bims-metlip 2025-01-12 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–01–12
24 papers selected by
Sofia Costa, Matterworks

Nextflow4MS-DIAL: A Reproducible Nextflow-Based Workflow for Liquid Chromatography-Mass Spectrometry Metabolomics Data Processing.
Analysis Profiling of 48 Endogenous Amino Acids and Related Compounds in Human Plasma Using Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry.
Advancements in Mass Spectrometry-Based Targeted Metabolomics and Lipidomics: Implications for Clinical Research.
Development and validation of an UPLC-MS/MS method with polarity switching for simultaneous determination of 14 antiepileptic drugs and 2 metabolites in human serum.
Development and validation of an LC-MS/MS method for quantifying total and unbound doravirine in human plasma.
Comprehensive, Quantitative Analysis of SRM 1950: the NIST Human Plasma Reference Material.
Development and validation of an LC-MS/MS method for quantification of Osimertinib and its two metabolites AZ7550 and AZ5104 in human plasma including long-time storage.
Modular comparison of untargeted metabolomics processing steps.
An Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Deoxycholic Acid and Application to a Pharmacokinetic Study in Human Plasma.
Atmospheric Pressure Mass Spectrometry Imaging Using Electrospray-Assisted Laser Desorption/Ionization with Gas Transportation through a Heated Tube and Minimal Sample Preparation.
Quantification of five antineoplastic agents in swab samples using UPLC-ESI-MS/MS: Method development and validation.
Repackable microfluidic molecularly imprinted solid-phase extraction coupled with mass spectrometry (μMISPE-MS) for rapid analysis of mycotoxin in agri-foods: an example of zearalenone.
Simultaneous quantitation of pentacyclic triterpenoids in fruit peels using liquid chromatography-single quadrupole mass spectrometry.
Determination of clostebol residues in the urine of slaughter animals using liquid chromatography-tandem mass spectrometry.
Toward Automated Preprocessing of Untargeted LC-MS-Based Metabolomics Feature Lists from Human Biofluids.
Signal Processing Workflow for Suspect Screening in LC × LC-HRMS: Efficient Extraction of Pure Mass Spectra for Identification of Suspects in Complex Samples Using a Mass Filtering Algorithm.
Validation of a liquid chromatography-high-resolution mass spectrometry method to quantify peptide-related impurities in teriparatide.
Enantioselective Analysis and Separation of Two β-Blockers via Derivatization Approach.
A fast region of interest algorithm for efficient data compression and improved peak detection in high-resolution mass spectrometry.
Simultaneous Quantification of Total Antibody and Conjugated Payload for DS001 in Rat Serum Using a Hybrid Immuno-Capture LC-MS/MS.
Ecofriendly and biocompatible biochars derived from waste-branches for direct and efficient solid-phase extraction of benzodiazepines in crude urine sample prior to LC-MS/MS.
Quantitative LC-MS/MS Analysis of Endogenous Pseudomonas aeruginosa Isomeric Metabolites Biliverdin IX Alpha, Beta, and Delta in Cell Culture Supernatant, Cell Pellet, and Lung Tissue.
UPLC-MS/MS High-Risk Screening for Sphingolipidoses Using Dried Urine Spots.
Serially coupled columns enhance rapid separation and predictive interaction understanding of 93 fentanyl analogs.

J Am Soc Mass Spectrom. 2025 Jan 05.

Nextflow4MS-DIAL: A Reproducible Nextflow-Based Workflow for Liquid Chromatography-Mass Spectrometry Metabolomics Data Processing.

Xinsong Du, Amanda Dobrowolski, Mathias Brochhausen, Timothy J Garrett, William R Hogan, Dominick J Lemas.

  Reproducibility in untargeted metabolomics data processing remains a significant challenge due to software limitations and the complex series of steps required. To address these issues, we developed Nextflow4MS-DIAL, a reproducible workflow for liquid chromatography-mass spectrometry (LC-MS) metabolomics data processing, validated with publicly available data from MetaboLights (MTBLS733). Nextflow4MS-DIAL automates LC-MS data processing to minimize human errors from manual data handling. The workflow supports software containerization, ensuring computational reproducibility and enabling collaborative research. Nextflow4MS-DIAL is compatible with any Unix-like system and supports multiple job schedulers, offering flexibility and ease of use. The Nextflow4MS-DIAL workflow is available under the permissive MIT license: https://github.com/Nextflow4Metabolomics/nextflow4ms-dial.

Keywords:  LC-MS; Metabolomics; Nextflow; Reproducibility; Workflow

DOI:  https://doi.org/10.1021/jasms.4c00364
Molecules. 2024 Dec 19. pii: 5993. [Epub ahead of print]29(24):

Analysis Profiling of 48 Endogenous Amino Acids and Related Compounds in Human Plasma Using Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry.

Xiongwei Yin, Irene Baldoni, Erwin Adams, Ann Van Schepdael.

  Analyzing and detecting endogenous amino acids in blood is of crucial importance for the diagnosis of medical conditions and scientific research. Considering the lack of UV chromophores in most of these analytes and the presence of several interfering substances in plasma, the quantification of quite a few amino acids and related compounds presents certain technical challenges. As a blank plasma matrix lacking these endogenous substances does not exist, the surrogate matrix method is used, as well as isotopic internal standards for calibration, to ensure the accuracy and reliability of the study. Method validation was conducted for 48 target analytes, giving the following results: linearity (R2 at least 0.99), limit of quantification (from 0.65 to 173.44 μM), precision (intra-day and inter-day RSD for LQC ranged from 3.2% to 14.2%, for MQC from 2.0% to 13.6%, and for HQC from 1.6% to 11.3%), accuracy, recovery, and stability of the method (all complied with the guidelines). To further investigate the applicability of this method to large-scale sample analysis, the method was successfully applied to the analysis of amino acids in plasma samples collected from 20 control individuals, demonstrating its wide application scope for clinical diagnosis and metabolic research.

