bims-metlip 2023-04-02 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023‒04‒02
37 papers selected by
Sofia Costa
Matterworks

Optimizing XCMS parameters for GC-MS metabolomics data processing: a case study.
A mass spectrum-oriented computational method for ion mobility-resolved untargeted metabolomics.
Description and validation of an equilibrium dialysis ID-LC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.
Mass Spectrometry Imaging for Single-Cell or Subcellular Lipidomics: A Review of Recent Advancements and Future Development.
MS2Query: reliable and scalable MS2 mass spectra-based analogue search.
Mechanistic Understanding of the Discrepancies between Common Peak Picking Algorithms in Liquid Chromatography-Mass Spectrometry-Based Metabolomics.
A Five-Year Update on Matrix Compounds for MALDI-MS Analysis of Lipids.
Development of a novel Ultra Performance Liquid Chromatography Tandem-Mass Spectrometry (UPLC-MS/MS) method to measure l-arginine metabolites in plasma.
Identifying Hair Biomarker Candidates for Alzheimer's Disease Using Three High Resolution Mass Spectrometry-Based Untargeted Metabolomics Strategies.
MAD HATTER Correctly Annotates 98% of Small Molecule Tandem Mass Spectra Searching in PubChem.
Systematic untargeted UHPLC-Q-TOF-MS based lipidomics workflow for improved detection and annotation of lipid sub-classes in serum.
Lipidomic Analysis of Mouse Brain to Evaluate the Efficacy and Preservation of Different Tissue Preparatory Techniques by IR-MALDESI-MSI.
Performance comparison of three scaling algorithms in NMR-based metabolomics analysis.
Development of an Untargeted Metabolomics Strategy to Study the Metabolic Rewiring of Dendritic Cells upon Lipopolysaccharide Activation.
A simple and sensitive LC-MS/MS method for therapeutic drug monitoring of digoxin in children.
High throughput FAMS - A fatty acid mass spectrometry method for monitoring polysorbate hydrolysis in QC.
Reliable quantification of citrate isomers and isobars with direct-infusion tandem mass spectrometry.
A High-Performance Liquid Chromatography-Mass Spectrometry Method for Simultaneous Determination of Vancomycin, Meropenem, and Valproate in Patients with Post-Craniotomy Infection.
Matrix Effects in GC-MS Profiling of Common Metabolites after Trimethylsilyl Derivatization.
Quantification of orelabrutinib in human plasma and cerebrospinal fluid by liquid chromatography tandem mass spectrometry.
Assessment of In Silico and In Vitro Selpercatinib Metabolic Stability in Human Liver Microsomes Using a Validated LC-MS/MS Method.
A Strategy for Uncovering the Serum Metabolome by Direct-Infusion High-Resolution Mass Spectrometry.
Recent advances in mass spectrometry-based computational metabolomics.
Cross-Platform Comparison of Amino Acid Metabolic Profiling in Three Model Organisms Used in Environmental Metabolomics.
Separation and Simultaneous Trapping of Multiply Charged and Singly Charged Ions for Mass Spectrometry: Application to Lipid Mixtures.
Analysis of N-nitrosamines and N-nitrosatable substances from baby bottle rubber teats by liquid chromatography tandem mass spectrometry.
A sensitive, robust and high-throughput isotope dilution LC-MS/MS method for quantifying three folate forms in serum.
A Simultaneous Extraction/Derivatization Strategy for Quantitation of Vitamin D in Dried Blood Spots Using LC-MS/MS: Application to Biomarker Study in Subjects Tested for SARS-CoV-2.
Impact of Matrix Species and Mass Spectrometry on Matrix Effects in Multi-Residue Pesticide Analysis Based on QuEChERS-LC-MS.
A simultaneous, high-throughput and sensitive method for analysing 13 neonicotinoids and metabolites in urine using a liquid chromatography-tandem mass spectrometry.
Targeted Desorption Electrospray Ionization Mass Spectrometry Imaging for Drug Distribution, Toxicity, and Tissue Classification Studies.
Development of tandem-column liquid chromatographic methods for pharmaceutical compounds using simulations based on hydrophobic subtraction model parameters.
Metabolite Fingerprinting for Identification of Panax ginseng Metabolites Using Internal Extractive Electrospray Ionization Mass Spectrometry.
In-depth profiling of di(2-ethylhexyl) phthalate metabolic footprints in rats using click chemistry-mass spectrometry probes.
Quantitative determination of lurbinectedin, its unbound fraction and its metabolites in human plasma utilizing ultra-performance LC-MS/MS.
Assessing the Effectiveness of Chemical Marker Extraction from Amazonian Plant Cupuassu (Theobroma grandiflorum) by PSI-HRMS/MS and LC-HRMS/MS.
A review on various analytical methods for the estimation of olanzapine - An antipsychotic drug.

