin Peets, Aristeidis Litos, Kai Dührkop, Daniel R Garza, Justin J J van der Hooft, Sebastian Böcker, Bas E Dutilh.

  Untargeted metabolomics can comprehensively map the chemical space of a biome, but is limited by low annotation rates (< 10%). We used chemical characteristics vectors, consisting of molecular fingerprints or chemical compound classes, predicted from mass spectrometry data, to characterize compounds and samples. These chemical characteristics vectors (CCVs) estimate the fraction of compounds with specific chemical properties in a sample. Unlike the aligned MS1 data with intensity information, CCVs incorporate the chemical properties of compounds, allowing chemical annotation to be used for sample comparison. Thus, we identified compound classes differentiating biomes, such as ethers which are enriched in environmental biomes, while steroids enriched in animal host-related biomes. In biomes with greater variability, CCVs revealed key clustering compound classes, such as organonitrogen compounds in animal distal gut and lipids in animal secretions. CCVs thus enhance the interpretation of untargeted metabolomic data, providing a quantifiable and generalizable understanding of the chemical space of natural biomes.

Keywords:  Bioinformatics; Cheminformatics; Computational metabolomics; Earth microbiome; Mass spectrometry; Nontargeted screening; Untargeted metabolomics

DOI:  https://doi.org/10.1186/s13321-025-01031-2
Talanta. 2025 May 14. pii: S0039-9140(25)00824-0. [Epub ahead of print]295 128334

Ultra-sensitive UHPLC-MS/MS method for simultaneous quantification of 31 neurochemicals in zebrafish larvae and brain.

Irene Romero-Alfano, Marija Stevanović, Júlia Goyenechea, Eva Prats, Carlos Barata, Demetrio Raldúa, Cristian Gómez-Canela.

  The precise determination of neurotransmitters and their metabolites in biological matrices is critical for research on neurological disorders, including those originated by the exposure to neurotoxic chemicals. This study presents an optimized liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for 31 neurochemicals, including neurotransmitters, their metabolites and precursors. The method is aimed at achieving lower limits of detection (LOD) and quantification (LOQ) compared to those currently available, while simultaneously expanding the number of compounds analyzed. The proposed approach achieves instrumental detection limits (IDLs) from 0.02 to 7.2 pg, except for homovanillic acid showing a value of 150 pg, with recovery values across all target analytes comprised between 50 and 140 % in zebrafish larvae and 55 and 150 % in adult zebrafish brain, when using internal standard calibration. Overall, the optimized method represents a significant improvement over previous methods, making it robust, accurate and sensitive enough for applications in studies requiring detailed analysis of neurochemicals in complex biological samples, such as zebrafish larvae and adult zebrafish brain. Adult zebrafish brain samples from adults intraperitoneally exposed to 5 doses of 1-methyl-4-phenyl,6-tetrahydropyridine (MPTP, 100 mg/kg-1 body weight (bw)) were accurately analyzed using the optimized UHPLC-MS/MS method, which provided insights into neurochemical alterations induced by this neurotoxin.

Keywords:  Adult zebrafish brain; Behavior; MPTP exposure; Neurotransmitters analysis; Parkinson's disease; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.talanta.2025.128334
Anal Chim Acta. 2025 Aug 01. pii: S0003-2670(25)00546-X. [Epub ahead of print]1361 344152

Automatised online SPE-LC-MS/MS method for the enantioselective determination of chiral β-blockers and antidepressants in wastewater.

Marina Arenas, Julia Martín, Juan Luis Santos, Irene Aparicio, Esteban Alonso.