Keywords:  HILIC-MS/MS; amino acids; non-derivatization; plasma; quantitative; surrogate matrix method

DOI:  https://doi.org/10.3390/molecules29245993
Molecules. 2024 Dec 16. pii: 5934. [Epub ahead of print]29(24):

Advancements in Mass Spectrometry-Based Targeted Metabolomics and Lipidomics: Implications for Clinical Research.

Nguyen Ky Anh, Nguyen Quang Thu, Nguyen Tran Nam Tien, Nguyen Phuoc Long, Huy Truong Nguyen.

  Targeted metabolomics and lipidomics are increasingly utilized in clinical research, providing quantitative and comprehensive assessments of metabolic profiles that underlie physiological and pathological mechanisms. These approaches enable the identification of critical metabolites and metabolic alterations essential for accurate diagnosis and precision treatment. Mass spectrometry, in combination with various separation techniques, offers a highly sensitive and specific platform for implementing targeted metabolomics and lipidomics in clinical settings. Nevertheless, challenges persist in areas such as sample collection, quantification, quality control, and data interpretation. This review summarizes recent advances in targeted metabolomics and lipidomics, emphasizing their applications in clinical research. Advancements, including microsampling, dynamic multiple reaction monitoring, and integration of ion mobility mass spectrometry, are highlighted. Additionally, the review discusses the critical importance of data standardization and harmonization for successful clinical implementation.

Keywords:  lipidomics; mass spectrometry; metabolomics; precision medicine; targeted assay

DOI:  https://doi.org/10.3390/molecules29245934
J Pharm Biomed Anal. 2025 Jan 02. pii: S0731-7085(24)00697-6. [Epub ahead of print]255 116655

Development and validation of an UPLC-MS/MS method with polarity switching for simultaneous determination of 14 antiepileptic drugs and 2 metabolites in human serum.

Xiao-Han Peng, Yan-Lin Zhao, Zhong Huang, Xin-Feng Xia, Kun Wang, Peng Jin, Yan Du, Dao-Quan Tang.

  Currently, treatment with antiepileptic drugs (AEDs) is still the first choice for epileptic patients, while monitoring their blood concentrations is undoubtedly beneficial for minimizing their adverse side effects and optimizing their therapeutic effects. In this study, an ultra-high performance liquid chromatography coupled with tandem mass spectrometry with polarity switching was developed and validated for simultaneous determination of 14 AEDs and 2 active metabolites in human serum. Olanzapine was selected as the internal standard. One-step protein precipitation using methanol containing 0.05 % formic acid was used to treat sample, and the supernatant was injected for analysis without further evaporation and reconstitution. Chromatographic separation was performed on an Aglient Zorbax Eclipse Plus C18 (50 mm × 2.1 mm, 1.8 μm) column with gradient methanol and 0.1 % formic acid in water as mobile phase. Multi-reaction monitoring was performed for quantification of 16 analytes in polarity switching mode. Matrix-matched calibration curves of 16 analytes presented good linearity within the test concentration range (r > 0.99). The intra- and inter-run accuracies and precisions at the lower limit of quantification, and low, medium and high quality control levels were all less than 20 % or 15 %, respectively. The extraction recovery, matrix effect, and stability were all acceptable under detected conditions. Finally, this method was successfully applied in the quantitation of target analytes in the serum of patients received AEDs.

Keywords:  Antiepileptic drugs; Human serum; Therapeutic drug monitoring; Ultra-high performance liquid chromatography-tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.jpba.2024.116655
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Dec 31. pii: S1570-0232(24)00448-3. [Epub ahead of print]1252 124439

Development and validation of an LC-MS/MS method for quantifying total and unbound doravirine in human plasma.

Rizqah Bernard, Sydwell P Maputla, Phiwe Zuma, Anton Joubert, Sandra Castel, Marthinus van der Merwe, Edda Zangenberg, Lubbe Wiesner.

  A robust LC-MS/MS method was developed to quantify total and unbound doravirine in plasma samples from patients receiving daily doses of 100 mg doravirine, in combination with lamivudine and tenofovir disoproxil fumarate, in a phase 3 clinical trial. The trial is ongoing, and sample analysis is planned to commence once all samples have been collected. The method was validated to quantify both total and unbound doravirine using a single calibration curve. Protein precipitation was used to obtain the total doravirine from plasma and ultrafiltration was used to obtain the unbound doravirine. A Kinetex 1.7 µm EVO C18 100 Å column was used for chromatography using a gradient mobile phase (0.1 % formic acid in water and 0.1 % formic acid acetonitrile) at a flow rate of 250 µL/min during a five minute runtime. The analyte was ionized by positive electrospray ionization and detection was by multiple reaction monitoring on a Sciex API3200 triple quadrupole mass spectrometer. Using calibration standards prepared in whole plasma, the calibration curve fitted a quadratic regression (weighted by 1/x) over a calibration range of 7.00-2000 ng/mL and was applied successfully for the measurement of both total and unbound doravirine. Validation experiments, conducted according to international guidelines, proved the accuracy and precision of the method over three consecutive days. The method demonstrated robustness in the presence of matrix components, different anticoagulants, hemolyzed blood (2 %), and concomitant medications, showing the necessary sensitivity and selectivity for the quantification of both total and unbound doravirine concentrations expected in the study samples.

Keywords:  LC-MS/MS; Protein precipitation; Total doravirine; Ultrafiltration; Unbound doravirine

DOI:  https://doi.org/10.1016/j.jchromb.2024.124439
Anal Chem. 2025 Jan 05.

Comprehensive, Quantitative Analysis of SRM 1950: the NIST Human Plasma Reference Material.

Rupasri Mandal, Jiamin Zheng, Lun Zhang, Eponine Oler, Marcia A LeVatte, Mark Berjanskii, Matthias Lipfert, Jun Han, Christoph H Borchers, David S Wishart.