Metabolomics. 2023 Mar 28. 19(4): 26

Optimizing XCMS parameters for GC-MS metabolomics data processing: a case study.

Emile Kelly Porto Dos Santos, Gisele André Baptista Canuto.

  BACKGROUND AND AIMS: Optimizing metabolomics data processing parameters is a challenging and fundamental task to obtain reliable results. Automated tools have been developed to assist this optimization for LC-MS data. GC-MS data require substantial modifications in processing parameters, as the chromatographic profiles are more robust, with more symmetrical and Gaussian peaks. This work compared an automated XCMS parameter optimization using the Isotopologue Parameter Optimization (IPO) software with manual optimization of GC-MS metabolomics data. Additionally, the results were compared to online XCMS platform.METHODS: GC-MS data from control and test groups of intracellular metabolites from Trypanosoma cruzi trypomastigotes were used. Optimizations were performed on the quality control (QC) samples.
RESULTS: The results in terms of the number of molecular features extracted, repeatability, missing values, and the search for significant metabolites showed the importance of optimizing the parameters for peak detection, alignment, and grouping, especially those related to peak width (fwhm, bw) and noise ratio (snthresh).
CONCLUSION: This is the first time that a systematic optimization using IPO has been performed on GC-MS data. The results demonstrate that there is no universal approach for optimization but automated tools are valuable at this stage of the metabolomics workflow. The online XCMS proves to be an interesting processing tool, helping, above all, in the choice of parameters as a starting point for adjustments and optimizations. Although the tools are easy to use, there is still a need for technical knowledge about the analytical methods and instruments used.

Keywords:  Data analysis; Data processing; Gas chromatography-mass spectrometry; IPO; XCMS

DOI:  https://doi.org/10.1007/s11306-023-01992-1
Nat Commun. 2023 Mar 31. 14(1): 1813

A mass spectrum-oriented computational method for ion mobility-resolved untargeted metabolomics.

Mingdu Luo, Yandong Yin, Zhiwei Zhou, Haosong Zhang, Xi Chen, Hongmiao Wang, Zheng-Jiang Zhu.

Ion mobility (IM) adds a new dimension to liquid chromatography-mass spectrometry-based untargeted metabolomics which significantly enhances coverage, sensitivity, and resolving power for analyzing the metabolome, particularly metabolite isomers. However, the high dimensionality of IM-resolved metabolomics data presents a great challenge to data processing, restricting its widespread applications. Here, we develop a mass spectrum-oriented bottom-up assembly algorithm for IM-resolved metabolomics that utilizes mass spectra to assemble four-dimensional peaks in a reverse order of multidimensional separation. We further develop the end-to-end computational framework Met4DX for peak detection, quantification and identification of metabolites in IM-resolved metabolomics. Benchmarking and validation of Met4DX demonstrates superior performance compared to existing tools with regard to coverage, sensitivity, peak fidelity and quantification precision. Importantly, Met4DX successfully detects and differentiates co-eluted metabolite isomers with small differences in the chromatographic and IM dimensions. Together, Met4DX advances metabolite discovery in biological organisms by deciphering the complex 4D metabolomics data.

