bims-metlip 2024-04-28 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–04–28
twenty-six papers selected by
Sofia Costa, Matterworks

SCALiR: A Web Application for Automating Absolute Quantification of Mass Spectrometry-Based Metabolomics Data.
Sample Preparation from Plant Tissue for Gas Chromatography-Mass Spectrometry (GC-MS)we.
Aza-Prilezhaev Aziridination-Enabled Multidimensional Analysis of Isomeric Lipids via High-Resolution U-Shaped Mobility Analyzer-Mass Spectrometry.
Methylation of phase II metabolites of endogenous anabolic androgenic steroids to improve analytical performance.
DIMet: An open-source tool for Differential analysis of targeted Isotope-labeled Metabolomics data.
Investigation of liquid chromatography-mass spectrometry analysis of a peptide aldehyde SJA6017 with identifying its hemiacetal, gem-diol, and enol ether.
Adjustment of Matrix Effects in Analysis of 36 Secondary Metabolites of Microbial and Plant Origin in Indoor Floor Dust Using Liquid Chromatography-Tandem Mass Spectrometry.
Liquid chromatography with tandem mass spectrometric method for determination of 52 antibiotics in human whole blood and urine and application to forensic cases.
Stable Isotope Labeling and Quantification of Photosynthetic Metabolites.
Fiber-in-tube SPME-CapLC-MS/MS method to determine Aβ peptides in cerebrospinal fluid obtained from Alzheimer's patients.
Monosaccharide Analysis After One-Pot Derivatization Followed by Reverse-Phase Liquid Chromatography Separation and UV/Vis Detection.
Novel dummy molecularly imprinted polymer for simultaneous solid-phase extraction of stanozolol metabolites from urine.
Development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of the antimalarial drug pyronaridine in human whole blood.
Development and validation of ivermectin quantification method in volumetric absorptive microsampling using liquid chromatography-tandem mass spectrometry.
Effect of Stool Sampling on a Routine Clinical Method for the Quantification of Six Short Chain Fatty Acids in Stool Using Gas Chromatography-Mass Spectrometry.
Challenges in the Metabolomics-Based Biomarker Validation Pipeline.
Single-Cell Untargeted Lipidomics Using Liquid Chromatography and Data-Dependent Acquisition after Live Cell Selection.
A validated method for simultaneous quantification of four antiretrovirals in dried blood spot and plasma using LC-MS/MS: Application to efavirenz therapeutic drug monitoring in pregnant patients.
Detection and Imaging of Exposure-Related Metabolites and Xenobiotics in Hard Tissues by Laser Sampling and Mass Spectrometry.
From molecules to visuals: Empowering drug discovery and development with mass spectrometry imaging.
Bioanalysis of Stress Biomarkers through Sensitive HILIC-MS/MS Method: A Stride toward Accurate Quantification of MDA, ACR, and CTA.
The Impact of Different Drying Methods on the Metabolomic and Lipidomic Profiles of Arthrospira platensis.
A specific and sensitive GC-MS/MS method for the quantitative determination of 2-phenoxyethanol and selected metabolites in human blood and urine.
Studying Venom Toxin Variation Using Accurate Masses from Liquid Chromatography-Mass Spectrometry Coupled with Bioinformatic Tools.
Abundance of Modifications in Mature miRNAs Revealed by LC-MS/MS Method Coupled with a Two-Step Hybridization Purification Strategy.
Labeling of Mucin-Type O-Glycans for Quantification Using Liquid Chromatography and Fluorescence Detection.

Anal Chem. 2024 Apr 20.

SCALiR: A Web Application for Automating Absolute Quantification of Mass Spectrometry-Based Metabolomics Data.

Luis F Ponce, Stephanie L Bishop, Soren Wacker, Ryan A Groves, Ian A Lewis.

Quantitative liquid chromatography-mass spectrometry (LC-MS)-based metabolomics is becoming an important approach for studying complex biological systems but presents several technical challenges that limit its widespread use. Computing metabolite concentrations using standard curves generated from standard mixtures of known concentrations is a labor-intensive process that is often performed manually. Currently, there are few options for open-source software tools that can automatically calculate metabolite concentrations. Herein, we introduce SCALiR (standard curve application for determining linear ranges), a new web-based software tool specifically built for this task, which allows users to automatically transform LC-MS signals into absolute quantitative data (https://www.lewisresearchgroup.org/software). SCALiR uses an algorithm that automatically finds the equation of the line of best fit for each standard curve and uses this equation to calculate compound concentrations from the LC-MS signal. Using a standard mix containing 77 metabolites, we show a close correlation between the concentrations calculated by SCALiR and the expected concentrations of each compound (R2 = 0.99 for a y = x curve fitting). Moreover, we demonstrate that SCALiR reproducibly calculates concentrations of midrange standards across ten analytical batches (average coefficient of variation 0.091). SCALiR can be used to calculate metabolite concentrations either using external calibration curves or by using internal standards to correct for matrix effects. This open-source and vendor agnostic software offers users several advantages in that (1) it requires only 10 s of analysis time to compute concentrations of >75 compounds, (2) it facilitates automation of quantitative workflows, and (3) it performs deterministic evaluations of compound quantification limits. SCALiR therefore provides the metabolomics community with a simple and rapid tool that enables rigorous and reproducible quantitative metabolomics studies.

DOI: https://doi.org/10.1021/acs.analchem.3c04988
Methods Mol Biol. 2024 ;2788 19-37

Sample Preparation from Plant Tissue for Gas Chromatography-Mass Spectrometry (GC-MS)we.

Rinku Dagar, Ashish Gautam, Kagolla Priscilla, Vinay Sharma, Prateek Gupta, Rakesh Kumar.

  Metabolites are intermediate products formed during metabolism. Metabolites play different roles, including providing energy, supporting structure, transmitting signals, catalyzing reactions, enhancing defense, and interacting with other species. Plant metabolomics research aims to detect precisely all metabolites found within tissues of plants through GC-MS. This chapter primarily focuses on extracting metabolites using chemicals such as methanol, chloroform, ribitol, MSTFA, and TMCS. The metabolic analysis method is frequently used according to the specific kind of sample or matrix being investigated and the analysis objective. Chromatography (LC, GC, and CE) with mass spectrometry and NMR spectroscopy is used in modern metabolomics to analyze metabolites from plant samples. The most frequently used method for metabolites analysis is the GC-MS. It is a powerful technique that combines gas chromatography's separation capabilities with mass spectrometry, offering detailed information, including structural identification of each metabolite. This chapter contains an easy-to-follow guide to extract plant-based metabolites. The current protocol provides all the information needed for extracting metabolites from a plant, precautions, and troubleshooting.