   BACKGROUND: Pharmacologically active compounds are emerging pollutants of greatest concern because of their continuous release to the aquatic media and potential effects on non-target organisms. Nevertheless, in spite that many pharmaceuticals are chiral compounds which enantiomers may have different environmental behaviour and effects, their enantiomeric determination has been scarcely evaluated. This fact can be explained by the great challenge to overcome when developing an analytical method for the individual determination of compounds with the same physical-chemical properties, as it is the case of enantiomers.
RESULTS: In this work, an automatised method based on online solid phase extraction (SPE) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was optimised and validated for the enantiomeric determination of highly prescribed (β-blockers (atenolol, metoprolol and propranolol), antidepressants (citalopram, fluoxetine, sertraline, duloxetine and venlafaxine) and two of their metabolites in influent and effluent wastewater. Three of them (metoprolol, citalopram and venlafaxine) have been recently included in the European Union Directive 2024/3019 as substances that should be measured in wastewater. Method quantification limits in the range from 0.1 to 50 ng L-1 for most compounds. Accuracy ranged from 60.8 to 114 % and precision, expressed as relative standard deviation, was lower than 12.5 % for all the compounds. Method application to wastewater samples revealed the presence of the target compounds at concentrations between 1.06 and 1213 ng L-1 and a preferential degradation of some enantiomers: S-(-)-atenolol, metoprolol-E1, venlafaxine-E1 and O-desmethylvenlafaxine-E1.
SIGNIFICANCE AND NOVELTY: This method is the first one for online SPE-chiral-LC-MS/MS determination of two therapeutic groups of chiral pharmaceuticals in wastewater. The method allows their automatised enantiomeric determination, including sample treatment, SPE column wash and conditioning, and LC-MS/MS determination, in 40 min with enantioresolution from 0.51 to 1.24. The online SPE method developed provides a fast way to obtain information about chiral compounds in wastewater reducing labour intensity, exposure to organic solvents, analyte loss and plastic waste.

Keywords:  Antidepressants; Chiral LC-MS/MS; Metabolites; Online SPE; Wastewater; β-Blockers

DOI:  https://doi.org/10.1016/j.aca.2025.344152
Environ Monit Assess. 2025 May 27. 197(6): 686

Comparison of per- and polyfluoroalkyl substance (PFAS) soil extractions and instrumental analysis: large-volume injection liquid chromatography-mass spectrometry, EPA Method 1633, and commercial lab results for 40 PFAS in various soils.

Morgan Eldridge, Jessica LaFond, Todd Anderson, Jennifer Guelfo, W Andrew Jackson.

  Quantifying per- and polyfluoroalkyl substances (PFAS) in soil is a crucial part of site evaluations. Several methods are currently used in commercial and academic labs to evaluate PFAS-affected soils, with differences in extraction solvent, extraction method, cleanup procedure, and instrumental analysis among laboratories. This study aims to compare the accuracy and efficiency of a legacy in-house soil extraction method for PFAS with EPA Method 1633 for sample extraction and analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS). An aqueous film-forming foam (AFFF)-impacted field soil (Soil A), a "clean soil" (Ottawa sand), and a certified reference soil were subjected to both extraction methods. Subsamples of these soils were also submitted to an accredited commercial lab. The commercial lab analyzed samples in accordance with EPA Method 1633 both for extraction and analysis. For comparison, our lab extracted the samples with both EPA Method 1633 and the in-house legacy soil extraction method, followed by a large-volume injection (LVI) adaptation of EPA Method 1633 instrumental analysis method. The EPA Method 1633 followed by LVI analysis quantified slightly more compounds without quality control flags than the legacy extraction method followed by LVI analysis for Soil A and the certified reference soil. Both in-house extractions had 76% of reportable compound concentrations within ± 15% relative standard deviation. The commercial small-volume injection results returned the least number of quality control flags, but quantified fewer compounds at low concentrations. Considering the time and cost of EPA Method 1633 and commercial analysis, this study supports the suitability of the legacy soil extraction method with LVI LC-MS/MS analysis for in-house soil analysis with comparable results to EPA Method 1633 as well as commercial analysis.

Keywords:  Large volume injection (LVI); Liquid chromatography tandem mass spectrometry (LC–MS/MS); Per- and polyfluoroalkyl substances (PFAS); Soil; Solid phase extraction (SPE)

DOI:  https://doi.org/10.1007/s10661-025-14138-8
Anal Chim Acta. 2025 Aug 08. pii: S0003-2670(25)00565-3. [Epub ahead of print]1362 344171

Equidistant regions of interest - multivariate curve resolution for clean spectra from plant-metabolomics by gas chromatography with high resolution mass spectrometry.