DOI: https://doi.org/10.1038/s41467-023-37539-0
Clin Chem Lab Med. 2023 Mar 31.

Description and validation of an equilibrium dialysis ID-LC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

Heleen I Jansen, Rob van der Steen, André Brandt, André J Olthaar, Hubert W Vesper, Eri Shimizu, Annemieke C Heijboer, Katleen Van Uytfanghe, Antonius E van Herwaarden.

  OBJECTIVES: Free thyroxine (FT4) in serum is routinely measured in clinical practice to diagnose and monitor thyroid disease. Due to its concentration in picomolar range and the delicate equilibrium of free and protein-bound T4, accurate measurement is challenging. As a consequence, large inter-method differences in FT4 results exists. Optimal method design and standardization of the FT4 measurement is therefore necessary. The IFCC Working Group for Standardization of Thyroid Function Tests proposed a reference system with a conventional reference measurement procedure (cRMP) for FT4 in serum. In this study, we describe our FT4 candidate cRMP and its validation in clinical samples.METHODS: This candidate cRMP is based on equilibrium dialysis (ED) combined with determination of T4 with an isotope-dilution liquid chromatography tandem mass-spectrometry (ID-LC-MS/MS) procedure and was developed according to the endorsed conventions. Its accuracy, reliability, and comparability was investigated using human sera.
RESULTS: It was shown that the candidate cRMP adhered to the conventions and its accuracy, precision, and robustness were adequate in serum of healthy volunteers.
CONCLUSIONS: Our candidate cRMP measures FT4 accurately and performs well in serum matrix.

Keywords:  LC-MS/MS; conventional reference measurement procedure; free thyroxine; standardization

DOI:  https://doi.org/10.1515/cclm-2022-1134
Molecules. 2023 Mar 17. pii: 2712. [Epub ahead of print]28(6):

Mass Spectrometry Imaging for Single-Cell or Subcellular Lipidomics: A Review of Recent Advancements and Future Development.

Dan Li, Zheng Ouyang, Xiaoxiao Ma.

  Mass Spectrometry Imaging (MSI) has emerged as a powerful imaging technique for the analysis of biological samples, providing valuable insights into the spatial distribution and structural characterization of lipids. The advancements in high-resolution MSI have made it an indispensable tool for single-cell or subcellular lipidomics. By preserving both intracellular and intercellular information, MSI enables a comprehensive analysis of lipidomics in individual cells and organelles. This enables researchers to delve deeper into the diversity of lipids within cells and to understand the role of lipids in shaping cell behavior. In this review, we aim to provide a comprehensive overview of recent advancements and future prospects of MSI for cellular/subcellular lipidomics. By keeping abreast of the cutting-edge studies in this field, we will continue to push the boundaries of the understanding of lipid metabolism and the impact of lipids on cellular behavior.

Keywords:  data analysis; deep learning; lipidomics; mass spectrometry imaging; matrix-assisted laser desorption ionization (MALDI); multimodal imaging; organelle; secondary ion mass spectrometry (SIMS); single-cell

DOI:  https://doi.org/10.3390/molecules28062712
Nat Commun. 2023 Mar 29. 14(1): 1752

MS2Query: reliable and scalable MS2 mass spectra-based analogue search.

Niek F de Jonge, Joris J R Louwen, Elena Chekmeneva, Stephane Camuzeaux, Femke J Vermeir, Robert S Jansen, Florian Huber, Justin J J van der Hooft.