Keywords:  Chromatogram; Derivatization; GC-MS; Metabolites; Plant tissue; Untargeted metabolomics

DOI:  https://doi.org/10.1007/978-1-0716-3782-1_2
Anal Chem. 2024 Apr 22.

Aza-Prilezhaev Aziridination-Enabled Multidimensional Analysis of Isomeric Lipids via High-Resolution U-Shaped Mobility Analyzer-Mass Spectrometry.

Yuling Li, Yiming Wang, Kang Guo, Kuo-Feng Tseng, Xiaoqiang Zhang, Wenjian Sun.

Unsaturated lipids constitute a significant portion of the lipidome, serving as players of multifaceted functions involving cellular signaling, membrane structure, and bioenergetics. While derivatization-assisted liquid chromatography tandem mass spectrometry (LC-MS/MS) remains the gold standard technique in lipidome, it mainly faces challenges in efficiently labeling the carbon-carbon double bond (C═C) and differentiating isomeric lipids in full dimension. This presents a need for new orthogonal methodologies. Herein, a metal- and additive-free aza-Prilezhaev aziridination (APA)-enabled ion mobility mass spectrometric method is developed for probing multiple levels of unsaturated lipid isomerization with high sensitivity. Both unsaturated polar and nonpolar lipids can be efficiently labeled in the form of N-H aziridine without significant side reactions. The signal intensity can be increased by up to 3 orders of magnitude, achieving the nM detection limit. Abundant site-specific fragmentation ions indicate C═C location and sn-position in MS/MS spectra. Better yet, a stable monoaziridination product is dominant, simplifying the spectrum for lipids with multiple double bonds. Coupled with a U-shaped mobility analyzer, identification of geometric isomers and separation of different lipid classes can be achieved. Additionally, a unique pseudo MS3 mode with UMA-QTOF MS boosts the sensitivity for generating diagnostic fragments. Overall, the current method provides a comprehensive solution for deep-profiling lipidomics, which is valuable for lipid marker discovery in disease monitoring and diagnosis.

DOI: https://doi.org/10.1021/acs.analchem.4c00481
Drug Test Anal. 2024 Apr 21.

Methylation of phase II metabolites of endogenous anabolic androgenic steroids to improve analytical performance.

Sandra Pfeffer, Guenter Gmeiner, Guro Forsdahl.

  The study of intact phase II metabolites of endogenous anabolic androgenic steroids (EAAS) gives important information about metabolism and has the potential to improve the detection of doping with testosterone. For analysis with liquid chromatography-mass spectrometry (LC-MS), chemical derivatization at the steroid moiety is a technique to improve the positive ionization efficiency of glucuronidated/sulfated EAAS under collision-induced dissociation (CID) conditions. However, regarding the chromatographic performance, there are still challenges to address, for example, poor peak shape, which is mainly caused by nondefined adsorption in the chromatographic system. Here, we show a novel derivatization technique for the analysis of selected phase II metabolites of EAAS, where the acidic moiety of the glucuronide/sulfate is methylated with different methylation reagents to reduce nondefined adsorption. The methylation reagent trimethylsilyl-diazomethane (TMSD) was preferred over the other tested reagents methyl iodide (MeI) and dimethyl sulfate (DMS). Glucuronidated and sulfated testosterone and epitestosterone were methylated, and their chromatographic performance and CID ion mass spectra obtained in positive ionization mode were investigated. The peak width and peak height were significantly improved for all substances. Methylated testosterone sulfate showed the best results with a 3.5 times narrower peak and 14 times increased intensity compared with underivatized testosterone sulfate. Furthermore, CID ion mass spectra obtained in positive ionization mode showed product ions characteristically for the steroidal backbone for all substances. This preliminary study shows the potential of methylation as a supplementary derivatization technique, which can assist in the development of more sensitive methods due to the improvements in method performance.

Keywords:  derivatization; doping; endogenous steroids; methylation; phase II metabolites

DOI:  https://doi.org/10.1002/dta.3694
Bioinformatics. 2024 Apr 24. pii: btae282. [Epub ahead of print]

DIMet: An open-source tool for Differential analysis of targeted Isotope-labeled Metabolomics data.

Johanna Galvis, Joris Guyon, Benjamin Dartigues, Helge Hecht, Björn Grüning, Florian Specque, Hayssam Soueidan, Slim Karkar, Thomas Daubon, Macha Nikolski.

MOTIVATION: Many diseases, such as cancer, are characterized by an alteration of cellular metabolism allowing cells to adapt to changes in the microenvironment. Stable isotope-resolved metabolomics and downstream data analyses are widely used techniques for unraveling cells' metabolic activity to understand the altered functioning of metabolic pathways in the diseased state. While a number of bioinformatic solutions exist for the differential analysis of Stable Isotope-Resolved Metabolomics data, there is currently no available resource providing a comprehensive toolbox.
RESULTS: In this work, we present DIMet, a one-stop comprehensive tool for differential analysis of targeted tracer data. DIMet accepts metabolite total abundances, isotopologue contributions, and isotopic mean enrichment, and supports differential comparison (pairwise and multi-group), time-series analyses, and labeling profile comparison. Moreover, it integrates transcriptomics and targeted metabolomics data through network-based metabolograms. We illustrate the use of DIMet in real SIRM datasets obtained from Glioblastoma P3 cell-line samples. DIMet is open-source, and is readily available for routine downstream analysis of isotope-labeled targeted metabolomics data, as it can be used both in the command line interface or as a complete toolkit in the public Galaxy Europe and Workfow4Metabolomics web platforms.
AVAILABILITY: DIMet is freely available at https://github.com/cbib/DIMet, and through https://usegalaxy.eu and https://workflow4metabolomics.usegalaxy.fr. All the datasets are available at Zenodo https://zenodo.org/records/10925786.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btae282
J Mass Spectrom. 2024 May;59(5): e5022

Investigation of liquid chromatography-mass spectrometry analysis of a peptide aldehyde SJA6017 with identifying its hemiacetal, gem-diol, and enol ether.