Zhuang Gao, Xianghui Yao, Yimin He, Shuyi Yu, Min He.

   BACKGROUND: The application of Gas Chromatography coupled with Orbitrap High-Resolution Mass Spectrometry (GC-Orbitrap HRMS) in plant metabolomics presents challenges including substantial data storage requirements, system complexity, and particularly the prevalence of co-eluted peaks in elution profiles. These limitations necessitate the integration of chemometric smart tools to extract interpretable mass spectra from complex chromatographic fingerprints. However, the direct applicability of multivariate resolution algorithms to GC-Orbitrap HRMS datasets remains limited. The existing deconvolution platforms exhibit operational constraints such as insensitivity toward embedded peaks and prerequisite data pretreatment.
RESULTS: Using Cyperus rotundus and Curcumae Radix as model medicinal plants, this study developed an equidistant Region of Interest (eROI) strategy to enhance metabolite annotation reliability. The eROI method systematically converts raw datasets into structured matrices with standardized dimensions, effectively preserving m/z reading for subsequent analytical phases. We implemented scenario-specific combinations of eROI with three multivariate curve resolution (MCR) techniques to isolate pure component spectra from co-eluted chromatographic features. Comprehensive metabolite annotation was achieved through systematic spectral interpretation of fragmentation patterns, supplemented by predictive mass spectral analysis when database matching proved inconclusive. Detailed validation using volatile-metabolomics fingerprints from both botanical species accompanies each methodological stage.
SIGNIFICANCE: Our work establishes a novel data reduction framework for GC-HRMS applications, enabling robust multi-modal chromatographic deconvolution. The integrated eROI-MCR methodology provides a validated solution for obtaining reliable qualitative and quantitative results in non-targeted plant-metabolomics studies.

Keywords:  Chemometric smart tools; Clean mass spectra; GC-Orbitrap HRMS; Metabolite annotation; eROI-MCR

DOI:  https://doi.org/10.1016/j.aca.2025.344171
Molecules. 2025 May 18. pii: 2204. [Epub ahead of print]30(10):

QuEChERS and UPLC-MS/MS-Based Quantification of Human Plasma of Eight Nucleoside Reverse Transcriptase Inhibitors and Platinum Anticancer Drugs for Hepatocellular Carcinoma.

Yanan Liu, Jiangning Peng, Yan Liang, Yilin Li, Xiaolan Zhen, Hui Li.

  Nucleoside reverse transcriptase inhibitors (NRTIs) and platinum-based chemotherapeutics are widely utilized in cancer treatment. Evidence suggests that drug plasma concentrations are closely linked to both therapeutic efficacy and the risk of adverse effects. Consequently, developing therapeutic drug monitoring (TDM) methods is essential. Here, an effective procedure utilizing QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) techniques for preparing samples and UPLC-MS/MS for simultaneously measuring eight NRTIs and platinum-based drugs in human plasma is described. Chromatographic separation was conducted with an Agilent Eclipse Plus C18 column (4.6 × 100 mm, 3.5 μm) with acetonitrile with 0.1% formic acid as Phase A and 0.1% formic acid in water as Phase B, achieving complete separation within 10 min. The target analytes-lamivudine, telbivudine, emtricitabine, entecavir, tenofovir, nedaplatin, oxaliplatin, and adefovir dipivoxil-exhibited strong linearity within the 10-1000 ng/mL and 1-100 ng/mL ranges, showing correlations (r2) ≥ 0.9962. The method demonstrated excellent accuracy (-6.72% to 7.82%) and selectivity (84.53%-110.49%), as well as satisfactory recovery and stability. Overall, this analytical approach can be used to detect the combination of eight NRTIs and platinum-based drugs in human plasma. This method enables plasma drug-level monitoring in real time, with applications for individualized treatment approaches.

