bims-metlip 2025-05-25 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–05–25
thirty-two papers selected by
Sofia Costa, Matterworks

Polyamine quantitation by LC-MS using isobutyl chloroformate derivatives.
[Determination of 22 organic ultraviolet absorbers in human serum by ultra performance liquid chromatography- tandem mass spectrometry].
Development and validation of an LC-MS/MS method for simultaneous determination of XZP-3287(bireociclib) and its metabolites in human plasma, and its clinical pharmacokinetics application.
Quality control in GC-MS analysis of amino acids in human urine and plasma: Possible implications for targeted and untargeted metabolomics.
[Simultaneous determination of 10 phthalate metabolites in urine by ultra performance liquid chromatography -tandem mass spectrometry].
A Spatial Metabolomics Annotation Workflow Leveraging Cyclic Ion Mobility and Machine Learning-Predicted Collision Cross Sections.
Liquid chromatography-tandem mass spectrometry-based pharmacokinetic and metabolic analysis of 4-bromo-2,5-dimethoxyphenethylamine and its metabolites in human plasma.
Quantitative ambient mass spectrometry imaging in plants: A perspective on challenges and future applications.
Rapid separation and quantification of imidazole dipeptides in meats using a PBr column packed with 3-(pentabromobenzyloxy)propyl group modified silica gel.
A comparison of SWATH-MS methods for measurement of residual host cell proteins in adeno-associated virus preparations.
Quantitative GC-NICI-MS and GC-NICI-MS/MS analysis by using isotopologs as internal standards: A tutorial review.
A simple surrogate approach for the quantitation of C4 (7α-hydroxy-4-cholesten-3-one) in human serum via LC-MS/MS and its application in a clinical study.
A Novel Liquid Chromatography-Tandem Mass Spectrometry Method for Monitoring Plasma l-Asparaginase Activity in Pediatric Patients With Acute Lymphoblastic Leukemia Receiving Pegaspargase Treatment.
An Offline SPE-LC-MS/MS Method for Simultaneous Quantification of Tacrolimus, Cyclosporine A, Kynurenine, Tryptophan, and Creatinine Using Volumetric Absorptive Microsampling Device Mitra.
Multiple Reaction Monitoring LC-MS/MS Quantification of N-Nitrosamine Drug Substance Related Impurities of Clonidine in Drug Substance and in Tablet Dosage Form.
Quantitative Mass Spectrometry Imaging Protocols for Spatially Heterogeneous Samples.
Development of a candidate reference method for the simultaneous quantification of betaine, choline and trimethylamine N-oxide in serum samples by two-dimensional liquid chromatography and isotope dilution tandem mass spectrometry.
Confirmation of a novel, stable estrogen metabolite, as 5a,6a-epoxy-estrone sulfate in stallion blood by LC-MS/MS.
Novel LC-MS/MS assay to quantify D,L-alpha-difluoromethylornithine (DFMO) in mouse plasma.
Determination of 25-Hydroxyvitamin D3 in Rat Brain by Derivatization-Assisted LC/ESI-MS/MS.
Quantification of five intracellular and extracellular methionine pathway intermediates using stable isotope dilution UHPLC-MS/MS.
Unveiling the dark matter of the metabolome: A narrative review of bioinformatics tools for LC-HRMS-based compound annotation.
Self-supervised learning of molecular representations from millions of tandem mass spectra using DreaMS.
Simultaneous Determination of 30 Flavonoids in Jinqiancao Granules by LC-MS/MS.
Development of Simultaneous Analytical Method of Three Polypeptide Toxins α-Amanitin, β-Amanitin and Phalloidin in Poisonous Mushrooms and Human Serum Using UHPLC-MS/MS.
Novel LC-MS/MS Method for Quantification of Famotidine and Metoprolol and Application to Pharmacokinetic Interaction.
Phase Modulation to Increase Ion Throughput for Ion Mobility-Time-of-Flight Experiments.
Opioid quantification via microsampling techniques to assess opioid use in human laboratory studies.
Quantification of sulbactam and durlobactam in saline and human plasma via ultra-performance liquid chromatography tandem mass spectrometry.
Stochastic dynamics mass spectrometric quantification and 3D molecular structural analysis of tricyclic antidepressant in marine dissolved organic matter.
LipidIN: a comprehensive repository for flash platform-independent annotation and reverse lipidomics.
Qualitative and quantitative determination of SIPI-2011 and its two major metabolites in human plasma.

Methods Enzymol. 2025 ;pii: S0076-6879(25)00086-2. [Epub ahead of print]715 437-458

Polyamine quantitation by LC-MS using isobutyl chloroformate derivatives.

Christine Isaguirre, Megan Gendjar, Kelsie M Nauta, Nicholas O Burton, Ryan D Sheldon.

  Polyamines are an important class of metabolites that are poorly covered in standard metabolomics workflows. Here, we describe a protocol for isobutyl-chloroformate derivatization that can be applied to metabolite extracts following other metabolomics applications. This simple procedure allows for quantitative measurement of thirteen polyamines and two internal standards in a short (15-minute) LC-MS method. We report at least two triple quadrupole mass spectrometer transitions for each compound. Among these are unique transitions for co-eluting isomers N1- and N8-acetylspermidine, enabling quantitation of each isomer individually. We further define the linear dynamic range for each compound, and present data in several biological sample types. This simple and robust method enables stand-alone and post-metabolomics polyamine analysis.

Keywords:  Acetylspermidine; Metabolism; Metabolomics; Reversed phase

DOI:  https://doi.org/10.1016/bs.mie.2025.01.063
Se Pu. 2025 Jun;43(6): 620-629

[Determination of 22 organic ultraviolet absorbers in human serum by ultra performance liquid chromatography- tandem mass spectrometry].

De-Jun Bao, Zhuang-Zhuang Feng, Xu Zhang, Qi Sun, Zhuo-Na Zhang, Xiao-Jian Hu, Ying Zhu, Xiao Lin.

  In this study， a method for the simultaneous determination of 22 organic ultraviolet absorbers （OUVs） in human serum was established by combining protein precipitation technology （PPT）， efficiency lipid removal technology （ELR） and ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）. The OUVs include five benzophenone compounds， five benzotriazole compounds， two cinnamate ester compounds and their three metabolites， two salicylate compounds， two camphor derivative compounds， one triazine compound， one dibenzoylmethane compound and one amino aminobenzoic acid derivative compound. Chromatography was performed using an Acquity BEH C18 column （100 mm×2.1 mm， 1.7 μm）， gradient elution was carried out using methanol-water and methanol-0.1% ammonia water as the mobile phases. Compounds were detected in both positive and negative electrospray ionization （ESI+/ESI‒） modes using multiple reaction monitoring （MRM）， and quantified using stable-isotope internal standards. The experimental results showed that the 22 target compounds exhibited good linear relationships within their respective linear ranges， with the correlation coefficients （r）≥0.999 3. The method detection limits （MDLs） ranged from 0.02 to 0.48 ng/mL， and the method quantification limits （MQLs） ranged from 0.02 to 1.60 ng/mL. At the three spiked levels of low， medium and high， the spiked recoveries of the 22 target analytes ranged from 79.9% to 136.1%， the intra-day precisions were from 1.5% to 25.4%， and the inter-day precisions were from 0.6% to 23.5%. After correction by the stable-isotope internal standard method， the matrix effects of the 22 target analytes in fetal bovine serum were 83.0%‒119.9%. The developed method was successfully used to detect 22 OUVs in 110 human serum samples. With the exception of 3-benzylidene camphor （3-BC）， 2-（2H-benzotriazol-2-yl）-4-methyl-6-（2-propenyl）phenol （UV-9）， 4-methylbenzylidene camphor （4-MBC）， 2，2'-dihydroxy-4-methoxybenzophenone （BP-8）， and 2，4-dihydroxybenzophenone （BP-1）， which were not detected， the remaining 17 substances were detected with overall detection rates of 0.9%-65.5% and the detection levels were <MQL‒11.7 ng/mL. The developed analytical method is simple， convenient， highly sensitive， and is expected to become an effective tool for quantifying 22 OUVs in human serum.