Zhiyang Zack Zou, Ming-Jie Han.

  The quantitative analysis of SJA6017, a peptide aldehyde inhibitor of calpain (Calpain Inhibitor VI), has encountered challenges in preclinical drug studies. The complex reverse-phase HPLC chromatographic behavior exhibits two peaks, each containing multiple species. An liquid chromatography-mass spectrometry (LC-MS/MS) study proposed an explanation for this phenomenon, caused by the amide aldehyde structure of SJA6017. Four chemical species corresponding to the two HPLC peaks have been identified as SJA6017 and its methyl hemiacetal, methyl enol ether, and gem-diol. In many instances of preclinical studies, methanol is favored as a substitute for DMSO. The hemiacetal is formed when the amide-activated peptide aldehyde reacts with methanol, which can then be further dehydrated in the mass spectrometer ion source under high temperature to form the methyl enol ether. The hemiacetal and gem-diol can also be decomposed to SJA6017 in the ion source. Additionally, the amide-activated peptide aldehyde can easily hydrate to the gem-diol of SJA6017 during sample incubation or sample preparation. The hemiacetal and gem-diol of SJA6017 are stable enough to have different retention times in the liquid chromatography, which explains why SJA6017 appears as two peaks, each containing multiple species. An LC-MS/MS tandem quadrupole mass spectrometer quantitative analysis method is proposed, enabling the analysis of these types of samples. This work serves as both an illustrative example and a cautionary note for mass analysis, sample incubations, and sample preparations involving compounds of peptide aldehyde, including similar aldehyde-containing metabolites, especially when methanol is present. This study provides the information needed to understand peptide aldehyde behavior at various steps of preclinical in vitro studies in the presence of methanol. It has assisted in the development of the SJA6017 bioanalysis method and will also aid in the development of bioanalysis methods for similar peptide aldehydes.

Keywords:  LC–MS/MS; enol ether; gem‐diol; hemiacetal; peptide aldehyde

DOI:  https://doi.org/10.1002/jms.5022
Buildings (Basel). 2023 ;13(5):

Adjustment of Matrix Effects in Analysis of 36 Secondary Metabolites of Microbial and Plant Origin in Indoor Floor Dust Using Liquid Chromatography-Tandem Mass Spectrometry.

Cornelius Rimayi, Ju-Hyeong Park.

  Exposure to microbial agents in water-damaged buildings is a major public health concern. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become a primary tool for testing environmental samples for microbial secondary metabolites (SMs); however, matrix effects can lead to inaccurate results in exposure assessment. Applying a universal internal standard (ISTD) and a matrix-matched calibration can adjust for matrix effects, as shown by our previous study. However, there are only few isotope-labeled internal standards for SMs available on the market. In this study, we determined the best-performing ISTDs among ten candidates (nine 13C-labeled isotopes and one unlabeled analogue) for each of 36 SMs. We analyzed school floor dust spiked with the 36 SMs to identify the best-performing ISTDs (initial experiment) and examined reproducibility with the selected ISTDs and the same spiked dust (validation 1). We also tested applicability for the selected ISTDs using spiked dust collected from different schools (validation 2). The three experiments showed that 26, 17, and 19 SMs had recoveries within the range 100 ± 40%. 13C-ochratoxin A and 13C-citrinin were most frequently selected as the best ISTDs for the 36 SMs, followed by deepoxy-deoxynivalenol, 13C-sterigmatocystin, and 13C-deoxynivalenol. Our study shows that using the identified, best-performing analogous ISTDs for those metabolites may improve testing accuracy for indoor dust and help better estimate exposure effects on potential health.

Keywords:  indoor floor dust; matrix effect adjustment; microbial secondary metabolite; recovery

DOI:  https://doi.org/10.3390/buildings13051112
Forensic Toxicol. 2024 Apr 20.

Liquid chromatography with tandem mass spectrometric method for determination of 52 antibiotics in human whole blood and urine and application to forensic cases.

Qianwen Shi, Gege Wang, Shuhui Wang, Chao Zhang, Zhiwen Wei, Zhongyuan Guo, Dan Zhang, Keming Yun, Shanlin Fu.

   PURPOSE: A rapid and reliable method was developed and validated for the simultaneous analysis of 52 antibiotics (cephalosporins, penicillins, carbapenems, lincosamides, quinolones, nitroimidazoles, macrolides, sulfonamides, tetracyclines, glycopeptide) in urine and whole blood by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).
METHOD: Analytes were extracted by dilution or protein precipitation and analyzed on an Agilent 1260 HPLC system coupled to an Agilent 6470 Triple Quadrupole Mass Spectrometer.
RESULTS: The method attended method validation criteria. The limits of detection were equal or lower than 2.0 ng/mL, whereas the limits of quantification ranged from 0.1 to 10.0 ng/mL, from 0.1 to 5.0 ng/mL, in urine and whole blood, respectively. For all analytes, the bias and intra- and inter-day precision values were less than 14.7%. The ranges of recovery values of all antibiotics were 76.5-124.5% in whole blood and 76.3-121.8% in urine, values of the effects were lower than 25% in two matrices. No evidence of carryover was observed. The study of sample stability showed that almost all analytes were stable at 24 °C for 24 h, all analytes were stable at -20 °C for 14 days and at -80 °C for 30 days. Freeze-thaw cycles stability showed that antibiotics were stable except for imipenem. Autosampler stability study showed that all analytes were stable for 24 h, except for imipenem and amoxicillin. Applicability was proven by analyzing authentic whole blood (n = 86) and urine (n = 79) samples from patients under antibiotics treatment. Therefore, this method was applied to the analysis 3 forensic allergy cases, which were positive for at least one analyte.
CONCLUSIONS: A simple, sensitive and high-throughput method for the simultaneous determination of different classes of antibiotics in urine and whole blood samples was developed and applied. This sensitive method was successfully applied to forensic cases.

Keywords:  Allergy case; Antibiotics; Forensic science; HPLC-MS/MS; Urine; Whole blood

DOI:  https://doi.org/10.1007/s11419-024-00688-y
Methods Mol Biol. 2024 ;2790 439-466

Stable Isotope Labeling and Quantification of Photosynthetic Metabolites.

Chiara Baccolini, Stéphanie Arrivault.