Keywords:  NRTIs; QuEChERS; UPLC-MS/MS; hepatocellular carcinoma; human plasma; platinum

DOI:  https://doi.org/10.3390/molecules30102204
Toxics. 2025 Apr 28. pii: 352. [Epub ahead of print]13(5):

Determination of Multiple Fluorescent Brighteners in Human Plasma Using Captiva EMR-Lipid Clean-Up and LC-MS/MS Analysis.

Yubing Yan, Bowen Liang, Jiawen Yang, Qing Deng, Xiaoying Liang, Hui Chen, Bibai Du, Lixi Zeng.

  Fluorescent brighteners (FBs) are a class of chemicals extensively used in industrial and consumer products. Their environmental occurrences and potential health risks have raised significant concerns. However, the lack of analytical methods for FBs in human samples has hindered the accurate assessment of internal exposure levels. Addressing this gap, this study developed and validated a novel method for the simultaneous determination of 13 FBs at trace levels in human plasma using solid-phase extraction combined with HPLC-MS/MS. The method employed EMR-Lipid SPE columns, which can selectively adsorb phospholipids for plasma sample pre-treatment. Detection was achieved through positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) modes. The results showed that all 13 FBs exhibited good linearity within their respective ranges, with correlation coefficients (R2) greater than 0.992. The method quantitation limits (MQLs) of the FBs ranged from 0.012 to 0.348 ng/mL, and the spiked recovery rates ranged from 61% to 98%. The method was successfully applied to analyze 10 adult plasma samples, detecting 10 FBs with total concentrations ranging from 0.221 to 0.684 ng/mL. This study provides a reliable analytical method for determining FBs in human plasma, providing a basis for further research on human internal exposure to FBs and associated health risk assessments.

Keywords:  fluorescent whitening agent; human blood; human exposure; lipid removal; new pollutant; solid-phase extraction (SPE)

DOI:  https://doi.org/10.3390/toxics13050352
Foods. 2025 May 08. pii: 1660. [Epub ahead of print]14(10):

High-Throughput Determination of Multiclass Chemical Hazards in Poultry Muscles and Eggs Using UPLC-MS/MS.

Rong Chen, Lan Chen, Mingyue Du, Qiaozhen Guo, Ciping Zhong, Jing Zhang, Xiaoqin Yu.

  A high-throughput method for the determination of a variety of chemical hazards in poultry muscle and egg samples was established via ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-QqQ-MS). The sample preparation procedure was developed based on this quick, easy, cheap, effective, rugged, and safe (QuEChERS) method and validated for 280 chemical hazards potentially present in poultry products. The target compounds in poultry samples were extracted with a 1% formic acid-acetonitrile solution (15:85, v/v), and the metal ions in the matrix were chelated by adding ethylenediaminetetraacetic acid disodium salt (Na2EDTA). The supernatant was purified using Enhanced Matrix Removal (EMR) lipid sorbent. Chromatographic gradient separation was performed on an ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm) column with multiple reaction monitoring (MRM) under both negative- and positive-ion mode. Internal standard calibration or matrix-matched calibration was used for the quantitation. The results showed that good linearity was achieved for each target compound with correlation coefficients (R2) ≥ 0.99. The limits of detection (LODs) ranged from 0.05 to 10 µg/kg, and the acceptable limits of quantification (LOQs) were determined to be 0.1-20 µg/kg for all 280 compounds. Approximately 90% of the target compounds exhibited mean recoveries ranging from 60% to 120%, with relative standard deviations (RSDs) within 16.2%. This method can be used for the high-throughput rapid detection of prohibited drug residues in poultry eggs due to its easy operation and high accuracy. It was applied in real sample detection, and 43 chemicals including metronidazole were found in 211 poultry samples, with a concentration range of 0.11-638 μg/kg.

Keywords:  UPLC–MS/MS; chemical hazards; high-throughput detection; poultry products

DOI:  https://doi.org/10.3390/foods14101660
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 May 15. pii: S1570-0232(25)00206-5. [Epub ahead of print]1262 124652

Quantitative analysis of DNDI-6174 using UPLC-MS/MS: A preclinical target site pharmacokinetic study.

Wietse M Schouten, Katrien Van Bocxlaer, Hilde Rosing, Alwin D R Huitema, Jos H Beijnen, Jadel M Kratz, Charles E Mowbray, Thomas P C Dorlo.