Many analytical methods have been developed for performing targeted metabolomics. By combining multiple analytical techniques, comprehensive coverage of the metabolome can be achieved. We combined multiple analytical techniques to comprehensively and quantitatively characterize the widely studied NIST human plasma reference material, SRM 1950. Our goal was to provide a large, well-validated list of confident metabolite concentration values (i.e., benchmarks) to assist the metabolomics community in its calibration and comparison efforts. We used four analytical platforms: high-resolution NMR spectroscopy, direct injection tandem MS (DI-MS/MS), liquid chromatography tandem MS (LC-MS/MS), and inductively coupled plasma MS (ICP-MS). Eight validated analytical assays were run, yielding accurate quantitative measurements for 728 unique metabolites or metabolite species. Through computer-aided literature mining, we identified another 330 unique metabolites previously quantified in SRM 1950. We compared NIST-certified values along with literature-derived concentrations/ranges to the metabolite concentrations measured by our four platforms and eight assays. From these assays/platforms, we generated a list of high-confidence concentration values of 1058 metabolites or metabolite species in SRM 1950 including data for 60 amino acids/related compounds, 48 bile acids, 72 amines/sugars/alcohols, 21 metals, 8 catecholamines, 11 vitamins, 92 organic acids, 40 fatty acids/steroids/nucleobases/indole derivatives, 5 polyfluorinated compounds, 7 carotenoids, 39 acylcarnitines, 76 oxylipins, 13 sterols, and 566 lipids/lipid species. This data set represents the most complete quantitative characterization of SRM 1950. An online database (SRM1950-DB) containing 1058 plasma metabolites/metabolite species in SRM 1950, their structures, HMDB IDs, mass, chemical class, concentrations, references, and reliability is freely available at https://srm1950-data.wishartlab.com.

DOI: https://doi.org/10.1021/acs.analchem.4c05018
J Pharm Biomed Anal. 2025 Jan 03. pii: S0731-7085(25)00003-2. [Epub ahead of print]255 116662

Development and validation of an LC-MS/MS method for quantification of Osimertinib and its two metabolites AZ7550 and AZ5104 in human plasma including long-time storage.

Eva Greibe, Boe Sorensen, Peter Meldgaard, Elke Hoffmann-Lücke.

  Osimertinib (AZD9291) is a widely used tyrosine kinase inhibitor for the treatment of non-small cell lung cancer patients with activating EGFR mutations. However, the correlation between dose and efficacy has been debated for several years. For this reason, there is a need for standardized methods for routine analysis, clinical studies on pharmacokinetics and dose-response relationships, and greater understanding of preanalytical conditions, such as sample storage stability. The objective of this study was to develop and validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of osimertinib and its two metabolites, AZ7550 and AZ5104, in human plasma and to investigate long-term storage stability of the analytes. Samples were prepared by protein precipitation and separated on a Kinetex EVO C18 column (2.1 × 150 mm, 2.6 µm). Electrospray ionization in positive mode and multiple reaction monitoring were used to monitor the ion transitions. The validated concentration ranges were from 1.25 to 3000 ng/mL. Interassay precisions and accuracies were all ≤ 15 %. Linearity, dilution integrity, and carry-over were also examined and satisfied the validation criteria. Stability was examined under different conditions, and the analytes were found to be stable for more than 3 years at -80°C (< 15 % decline). Finally, the analytical method was successfully applied in a clinical setting on plasma samples from 30 patients with non-small cell lung cancer in treatment with osimertinib, demonstrating its suitability for use in clinical studies and its potential for therapeutic drug monitoring.

Keywords:  AZ5104; AZ7550; High-performance liquid chromatography tandem mass spectrometry (LC-MS/MS); Osimertinib; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.jpba.2025.116662
Anal Chim Acta. 2025 Jan 22. pii: S0003-2670(24)01292-3. [Epub ahead of print]1336 343491

Modular comparison of untargeted metabolomics processing steps.

Markus Aigensberger, Christoph Bueschl, Ezequias Castillo-Lopez, Sara Ricci, Raul Rivera-Chacon, Qendrim Zebeli, Franz Berthiller, Heidi E Schwartz-Zimmermann.

   BACKGROUND: Untargeted metabolomics requires robust and reliable strategies for data processing to extract relevant information form the underlying raw data. Multiple platforms for data processing are available, but the choice of software tool can have an impact on the analysis. This study provides a comprehensive evaluation of four workflows based on commonly used metabolomics software tools: XCMS, Compound Discoverer, MS-DIAL, and MZmine. These tools were applied to a dataset derived from bovine saliva samples spiked with small polar molecules analyzed by anion exchange chromatography coupled to high resolution mass spectrometry.
RESULTS: The analysis revealed significant differences in the number and overlap of detected features, with only approximately 8 % of the features included in all four peak tables. Among the overlapping features, MS-DIAL demonstrated the greatest similarity to manual integration, while XCMS and MZmine also performed well. In contrast, Compound Discoverer had issues to reliably integrate high baseline peaks. This study also explores various post-processing strategies, including missing value imputation, transformation, scaling, and filtering. The assessment of missing values indicated that they primarily originated from low abundance, making imputation with small values the most effective approach. No clear evidence suggested that transformation is necessary for downstream statistical analyses. Auto scaling emerged as the most suitable strategy for data scaling. Low thresholds for blank filtering were found to be the most effective in enhancing data quality. The optimization of filtering thresholds required a careful balance to remove unnecessary information while retaining vital data.
SIGNIFICANCE AND NOVELTY: This work provides an overview of commonly applied strategies in untargeted metabolomics analysis, emphasizing the importance of careful workflow selection and optimization. It serves as a resource for refining data processing strategies to achieve accurate and reliable results, while also offering fresh insights into the challenges encountered throughout the untargeted metabolomics processing pipeline.