  Stable isotope labeling with 13CO2 coupled with mass spectrometry allows monitoring the incorporation of 13C into photosynthetic intermediates and is a powerful technique for the investigation of the metabolic dynamics of photosynthesis. We describe here a protocol for 13CO2 labeling of large leaved plants and of Arabidopsis thaliana rosette, and a method for quantitative mass spectrometry analyses to uncover the labeling pattern of Calvin-Benson cycle sucrose, and starch synthesis as well as carbon-concentrating mechanism metabolites.

Keywords:  13C enrichment; 13CO2 labeling; Calvin-Benson Cycle; Carbon-concentrating mechanism; Isotopomer distribution; LC-MS/MS; Mass spectrometry; Metabolite quantification; Photosynthesis; Pulse-chase

DOI:  https://doi.org/10.1007/978-1-0716-3790-6_24
J Chromatogr A. 2024 May 24. pii: S0021-9673(24)00287-5. [Epub ahead of print]1723 464913

Fiber-in-tube SPME-CapLC-MS/MS method to determine Aβ peptides in cerebrospinal fluid obtained from Alzheimer's patients.

Israel Donizeti de Souza, Fernando M Lanças, Jaime E Cecílio Hallak, Maria E Costa Queiroz.

  Mass spectrometry is characterized by its high sensitivity, ability to measure very low analyte concentrations, specificity to distinguish between closely related compounds, availability to generate high-throughput methods for screening, and high multiplexing capacity. This technique has been used as a platform to analyze fluid biomarkers for Alzheimer's disease. However, more effective sample preparation procedures, preferably antibody-independent, and more automated mass spectrometry platforms with improved sensitivity, chromatographic separation, and high throughput are needed for this purpose. This short communication discusses the development of a fiber-in-tube SPME-CapLC-MS/MS method to determine Aβ peptides in cerebrospinal fluid obtained from Alzheimer's disease patients. To obtain the fiber-in-tube SPME capillary, we longitudinally packed 22 nitinol fibers coated with a zwitterionic polymeric ionic liquid into the same length of the PEEK tube. In addition, this communication compares this fiber-in-tube SPME method with the conventional HPLC scale (HPLC-MS/MS) and when directly coupled to CapESI-MS/MS without chromatographic separation, and, as a case study, discusses the benefits and challenges inherent in miniaturizing the flow scale of the sample preparation technique (fiber-in-tube SPME) to the CapLC-MS/MS system. Fiber-in-tube SPME-CapLC-MS/MS provided LLOQ ranging from 0.09 to 0.10 ng mL-1, accuracy ranging from 91 to 117 % (recovery), and reproducibility of less than 18 % (RSD). Analysis of the cerebrospinal fluid samples obtained from Alzheimer's disease patients evidenced that the method is robust. At the capillary scale (10 µL min-1), this innovative method presented higher analytical sensitivity than the conventional HPLC-MS/MS scale. Although fiber-in-tube SPME directly coupled to CapESI-MS/MS offers advantages in terms of high throughput, the sample was dispersed and non-quantitatively desorbed from the capillary at low flow rate. These results highlighted that chromatographic separation is important to decrease the matrix effect and to achieve higher detectability, which is indispensable for bioanalysis.

Keywords:  Biological samples; Biomarkers; In-tube SPME; Mass spectrometry; Miniaturization

DOI:  https://doi.org/10.1016/j.chroma.2024.464913
Methods Mol Biol. 2024 ;2788 67-79

Monosaccharide Analysis After One-Pot Derivatization Followed by Reverse-Phase Liquid Chromatography Separation and UV/Vis Detection.

José Manuel Cruz-Rubio, Renate Loeppert, Werner Praznik.

  Derivatization of monosaccharides with 1-phenyl-3-methyl-5-pyrazolone (PMP) introduces two chromophores per sugar molecule. Their separation on a superficially porous C18 reverse-phase column, using common liquid chromatography equipment, results in short analysis times (under 20 min) and high sensitivity (limit of quantitation 1 nmol). This method allows for complex monosaccharide mixtures to be separated and quantified using a reasonably simple and safe derivatization procedure.

Keywords:  1-Phenyl-3-methyl-5-pyrazolone; Carbohydrate analysis; Chromatography; Monosaccharide chromophores; Monosaccharide derivatization; PMP; Superficially porous columns

DOI:  https://doi.org/10.1007/978-1-0716-3782-1_5
Anal Bioanal Chem. 2024 Apr 25.

Novel dummy molecularly imprinted polymer for simultaneous solid-phase extraction of stanozolol metabolites from urine.

Yomna G Farag, Rasha S Hanafi, Mennatallah A Hammam.

  Stanozolol, a synthetic derivative of testosterone, is one of the common doping drugs among athletes and bodybuilders. It is metabolized to a large extent and metabolites are detected in urine for a longer duration than the parent compound. In this study, a novel dummy molecularly imprinted polymer (DMIP) is developed as a sorbent for solid-phase extraction of stanozolol metabolites from spiked human urine samples. The optimized DMIP is composed of stanozolol as the dummy template, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker in a ratio of 1:10:80. The extracted analytes were quantitively determined using a newly developed and validated ultrahigh-performance liquid chromatography tandem mass spectrometry method, where the limits of detection and quantitation were 0.91 and 1.81 ng mL-1, respectively, fulfilling the minimum required performance limit decided on by the World Anti-Doping Agency. The mean percentage extraction recoveries for 3'-hydroxystanozolol, 4β-hydroxystanozolol, and 16β-hydroxystanozolol are 97.80% ± 13.80, 83.16% ± 7.50, and 69.98% ± 2.02, respectively. As such, the developed DMISPE can serve as an efficient cost-effective tool for doping and regulatory agencies for simultaneous clean-up of the stanozolol metabolites prior to their quantification.

Keywords:  Bulk polymerization; Molecularly imprinted polymer; Solid-phase extraction; Stanozolol metabolites; UHPLC-MS/MS

DOI:  https://doi.org/10.1007/s00216-024-05285-x
J Pharm Biomed Anal. 2024 Apr 16. pii: S0731-7085(24)00194-8. [Epub ahead of print]245 116154

Development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of the antimalarial drug pyronaridine in human whole blood.

Wietse M Schouten, Ignace C Roseboom, Luc Lucas, Japhet Kabalu Tshiongo, Hypolite Muhindo Mavoko, Kassoum Kayentao, Hilde Rosing, Alwin D R Huitema, Jos H Beijnen, Thomas P C Dorlo.