Metabolomics-driven discoveries of biological samples remain hampered by the grand challenge of metabolite annotation and identification. Only few metabolites have an annotated spectrum in spectral libraries; hence, searching only for exact library matches generally returns a few hits. An attractive alternative is searching for so-called analogues as a starting point for structural annotations; analogues are library molecules which are not exact matches but display a high chemical similarity. However, current analogue search implementations are not yet very reliable and relatively slow. Here, we present MS2Query, a machine learning-based tool that integrates mass spectral embedding-based chemical similarity predictors (Spec2Vec and MS2Deepscore) as well as detected precursor masses to rank potential analogues and exact matches. Benchmarking MS2Query on reference mass spectra and experimental case studies demonstrate improved reliability and scalability. Thereby, MS2Query offers exciting opportunities to further increase the annotation rate of metabolomics profiles of complex metabolite mixtures and to discover new biology.

DOI: https://doi.org/10.1038/s41467-023-37446-4
Anal Chem. 2023 Mar 27.

Mechanistic Understanding of the Discrepancies between Common Peak Picking Algorithms in Liquid Chromatography-Mass Spectrometry-Based Metabolomics.

Jian Guo, Tao Huan.

Inconsistent peak picking outcomes are a critical concern in processing liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics data. This work systematically studied the mechanisms behind the discrepancies among five commonly used peak picking algorithms, including CentWave in XCMS, linear-weighted moving average in MS-DIAL, automated data analysis pipeline (ADAP) in MZmine 2, Savitzky-Golay in El-MAVEN, and FeatureFinderMetabo in OpenMS. We first collected 10 public metabolomics datasets representing various LC-MS analytical conditions. We then incorporated several novel strategies to (i) acquire the optimal peak picking parameters of each algorithm for a fair comparison, (ii) automatically recognize false metabolic features with poor chromatographic peak shapes, and (iii) evaluate the real metabolic features that are missed by the algorithms. By applying these strategies, we compared the true, false, and undetected metabolic features in each data processing outcome. Our results show that linear-weighted moving average consistently outperforms the other peak picking algorithms. To facilitate a mechanistic understanding of the differences, we proposed six peak attributes: ideal slope, sharpness, peak height, mass deviation, peak width, and scan number. We also developed an R program to automatically measure these attributes for detected and undetected true metabolic features. From the results of the 10 datasets, we concluded that four peak attributes, including ideal slope, scan number, peak width, and mass deviation, are critical for the detectability of a peak. For instance, the focus on ideal slope critically hinders the extraction of true metabolic features with low ideal slope scores in linear-weighted moving average, Savitzky-Golay, and ADAP. The relationships between peak picking algorithms and peak attributes were also visualized in a principal component analysis biplot. Overall, the clear comparison and explanation of the differences between peak picking algorithms can lead to the design of better peak picking strategies in the future.

DOI: https://doi.org/10.1021/acs.analchem.2c04887
Biomolecules. 2023 03 16. pii: 546. [Epub ahead of print]13(3):

A Five-Year Update on Matrix Compounds for MALDI-MS Analysis of Lipids.

Jenny Leopold, Patricia Prabutzki, Kathrin M Engel, Jürgen Schiller.

  Matrix-assisted laser desorption and ionization (MALDI) is a widely used soft-ionization technique of modern mass spectrometry (MS). MALDI enables the analysis of nearly all chemical compounds-including polar and apolar (phospho)lipids-with a minimum extent of fragmentation. MALDI has some particular advantages (such as the possibility to acquire spatially-resolved spectra) and is competitive with the simultaneously developed ESI (electrospray ionization) MS. Although there are still some methodological aspects that need to be elucidated in more detail, it is obvious that the careful selection of an appropriate matrix plays the most important role in (lipid) analysis. Some lipid classes can be detected exclusively if the optimum matrix is used, and the matrix determines the sensitivity by which a particular lipid is detected within a mixture. Since the matrix is, thus, crucial for optimum results, we provide here an update on the progress in the field since our original review in this journal in 2018. Thus, only the development during the last five years is considered, and lipids are sorted according to increasing complexity, starting with free fatty acids and ending with cardiolipins and phosphoinositides.