  Leishmaniasis is a neglected parasitic infection that continues to pose a significant global health challenge, with currently limited effective treatment options. DNDI-6174 is a novel orally-active, investigational drug with antileishmanial properties. Herein, a novel ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify DNDI-6174 in relevant murine biomatrices, i.e., K2EDTA plasma and enzymatically-homogenized skin, spleen and liver to support the translational pharmacokinetic-pharmacodynamic model-informed drug development. The chromatographic system consisted of a gradient elution on a standard C18 column connected to a triple quadrupole MS, operating in positive ionization mode. Pre-processing of murine tissues with collagenase A led to a superior homogenization and analyte extraction compared to mechanical disruption. Human K2EDTA plasma served as a surrogate matrix, enabling accurate (bias between -12.0 % and 9.8 %) and precise (relative standard deviation (RSD) ≤ 12.5 %) quantification of DNDI-6174 in the various murine biomatrices. Sample processing with tert-methylbutyl ether resulted in a reproducible recovery between 70.0 % and 93.8 % (RSD ≤ 4.0 %) with an absolute matrix factor between 0.89 and 1.00 for all biomatrices. DNDI-6174 was stable under various conditions, including under tissue homogenization conditions, in all biomatrices investigated. This method was successfully applied in a translational study using a murine cutaneous leishmaniasis skin infection model to assess the target site pharmacokinetics of DNDI-6174, supporting its development as clinical candidate.

Keywords:  DNDI-6174; Leishmaniasis; Murine tissues; Target site pharmacokinetics; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2025.124652
Crit Rev Anal Chem. 2025 May 29. 1-15

Liquid Chromatography Coupled to High-Resolution Mass Spectrometry for High-Throughput Pesticide Residue Detection: Methodological Advances and Insights for Herbal Medicine Analysis.

Huiqin Pan, Guyu Zhao, Yannan Tan, Shui Miao, Xiuhong Mao, Shen Ji, Heng Zhou, Qing Hu.

  The safety evaluation of herbal medicines faces a critical challenge due to exogenous pesticide residues, where traditional detection methods struggle to address the dual complexity of highly variable phytochemical matrices and trace-level multi-residue contaminants. Recent regulatory shifts emphasizing large-scale detection have positioned liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) as a transformative solution. This review systematically examines cutting-edge LC-HRMS workflows tailored for complex herbal matrices, with three pivotal technical dimensions: (1) Matrix-specific optimization spanning from QuEChERS-based sample preparation to chromatographic separation protocols that reduce matrix interferences; (2) Intelligent data acquisition strategies balancing selectivity and coverage through adaptive MS/MS triggering and narrow-window fragmentation; (3) Integrated analytical frameworks combining targeted screening with expanding pesticide databases and non-targeted approaches leveraging retrospective HRMS data mining. While LC-HRMS has demonstrated exceptional performance in food safety domains, its application in herbal medicine analysis remains constrained by insufficient method harmonization and underutilized data potential. We critically evaluate how emerging techniques, including comprehensive two-dimensional liquid chromatography, ion mobility mass spectrometry, structure-informed parameter prediction, suspect screening based on biotransformation, and metabolomics-driven non-targeted screening, could overcome current limitations in compound identification confidence and pesticide coverage. By bridging technological advancements with the challenges faced in practical residue analysis of herbal medicines, this review provides actionable guidelines to empower researchers in developing robust, future-proof analytical schemes that meet evolving regulatory standards and public health expectations.

Keywords:  Herbal medicines; liquid chromatography-mass spectrometry; non-targeted analysis; pesticide residues; targeted analysis

DOI:  https://doi.org/10.1080/10408347.2025.2511140
Toxics. 2025 Apr 23. pii: 329. [Epub ahead of print]13(5):

TD-ESI-MS/MS for High-Throughput Screening of 13 Common Drugs and 4 Etomidate Analogs in Hair: Method Validation and Forensic Applications.

Meng Li, Jinbo Li, Binling Zhu.