  Malaria remains a major health concern, aggravated by emerging resistance of the parasite to existing treatments. The World Health Organization recently endorsed the use of artesunate-pyronaridine to treat uncomplicated malaria. However, there is a lack of clinical pharmacokinetic (PK) data of pyronaridine, particularly in special populations such as children and pregnant women. Existing methods for the quantification of pyronaridine in biological matrices to support PK studies exhibit several drawbacks. These include limited sensitivity, a large sample volume required, and extensive analysis time. To overcome these limitations, an ultra-performance reversed-phase liquid chromatography tandem-mass spectrometry method to determine pyronaridine was developed and validated according to international guidelines. The method enabled fast and accurate quantification of pyronaridine in whole blood across a clinically relevant concentration range of 0.500-500 ng/mL (r2 ≥ 0.9963), with a required sample volume of 50 µL. Pyronaridine was extracted from whole blood using liquid-liquid extraction, effectively eliminating the matrix effect and preventing ion enhancement or suppression. The method achieved a satisfactory reproducible sample preparation recovery of 77%, accuracy (as bias) and precision were within ±8.2% and ≤5.3%, respectively. Stability experiments demonstrated that pyronaridine was stable for up to 315 days when stored at -70°C. Adjustments to the chromatographic system substantially reduced carry-over and improved sensitivity compared to prior methods. The method was successfully applied to quantify pyronaridine in whole blood samples from a selection of pregnant malaria patients participating in the PYRAPREG clinical trial (PACTR202011812241529) in the Democratic Republic of the Congo, demonstrating its suitability to support future PK studies. Furthermore, the enhanced sensitivity allows for the determination of pyronaridine up to 42 days post-treatment initiation, enabling assessment of the terminal elimination half-life.

Keywords:  Liquid-liquid extraction; Pharmacokinetics; Pyronaridine; UPLC-MS/MS; Whole blood

DOI:  https://doi.org/10.1016/j.jpba.2024.116154
Heliyon. 2024 Apr 30. 10(8): e29606

Development and validation of ivermectin quantification method in volumetric absorptive microsampling using liquid chromatography-tandem mass spectrometry.

Yahdiana Harahap, Salsabila Salsabila, Febrina Amelia Saputri.

   Background: Ivermectin is a broad-spectrum anthelmintic used to control onchocerciasis from nematode parasites. As an anthelmintic, ivermectin is designed to have high levels in the gastrointestinal tract, so that the systemic intake is relatively low. Due to the very small concentration of ivermectin, a selective and sensitive approach is needed for the analysis of ivermectin in blood. Several methods have been developed using plasma and Dried Blood Spots, but there are still shortcomings due to hematocrit effects. Therefore, this study was conducted to establish a validated ivermectin analysis method with doramectin as the internal standard in using Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry.
Methods: Mass spectrometry equipped with triple quadrupole and positive electrospray ionization mode was used to conduct the analysis. For the biological matrix, whole blood was used by Volumetric Absorptive Microsampling and extracted using a protein precipitation technique with a combination of acetonitrile and methanol (1:1). VAMS has some advantages such as not being affected by hematocrit, requires a small and fixed volume of sample, also a more efficient sampling process.
Results: The optimum conditions were achieved with an Acquity® UPLC BEH C18 column (1,7 μm; 2.1 × 100 mm); extracted-flow rate was 0,2 mL/min; mobile phase was 5 mM ammonium formate pH 3.00 and acetonitrile (10:90) with isocratic elution. Multiple Reaction Monitoring (MRM) detection by m/z values was 892.41 > 569.5 for ivermectin and 916,41 > 331,35 for doramectin.
Conclusion: The method has been appropriately validated in compliance with the 2018 guidelines laid out by the US Food and Drug Administration. Resulting the minimum detection (LLOQ) was 1 ng/mL with a linear concentration range spanning from 1 to 150 ng/mL.

Keywords:  Doramectin; Ivermectin; LC-MS/MS; Onchocerciasis; VAMS

DOI:  https://doi.org/10.1016/j.heliyon.2024.e29606
Microorganisms. 2024 Apr 19. pii: 828. [Epub ahead of print]12(4):

Effect of Stool Sampling on a Routine Clinical Method for the Quantification of Six Short Chain Fatty Acids in Stool Using Gas Chromatography-Mass Spectrometry.

Tarek Mahdi, Aurore Desmons, Pranvera Krasniqi, Jean-Marc Lacorte, Nathalie Kapel, Antonin Lamazière, Salma Fourati, Thibaut Eguether.

  Short chain fatty acids (SCFAs) are primarily produced in the caecum and proximal colon via the bacterial fermentation of undigested carbohydrates that have avoided digestion in the small intestine. Increasing evidence supports the critical role that SCFAs play in health and homeostasis. Microbial SCFAs, namely butyric acid, serve as a principal energy source for colonocytes, and their production is essential for gut integrity. A direct link between SCFAs and some human pathological conditions, such as inflammatory bowel disease, irritable bowel syndrome, diarrhea, and cancer, has been proposed. The direct measurement of SCFAs in feces provides a non-invasive approach to demonstrating connections between SCFAs, microbiota, and metabolic diseases to estimate their potential applicability as meaningful biomarkers of intestinal health. This study aimed to adapt a robust analytical method (liquid-liquid extraction, followed by isobutyl chloroformate derivatization and GC-MS analysis), with comparable performances to methods from the literature, and to use this tool to tackle the question of pre-analytical conditions, namely stool processing. We focused on the methodology of managing stool samples before the analysis (fresh stool or dilution in either ethanol/methanol, lyophilized stool, or RNAlater®), as this is a significant issue to consider for standardizing results between clinical laboratories. The objective was to standardize methods for future applications as diagnostic tools. In this paper, we propose a validated GC-MS method for SCFA quantification in stool samples, including pre- and post-analytical comparison studies that could be easily used for clinical laboratory purposes. Our results show that using lyophilization as a stool-processing method would be the best method to achieve this goal.

Keywords:  GC-MS; gut microbiome; metabolomic; short chain fatty acids; stool sampling

DOI:  https://doi.org/10.3390/microorganisms12040828
Metabolites. 2024 Apr 03. pii: 200. [Epub ahead of print]14(4):

Challenges in the Metabolomics-Based Biomarker Validation Pipeline.