Keywords:  efficiency lipid removal technology （ELR）; human biomonitoring; organic ultraviolet absorbents （OUVs）; protein precipitation technology （PPT）; serum; ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）

DOI:  https://doi.org/10.3724/SP.J.1123.2024.10031
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 May 16. pii: S1570-0232(25)00212-0. [Epub ahead of print]1261 124658

Development and validation of an LC-MS/MS method for simultaneous determination of XZP-3287(bireociclib) and its metabolites in human plasma, and its clinical pharmacokinetics application.

Bohan Liang, Yanjie Li, Lingmei Xu, Li Wang, Quankun Zhuang, Shiqi Dong, Huirong Fan.

  XZP-3287(bireociclib) is a novel and selective inhibitor of the cell cyclin-dependent kinases 4/6 (CDK4/6), which is primarily employed for the treatment of breast cancer in clinical trials. In this study, a novel and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of XZP-3287 and its metabolites XZP-5286, XZP-3584 and XZP-5736 in human plasma in accordance with international conference on harmonization of technical requirements for registration of Pharmaceuticals for Human use (ICH) guideline R3 (M10) guideline. The multiple reaction monitoring mode (MRM) of mass spectrometer was used and all compounds were monitored in electrospray ionization (ESI+) mode. The correlation coefficients (R2) of all calibration curves for linear regression were greater than 0.99. The intra- and inter-day precision of XZP-3287 and its metabolites XZP-5286, XZP-3584 and XZP-5736 were determined to be 5.2 %-5.5 %, 14.9 %-10.1 %, 6.9 %-13.8 % and 7.3 %-5.6 %, and their accuracy were determined to be 5.2 %-6.0 % 6.9 %-4.4 %, 11.1 %-5.0 % and 7.4 %-5.6 %, respectively. In conclusion, a method for the simultaneous detection of the pharmacokinetic profiles of XZP-3287 and its metabolites in human plasma had been successfully developed. The results demonstrated the efficacy, sensitivity, and reliability of this method.

Keywords:  Inhibitor of the CDK4/6; LC-MS/MS method; Pharmacokinetics; XZP-3287(bireociclib)

DOI:  https://doi.org/10.1016/j.jchromb.2025.124658
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 May 19. pii: S1570-0232(25)00215-6. [Epub ahead of print]1262 124661

Quality control in GC-MS analysis of amino acids in human urine and plasma: Possible implications for targeted and untargeted metabolomics.

Alexander Bollenbach, Bibiana Beckmann, Dimitrios Tsikas.

  In metabolomics, LC-MS(/MS) is currently used about two times more frequently than GC-MS(/MS) since about 2005, perhaps the year of appearance of metabolomics as an individual analytical approach in the life sciences. LC-MS(/MS) and GC-MS(/MS) share many common challenges in targeted and untargeted metabolomics, the Janus face of metabolomics. Especially the importance of the key issue of quality assurance (QA) and quality control (QC) has been recognized and is increasingly addressed by individual researchers and consortia. In previous work, our group has proposed a QC system for the quantitative GC-MS analysis of amino acids in human plasma samples. In the present study, we investigated the utility of such a QC approach for the quantitative (targeted) GC-MS analysis of amino acids in human urine samples by using previously validated methods. Endogenous (unlabeled) amino acids were analyzed in 10-μL aliquots of study urine samples and in QC urine samples as methyl ester pentafluoropropionyl derivatives (d0Me-PFP) using a mixture of in-situ prepared trideuteromethyl esters for use as internal standards, which were then converted into their PFP derivatives (i.e., d3Me-PFP). GC-MS analysis of 38 study urine samples and 8 QC urine samples was performed in the negative-ion chemical ionization (NICI) mode by selected-ion monitoring (SIM) of characteristic ions of d0Me-PFP and d3Me-PFP within a single run by using an oven temperature program. For direct comparison, analysis of 35 study plasma samples and 8 QC plasma samples of the same clinical study was performed. Closely comparable experimental and instrumental conditions were used in the analyses, and the same staff was involved in the entirely analytical process. Chromatographic H/D isotope effects and peak area values were determined and examined with respect to qualitative and quantitative GC and MS parameters including accuracy and precision. Study and QC plasma behaved similarly. On a molar basis, the amino acid derivatives have different peak area values. Yet, this does not affect the accuracy of the GC-MS method. Our study suggests that untargeted GC-MS metabolomics studies on amino acids in biological samples are inappropriate for quantitative GC-MS analysis. Targeted metabolomics, i.e., use of isotopologs are indispensable for reliable quantitative GC-MS analysis of amino acids in biological samples. It is reasonable to assume that our findings will also apply to other classes of analytes and types of biological samples.

Keywords:  Amino acids; Comparison; Derivatization; OMICS; Plasma; Quality control; Urine

DOI:  https://doi.org/10.1016/j.jchromb.2025.124661
Se Pu. 2025 Jun;43(6): 640-649

[Simultaneous determination of 10 phthalate metabolites in urine by ultra performance liquid chromatography -tandem mass spectrometry].

Xiao-Ying Zhao, Hui Kang, Yan-Yan Wang, Wen-Hui Li, Ming Chen, Chan Ke, Feng Qin, Xi-Xiong Kang.

  Phthalates （PAEs） are widely employed as plasticizers in plastic products that are used in industrial， agricultural， food， medical， and other fields. PAEs are relatively weakly bonded to plastic products through non-covalent interactions. Consequently， PAEs can easily leak from the product into the environment， which exposes the public to PAEs through food intake， skin absorption from personal care products， and by inhaling air. Related studies have shown that PAEs are endocrine-disrupting substances and that long-term exposure to PAEs may result in diseases of the nervous， reproductive， cardiovascular and immune systems. In addition， excessive exposure to PAEs may trigger inflammatory responses and induce tumors. Therefore， establishing a highly sensitive assay for determining PAE levels in the human body following exposure is an important objective. PAEs generally have half-lives of less than 24 h； they are rapidly metabolized through enzymatic hydrolysis after entering the human body and excreted through urine. Therefore， most studies have focused on PAE metabolites as target compounds； hence， human body exposure to PAEs can be assessed by analyzing the types and levels of these metabolites. Herein， we established a method for simultaneously determining ten phthalate （PAE） metabolites in human urine using ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）. The ten PAE metabolites in urine were separated using an ACQUITY UPLC BEH Phenyl column （50 mm×2.1 mm， 1.7 μm）. Gradient elution was performed using 0.1% formic acid aqueous solution and 0.1% formic acid in acetonitrile as the mobile phases， at a flow rate of 0.5 mL/min， a column temperature of 40 ℃， and a sample size of 20 μL. Data were acquired in negative-ion electrospray ionization （ESI） and multiple reaction monitoring （MRM） modes， and quantified using the isotope internal standard method. The method was found to be highly specific， with the ten PAE metabolites exhibiting good linearities in their linear ranges， with limits of detection （LODs） and quantification （LOQs） of 0.03-0.3 and 0.1-1 ng/mL， respectively. Under the four quality control （QC） levels， the intra-day and inter-day precisions of the ten PAE metabolites were all ≤8.3%， and the accuracy ranged from ‒10.5% to 7.3%. The method was used to assess the exposure levels of PAE metabolites in the urine samples of 60 volunteers， with 1‒6 kinds of PAE metabolites detected in the urine of each volunteer. This method is sensitive， accurate， simple， efficient， and suitable for the large-scale biological monitoring of PAE metabolites.

Keywords:  phthalate metabolites; plasticizer; ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）; urine samples

DOI:  https://doi.org/10.3724/SP.J.1123.2024.04002
J Am Soc Mass Spectrom. 2025 May 21.

A Spatial Metabolomics Annotation Workflow Leveraging Cyclic Ion Mobility and Machine Learning-Predicted Collision Cross Sections.

Dmitry Leontyev, Eric C Gier, Viraj A Master, Rebecca S Arnold, John A Petros, Facundo M Fernández.