  This study established a dual analytical workflow integrating thermal desorption-electrospray ionization-tandem mass spectrometry (TD-ESI-MS/MS) for rapid qualitative screening and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for confirmatory quantification of 17 psychoactive substances and metabolites across six classes (opioids, amphetamine-type stimulants, cocaine, ketamine-type drugs, cannabinoids, and etomidate analogs) in hair matrices. Validation of the TD-ESI-MS/MS method demonstrated its sensitivity (limits of detection: 0.1-0.2 ng/mg) and precision (<19.3%), with matrix effects controlled to <19.6%. The TD-ESI-MS/MS method achieved an analysis time of 1 min per sample, enabling high-throughput screening with a sensitivity >85.7% and a specificity >89.7% for the 17 analytes. UPLC-MS/MS confirmation validated the screening results with accuracy rates of 89.7-99.8%. An analysis of specimens confirmed positive identified etomidate analogs as the predominant psychoactive substances (73.6%), with a lower prevalence of amphetamine-type stimulants (12.5%), ketamine-type drugs (9.0%), and opioids (2.8%). The polydrug use patterns identified concurrent etomidate-amphetamine consumption (n = 5) and complex analog combinations (etomidate-isopropoxate-metomidate, n = 13), suggesting evolving abuse trends. Despite limitations in the temporal resolution and representativeness of the cohort, this study demonstrated the viability of TD-ESI-MS/MS for bridging forensic and public health priorities. Future work should focus on optimizing the durability of the ion source for TD-ESI and validating this method across diverse populations to enhance its generalizability.

Keywords:  TD-ESI-MS/MS; drugs of abuse; etomidate analogs; forensic applications; high-throughput screening

DOI:  https://doi.org/10.3390/toxics13050329
Anal Chem. 2025 May 29.

TopLib: Building and Searching Top-Down Mass Spectral Libraries for Proteoform Identification.

Kun Li, Haixu Tang, Xiaowen Liu.

Mass spectral library search is a widely used approach for spectral identification in mass spectrometry (MS)-based proteomics. While numerous methods exist for building and searching bottom-up mass spectral libraries, there is a lack of software tools for top-down mass spectral libraries. To fill the gap, we introduce TopLib, a new software package designed for building and searching top-down spectral libraries. TopLib utilizes an efficient spectral representation technique to reduce database size and improve query speed and performance. We systematically evaluated various spectral representation techniques and scoring functions for top-down spectral clustering and search. Our results demonstrate that TopLib is significantly faster and yields higher reproducibility in proteoform identification compared to conventional database search methods in top-down MS.

DOI: https://doi.org/10.1021/acs.analchem.4c06627
JACS Au. 2025 May 26. 5(5): 2379-2387

Developing a Cell Quenching Method to Facilitate Single Cell Mass Spectrometry Metabolomics Studies.

Shakya Wije Munige, Deepti Bhusal, Zongkai Peng, Dan Chen, Zhibo Yang.

  Single-cell mass spectrometry (SCMS) has emerged as a powerful tool for analyzing metabolites in individual cells, including live cells. However, cell metabolites have a rapid turnover rate, whereas maintaining metabolites' profiles of live cells during sample transport, storage, or extended measurements can be challenging. In this study, a cell preparation method, which integrates cell washing by volatile salt solution, rapid liquid nitrogen (LN2) quenching, freeze-drying in vacuum, and freezer storage at -80 °C, to preserve cell metabolites for SCMS measurement is discussed. Experimental results revealed that LN2 quenching preserved the overall cell metabolome, whereas storage at -80 °C for 48 h slightly changed the metabolite profiles in quenched cells. However, metabolites in unquenched cells were changed regardless of low-temperature storage. The influence of omission of quenching and low-temperature storage on cell metabolites and relevant pathways were investigated. Results from this work indicate that cell quenching is necessary, but low-temperature storage time should be minimized to preserve cell metabolites. The method developed in the current work can be readily adopted by SCMS techniques with storage remaining largely unaltered, allowing for extended SCMS studies.