Shenghan Li, Nikita Looby, Vinod Chandran, Vathany Kulasingam.

  As end-products of the intersection between the genome and environmental influences, metabolites represent a promising approach to the discovery of novel biomarkers for diseases. However, many potential biomarker candidates identified by metabolomics studies fail to progress beyond analytical validation for routine implementation in clinics. Awareness of the challenges present can facilitate the development and advancement of innovative strategies that allow improved and more efficient applications of metabolite-based markers in clinical settings. This minireview provides a comprehensive summary of the pre-analytical factors, required analytical validation studies, and kit development challenges that must be resolved before the successful translation of novel metabolite biomarkers originating from research. We discuss the necessity for strict protocols for sample collection, storage, and the regulatory requirements to be fulfilled for a bioanalytical method to be considered as analytically validated. We focus especially on the blood as a biological matrix and liquid chromatography coupled with tandem mass spectrometry as the analytical platform for biomarker validation. Furthermore, we examine the challenges of developing a commercially viable metabolomics kit for distribution. To bridge the gap between the research lab and clinical implementation and utility of relevant metabolites, the understanding of the translational challenges for a biomarker panel is crucial for more efficient development of metabolomics-based precision medicine.

Keywords:  analytical validation; biomarker; kit development; metabolomics; pre-analytical factors; regulatory guideline

DOI:  https://doi.org/10.3390/metabo14040200
Anal Chem. 2024 Apr 23.

Single-Cell Untargeted Lipidomics Using Liquid Chromatography and Data-Dependent Acquisition after Live Cell Selection.

Johanna von Gerichten, Kyle D G Saunders, Anastasia Kontiza, Carla F Newman, George Mayson, Dany J V Beste, Eirini Velliou, Anthony D Whetton, Melanie J Bailey.

We report the development and validation of an untargeted single-cell lipidomics method based on microflow chromatography coupled to a data-dependent mass spectrometry method for fragmentation-based identification of lipids. Given the absence of single-cell lipid standards, we show how the methodology should be optimized and validated using a dilute cell extract. The methodology is applied to dilute pancreatic cancer and macrophage cell extracts and standards to demonstrate the sensitivity requirements for confident assignment of lipids and classification of the cell type at the single-cell level. The method is then coupled to a system that can provide automated sampling of live, single cells into capillaries under microscope observation. This workflow retains the spatial information and morphology of cells during sampling and highlights the heterogeneity in lipid profiles observed at the single-cell level. The workflow is applied to show changes in single-cell lipid profiles as a response to oxidative stress, coinciding with expanded lipid droplets. This demonstrates that the workflow is sufficiently sensitive to observing changes in lipid profiles in response to a biological stimulus. Understanding how lipids vary in single cells will inform future research into a multitude of biological processes as lipids play important roles in structural, biophysical, energy storage, and signaling functions.

DOI: https://doi.org/10.1021/acs.analchem.3c05677
Clin Biochem. 2024 Apr 20. pii: S0009-9120(24)00059-6. [Epub ahead of print]127-128 110765

A validated method for simultaneous quantification of four antiretrovirals in dried blood spot and plasma using LC-MS/MS: Application to efavirenz therapeutic drug monitoring in pregnant patients.

Maira Ludna Duarte, Aurylanne Mikaelle Brandão Silva, José Wellithom Viturino da Silva, Davi Pereira Santana, Whocely Victor de Castro, Luiz Cláudio Arraes de Alencar, Danilo César Galindo Bedor, Leila Bastos Leal.

   INTRODUCTION: The specific physiological background induced by pregnancy leads to significant changes in maternal pharmacokinetics, suggesting potential variability in plasma concentrations of antiretrovirals. Pregnant HIV patients exposed to subtherapeutic doses, particularly in the last trimester of the pregnancy, have higher chances to transmit the infection to their children. Therefore, the therapeutic drug monitoring of antiretrovirals in HIV pregnant patients would be of great value.
OBJECTIVES: This study aimed to develop and validate a sensitive liquid chromatograph tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of efavirenz, raltegravir, atazanavir, and ritonavir in dried blood spots (DBS) and plasma.
DESIGN AND METHODS: The analytes were extracted from the DBS punch and plasma with a mixture of methanol:zinc sulfate 200 mM (50:50, v/v) and 100 % methanol, respectively. For the chromatographic separation a Shim-pack® C18, 4.6 mm × 150 mm, 5 μm column was used. Detection was performed in a 3200-QTRAP® mass spectrometer, with a run time of 6 min.
RESULTS: The assay was linear in the range of 15-1,000 ng/mL for raltegravir, 50-10,000 ng/mL for both atazanavir and ritonavir, 50-5,000 ng/mL for efavirenz. Precision and accuracy at these concentrations were less than 15 % for all analytes. Raltegravir, atazanavir, and ritonavir were stable for seven days at 23 °C and 40 °C, whereas efavirenz was stable for twenty-four hours at the same conditions.
CONCLUSIONS: The method was successfully applied to quantify efavirenz in DBS samples obtained from HIV-1 infected pregnant volunteers under antiretroviral therapy. The concentrations of efavirenz in DBS and plasma were comparable according to Passing-Bablok regression and Bland-Altman analysis.

Keywords:  Antiretrovirals; Dried Blood Spot; HIV; Pregnancy; Therapeutic Drug Monitoring

DOI:  https://doi.org/10.1016/j.clinbiochem.2024.110765
Anal Chem. 2024 Apr 26.

Detection and Imaging of Exposure-Related Metabolites and Xenobiotics in Hard Tissues by Laser Sampling and Mass Spectrometry.

Marjan Dolatmoradi, Joanna Ellis, Christine Austin, Manish Arora, Akos Vertes.