In nontargeted spatial metabolomics, accurate annotation is crucial for understanding metabolites' biological roles and spatial patterns. MS2 mass spectrometry imaging (MSI) coverage is often incomplete or nonexistent, resulting in many unknown features that represent an untapped source of biological information. Ion mobility-derived collision cross sections (CCS) have been leveraged as valuable descriptors for confirming putative metabolite annotations, distinguishing isomers, and aiding in unknown structural elucidation. In this study, desorption electrospray ionization cyclic ion mobility mass spectrometry imaging (DESI-cIM-MSI) data from human renal cell carcinoma (RCC) tissues is used as a testbed to explore the extent to which CCS measurements enhance MSI lipid annotation confidence when combined with machine learning CCS predictions and SIRIUS analysis of MS2 data. Multipass IM experiments yielded excellent CCS accuracy (<0.4%) relative to database values for differential lipids found in RCC tissues, improving the filtering threshold used in previous CCS-based annotation workflows. High-accuracy multipass CCS measurements enabled the correct annotation of isobaric lipid database matches, even in the absence of MS2 data. Additionally, MS2 data from differential RCC features were uploaded to SIRIUS, and the predicted CCS values for SIRIUS candidates were compared to experimental CCS data to filter out unlikely candidates. Finally, CCS measurements contributed to the annotation of two spatially correlated unknown features, differential between tumor and control kidney tissues. Both features were assigned to rocuronium, a surgical muscle relaxant that had not been previously reported in MSI studies. Overall, these results underscore the potential of high-accuracy CCS values to enhance metabolite annotations in MSI-based spatial metabolomics.

DOI: https://doi.org/10.1021/jasms.5c00090
Drug Metab Dispos. 2025 Apr 28. pii: S0090-9556(25)09095-6. [Epub ahead of print]53(6): 100086

Liquid chromatography-tandem mass spectrometry-based pharmacokinetic and metabolic analysis of 4-bromo-2,5-dimethoxyphenethylamine and its metabolites in human plasma.

Jan Thomann, Deborah Rudin, Selina Kraus, Denis Arikci, Friederike Holze, Matthias E Liechti, Dino Luethi.

  4-Bromo-2,5-dimethoxyphenethylamine (2C-B) is widely used recreationally and has recently gained interest as a treatment for mental health disorders. In this study, a liquid chromatography-tandem mass spectrometry method to quantify 2C-B and its metabolites 4-bromo-2,5-dimethoxyphenylacetic acid (BDMPAA) and 4-bromo-2-hydroxy-5-methoxyphenylacetic acid (B-2-HMPAA) in human plasma was developed and validated. Moreover, pharmacokinetic analysis was performed on samples from clinical study participants who received 30 mg of 2C-B. The metabolic degradation of 2C-B and its metabolites via monoamine oxidases (MAOs), cytosolic enzymes, and cytochrome P450 enzymes was assessed and their activation potencies at the serotonin 2A receptor were investigated. Optimal chromatographic separation was achieved using a Kinetex 2.6 μm XB-C18 analytical column and a mobile phase gradient of water and acetonitrile supplemented with 0.1% formic acid. Using electrospray ionization, a linear range of 0.5-100 ng/mL for 2C-B, 2.5-1000 ng/mL for BDMPAA, and 0.5-1000 ng/mL for B-2-HMPAA was achieved. The method demonstrated high accuracy, precision, and extraction recovery with minimal matrix effects. MAO-A, MAO-B, cytosolic enzymes, and CYP2D6 were identified as key enzymes involved in the metabolic degradation of 2C-B. Unlike 2C-B, BDMPAA and B-2-HMPAA did not activate the human serotonin 2A receptor, suggesting that these metabolites do not contribute to the psychedelic effect. This study provides insights into the pharmacokinetics, metabolism, and pharmacological activity of 2C-B and its metabolites. The validated liquid chromatography-tandem mass spectrometry method offers a reliable tool for future clinical studies investigating the therapeutic potential and metabolism of 2C-B. SIGNIFICANCE STATEMENT: A rapid and nonlaborious liquid chromatography-tandem mass spectrometry method was developed and validated for pharmacokinetic analysis of 4-bromo-2,5-dimethoxyphenethylamine (2C-B) and its metabolites 4-bromo-2,5-dimethoxyphenylacetic acid and 4-bromo-2-hydroxy-5-methoxyphenylacetic acid in human plasma. To assess the metabolites' relevance in psychedelic drug action, serotonin 2A receptor activity was studied. Unlike 2C-B, the metabolites failed to activate the receptor. Monoamine oxidase A and B, and cytosolic enzymes were confirmed in 4-bromo-2,5-dimethoxyphenylacetic acid formation, whereas CYP2D6 was found to metabolize 2C-B through a minor pathway.

Keywords:  Bioanalysis; Metabolism; Method validation; Pharmacokinetics; Receptor activation

DOI:  https://doi.org/10.1016/j.dmd.2025.100086
Curr Opin Plant Biol. 2025 May 19. pii: S1369-5266(25)00050-0. [Epub ahead of print]85 102736

Quantitative ambient mass spectrometry imaging in plants: A perspective on challenges and future applications.

Sarah E Noll, Andrea M Sama, Abigail Tripka, Alexandra J Dickinson.

  Mass spectrometry imaging (MSI) is a powerful approach to understanding plant chemistry in a native context because it retains key spatial information that is otherwise averaged out, permitting chemical compounds to be mapped to specific tissue structures. Identifying the spatial localization of compounds in plant tissues has provided insights into the synthesis and functional role of a wide range of endogenous molecules. The power and utility of MSI is being further expanded through the development of quantitative methodologies, which enable relative and absolute quantification of target analytes. Here, we briefly summarize applications of MSI in plant studies. We then turn our discussion to the challenges and developments in quantitative MSI, with a particular focus on ambient liquid extraction-based methods. Quantitative MSI is an emerging discipline in plant studies and holds great promise for revealing new information about the molecular composition of plant tissues and the pathways that regulate plant physiology.

Keywords:  Mass spectrometry imaging; Plant metabolites; Quantitative analysis

DOI:  https://doi.org/10.1016/j.pbi.2025.102736
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 May 16. pii: S1570-0232(25)00214-4. [Epub ahead of print]1262 124660

Rapid separation and quantification of imidazole dipeptides in meats using a PBr column packed with 3-(pentabromobenzyloxy)propyl group modified silica gel.

Makoto Ozaki, Tomomi Nakade, Yasunari Yamada, Tsunehisa Hirose, Motoshi Shimotsuma, Shozo Tomonaga.

  Meats are rich in imidazole dipeptides (IDPs) such as carnosine, anserine, and balenine, known for their antioxidant and antifatigue properties. The concentrations and types of these IDPs vary significantly among different animal species, necessitating a quantitative method for the precise measurement of individual IDPs. Simultaneous analysis of multiple compounds is typically conducted using reversed-phase high-performance liquid chromatography (RP-HPLC). However, C18 columns, which are commonly employed in RP-HPLC, fail to adequately retain highly hydrophilic IDPs, making separation and quantification challenging. Previously, we developed a PBr column packed with 3-(pentabromobenzyloxy)propyl group modified silica gel, which effectively retains various highly hydrophilic compounds in RP mode. In this study, we established a method for the rapid separation and quantification of IDPs within 10 min under simplified conditions (isocratic mode) using a single quadrupole liquid chromatography-mass spectrometer (LC-MS) equipped with a PBr column, without the need for derivatization. Linear calibration curves for each IDP were generated using glycyl-L-leucine as the internal standard, with the desolvation temperature of the MS instrument set at 500 °C. The proposed method achieved extraction and recovery rates of IDPs ranging from 100.0 % to 113.5 % at three spiking levels, with no carryover observed, even in samples with high concentrations. Additionally, matrix effects ranged from 95.5 % to 109.6 %, with negligible ion suppression and enhancement effects. Furthermore, the method enabled accurate analysis of IDPs with a relative standard deviation of <15 % in meats from various animal species, including chicken, pork, beef, lamb, mutton, deer, horse, and kangaroo.

Keywords:  Animal; Anserine; Balenine; Carnosine; Imidazole dipeptides; LC–MS; Meats

DOI:  https://doi.org/10.1016/j.jchromb.2025.124660
Front Bioeng Biotechnol. 2025 ;13 1579098

A comparison of SWATH-MS methods for measurement of residual host cell proteins in adeno-associated virus preparations.

Thomas M Leibiger, Lie Min, Kelvin H Lee.