Keywords:  Anion exchange chromatography; Data treatment; Mass spectrometry; Metabolomics; Processing

DOI:  https://doi.org/10.1016/j.aca.2024.343491
Biomed Chromatogr. 2025 Feb;39(2): e6076

An Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Deoxycholic Acid and Application to a Pharmacokinetic Study in Human Plasma.

Wanlin Xi, Diyi Fu, Ni Wu, Fei Liu, Cuixia Zhang, Ruijie Wan, Zhen Wu, Rui Chen, Qian Zhao.

  Deoxycholic acid (DCA) injection is applied in treating moderate to severe submental bulge or facial fullness caused by excessive submental fat accumulation. Using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology, which was swiftly, precisely, and reliably confirmed, DCA was determined in human plasma with low quantification limits of 56 ng/mL. We selected six healthy individual blank human plasma with low concentrations of endogenous DCA and mixed them to prepare standard curve samples. The samples were purified by the protein precipitation technique and then separated using a BEH C18 column (2.1 × 50 mm, 1.7 μm). Using multiple reaction monitoring (MRM) and electrospray ionization (ESI) sources operating in negative mode, the mass was identified and measured. The precursor-to-product ion transitions were observed at m/z 391.2- → 345.2- and m/z 395.2- → 349.2- for DCA and DCA-d4 (isotope internal standard), respectively. This method was thoroughly validated, encompassing assessments of linearity, sensitivity, precision, selectivity, stability, matrix effect, accuracy, carryover, and recovery. In a word, the validation results demonstrated that this method exhibited sensitivity, accuracy, and reproducibility and could effectively be utilized for studying the pharmacokinetic properties of DCA in a randomized, parallel, controlled, Phase I clinical trial.

Keywords:  UPLC–MS/MS; deoxycholic acid; human plasma; pharmacokinetic

DOI:  https://doi.org/10.1002/bmc.6076
Mass Spectrom (Tokyo). 2024 ;13(1): A0167

Atmospheric Pressure Mass Spectrometry Imaging Using Electrospray-Assisted Laser Desorption/Ionization with Gas Transportation through a Heated Tube and Minimal Sample Preparation.

Riku Hirotani, Yuto Miyoshi, Varun Sendilraj, Hisanao Hazama.

  Mass spectrometry (MS) is a valuable tool that enables label-free analysis and the ability to measure multiple molecules. The atmospheric pressure MS imaging (MSI) method usually requires tedious sample preparation. A simple ionization method with minimal sample preparation is needed for high-throughput analysis. We have developed an ion source that does not require sample preparation such as thinning, curing, planarization, or addition of matrix by the electrospray-assisted laser desorption/ionization with gas transportation (ELDI-GT). The sample is transported with nitrogen gas through a heated tube to the electrospray. The ion signal of protonated caffeine was measured under different transport conditions. The ion signal intensity was found to increase 11-fold by changing the flow rate and tube temperature from 2.8 cm3/s and 473 K to 25 cm3/s and 673 K. ELDI-GT was able to visualize the localization of caffeine crystals at a pixel size of 50 µm using MSI because of the effective GT using the heated tube. The dependence of the ion signal intensity was discussed on the amount of heat applied to the sample in the heated tube. ELDI-GT allowed accurate localization of caffeine at a pixel size of 50 µm without the need to apply thinning and matrix to a sample.

Keywords:  atmospheric pressure; focal distance; minimal sample preparation

DOI:  https://doi.org/10.5702/massspectrometry.A0167
Anal Chim Acta. 2025 Jan 22. pii: S0003-2670(24)01293-5. [Epub ahead of print]1336 343492

Quantification of five antineoplastic agents in swab samples using UPLC-ESI-MS/MS: Method development and validation.

Eline Verscheure, Matteo Creta, Katrien Poels, Jeroen Vanoirbeek, Manosij Ghosh, Lode Godderis.

   BACKGROUND: Antineoplastic agents are hazardous drugs used in cancer treatment and consequently can be present at the workplace (e.g. hospital), but also in a home-setting in case of treatment at home. Given the serious health effects, it is important to monitor environmental contamination on a regular base. As currently, no method allows for simultaneous quantification of cyclophosphamide, etoposide, mitomycin C, imatinib and 5-fluorouracil, we developed and validated a method for simultaneous quantification of multiple antineoplastic agents in swab samples.
RESULTS: Sample collection was optimised and samples were extracted using solely an extraction solvent (water or water/acetonitrile), shaking and sonicating. Ultra performance liquid chromatography coupled to tandem mass spectrometry with electrospray ionisation was used for detection of the compounds. Selectivity, linearity, sensitivity, precision and accuracy were determined. The LLOQs were 0.3 ng/swab (cyclophosphamide, mitomycin C), 1 ng/swab (etoposide) and 5 ng/swab (imatinib, 5-fluorouracil). After a spill, quantifiable concentrations of antineoplastic agents could be measured in swabs using the developed methods. After cleaning, all concentrations fell below the LLOQ.
SIGNIFICANCE: Two methods allowing for safe collection, rapid preparation and sensitive quantification were developed and can be applied in the field to measure environmental contamination. One for simultaneous quantification of cyclophosphamide, etoposide, mitomycin C and imatinib, one for quantification of 5-fluorouracil.

Keywords:  Anticancer drug; Antineoplastic agents; Chemical contamination; Environmental monitoring; Liquid chromatography; Mass spectrometry; Swabs; Trace analysis

DOI:  https://doi.org/10.1016/j.aca.2024.343492
Lab Chip. 2025 Jan 08.

Repackable microfluidic molecularly imprinted solid-phase extraction coupled with mass spectrometry (μMISPE-MS) for rapid analysis of mycotoxin in agri-foods: an example of zearalenone.

Marti Z Hua, Jinxin Liu, Tianqi Li, David R McMullin, Yaxi Hu, Xiaonan Lu.