The utility of two novel laser-based methods, laser ablation electrospray ionization (LAESI) and laser desorption ionization (LDI) from silicon nanopost array (NAPA), is explored via local analysis and mass spectrometry imaging (MSI) of hard tissues (tooth and hair) for the detection and mapping of organic components. Complex mass spectra are recorded in local analysis mode from tooth dentin and scalp hair samples. Nicotine and its metabolites (cotinine, hydroxycotinine, norcotinine, and nicotine) are detected by LAESI-MS in the teeth of rats exposed to tobacco smoke. The intensities of the detected metabolite peaks are proportional to the degree of exposure. Incorporating ion mobility separation in the LAESI-MS analysis of scalp hair enables the detection of cotinine in smoker hair along with other common molecular species, including endogenous steroid hormones and some lipids. Single hair strands are imaged by MALDI-MSI and NAPA-LDI-MSI to explore longitudinal variations in the level of small molecules. Comparing spectra integrated from NAPA-LDI-MSI and MALDI-MSI images reveals that the two techniques provide complementary information. There were 105 and 82 sample-related peaks for MALDI and NAPA, respectively, with an overlap of only 16 peaks, indicating a high degree of complementarity. Enhanced molecular coverage and spatial resolution offered by LAESI-MS and NAPA-LDI-MSI can reveal the distributions of known and potential biomarkers in hard tissues, facilitating exposome research.

DOI: https://doi.org/10.1021/acs.analchem.4c00224
J Mass Spectrom. 2024 May;59(5): e5029

From molecules to visuals: Empowering drug discovery and development with mass spectrometry imaging.

Bingming Chen, Marissa Vavrek, Mark T Cancilla.

  Over the past three decades, mass spectrometry imaging (MSI) has emerged as a valuable tool for the spatial localization of drugs and metabolites directly from tissue surfaces without the need for labels. MSI offers molecular specificity, making it increasingly popular in the pharmaceutical industry compared to conventional imaging techniques like quantitative whole-body autoradiography (QWBA) and immunohistochemistry, which are unable to distinguish parent drugs from metabolites. Across the industry, there has been a consistent uptake in the utilization of MSI to investigate drug and metabolite distribution patterns, and the integration of MSI with omics technologies in preclinical investigations. To continue the further adoption of MSI in drug discovery and development, we believe there are two key areas that need to be addressed. First, there is a need for accurate quantification of analytes from MSI distribution studies. Second, there is a need for increased interactions with regulatory agencies for guidance on the utility and incorporation of MSI techniques in regulatory filings. Ongoing efforts are being made to address these areas, and it is hoped that MSI will gain broader utilization within the industry, thereby becoming a critical ingredient in driving drug discovery and development.

Keywords:  drug discovery and development; drug metabolism and pharmacokinetics; mass spectrometry imaging

DOI:  https://doi.org/10.1002/jms.5029
J Am Soc Mass Spectrom. 2024 Apr 25.

Bioanalysis of Stress Biomarkers through Sensitive HILIC-MS/MS Method: A Stride toward Accurate Quantification of MDA, ACR, and CTA.

Harsh Thakkar, Shriya Gangakhedkar, Ravi P Shah.

  Quantifying reactive aldehyde biomarkers, such as malondialdehyde, acrolein, and crotonaldehyde, is the most preferred approach to determine oxidative stress. However, reported analytical methods lack specificity for accurately quantifying these aldehydes as certain methodologies may produce false positive results due to harsh experimental conditions. Thus, in this research work, a novel HILIC-MS/MS method with endogenous histidine derivatization is developed, which proves to have higher specificity and reproducibility in quantifying these aldehydes from the biological matrix. To overcome the reactivity of aldehyde, endogenous histidine is used for its derivatization. The generated adduct is orthogonally characterized by NMR and LC-HRMS. The method employed a hydrophilic HILIC column and multiple reaction monitoring (MRM) to accurately quantify these reactive aldehydes. The developed method is an unequivocal solution for quantifying stress in in vivo and in vitro studies.

Keywords:  HILIC-Triple Quad LC-MS; acrolein; crotonaldehyde; malondialdehyde; stress biomarkers

DOI:  https://doi.org/10.1021/jasms.3c00431
Molecules. 2024 Apr 12. pii: 1747. [Epub ahead of print]29(8):

The Impact of Different Drying Methods on the Metabolomic and Lipidomic Profiles of Arthrospira platensis.

Marika Mróz, Karol Parchem, Joanna Jóźwik, M Rosário Domingues, Barbara Kusznierewicz.

  Drying is an inseparable part of industrial microalgae production. In this work, the impacts of eight different drying methods on the metabolome and lipidome of Arthrospira platensis were investigated. The studied drying methods were freeze drying (FD), sun drying (SD), air drying at 40 and 75 °C (AD' and AD″), infrared drying at 40 and 75 °C (IRD' and IRD″), and vacuum drying at 40 and 75 °C (VD' and VD″). Results gathered by reversed-phase liquid chromatography separation coupled with high-resolution tandem mass spectrometry with electrospray ionization (RP-LC-ESI-Orbitrap HRMS/MS) analysis allowed researchers to identify a total of 316 metabolites (including lipids) in aqueous and ethanolic extracts. The compounds identified in ethanolic extracts were mainly lipids, such as neutral and polar lipids, chlorophylls and carotenoids, while the compounds identified in the aqueous extracts were mainly amino acids and dipeptides. Among the identified compounds, products of enzymatic and chemical degradation, such as pyropheophytins, monoacylglycerols and lysophosphatidylcholines were also identified and their amounts depended on the drying method. The results showed that except for FD method, recognized as a control, the most protective method was AD'. Contrary to this, VD' and VD″, under the conditions used, promoted the most intense degradation of valuable metabolites.

Keywords:  Arthrospira platensis; HPLC-HRMS/MS; drying; lipidomics; metabolomics; microalgae

DOI:  https://doi.org/10.3390/molecules29081747
J Anal Toxicol. 2024 Apr 24. pii: bkae037. [Epub ahead of print]

A specific and sensitive GC-MS/MS method for the quantitative determination of 2-phenoxyethanol and selected metabolites in human blood and urine.

Thomas Jäger, Elisabeth Eckert, Edgar Leibold, Michael Bader.