   Introduction: Analysis of residual host cell proteins in adeno-associated virus (AAV) preparations is challenging due to low availability and high complexity of samples. One strategy to address these challenges is through development of improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods with greater sensitivity and reduced sample requirement.
Methods: In this work, we compare the performance of four sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) methods for identification and quantitation of residual HCPs in rAAV2, -5, -8, and -9 preparations produced with human embryonic kidney 293 (HEK293) cells and purified using immunoaffinity chromatography. Key SWATH-MS parameters including spectral library construction (data dependent vs. in silico), data processing software (DIA-NN vs. Skyline), and mass spectrometer instrument (Sciex TripleTOF 6600 vs. Sciex ZenoTOF 7600) were assessed. Method attributes including sample requirement and processing time, and method outputs including protein and precursor identifications, host cell protein quantitation comparisons across methods, and quantitation coefficients of variance (CV) were considered to help establish a SWATH-MS workflow well-suited for rAAV HCP analytics.
Results: A 78% increase in HCP identifications, 80% reduction in sample requirement, and 70% reduction in instrument runtime was achieved with an in silico spectral library, data processing in DIA-NN, and data collection with the Sciex ZenoTOF 7600 instrument (DIA-NN-7600 method) compared to a previously established method using a DDA-derived spectral library, data processing in Skyline, and data collection with the Sciex TripleTOF 6600 instrument (Skyline-DDA-6600 method). Additionally, the DIA-NN-7600 method shows median HCP quantitation CV below 10% for triplicate data acquisitions, and comparable quantitation to other methods for a panel of highly abundant residual HCPs previously identified in rAAV downstream processing.
Discussion: This work highlights a SWATH-MS method with data collection and processing specifically tailored for rAAV residual HCP analysis.

Keywords:  DIA-NN; SWATH-MS; adeno-associated virus (AAV); data independent acquisition (DIA); host cell proteins (HCPs); liquid chromatography-tandem mass spectrometry (LC-MS/MS); mass spectrometry

DOI:  https://doi.org/10.3389/fbioe.2025.1579098
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 May 15. pii: S1570-0232(25)00202-8. [Epub ahead of print]1262 124648

Quantitative GC-NICI-MS and GC-NICI-MS/MS analysis by using isotopologs as internal standards: A tutorial review.

Dimitrios Tsikas.

  The use of analytes labelled with stable-isotopes of 2H, 13C, 15N, 17O and/or 18O, i.e., the isotopologs, as internal standards is unique and considered the Golden Standard is quantitative analyses based on mass spectrometry. However, the handling with isotopologs deserves a great extent of care and attentiveness from the very begin of the analytical process. Many issues need to be considered in order to create an analytical method that generates close-to-reality concentrations of a certain analyte or of group of analytes of the same or different chemical classes in complex biological samples. They including isotopic purity, stability through the entire analytical process including sampling, derivatization, ionization and collision-induced dissociation (CID). The present work deals specifically with the use of isotopologs as internal standards for the quantitative analysis of endogenous and exogenous substances in plasma, serum and urine samples. It focuses on GC-MS and GC-MS/MS, negative-ion chemical ionization (NICI) with methane as the reagent gas, and selected-ion monitoring (SIM) or selected-reaction monitoring (SRM) using argon as the collision gas. Special attention has been paid to purity of isotopologs, cross-contribution of analyte-internal standard, and stability of isotopologs during NICI and CID, to linearity of analyte-isotope detector response upon the analyte concentration. This tutorial review re-examines and discusses exemplarily previously reported validated GC-MS and GC-MS/MS methods, and gives recommendations regarding the handling with stable-isotope labelled analogs in quantitative analyses.

Keywords:  Derivatization; Kinetic and chromatographic H/D isotope effects; Negative-ion chemical ionization

DOI:  https://doi.org/10.1016/j.jchromb.2025.124648
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 May 14. pii: S1570-0232(25)00205-3. [Epub ahead of print]1261 124651

A simple surrogate approach for the quantitation of C4 (7α-hydroxy-4-cholesten-3-one) in human serum via LC-MS/MS and its application in a clinical study.

Feng Yin, Mani Deepika Vakkalanka, Walter Wiley, M Shane Woolf, Yousef Basir, Kumar Shah, Aaron M Wheeler, Moucun Yuan, William R Mylott, Mike Baratta.

  We present a validated LC-MS/MS assay for the quantitation of 7α-hydroxy-4-cholesten-3-one (C4), a key intermediate in the bile acid synthesis pathway from cholesterol, in human serum. A surrogate matrix approach was employed to overcome the challenges posed by the endogenous C4 levels in the biological matrix. Human serum samples were spiked with stable isotope labeled internal standard (SIL-IS), processed using supported liquid extraction (SLE), and analyzed by LC-MS/MS. Parallelism was successfully demonstrated between human serum (authentic matrix) and 5 % bovine serum albumin in phosphate buffered saline containing 0.1 % tween 20 (5 % BSA in PBST) (surrogate matrix). The assay's linear analytical range was established from 0.200 to 200 ng/mL. This validated LC-MS/MS method exhibited excellent accuracy and precision. The overall accuracy was between 97.9 % and 101 % with %CV less than 4.0 % for C4 in human serum. C4 was found to be stable in human serum for up to 24.7 h at room temperature, up to 34 days when stored at -25 °C or - 80 °C, and after five freeze/thaw cycles. The assay has been successfully applied to human serum samples to support a clinical study.

Keywords:  7α-Hydroxy-4-cholesten-3-one; Biomarker; C4; Human serum; LC-MS/MS; Validated

DOI:  https://doi.org/10.1016/j.jchromb.2025.124651
Ther Drug Monit. 2025 May 23.

A Novel Liquid Chromatography-Tandem Mass Spectrometry Method for Monitoring Plasma l-Asparaginase Activity in Pediatric Patients With Acute Lymphoblastic Leukemia Receiving Pegaspargase Treatment.

Hengyi Yu, Qun Hu, Lu Liu, Dong Xiang, Guangjie Yang, Yaqin Wang, Zhen Wang, Dong Liu, Aiguo Liu, Xuepeng Gong.

   BACKGROUND: Acute lymphoblastic leukemia (ALL) is the predominant malignancy in pediatric patients. As a crucial constituent of ALL chemotherapy, l-asparaginase is recognized as an integral element of treatment with a threshold concentration of 0.1 IU/mL used in treatment protocols. This study presents a novel liquid chromatography-tandem mass spectrometry method for evaluating plasma l-asparaginase activity in pediatric patients with ALL undergoing pegaspargase therapy.
METHODS: Initially, an enzyme incubation was conducted using 20 μL of plasma and 100 μL of l-asparagine (0.1 mol/L) at 37°C for 15 minutes. The reaction was stopped by adding sulfosalicylic acid and methanol. After the addition of isotope internal standard l-aspartic 13C4 acid, subsequent centrifugation, and dilution, the plasma samples underwent analysis by liquid chromatography-tandem mass spectrometry on a hydrophilic interaction liquid chromatography analytical column. A flow rate of 0.3 mL/min was used during isocratic elution using a mobile phase consisting of methanol-water (95:5, vol/vol) with 0.2% formic acid within a 5-minute run.
RESULTS: The calibration curves exhibited excellent linearity ranging from 0.1 to 15 IU/mL, with determination coefficients (r2) exceeding 0.99. The precision and accuracy ranged from 1% to 7% and from 93% to 110%, respectively. The relative recovery fell within the range of 98%-100%, and the internal standard-normalized matrix effect ranged from 95% to 101%. The stability was satisfactory across various conditions.
CONCLUSIONS: This method was fully validated and successfully applied to quantify l-asparaginase activity in plasma samples of 15 children with ALL, enabling the monitoring of l-asparaginase activity with mass spectrometry.

Keywords:  ; LC-MS/MS; acute lymphoblastic leukemia; pediatric patient; pegaspargase

DOI:  https://doi.org/10.1097/FTD.0000000000001345
Ther Drug Monit. 2025 May 22.

An Offline SPE-LC-MS/MS Method for Simultaneous Quantification of Tacrolimus, Cyclosporine A, Kynurenine, Tryptophan, and Creatinine Using Volumetric Absorptive Microsampling Device Mitra.

Kajetan Nierychlewski, Katharina Habler, Stephan Kemmner, Tobias Seibt, Michael Fischereder, Markus Schwarz.