Keywords:  Single-probe; cell quenching; freeze-drying; metabolomics; single-cell mass spectrometry; −80 °C storage

DOI:  https://doi.org/10.1021/jacsau.5c00327
Metabolomics. 2025 May 27. 21(3): 71

Comprehensive profiling of folates across polyglutamylation and one-carbon states.

Sevcan Erşan, Yu Chen, Junyoung O Park.

   INTRODUCTION: One-carbon metabolism is central to carbon fixation, methylation, and biosynthesis of amino acids, lipids, and nucleotides. Folates are organic cofactors that harbor one-carbon units and shunt them across these metabolic pathways. Despite its essentiality to all life forms, the diverse nature of folate species with various polyglutamylation and one-carbon states makes their measurement challenging.
OBJECTIVES: We aim to illuminate one-carbon metabolism by streamlining comprehensive profiling of folate polyglutamates.
METHODS: We analyze folate standards and cellular extracts containing diverse folates species by liquid chromatography-mass spectrometry (LC-MS).
RESULTS: We observe that Escherichia coli cells possess diverse folate polyglutamates with one to ten terminal glutamates. Interestingly, most folate polyglutamates form doubly charged ions as well as singly charged ions in LC-MS. Folates also undergo in-source fragmentation. The disparate fates of folates in MS make their quantitation prone to underestimation. Fragmentation by in-source collision-induced dissociation (CID) and LC separation circumvent this issue and facilitate robust and sensitive quantification of folates. In-source CID of folates generates reporter fragment ions that yield higher signals in the mass-to-charge ratio (m/z) range near the maximal mass resolution of Orbitrap MS. Our LC methods complement MS by effectively separating folates based on their polyglutamylation and one-carbon states.
CONCLUSION: Our metabolomics approach tailored to folate polyglutamates reveals multiple layers of one-carbon metabolism organized by the lengths of polyglutamate tails in folates. Our analytical workflow is broadly applicable to folate profiling across various cell types to advance our knowledge of one-carbon metabolism as well as biotechnology and medicine.

Keywords:  Folate; LC-MS; Metabolomics; One-carbon metabolism; Polyglutamylation

DOI:  https://doi.org/10.1007/s11306-025-02269-5
Clin Breast Cancer. 2025 May 08. pii: S1526-8209(25)00118-1. [Epub ahead of print]

Metabolomics Biomarkers for Breast Cancer: An updated perspective.

Christina Jane Vellan, Vijayakumar Natesan, Jaime Jacqueline Jayapalan.

  Breast cancer (BC) remains a significant global health concern, emphasising the need for accurate diagnostic and prognostic tools. Metabolomics, the study of small molecules involved in cellular processes, has emerged as a promising approach for identifying BC biomarkers. This review provides an updated overview of metabolomic biomarkers for BC, focusing on early detection, diagnosis, prognosis prediction, and subtype-specific markers. It covers the various sources of biological samples used in biomarker investigations and common methodologies such as liquid chromatography-mass spectrometry and nuclear magnetic resonance. This review also highlights key classes of metabolites, including amino acids, lipids, and carbohydrates, which exhibit consistent alterations in BC patients and are integral to crucial oncogenic pathways, such as energy metabolism, redox balance, immune modulation, and membrane remodeling. Notably, several metabolite panels have demonstrated high sensitivity and specificity, showing promise for effectively stratifying patients according to tumor subtype. Despite the promising potential of metabolomics, challenges remain in standardizing analytical techniques, validating biomarkers, and integrating metabolomics with other omics approaches. Addressing these will be essential for harnessing the full potential of metabolomics in advancing precision oncology for BC.

Keywords:  Detection; Diagnosis; Metabolites; Prognosis; Treatment response

DOI:  https://doi.org/10.1016/j.clbc.2025.05.005
J Chromatogr A. 2025 May 24. pii: S0021-9673(25)00439-X. [Epub ahead of print]1756 466092

Data-independent profiling of phenolic constituents in shea using comprehensive two-dimensional liquid chromatography (RPLC × HILIC) hyphenated to cyclic ion mobility-quadrupole-time-of-flight mass spectrometry.