  2-Phenoxyethanol (PhE) is widely used as a preservative in consumer products such as cosmetics as well as at the workplace as a component of metal-working fluids and hydraulic fluids. Therefore, both industry workers and consumers may potentially be exposed to PhE. An analytical method for the quantification of PhE and three selected metabolites, namely phenoxyacetic acid (PhAA), 4-hydroxyphenoxyacetic acid (4-OH-PhAA), and 4-hydroxyphenoxyethanol (4-OH-PhE) in human urine and blood was developed and validated. The sample preparation includes enzymatic hydrolysis of urine samples or protein precipitation of blood samples, followed by liquid-liquid extraction and silylation of the target analytes. Analyses of the extracts were carried out by gas chromatography with tandem mass spectrometry (GC-MS/MS). 3,4-Hydroxyphenoxyethanol, a probably minor PhE metabolite could not reliable be analyzed due to its instability. The limits of quantification (LOQ) of the analytes ranged between 0.5 and 6.1 μg/L and 2.0 and 3.9 μg/L in urine and blood, respectively. The method was successfully applied to spot urine samples of 50 individuals without occupational exposure to PhE and additionally to blood samples from seven volunteers. In urine, PhAA and 4-OH-PhAA could be quantified in all analyzed samples, whereas 4-OH-PhE and unchanged PhE were found in 36 % and 32 % of the samples, respectively. In blood, PhAA was also found in every sample in levels above the LOQ, whereas PhE itself was detected in three of seven samples only. Neither 4-OH-PhAA nor 4-OH-PhE were found in any of the analyzed blood samples. The developed method promises to be a valuable tool for PhE monitoring of urine and blood samples and may also enable an advanced investigation of PhE biotransformation pathways in humans.

Keywords:  2-phenoxyethanol; biological matrices; biotransformation; broad-spectrum preservative; human biomonitoring (HBM)

DOI:  https://doi.org/10.1093/jat/bkae037
Toxins (Basel). 2024 Apr 07. pii: 181. [Epub ahead of print]16(4):

Studying Venom Toxin Variation Using Accurate Masses from Liquid Chromatography-Mass Spectrometry Coupled with Bioinformatic Tools.

Luis L Alonso, Jory van Thiel, Julien Slagboom, Nathan Dunstan, Cassandra M Modahl, Timothy N W Jackson, Saer Samanipour, Jeroen Kool.

  This study provides a new methodology for the rapid analysis of numerous venom samples in an automated fashion. Here, we use LC-MS (Liquid Chromatography-Mass Spectrometry) for venom separation and toxin analysis at the accurate mass level combined with new in-house written bioinformatic scripts to obtain high-throughput results. This analytical methodology was validated using 31 venoms from all members of a monophyletic clade of Australian elapids: brown snakes (Pseudonaja spp.) and taipans (Oxyuranus spp.). In a previous study, we revealed extensive venom variation within this clade, but the data was manually processed and MS peaks were integrated into a time-consuming and labour-intensive approach. By comparing the manual approach to our new automated approach, we now present a faster and more efficient pipeline for analysing venom variation. Pooled venom separations with post-column toxin fractionations were performed for subsequent high-throughput venomics to obtain toxin IDs correlating to accurate masses for all fractionated toxins. This workflow adds another dimension to the field of venom analysis by providing opportunities to rapidly perform in-depth studies on venom variation. Our pipeline opens new possibilities for studying animal venoms as evolutionary model systems and investigating venom variation to aid in the development of better antivenoms.

Keywords:  LC-MS; data analysis; high throughput; snake venom

DOI:  https://doi.org/10.3390/toxins16040181
Anal Chem. 2024 Apr 22.

Abundance of Modifications in Mature miRNAs Revealed by LC-MS/MS Method Coupled with a Two-Step Hybridization Purification Strategy.

Chenchen Lü, Hua Xu, Pengxia Gao, Aixue Huang, Minmin Qu, Weiwei He, Haijiang Wu, Jia Chen, Bin Xu, Lei Guo, Jianwei Xie.

Accurate detection of endogenous miRNA modifications, such as N6-methyladenosine (m6A), 7-methylguanosine (m7G), and 5-methylcytidine (m5C), poses significant challenges, resulting in considerable uncertainty regarding their presence in mature miRNAs. In this study, we demonstrate for the first time that liquid chromatography coupled with a tandem mass spectrometry (LC-MS/MS) nucleoside analysis method is a practical tool for quantitatively analyzing human miRNA modifications. The newly designed liquid-solid two-step hybridization (LSTH) strategy enhances specificity for miRNA purification, while LC-MS/MS offers robust capability in recognizing modifications and sufficient sensitivity with detection limits ranging from attomoles to low femtomoles. Therefore, it provides a more reliable approach compared to existing techniques for revealing modifications in endogenous miRNAs. With this approach, we characterized m6A, m7G, and m5C modifications in miR-21-5p, Let-7a/e-5p, and miR-10a-5p isolated from cultured cells and observed unexpectedly low abundance (<1% at each site) of these modifications.

DOI: https://doi.org/10.1021/acs.analchem.4c01326
ACS Meas Sci Au. 2024 Apr 17. 4(2): 223-230

Labeling of Mucin-Type O-Glycans for Quantification Using Liquid Chromatography and Fluorescence Detection.

Marc Safferthal, Leïla Bechtella, Andreas Zappe, Gaël M Vos, Kevin Pagel.

O-glycosylation is a common post-translational modification that is essential for the defensive properties of mucus barriers. Incomplete and altered O-glycosylation is often linked to severe diseases, such as cancer, cystic fibrosis, and chronic obstructive pulmonary disease. Originating from a nontemplate-driven biosynthesis, mucin-type O-glycan structures are very complex. They are often present as heterogeneous mixtures containing multiple isomers. Therefore, the analysis of complex O-glycan mixtures usually requires hyphenation of orthogonal techniques such as liquid chromatography (LC), ion mobility spectrometry, and mass spectrometry (MS). However, MS-based techniques are mainly qualitative. Moreover, LC separation of O-glycans often lacks reproducibility and requires sophisticated data treatment and analysis. Here we present a mucin-type O-glycomics analysis workflow that utilizes hydrophilic interaction liquid chromatography for separation and fluorescence labeling for detection and quantification. In combination with mass spectrometry, a detailed analysis on the relative abundance of specific mucin-type O-glycan compositions and features, such as fucose, sialic acids, and sulfates, is performed. Furthermore, the average number of monosaccharide units of O-glycans in different samples was determined. To demonstrate universal applicability, the method was tested on mucins from different tissue types and mammals, such as bovine submaxillary mucins, porcine gastric mucins, and human milk mucins. To account for day-to-day retention time shifts in O-glycan separations and increase the comparability between different instruments and laboratories, we included fluorescently labeled dextran ladders in our workflow. In addition, we set up a library of glucose unit values for all identified O-glycans, which can be used to simplify the identification process of glycans in future analyses.

DOI: https://doi.org/10.1021/acsmeasuresciau.3c00071