   BACKGROUND: Therapeutic drug monitoring of immunosuppressants is critical in balancing insufficient immunosuppression due to underdosing, and severe adverse effects due to overdosage. For a more comprehensive therapeutic drug monitoring and follow-up of transplant patients, the aim was to develop a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of tacrolimus, cyclosporine A, tryptophan, kynurenine, and creatinine using a volumetric absorptive microsampling device.
METHODS: Venous and capillary blood samples were simultaneously collected using a volumetric absorptive microsampling device called Mitra. The method involved protein precipitation followed by offline solid-phase extraction using a positive pressure manifold. Chromatographic separation was achieved by a formic acid-ammonium formate-methanol gradient on a Synergi Polar reversed-phase column. Multiple reaction monitoring in the positive ion mode and stable isotope-labeled internal standards were used for quantification. Validation was performed according to the European Medicines Agency and US Food and Drug Administration (FDA) guidelines.
RESULTS: Validation was successful, meeting European Medicines Agency and FDA guidelines. Investigation of selectivity, accuracy, and precision met the required criteria of a deviation <15%. Internal standards successfully compensated potential matrix effects. A comparison of 26 anonymized samples from transplant patients on Mitra with venous blood controls demonstrated the method's suitability.
CONCLUSIONS: For the first time, we herein describe a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of tacrolimus, cyclosporine A, tryptophan, kynurenine, and creatinine on Mitra. Self-collection of samples may facilitate therapeutic monitoring. Simultaneous determination of creatinine may help monitor kidney function, while tryptophan and kynurenine may serve as a biomarker for early detection of transplant rejection.

Keywords:  high-performance liquid chromatography–tandem mass spectrometry; immunosuppressants; kynurenine pathway; therapeutic drug monitoring; volumetric absorptive microsampling device

DOI:  https://doi.org/10.1097/FTD.0000000000001341
Biomed Chromatogr. 2025 Jul;39(7): e70123

Multiple Reaction Monitoring LC-MS/MS Quantification of N-Nitrosamine Drug Substance Related Impurities of Clonidine in Drug Substance and in Tablet Dosage Form.

Bhaskar Vallamkonda, Srinivasa Rao Yarguntla, Ranadheer Reddy Challa.

  N-nitrosamine drug substance related impurities (NDSRIs) present recent concerns for manufacturers of both drug substances and products. The regulatory authorities prioritize understanding the chemical properties, potential formation, and control methods of these impurities due to their adverse effects. Clonidine is a medication for hypertension and attention deficit hyperactivity disorder, characterized by a secondary amine in its structure and a propensity to generate NDSRIs. Its chemical structural evaluation reveals a possible risk for the formation of two separate NDSRIs, namely, N-nitroso and N-dinitroso clonidine. The carcinogenic/mutagenic category was established using carcinogenic potency categorization approach method, and the corresponding acceptable intake values were implemented. A liquid chromatography-tandem mass spectrometry method in a multiple reaction monitoring mode was established to quantify both impurities in its drug substance and tablet dosage. A Poroshell C18 column of 150 × 4.6 mm, and with particle size of 2.7 μm was employed at a temperature of 30°C. A gradient elution method opted with water and acetonitrile as the mobile phases, containing formic acid as a buffering agent. Method validation conducted as per regulatory standards and demonstrated superior specificity, linearity, accuracy, precision, and robustness. As a result, this method is regarded as suitable for its intended purpose.

Keywords:  ADHD; CPCA; LC‐MS/MS; NDSRIs; clonidine; method validation

DOI:  https://doi.org/10.1002/bmc.70123
Anal Chem. 2025 May 22.

Quantitative Mass Spectrometry Imaging Protocols for Spatially Heterogeneous Samples.

Reza Shariatgorji, Michael Niehues, Anna Nilsson, Tina Angerer, Nikolas Stroth, Wojciech Paslawski, Sandra Jabre, Per Svenningsson, Per E Andrén.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a promising tool for the spatial quantitation of endogenous and exogenous compounds directly in biological tissue sections. However, precise quantitation may be hampered due to matrix effects and variations in ionization efficiency, especially in spatially heterogeneous samples such as brain tissue. In this study, we developed and implemented two advanced MALDI-MSI protocols to address these limitations by employing a standard addition approach. The protocols involved the homogeneous spraying of standard solutions onto tissue sections to minimize the matrix effects associated with heterogeneous samples. The first method utilized spraying of deuterated analogues of neurotransmitters across all tissue sections for normalization, while calibration standards were applied in a quantitative manner to consecutive tissue sections. The second method employed two stable isotope-labeled compounds: one for calibration and the other for normalization. Both methods were applied to quantify neurotransmitters and their metabolites, e.g., dopamine, norepinephrine, and 3-methoxytyramine, in rodent brain tissue. The results showed strong linearity between signal intensities and analyte concentrations across brain tissue sections with values comparable to those obtained using high-performance liquid chromatography-electrochemical detection. The standard addition approach significantly enhanced the quantitation accuracy by accounting for tissue-specific matrix effects, providing a robust method for the spatial quantification of neurotransmitters in complex brain tissue environments.

DOI: https://doi.org/10.1021/acs.analchem.5c00677
Anal Bioanal Chem. 2025 May 23.

Development of a candidate reference method for the simultaneous quantification of betaine, choline and trimethylamine N-oxide in serum samples by two-dimensional liquid chromatography and isotope dilution tandem mass spectrometry.

Daniela Pineda-Cevallos, María Castañón Apilánez, Elena Cancio-López, Belén Prieto García, J Ignacio García Alonso, Pablo Rodríguez-González.

  Serum choline, betaine and trimethylamine N-oxide levels are associated with the risk of cardiovascular events. However, no reference procedure for the determination of these compounds in serum has been developed so far. This work describes the combination of IDMS and two-dimensional liquid chromatography operating in multiple heart-cutting mode for the simultaneous quantification of the three compounds in human serum by isotope dilution tandem mass spectrometry. A reversed-phase separation is proposed as the first dimension and coupled with cation exchange chromatography in the second dimension. The online isolation of the single fraction in which the three analytes co-elute from the first dimension enables a rapid chromatographic separation in the second dimension through cation exchange. The method was validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines and applied to the analysis of 74 serum samples from patients who had suffered from an ischemic stroke in the past 24 h for further study of these metabolites as potential biomarkers to predict an ischemic stroke patient's prognosis.

Keywords:  Bidimensional chromatography; Isotope dilution; Stroke biomarkers; Tandem mass spectrometry

DOI:  https://doi.org/10.1007/s00216-025-05914-z
J Steroid Biochem Mol Biol. 2025 May 20. pii: S0960-0760(25)00116-5. [Epub ahead of print] 106788

Confirmation of a novel, stable estrogen metabolite, as 5a,6a-epoxy-estrone sulfate in stallion blood by LC-MS/MS.

James I Raeside, Heather L Christie, Tracey Chenier, Dyanne Brewer, Armen Charchoglyan.

  Mass spectrometry (MS) has become pivotal for accurately delineating intricate molecular structures for steroids present in minute quantities within biological samples. This study utilized liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) to identify and characterize a 'new' estrogen metabolite, 5α,6α-epoxy-estrone sulfate, in stallion serum from three animals. The estrogen structure was predicted previously using radiolabeled steroids. HRMS/MS, in combination with a seamless sample preparation involving liquid-liquid extraction and chromatographic separation, enabled accurate mass spectrometric identification of the target metabolite. A distinct chromatographic peak corresponding to the metabolite displayed a fragmentation pattern consistent with its predicted structure. Fragment ions at m/z 79.9 and 285.1 resulting from precursor ion m/z 365.5 [M-H]- suggested the presence of a sulfated group and epoxy form of estrone, with an additional oxygen atom when compared with those for a reference standard of estrone sulfate. The assignment of other fragment ions from the target ion further elucidated the predicted structure. Evidence for a structure unique from any other estrogen metabolite on record was demonstrated on two different LC-QTOF instruments. Its identification in the blood circulation ensures distribution throughout the body. The potential significance for future physiological/pathological investigations is discussed.

Keywords:  LC-HRMS/MS; epoxide sulfate; estrone metabolite; serum; stallion

DOI:  https://doi.org/10.1016/j.jsbmb.2025.106788
Methods Enzymol. 2025 ;pii: S0076-6879(25)00043-6. [Epub ahead of print]715 423-436

Novel LC-MS/MS assay to quantify D,L-alpha-difluoromethylornithine (DFMO) in mouse plasma.

Matthew A Swanson, Carlye Szarowicz, Schuyler T Pike, Chad R Schultz, André S Bachmann, Thomas C Dowling.