Mycotoxins are detectable in 60-80% of food crops, posing significant threats to human health and food security, and causing substantial economic losses. Most mitigation approaches focus on detecting mycotoxins with standard methods based on liquid chromatography coupled with mass spectrometry (LC-MS). Typical MS methods require extensive sample preparation and clean-up due to the matrix effect, followed by time-consuming LC separation, complicating the analysis process and limiting analytical throughput. This study reports the development of a repackable microfluidic molecularly imprinted solid-phase extraction coupled with mass spectrometry (μMISPE-MS) method for rapid detection of zearalenone in agri-food samples. Silica microspheres coated with molecularly imprinted polymers were synthesized as the sorbent for analyte enrichment and sample clean-up. A cost-effective microfluidic chip was designed and fabricated as the μMISPE platform with fully automated operation, including on-line microcolumn packing and unpacking. With optimized solvent conditions and on-chip μMISPE protocol, the entire analytical process from sample to answer was completed within 15 min and achieved high recoveries (71-94%) for corn and rice samples at residue levels of 0.05-0.5 ppm (within Canadian regulatory limits of 0.2-10 ppm). This μMISPE-MS method provides a promising tool for improving mycotoxin monitoring in agri-food systems and is generalizable to other rapid analyses of targeted chemicals in complex matrices.

DOI: https://doi.org/10.1039/d4lc00760c
Food Chem. 2025 Jan 05. pii: S0308-8146(25)00031-7. [Epub ahead of print]471 142781

Simultaneous quantitation of pentacyclic triterpenoids in fruit peels using liquid chromatography-single quadrupole mass spectrometry.

Keiji Yabukawa, Ryouichi Izuchi.

  The simultaneous determination of pentacyclic triterpenoids, which are secondary metabolites in plants, is difficult because of the similarity in structure and minimal differences in polarity. In this study, we established a simple and sensitive method for the simultaneous determination of pentacyclic triterpenoids based on liquid chromatography-single-quadrupole mass spectrometry (LC-MS) using a C30 column. The established method completely separated 13 pentacyclic triterpenoids based on their different retention times. Furthermore, the molecular weight and reliability were confirmed for ten compounds. This method was used to quantify pentacyclic triterpenoids in acetone extracts of the peels of ten varieties of apple and eight varieties of persimmon. Therefore, this method is expected to be used in a comprehensive search for pentacyclic triterpenoids in fruit-processing residues.

Keywords:  Apples; Liquid chromatography; Peels; Pentacyclic triterpenoid; Persimmons; Simultaneous quantitation; Single quadrupole mass spectrometry

DOI:  https://doi.org/10.1016/j.foodchem.2025.142781
J Vet Res. 2024 Dec;68(4): 611-621

Determination of clostebol residues in the urine of slaughter animals using liquid chromatography-tandem mass spectrometry.

Iwona Matraszek-Żuchowska, Alicja Kłopot, Justyna Grzelak, Paulina Zdonek.

   Introduction: Synthetic anabolic hormones, which may pose a potential risk to human health, should not be used in fattening food-producing animals. Because of the hormonal effects they cause, growth-promoting compounds are banned by legislation in the EU for use in animal husbandry. Consequently, all EU member states are required to conduct monitoring tests on the content and residues of these compounds in prescribed biological matrices to ensure the safety of food consumers. The aim of this research was to develop a liquid chromatography-tandem mass spectrometry method for the detection of the residue of one such anabolic hormone, clostebol in food animal urine.
Material and Methods: Clostebol and its marker metabolite residues were determined by a method involving enzymatic hydrolysis, isolation of compounds from urine on a C18 solid-phase extraction (SPE) column, purification of the extract by liquid-liquid extraction using n-pentane and a NH2 SPE column, and detection by liquid chromatography-tandem mass spectrometry.
Results: No traces of this anabolic steroid hormone or its metabolites were found in any of the samples tested. The method was validated in accordance with the current requirements for confirmatory methods, and the determined parameters of the decision limits necessary for assessing sample compliance met the specified criteria.
Conclusion: In 2023, the method was introduced for testing under the National Control Plan in Poland. Up to July 19, 2024, 53 urine samples from different animal species had been tested.

Keywords:  LC-MS/MS; clostebol; hormone residue; urine

DOI:  https://doi.org/10.2478/jvetres-2024-0070
Anal Chem. 2025 Jan 06.

Toward Automated Preprocessing of Untargeted LC-MS-Based Metabolomics Feature Lists from Human Biofluids.

Amy Hughes, Pablo Vangeenderhuysen, Marilyn De Graeve, Beata Pomian, Tim S Nawrot, Jeroen Raes, Simon J S Cameron, Lynn Vanhaecke.

Maximizing the extraction of true, high-quality, nonredundant features from biofluids analyzed via LC-MS systems is challenging. Here, the R packages IPO and AutoTuner were used to optimize XCMS parameter settings for the retrieval of metabolite or lipid features in both ionization modes from either faecal or urine samples from two cohorts (n = 621). The feature lists obtained were compared with those where the parameter values were selected manually. Three categories were used to compare feature lists: 1) feature quality through removing false positives, 2) tentative metabolite identification using the Human Metabolome Database (HMDB) and 3) feature utility such as analyzing the proportion of features within intensity threshold bins. Furthermore, a PCA-based approach to feature filtering using QC samples and variable loadings was also explored under this category. Overall, more features were observed after automated selection of parameter values for all data sets (1.3- to 3.7-fold), which propagated through comparative exercises. For example, a greater number of features (on average 51 vs 45%) had a coefficient of variation (CV) < 30%. Additionally, there was a significant increase (7.6-10.4%) in the number of faecal metabolites that could be tentatively annotated, and more features were present in higher intensity threshold bins. Considering the overlap across all three categories, a greater number of features were also retained. Automated approaches that guide selection of optimal parameter values for preprocessing are important to decrease the time invested for this step, while taking advantage of the wealth of data that LC-MS systems provide.