Nikoline J Nielsen, Oskar M Kronik, Romina A Forte Neran, Jan H Christensen, Tore K Ravn, André de Villiers.

  A total of 50 secondary metabolites were profiled in shea kernels and shells using two-dimensional liquid chromatography (RPLC × HILIC) coupled with electrospray cyclic ion mobility quadrupole time-of-flight mass spectrometry (ESI-Q-cIMS-qTOF) and data-independent acquisition (DIA). The 2D chromatograms revealed distinct regions occupied by specific chemical classes, facilitating compound annotation based on high-resolution mass spectra and collision cross-section (CCS) data. CCS values showed maximum deviation of 2.9 %, and equivalent to the inherent CCS calibration error, from experimental values from the AllCCS database, and deviation of 0.1-6.6 % (mean 3.4 %) from predicted values, confirming compound identities. Key findings included the first-time identification of epitaxifolin, procyanidins B1-B4, prodelphinidins B1-B4, isoorientin and isoquercitrin in shea. The flavone, flavanones and flavanonols were 1.3-9 times more abundant in shells, while flavone glycosides, flavan-3-ols and their dimers, including mono- and di-galloylated forms, were 1.9-50 times higher in kernels. Notably, quercetin-species levels in shells were 4-15 times higher than in kernels. The analytical platform demonstrated high spectral purity in DIA mode due to the arrival time separation of precursor ions prior to fragmentation. The single pass cIMS had, in certain cases, the potential to separate isobaric (isoorientin and quercitrin) and near-isobaric ions (ellagic acid and quercetin). However, accessing the fully resolved and structured 4D data required customized computing tools due to a lack of fit-for-purpose software, highlighting a barrier to broader adoption. These findings support the further valorization of both shea kernels and shells for nutraceutical and cosmetic applications.

Keywords:  2D-LC; Arrival time separation; Flavonoids; Kernels; Proanthocyanidins; Secondary metabolites; Shells

DOI:  https://doi.org/10.1016/j.chroma.2025.466092
Malar J. 2025 May 27. 24(1): 168

Multiplex LC-MS/MS assay for simultaneous quantification of artesunate and its active metabolite dihydroartemisinin with pyronaridine, proguanil, cycloguanil, and clindamycin in pharmacokinetic studies.

Christoph Pfaffendorf, Johannes Mischlinger, Jean Claude Dejon-Agobé, Oumou Maïga-Ascofaré, Ebenezer Ahenkan, Ayôla Akim Adegnika, Michael Ramharter, Sebastian G Wicha.

BACKGROUND: Malaria, especially caused by Plasmodium falciparum, remains a major global health concern, particularly in sub-Saharan Africa. To combat rising drug resistance, innovative treatment approaches like triple artemisinin-based combination therapy (TACT) and multi-drug antimalarial combination therapies (MDACTs) are being explored.
METHODS: This study introduces a robust and validated multiplex LC-MS/MS assay for the simultaneous quantification of key antimalarial drugs and their metabolites, including artesunate, dihydroartemisinin, pyronaridine, proguanil, cycloguanil, and clindamycin. Developed in accordance with EMA guidelines, the assay ensures high accuracy, sensitivity, and stability. Serum samples were prepared through a process of protein precipitation with acetonitrile, followed by the evaporation of the supernatant. The resulting residues were then reconstituted in a 50/50 mixture of aqueous 20 mM ammonium formate buffer and methanol for the analysis.
RESULTS: The assay achieves lower limits of quantifications of 1 ng/mL for proguanil, 0.2 ng/mL for cycloguanil, 1 ng/mL for artesunate, 4 ng/mL for dihydroartemisinin, 2 ng/mL for pyronaridine, and 5 ng/mL for clindamycin. The assay was successfully applied in a pharmacokinetic study conducted as part of a clinical trial in Gabon and Ghana, assessing novel drug combinations in both children and adults against a standard of care artemisinin-based combination therapy.
CONCLUSIONS: The developed assay can support the further clinical development of these TACTs and MDACTs, ultimately contributing to enhanced malaria treatment strategies.

DOI: https://doi.org/10.1186/s12936-025-05387-6