  D,L-alpha-difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase (ODC) that is being investigated to treat cancers such as pediatric neuroblastoma. A novel and sensitive LC-MS/MS assay was developed and validated to quantify DFMO concentrations in support of pre-clinical pharmacokinetic studies in mice. The study was performed using a Shimadzu triple quad LC-MS/MS equipped with an Atlantis HILIC Silica 3 µm 2.1 × 100 mm column, and an isocratic mobile phase (75:25 acetonitrile and 0.2 % formic acid) at a flow rate of 0.5 mL/min. Multiple Reaction Monitoring (MRM) was used to identify the precursor ion (183 m/z) with quantification of daughter ions at transitions of 183 > 120.10, 183 > 166.10, and 183 > 80.05. Plasma standards and quality control samples (20 µL) were processed using protein precipitation with cold acetonitrile. The lower limit of detection (LLOQ) was 5 ng/mL. Assay performance was determined from multiple runs (n = 10) with standards ranging from 250-50,000 ng/mL and three levels of quality control (500, 4000, and 40,000 ng/mL). Standard curves were linear with r2 values between 0.9960 and 0.9999. Quality control samples were stable and exhibited maximum inter-day % bias of ≤3 % and CV% of ≤0.7 %. The assay was successfully applied to an in vivo study to determine the pharmacokinetics of DFMO in athymic nu/nu mice.

Keywords:  D,L-alpha-difluoromethylornithine; DFMO; Eflornithine; Mass spectroscopy; Ornithine decarboxylase; Pharmacokinetics

DOI:  https://doi.org/10.1016/bs.mie.2025.01.036
Biomed Chromatogr. 2025 Jul;39(7): e70119

Determination of 25-Hydroxyvitamin D3 in Rat Brain by Derivatization-Assisted LC/ESI-MS/MS.

Toma Shibuya, Fuwari Shishikura, Natsuki Yoshida, Shoujiro Ogawa, Tatsuya Higashi.

  Recent studies have suggested that vitamin D deficiency may have relations with various neuropsychiatric diseases as well as bone diseases. However, the concentrations of vitamin D metabolites in the brain and the relationship between their brain and serum concentrations remain poorly understood. To answer these questions, we developed and validated an LC/ESI-MS/MS method for quantifying 25-hydroxyvitamin D3 [25(OH)D3], an established marker for assessing vitamin D sufficiency/deficiency, in the rat brain and compared the brain concentrations with the serum concentrations. To enhance the assay sensitivity and specificity, the 25(OH)D3 was derivatized with 4-[4-(1-pipelidinyl)phenyl]-1,2,4-triazoline-3,5-dione (PIPTAD) after purification of the brain sample by a two-step solid-phase extraction. A good linearity was obtained within the range of 20-1000 pg/g tissue, and the intra-assay and interassay precision and accuracy were acceptable. In normal rats (n = 6), the brain 25(OH)D3 concentrations ranged from 128 to 175 pg/g tissue, which were extremely low (approximately 1/100) compared to the serum concentrations. The bile duct ligation caused the decreased serum 25(OH)D3 level, which produced the subsequent decreased brain 25(OH)D3 level (44-79 pg/g, n = 6). These results strongly suggested that the serum 25(OH)D3 concentration has a significant effect on its brain level.

Keywords:  25‐hydroxyvitamin D3; LC/ESI‐MS/MS; derivatization; rat brain; serum

DOI:  https://doi.org/10.1002/bmc.70119
J Chromatogr A. 2025 May 10. pii: S0021-9673(25)00384-X. [Epub ahead of print]1755 466036

Quantification of five intracellular and extracellular methionine pathway intermediates using stable isotope dilution UHPLC-MS/MS.

Lyuye Zhang, Ziyu Liang, Weibing Zhang, Hailin Wang.

  Unraveling the intricate mechanisms underlying methionine metabolism reprogramming and its extracellular release during apoptosis requires robust and sensitive quantification of key intermediates in both intracellular and extracellular compartments. Here, we developed a highly sensitive and precise stable isotope-dilution UHPLC-MS/MS method for simultaneous quantification of five critical intermediates in methionine cycle and methionine salvage pathway: l-methionine (Met), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), l-homocysteine (Hcy), and 5'-methylthioadenosine (5'-MTA). Through optimizing mobile phase additives and implementing an effective dilution strategy, this method minimizes matrix effects. The recovery rates of the five compounds exceeds 71.6 % in cell cytosol and cell medium samples, under a wide concentration range. The validated method presents good precision and linearity, with limits of detection (LODs) ranging from 1.9 fmol/10⁵ cells (SAH) to 555.4 fmol/10⁵ cells (Hcy) in cytosolic fractions, and from 0.7 fmol/10⁵ cells (SAH) to 52.1 fmol/10⁵ cells (Met) in culture media. Application of this validated method to UV-induced apoptosis in human promyelocytic leukemia HL60 cells revealed significant dynamic alterations in both intracellular and extracellular release of methionine pathway intermediates during apoptotic progression. The quantification method provides a robust tool for investigating the regulation and functions of methionine metabolism across basic research and clinical applications.

Keywords:  5′-MTA; Apoptosis; Methionine metabolism; SAH; SAM; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.chroma.2025.466036
Talanta. 2025 May 14. pii: S0039-9140(25)00817-3. [Epub ahead of print]295 128327

Unveiling the dark matter of the metabolome: A narrative review of bioinformatics tools for LC-HRMS-based compound annotation.

Rui Xu, Jiangjiang Zhu.

  Compound annotation, including the unveiling of dark matter in the metabolomics study represents a pivotal undertaking within the metabolomics field, serving as the linchpin for unraveling the identities and attributes of chemical entities. This narrative review examines the evolution of widely adopted compound annotation tools tailored for liquid chromatography-mass spectrometry (LC-MS) data analysis over the past two decades, which has been characterized by a transition from library-based search methodologies to advanced high-throughput approaches. Furthermore, emerging tools originating from both LC and MS domains were summarized. The synergistic partnership between quantitative structure-retention relationship (QSRR) models and machine learning (ML) techniques is explored, encompassing both conventional methodologies and advanced convolutional neural networks (CNNs). This collaborative framework has played a pivotal role in the precise prediction of retention times. Additionally, the enhanced applicability and extensibility of retention order prediction are emphasized, particularly under the constraints of experimental configurations. Within the domain of mass spectra-based annotation, the foundational task of mapping compound structures to mass spectra is examined-traditionally accomplished by aligning experimental data with established standards and libraries. Recent advancements highlight emerging tools that adopt multi-tiered mapping strategies, such as molecular networks and fragmentation trees, or incorporate machine learning to capture complex mapping patterns. This comprehensive examination underscores the pivotal role of compound annotation tools in advancing our understanding of complex LC-MS data matrix to further assist the annotation of dark matter in metabolome.

Keywords:  Compound annotation; Liquid chromatography-mass spectrometry; Machine learning; Mass spectra similarity; Retention time prediction; Structure-spectra mapping

DOI:  https://doi.org/10.1016/j.talanta.2025.128327
Nat Biotechnol. 2025 May 23.

Self-supervised learning of molecular representations from millions of tandem mass spectra using DreaMS.

Roman Bushuiev, Anton Bushuiev, Raman Samusevich, Corinna Brungs, Josef Sivic, Tomáš Pluskal.

Characterizing biological and environmental samples at a molecular level primarily uses tandem mass spectroscopy (MS/MS), yet the interpretation of tandem mass spectra from untargeted metabolomics experiments remains a challenge. Existing computational methods for predictions from mass spectra rely on limited spectral libraries and on hard-coded human expertise. Here we introduce a transformer-based neural network pre-trained in a self-supervised way on millions of unannotated tandem mass spectra from our GNPS Experimental Mass Spectra (GeMS) dataset mined from the MassIVE GNPS repository. We show that pre-training our model to predict masked spectral peaks and chromatographic retention orders leads to the emergence of rich representations of molecular structures, which we named Deep Representations Empowering the Annotation of Mass Spectra (DreaMS). Further fine-tuning the neural network yields state-of-the-art performance across a variety of tasks. We make our new dataset and model available to the community and release the DreaMS Atlas-a molecular network of 201 million MS/MS spectra constructed using DreaMS annotations.