DOI: https://doi.org/10.1021/acs.analchem.4c03124
Anal Chem. 2025 Jan 07.

Signal Processing Workflow for Suspect Screening in LC × LC-HRMS: Efficient Extraction of Pure Mass Spectra for Identification of Suspects in Complex Samples Using a Mass Filtering Algorithm.

Paul-Albert Schneide, Oskar Munk Kronik.

The data processing workflows for comprehensive two-dimensional liquid chromatography (LC × LC) hyphenated to high-resolution mass spectrometry (HRMS) operated in data-independent acquisition (DIA) are limited compared to their one-dimensional counterparts. A two-step workflow is proposed to extract pure mass spectra from LC × LC-HRMS. First, a mass filtering (MF) algorithm groups ions belonging to the same compound based on their elution profile similarity in the first (1D) and second dimension (2D). Second, the filtered data are deconvoluted using multivariate curve resolution (MCR) to address potential coelution. The presented workflow is termed MF + MCR and was tested on pulsed elution-LC × LC-HRMS data from a wastewater effluent extract. The proposed workflow was benchmarked to the following three data processing strategies for mass spectra extraction: peak apex (PAM), using the MF approach alone, or using MCR without prior MF. The MF + MCR workflow identified 25 suspect compounds, compared to 23, 16, and 10 identified by MF, MCR, and PAM, respectively. The nine suspects that could not be identified using MCR compared to the MF + MCR all had low total signal contributions, i.e., low intensities compared to the TIC. This showed that adequate preprocessing prior to MCR is essential for trace level analysis. Additionally, it was shown that the MF + MCR workflow extracted statistically significantly purer mass spectra compared to PAM (p-value: 0.003) and MCR (p-value: 0.04) from a spiked blank sample. The results highlight that by utilizing the elution profiles in both chromatographic dimensions, clean mass spectra of analytes at trace levels measured in DIA can be extracted, allowing for more reliable compound identification compared to the workflows that were used for benchmarking.

DOI: https://doi.org/10.1021/acs.analchem.4c04288
J Pharm Biomed Anal. 2024 Dec 28. pii: S0731-7085(24)00696-4. [Epub ahead of print]255 116654

Validation of a liquid chromatography-high-resolution mass spectrometry method to quantify peptide-related impurities in teriparatide.

Daniel A Weisz, Sarah M Rogstad, Kui Zeng, Eric Pang, Ilan Geerlof-Vidavsky.

  With recent advances in quantitative high-resolution mass spectrometry (HRMS), there is growing interest in developing liquid chromatography (LC)-HRMS methods that can simultaneously quantify numerous critical impurities in a peptide or protein drug. This approach is attractive as it could reduce the total number of methods and instruments required during product development and quality control testing, while taking advantage of the technique's high specificity and sensitivity. To investigate the feasibility of this approach for peptide drugs, an LC-HRMS method was validated for the quantification of six peptide-related impurities in teriparatide, the 34-amino acid active ingredient in Forteo. External calibration curves were constructed to correlate the peak area ratio of impurity-to-teriparatide to a known impurity abundance. The method displayed good specificity, sensitivity, linearity, accuracy, repeatability, intermediate precision, and robustness. The lower limits of quantification were 0.02 % or 0.03 % of teriparatide, below the regulatory reporting threshold of 0.10 %. It was found that quantification using three isotopic peaks per peptide did not provide a significant benefit over quantification with one isotopic peak. The method was validated successfully without the impractical inclusion of an isotopically-labeled internal standard for each impurity. Future studies will be conducted to determine the method's longer-term reproducibility.

Keywords:  Impurity; Mass spectrometry; Method validation; Peptide therapeutic

DOI:  https://doi.org/10.1016/j.jpba.2024.116654
Chirality. 2025 Jan;37(1): e70010

Enantioselective Analysis and Separation of Two β-Blockers via Derivatization Approach.

Vinod, Shubham Sharma, Rajasekhar V S R Pullabhotla, Ankit Mittal.

  Enantiomeric analysis of chiral drugs is very significant, as their enantiomers display different pharmacological or toxicological behavior towards living systems. Among these drugs, β-blockers are available as racemates, where their enantiomers display different pharmacological effects. Herein, we report enantioselective separation of two β-blockers, namely, atenolol and sotalol, using a derivatization approach. The analytes were derivatized with "(S)-1-[1H-benzo(d)(1,2,3)triazol-1-yl]-2-[6-methoxynaphthalen-2-yl-propan-1-one]" {(S)-BTMNP} in a straightforward derivatization step. The resulting diastereomers were separated on a reverse-phase HPLC C18 column with a mobile phase composed of acetonitrile and TEAP buffer (75:25, v/v, pH = 3.5) and detection at 230 nm. This method achieved successful enantiomer separation for both drugs within 20 min, yielding resolution values greater than 3.8. The detection limits were determined to be 6.4 and 4.6 ng mL-1 for atenolol and sotalol, respectively, which indicated sensitivity and effectiveness of the method for the analysis of two β-blockers from their dosage formulations.

Keywords:  (S)‐1‐[1H‐benzo(d)(1,2,3)triazol‐1‐yl]‐2‐[6‐methoxynaphthalen‐2‐yl‐propan‐1‐one; (S)‐naproxen; HPLC; atenolol; chiral derivatizing reagent; enantioresolution; sotalol

DOI:  https://doi.org/10.1002/chir.70010
Anal Bioanal Chem. 2025 Jan 09.

A fast region of interest algorithm for efficient data compression and improved peak detection in high-resolution mass spectrometry.

Oskar Munk Kronik, Jan H Christensen, Nikoline Juul Nielsen, Selina Tisler, Giorgio Tomasi.

  Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) is commonly used for identification of compounds in complex samples due to the high chromatographic and mass spectral resolution provided. In subsequent data processing workflows, it is imperative to preserve this resolution to fully exploit the data. "Region of interest" (ROI) algorithms were introduced as a better alternative to equidistant binning for compressing HRMS data because they better preserve the mass spectral resolution. In this paper, we present a new ROI algorithm that improves on the selection of contiguous m/z traces, amongst others by introducing the concept of chromatographic filter, allows for an automated approach to optimise the admissible mass-to-charge deviation (δm/z) and can be used to match ROIs across multiple samples. The algorithm was tested on a LC-HRMS dataset comprised of 21 replicate injections of a wastewater effluent extract and assessed on its ability to correctly retrieve the ROI's relative to 57 compounds and match them across all injections. In summary, it achieved a ten-fold compression rate in on-disk storage at a noise threshold of 200 counts, and the median ROI length matched the observed chromatographic peak width (12-23 points). Correct ROI matching with a mass accuracy of 9 ppm was observed for 52 compounds across all 21 injections with only one compound split between two adjacent m/z traces in six runs. Overall, the new algorithm performed favourably compared to the ROI algorithm currently used in the well-established ROI-MCR (multivariate curve resolution) workflow for deconvolution of HRMS chromatographic data.

Keywords:  Chromatography; Data preprocessing; High-resolution mass spectrometry; Non-target screening; Objective parameterisation; Region of interest

DOI:  https://doi.org/10.1007/s00216-024-05718-7
AAPS J. 2025 Jan 07. 27(1): 23

Simultaneous Quantification of Total Antibody and Conjugated Payload for DS001 in Rat Serum Using a Hybrid Immuno-Capture LC-MS/MS.

Xiong Yu, Weiqiang Li, Wensi Huang, Bo Xiao, Jing Long, Qi Wang, Guifeng Wang, Chunhe Wang, Mingming Yu, Jinghua Yu, Xingxing Diao.

  Antibody-drug conjugates (ADCs) are intricate compounds that pose significant challenges in bioanalytical characterization. Therefore, multiple bioanalytical methods are required to comprehensively elucidate their pharmacokinetic (PK) profiles. In this study, we investigated DS001, an ADC consisting of a humanized monoclonal antibody (hRS7), a cleavable chemical linker, and the microtubule inhibitor monomethyl auristatin E (MMAE), with a drug-to-antibody ratio (DAR) of 8. This study established a rapid and sensitive hybrid immunoaffinity liquid-chromatography-tandem-mass-spectrometry (LC-MS/MS) approach for the simultaneous quantification of the total antibody and the enzymatically cleavable conjugated payload of DS001. This method is capable of monitoring fluctuations in average DAR values during PK assessments. The sample preparation procedure involved immunocapture, denaturation, trypsin digestion, papain digestion, and termination, all completed within a total processing time of less than 4 h. The method demonstrated linearity for the total antibody in the range of 100 ng/mL (lower-limit-of-quantification, LLOQ) to 100,000 ng/mL, and for the conjugated payload from 3.495 ng/mL (LLOQ) to 3495 ng/mL in rat serum. Both analytes exhibited standard curve correlation coefficients (r) greater than 0.990 within their respective linear ranges. The precision and accuracy of the method were within ± 15% (± 20% for LLOQ). The verified LC-MS/MS approach was successfully employed in the PK analysis following intravenous administration of 0.2 mg/kg DS001 in rats via tail vein injection.

Keywords:  ADC; LC–MS/MS; payload; simultaneous quantification; total antibody

DOI:  https://doi.org/10.1208/s12248-024-01007-4
Mikrochim Acta. 2025 01 10. 192(2): 66

Ecofriendly and biocompatible biochars derived from waste-branches for direct and efficient solid-phase extraction of benzodiazepines in crude urine sample prior to LC-MS/MS.

Shi-Qi Fu, Bing-Dan Wang, Yuan-Chun Li, Zhi-Xin Huang, Zhao-Wei Shi, Gui-Fu Zuo, Jun-Jian Zhao, Hou-Jun Xu, Man-Man Wang.

  Biochars (BCs) derived from waste-branches of apple tree, grape tree, and oak were developed for direct solid-phase extraction (SPE) of five benzodiazepines (BZDs) in crude urine samples prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination. Scanning electron microscopy, elemental analyzer, X-ray diffractometry, N2 adsorption/desorption experiments, and Fourier transform infrared spectrometry characterizations revealed the existence of their mesoporous structure and numerous oxygen-containing functional groups. The obtained BCs not only possessed high affinity towards BZDs via π-π and hydrogen bond interactions, but also afforded the great biocompatibility of excluding interfering components from undiluted urine samples when using SPE adsorbents. Variables affecting SPE of target analytes were systematically optimized including pH, ionic strength, dilution ratio, washing solution, desorption solvent, and its volume. The method of BC-based SPE combined with LC-MS/MS exhibited a wide linear range of 0.03-100 ng/mL, a low detection limit of 0.01-0.08 ng/mL, and satisfactory recovery of 77.6-106% for the studied BZDs. Notably, this method allowed the possibility of direct loading of undiluted urine samples and avoided tedious filtration and dilution steps, which significantly simplified the pretreatment process. Additionally, these BC sorbents derived from waste-branches were ecofriendly and cost-effective, providing a sustainable alternative for the traditional SPE sorbents. Thus, the proposed method has promising application for ecofriendly, simple, efficient, and reliable monitoring of BZDs in urine samples.

Keywords:  Benzodiazepines; Biochar; Liquid chromatography tandem mass spectrometry; Solid-phase extraction; Urine analysis

DOI:  https://doi.org/10.1007/s00604-024-06912-1
J Proteome Res. 2025 Jan 10.

Quantitative LC-MS/MS Analysis of Endogenous Pseudomonas aeruginosa Isomeric Metabolites Biliverdin IX Alpha, Beta, and Delta in Cell Culture Supernatant, Cell Pellet, and Lung Tissue.