DOI: https://doi.org/10.1038/s41587-025-02663-3
J Chromatogr Sci. 2025 May 07. pii: bmaf028. [Epub ahead of print]63(5):

Simultaneous Determination of 30 Flavonoids in Jinqiancao Granules by LC-MS/MS.

Jiajie Wang, Yimeng Liu, Shuang Xie, Luyao Wang, Beibei Yao, Xu Zhang, Wanwan Zhang, Chunying Wang.

In order to determine the content of flavonoid components in Jinqiancao granules efficiently and accurately, a high-efficiency quantitative analysis method utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed. As per the guidelines from the International Council for Harmonization, a comprehensive method validation was conducted for this method. Experimental data demonstrated that the method exhibited excellent accuracy, specificity and reproducibility in the multiple reaction monitoring mode, facilitating the swift and accurate quantification of flavonoid content. Through chemometrics analysis, significant differences in the content of flavonoid compounds were observed among different batches of Jinqiancao granules samples. Isoschaftoside, Vicenin-2, Schaftoside and myricitrin can be used as the main quality markers for Jinqiancao granules. The research results provided a scientific basis for the quality control of Jinqiancao granules, which helps enhance the consistency and reliability of the product, thereby ensuring the superior quality of the preparation.

DOI: https://doi.org/10.1093/chromsci/bmaf028
J Mass Spectrom. 2025 Jun;60(6): e5145

Development of Simultaneous Analytical Method of Three Polypeptide Toxins α-Amanitin, β-Amanitin and Phalloidin in Poisonous Mushrooms and Human Serum Using UHPLC-MS/MS.

Hang-Ji Ok, Eun-Young Park, Yongho Shin, Jeong-Han Kim, Min-Ho Song, Ji-Ho Lee.

  Accidental ingestion of toxic mushrooms remains a global public health concern because of the presence of highly potent peptide toxins such as α-amanitin, β-amanitin, and phalloidin. These compounds exhibit strong hepatotoxicity and can lead to acute liver failure and death. However, their rapid detection in biological and food matrices remains analytically challenging. Existing methods often require extensive sample preparation and are not suitable for urgent diagnostic applications. This study presents the development and validation of a rapid and sensitive analytical method for the simultaneous quantitation of α-amanitin, β-amanitin, and phalloidin in poisonous mushrooms and human serum. Among several preparation strategies evaluated, a method following direct extraction with 1% formic acid in methanol was selected for its speed, simplicity, and effectiveness in minimizing matrix interference. The method demonstrated excellent linearity (r2 ≥ 0.99), low quantitation limits (10-50 ng/mL), and satisfactory recovery (72%-117%) and precision (RSD ≤ 19%) in both food and biological matrices. When applied to field-collected Amanita virosa, α-amanitin and β-amanitin were detected at 39 and 145 mg/kg, respectively, whereas no toxins were found in Amanita volvata. These findings demonstrate that the established method enables rapid and reliable detection of lethal peptide toxins with minimal sample preparation. The protocol is suitable for forensic investigations, clinical toxicology, and food safety monitoring. Its applicability in emergency settings underscores its potential as a practical tool for improving public health responses to mushroom poisoning incidents.

Keywords:  UHPLC–MS/MS; mushroom; quantitative analysis; serum; toxins

DOI:  https://doi.org/10.1002/jms.5145
Biomed Chromatogr. 2025 Jul;39(7): e70114

Novel LC-MS/MS Method for Quantification of Famotidine and Metoprolol and Application to Pharmacokinetic Interaction.

Ran Xu, En-Fu Feng, Sun-Jun Yin, Ping Wang, Rui Meng, Xue-Sha Zhang, Jian-Mei Pan, Gong-Hao He.

  A rapid and sensitive LC-MS/MS method was developed and validated to simultaneously determine famotidine (FAM) and metoprolol (MET) in rat plasma and applied to study the pharmacokinetic drug-drug interaction between these two drugs in rats. In this method, D4-famotine (D4-FAM) and D6-metoprolol (D6-MET) were used as the internal standard and methanol protein precipitation method was used for sample preparation. After extraction, the samples were carried on an Agilent Gemini-NX C18 column and subjected to a gradient elution process using a mixture of methanol and water containing 0.1% formic acid at a flow rate of 0.4 mL/min within 8 min. The monitored transitions were m/z 338.1 → 189.1 for FAM, 268.2 → 116.1 for MET, 342.1 → 190 for D4-FAM, and 274.2 → 122.1 for D6-MET. The analytes had good linearity in the range of 1-200 ng/mL for FAM and 1-400 ng/mL for MET, with the lower limit of quantitation of 1 ng/mL for both drugs. The validated method was verified to meet the determination requirements of biological samples. It was the first time to study the pharmacokinetics interaction between FAM and MET successfully, which would be necessary and beneficial to explore the clinical safety and efficacy of the combination of these two drugs in the treatment of HF.

Keywords:  LC‐MS/MS; drug–drug interactions; famotidine; metoprolol; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.70114
J Am Soc Mass Spectrom. 2025 May 19.

Phase Modulation to Increase Ion Throughput for Ion Mobility-Time-of-Flight Experiments.

Nathan W Buzitis, Daniel Wu, Zackary Kinlein, Brian H Clowers.

  Implementing a phase-based gating scheme with structures for lossless ion manipulation and a time-of-flight mass analyzer significantly enhances analytical efficiency. Phased ion mobility spectrometry substantially reduces the experimental analysis duration compared to single injection, signal-averaged structures for lossless ion manipulation (SLIM), without compromising separative capabilities. Notably, the observed duty cycle in phased ion mobility spectrometry increases 10x compared to signal-averaged SLIM experiments. A 6-m SLIM system, integrated with a time-of-flight mass analyzer, demonstrates equivalent separative capabilities and resolving power in both signal-averaged and phased-ion mobility modes. However, substantial duty cycle and ion efficiency improvements are evident during phased ion mobility spectrometry. The determination of the numerical integer required for accurate arrival times can be achieved algebraically or directly derived from measured arrival times in signal-averaged experiments. This methodology seamlessly integrates into existing single-gated SLIM or drift tube ion mobility-mass spectrometry instrumentation with minimal experimental adjustments, directly enhancing the observed duty cycle and reducing experimental analysis times.

Keywords:  Instrumentation; Ion Mobility Spectrometry; Multiplexing; Structures for Lossless Ion Manipulation; Time of Flight MS; Traveling Wave

DOI:  https://doi.org/10.1021/jasms.5c00044
Sci Rep. 2025 May 21. 15(1): 17678

Opioid quantification via microsampling techniques to assess opioid use in human laboratory studies.

Ramisa Fariha, Emma Rothkopf, Carolina L Haass-Koffler, Anubhav Tripathi.

Despite progress in neurobiological studies with human subjects, sample availability remains a challenge. Urine samples, widely used for screening, suffer from false-positive results due to immunoassay cross-reactivity. Serum, used for confirmatory testing, offers advantages but faces limitations due to blood collection. Microsamples, with a working volume less than 50 μL, present an ideal strategy for robust quantitative data collection in investigations and human laboratory studies. We developed, validated, and automated a serum-based LC-MS/MS assay for accurate quantification of six opioids using only 20 μL of patient samples. Our method, applied in a clinical trial with patients with opioid use disorder (N = 20) receiving intranasal oxytocin, or placebo, for one week in addition to opioid agonist therapy (buprenorphine or methadone). We quantified six different opioids, undetected by urine strip tests, that were used by patients during the treatment phase. Our high-throughput, automated approach surpasses existing methods in literature, enhancing efficiency in multi-matrix studies.

DOI: https://doi.org/10.1038/s41598-025-99130-5
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 May 14. pii: S1570-0232(25)00208-9. [Epub ahead of print]1261 124654

Quantification of sulbactam and durlobactam in saline and human plasma via ultra-performance liquid chromatography tandem mass spectrometry.

Hanna F Roenfanz, David P Nicolau, Joseph L Kuti.

  Sulbactam-durlobactam is a combination β-lactam/β-lactamase inhibitor antibiotic approved in the United States for the treatment of adult patients with hospital-acquired and ventilator-associated bacterial pneumonia due to susceptible isolates of Acinetobacter baumannii-calcoaceticus complex. A liquid chromatography tandem mass spectrometry method for the quantification of sulbactam and durlobactam in human plasma and aqueous matrices has been developed and validated. The standard curves for each drug were linear over the range 0.5 to 50 μg/mL and use the isotopic analogs sulbactam-d5 and [13C2, 15N2]-durlobactam as internal standards for their respective analytes. This simple, reproducible method for the determination of sulbactam and durlobactam concentrations was developed with the intent to conduct future pharmacokinetic analyses and to guide clinical laboratories in the development of a therapeutic drug monitoring assay.

Keywords:  Acinetobacter; Drug concentrations; Pharmacokinetics; Β-Lactam/β-lactamase inhibitor

DOI:  https://doi.org/10.1016/j.jchromb.2025.124654
Environ Geochem Health. 2025 May 17. 47(6): 211

Stochastic dynamics mass spectrometric quantification and 3D molecular structural analysis of tricyclic antidepressant in marine dissolved organic matter.

Bojidarka Ivanova.

  Antidepressants show widespread administration and higher market demand causing for their increasing concentrations in the environment; thus, highlighting the aquatic systems. This further leads to synergistic effects when antidepressants and metal ions interact simultaneously on free-living aquatic organisms. Therefore, the monitoring of the environmental pollution by antidepressants is imperative to develop proper management strategies for eco-toxicological human health risk assessment. The study applies innovative stochastic dynamics equations for exact processing of mass spectrometric variables to (i) identify and quantify carbamazepine, amitriptyline, and scandoside in marine dissolved organic matter in presence of their oxidized products and complex sample matrix effect; and (ii) determine them 3D molecular structurally; thus, increasing the selectivity and sensitivity of the mass spectrometric protocol. The development of novel methods for mass spectrometric quantitative and structural analysis is of significant importance. Available methods for database matching algorithms and standard spectral library show low cosine similarity values (0.8-0.88) due to the fact that marine organic matter is the most complex natural chemical mixture reducing the method performances of the available protocols. The routinely used selected reaction monitoring approach detecting only analytes of interest could not detect interfering antidepressants due to their environmental chemical transformation. Furthermore, there is difference in retention times ΔRTs = 0.05 min; thus, also presenting reliable co-chromatographic analysis. There are utilised ultra-high resolution electrospray ionization mass spectrometric and co-chromatographic methods, high accuracy computational quantum chemical approaches, and chemometrics. The provided empirical proof of the novel tool using the linear equation D"SD = f(conc.), which drastically assists capability and performances of the analytical mass spectrometry; furthermore, at very low concentration levels within 0.28 ng L-1-1 ng.(mL-1). The quantification of carbamazepine in wastewater waste water effluent via traditional methods show |r|= 0.99448, while the application to the novel formulas D''SD = f(conc.) cause for |r|= 0.99961 within linear dynamic range 5-200 ng mL-1. The method is accurate showing parameters 237.1764; sd(yEr ±) = 0-0.01356, and se(yEr ±) = 0-0.00959 depending on the sample matrix effect. It is also precise, showing m/z 237.267 ± 0.088 and m/z 194.21913 ± 0.03842 via single and tandem operation modes. The chromatographic performances show RT = 18.328, sd(yEr ±) = 0.013, and se(yEr ±) = 0.0075. The sample matrix effect is evaluated via pre- and post extraction spike data both using selected reaction monitoring and full scan operation modes. Two approaches assessing the matrix effect are discussed. The quantitative determining and 3D structural analysis of species within the innovative approach assessing the relation D''SD = f(DQC) shows the following performances: |r|= 0.99797 (carbamazepine), |r|= 0.99962 (scandoside), and |r|= 0.99999 (amitriptyline) in marine organic matter, respectively. The study solves the most enduring problem of monitoring of pharmaceutics pollution causing for eco-toxicological and human health risk of antibiotic resistance and affect on microbial communities; thus, illustrating best performances reported, so far of |r|= 0.99999-0.99962 via innovative stochastic dynamics equations for exact processing of mass spectrometric variables and obtaining of highly reliable quantitative and 3D structural analyses of degradation and synthetic carbamazepines exhibiting diverse oxidized products at very low analyte concentrations within 0.28 ng L-1-1 ng.(mL-1) in marine organic matter which is the most complex natural chemical mixture.

Keywords:  Amitriptyline; Carbamazepine; Chromatography; Marine dissolved organic matter; Mass spectrometry; Quantum chemistry; Stochastic dynamics

DOI:  https://doi.org/10.1007/s10653-025-02519-4
Nat Commun. 2025 May 16. 16(1): 4566

LipidIN: a comprehensive repository for flash platform-independent annotation and reverse lipidomics.

Hao Xu, Tianhang Jiang, Yuxiang Lin, Lei Zhang, Huan Yang, Xiaoyun Huang, Ridong Mao, Zhu Yang, Changchun Zeng, Shuang Zhao, Lijun Di, Wenbin Zhang, Jun Zeng, Zongwei Cai, Shu-Hai Lin.

Improving annotation accuracy, coverage, speed and depth of lipid profiles remains a significant challenge in traditional lipid annotation. We introduce LipidIN, an advanced framework designed for flash platform-independent annotation. LipidIN features a 168.5-million lipid fragmentation hierarchical library that encompasses all potential chain compositions and carbon-carbon double bond locations. The expeditious querying module achieves speeds exceeding one hundred billion queries per second across all mass spectral libraries. The lipid categories intelligence model is developed using three relative retention time rules, reducing false positive annotations and predicting unannotated lipids with a 5.7% estimated false discovery rate, covering 8923 lipids cross various species. More importantly, LipidIN integrates a Wide-spectrum Modeling Yield network for regenerating lipid fragment fingerprints to further improve accuracy and coverage with a 20% estimated recall boosting. We further demonstrate the utility of LipidIN in multiple tasks for lipid annotation and biomarker discovery in clinical cohorts.

DOI: https://doi.org/10.1038/s41467-025-59683-5
J Pharm Biomed Anal. 2025 May 15. pii: S0731-7085(25)00299-7. [Epub ahead of print]264 116958

Qualitative and quantitative determination of SIPI-2011 and its two major metabolites in human plasma.

Jie Fang, Jiao Jing, Guangyao Li, Yongbin Wang, Binjiang Zhao, Yan Zhan, Lei Zhou, Ying Liu, Wei Zhang, Ni Peng, Xiaoyan Chen.

  SIPI-2011, a structural modification of isoquinoline alkaloid, is under investigation for treating arrhythmias. To characterize the safety and tolerability, the pharmacokinetics and metabolism of SIPI-2011 were investigated in humans. After an oral administration of 600 mg SIPI-2011, a total of 32 metabolites were detected in human plasma by UPLC-UV/Q-TOF mass spectrometry utilizing mass defect filter method. The principal biotransformation pathways included di-dehydrogenation (M8-1), dehydrogenation (M9-2), and oxidation and dehydrogenation (M10-4). Afterward, a sensitive LC-MS/MS method was developed to simultaneously determine SIPI-2011 and its two major metabolites M8-1 and M9-2 in human plasma. The isotopically labeled internal standards of the metabolites were obtained by incubating deuterated SIPI-2011 with rat liver homogenates. To achieve effective chromatographic retention and separation, three analytes were eluted on an XDB-phenyl column with alkaline mobile phase, and detected by multiple reaction monitoring (MRM) with positive electrospray ionization source. To reduce the interference from the isotope signals of M8-1 and M9-2 in the higher calibration point, [M+H+ 1]+ ions were selected as precursor ions of M9-2 and SIPI-2011 for MRM analysis. The assay was linear in the concentration range 15.0-3000 ng/mL for SIPI-2011, 0.500-100 ng/mL for M8-1 and 1.00-200 ng/mL for M9-2. The parameters of the method validation all met the acceptance criteria. The pharmacokinetic study indicated that SIPI-2011 was rapidly absorbed with a median Tmax of 0.65 h and a terminal half-life of 15.4 h when healthy volunteers were administered a single dose of 300 mg SIPI-2011. And the plasma exposures of the two metabolites M8-1 and M9-2 were less than 10 % of that of the parent drug.

Keywords:  LC-MS/MS; Metabolic Profiling; Pharmacokinetics; SIPI-2011; UPLC-UV/Q-TOF

DOI:  https://doi.org/10.1016/j.jpba.2025.116958