Samuel A Krug, Saba Shahzad, William T Witt, Mariette Barbier, Angela Wilks, Maureen A Kane.

  Pseudomonas aeruginosa (Pa) utilizes heme as an iron source from the host during infection. Biliverdin beta and delta (BVIXβ and BVIXδ) are generated by HemO, specific to Pa, while biliverdin alpha is generated from the bacterial BphO system and by mammalian heme oxygenases. Here, we have developed and characterized a quantitative LC-MS/MS assay for the separation of three endogenous isomers, BVIXα, BVIXβ, and BVIXδ. The assay was validated for accuracy, precision, linearity, extraction recovery, solution stability, freeze-thaw stability, benchtop stability, postextraction stability, and nonspecific oxidation of BVIX. The addition of an antioxidant, butylated hydroxytoluene, during sample preparation is needed in order to prevent coupled oxidation from inflating quantitative values of BVIX. The assay development included optimization of a liquid-liquid extraction for bacterial culture supernatants and sample preparation procedures for cell pellets and tissue homogenate to reduce sample demand and automate the extraction procedure in a 96-well format, to enhance extraction throughput. This method was applied to analyze isomer distribution in Pa supernatant, bacterial pellet, and infected lung tissue from Pa-challenged mice. This method can be used in the future for low-volume culture samples, as well as tissue samples, to understand the mechanisms of virulence and inform future drug development.

Keywords:  Pseudomonas aeruginosa; bacterial cell culture; biliverdin IX; heme; isomer separation; lung tissue; quantitative LC−MS/MS

DOI:  https://doi.org/10.1021/acs.jproteome.4c00750
Biomolecules. 2024 Dec 17. pii: 1612. [Epub ahead of print]14(12):

UPLC-MS/MS High-Risk Screening for Sphingolipidoses Using Dried Urine Spots.

Tristan Martineau, Bruno Maranda, Christiane Auray-Blais.

   BACKGROUND: Early detection of sphingolipidoses is crucial to prevent irreversible complications and improve patient outcomes. The use of urine samples dried on filter paper (DUS) is a non-invasive strategy that simplifies the collection, storage, and shipping of samples compared to using liquid urine specimens.
OBJECTIVES: (1) Develop and validate a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methodology using DUS to quantify twenty-one lysosphingolipids normalized to creatinine for eight different sphingolipidoses. (2) Establish normal reference values to evaluate the clinical utility of the methodology.
METHODS: Samples were eluted from a 5 cm filter paper disk (~1 mL of urine) and extracted on Oasis MCX solid-phase extraction cartridges prior to injection in the UPLC-MS/MS system.
RESULTS: Urinary lysosphingolipids were stable on DUS at -80 °C and -30 °C for 117 days, at 21.5 °C and 4 °C for at least 26 days, and at 35 °C for 3 days. Globotriaosylsphingosine, glucosylsphingosine, and their analogs were elevated in patients with Fabry disease and Gaucher disease, respectively, compared to controls (p-value < 0.0001). The analysis of related analog profiles suggests a better overall reliability in detecting patients early, especially for Fabry patients.
CONCLUSIONS: This approach is feasible and might be useful for the early detection, monitoring, and follow-up of patients with sphingolipidoses.

Keywords:  Fabry disease; Gaucher disease; lysosomal storage disorders; lysosphingolipids; sphingolipidoses; tandem mass spectrometry; urine dried on filter paper

DOI:  https://doi.org/10.3390/biom14121612
Anal Chim Acta. 2025 Jan 22. pii: S0003-2670(24)01280-7. [Epub ahead of print]1336 343479

Serially coupled columns enhance rapid separation and predictive interaction understanding of 93 fentanyl analogs.

Jiyu Kim, Sumin Seo, Chohee Jeong, Eunbin Bae, Donghee Lee, Juhyeon Kim, Eunjin Ko, Hamin Choi, Sang Beom Han.

   BACKGROUND: The simplicity of synthesis methods has facilitated the illegal manufacture of various fentanyl analogs, leading to numerous fatal overdoses worldwide, particularly in North America. Fentanyl analogs with similar structures are difficult to distinguish due to their fragmentation patterns, making separation using chromatography essential. Additionally, because fentanyl analogs are lethal even in trace amounts, they are easily smuggled, and commonly used fentanyl test strips often fail to detect them due to their low sensitivity. Therefore, the urgent need for analytical methods that can simultaneously identify multiple analogs and swiftly detect them at low concentrations.
RESULTS: In this study, liquid chromatography-tandem mass spectrometry was conducted to screen 93 types of fentanyl analogs among the illegal fentanyl substances. The phenyl-hexyl columns enhance fentanyl analog separation through strong π-π interactions. The serially coupled column system increased the separation efficiency and mitigated peak distortion, particularly those of polar fentanyl analogs. The selectivity varied significantly, depending on the interactions with the combined columns. The phenyl-hexyl column's superior ability to predict fentanyl analog interactions based on molecular structure was confirmed by retention factor analysis. The resolution of fentanyl isomers increased significantly when methanol was used instead of acetonitrile as an organic modifier in the mobile phase. The approach was validated by determining the limits of detection and quantification, specificity, detection capability, recovery, and relative ion intensity.
SIGNIFICANCE: The fentanyl analogs, including 23 sets of isomeric and isobaric compounds, were analyzed via separation using a phenyl-hexyl column serially coupled with a cyano column. The serially coupled column system increased the separation efficiency and mitigated peak distortion, particularly those of polar fentanyl analogs. The proposed strategy can be adopted in exploring methods of effectively separating mixtures with diverse properties, aiding the prevention of drug abuse and bolstering public health and safety efforts.

Keywords:  Fentanyl analogs; Liquid chromatography (LC); Mass spectrometry (MS); Serially coupled columns; π–π interactions

DOI:  https://doi.org/10.1016/j.aca.2024.343479