Keywords:  MALDI-MS; lipid; matrix; phospholipid

DOI:  https://doi.org/10.3390/biom13030546
Clin Chim Acta. 2023 Mar 27. pii: S0009-8981(23)00108-0. [Epub ahead of print]543 117306

Development of a novel Ultra Performance Liquid Chromatography Tandem-Mass Spectrometry (UPLC-MS/MS) method to measure l-arginine metabolites in plasma.

Lavinia Santucci, Sara Lomuscio, Aniello Primiano, Riccardo Calvani, Silvia Persichilli, Federica Iavarone, Anna Picca, Francesca Canu, Andrea Urbani, Jacopo Gervasoni.

  INTRODUCTION: Arginine metabolism is involved in the regulation of several biological processes. Many liquid chromatography tandem-mass spectrometry methods for the determination of arginine and its metabolites have been developed but they are time consuming and imply long pre-analytical procedures. The purpose of this study was to develop a rapid method for the simultaneous determination of arginine, citrulline, ornithine, symmetric and asymmetric dimethylarginine and monomethylarginine in human plasma.MATERIALS AND METHODS: The pre-analytical procedure consisted in a simple deproteinization. The chromatographic separation was performed using hydrophilic interaction liquid chromatography. Analytes detection was performed with a triple quadrupole equipped with electrospray ion source operating in positive ion mode. Mass spectrometry experiments were conducted in multiple reaction monitoring mode.
RESULTS AND CONCLUSIONS: Recovery ranged from 92.2 to 108.0%. The within-run imprecision and between-run imprecision ranged from 1.5 to 6.8 % and 3.8 to 11.9%, respectively. Carry over and matrix effect did not affect quantitative analysis. Extraction recovery was between 95 and 105 %. Stability after pre-analytical procedure was tested and all the metabolites were stable after 48 h at 4 °C. In conclusion, our novel method allow a rapid and easy determination of arginine and its metabolites both for research and clinical routine use.

Keywords:  Arginine; HILIC; Nitric Oxide Synthase; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.cca.2023.117306
J Am Soc Mass Spectrom. 2023 Mar 27.

Identifying Hair Biomarker Candidates for Alzheimer's Disease Using Three High Resolution Mass Spectrometry-Based Untargeted Metabolomics Strategies.

Chih-Wei Chang, Jen-Yi Hsu, Ping-Zu Hsiao, Yuan-Chih Chen, Pao-Chi Liao.

  High-resolution mass spectrometry (HRMS)-based untargeted metabolomics strategies have emerged as an effective tool for discovering biomarkers of Alzheimer's disease (AD). There are various HRMS-based untargeted metabolomics strategies for biomarker discovery, including the data-dependent acquisition (DDA) method, the combination of full scan and target MS/MS, and the all ion fragmentation (AIF) method. Hair has emerged as a potential biospecimen for biomarker discovery in clinical research since it might reflect the circulating metabolic profiles over several months, while the analytical performances of the different data acquisition methods for hair biomarker discovery have been rarely investigated. Here, the analytical performances of three data acquisition methods in HRMS-based untargeted metabolomics for hair biomarker discovery were evaluated. The human hair samples from AD patients (N = 23) and cognitively normal individuals (N = 23) were used as an example. The most significant number of discriminatory features was acquired using the full scan (407), which is approximately 10-fold higher than that using the DDA strategy (41) and 11% higher than that using the AIF strategy (366). Only 66% of discriminatory chemicals discovered in the DDA strategy were discriminatory features in the full scan dataset. Moreover, compared to the deconvoluted MS/MS spectra with coeluted and background ions from the AIF method, the MS/MS spectrum obtained from the targeted MS/MS approach is cleaner and purer. Therefore, an untargeted metabolomics strategy combining the full scan with the targeted MS/MS method could obtain most discriminatory features along with a high quality MS/MS spectrum for discovering the AD biomarkers.

Keywords:  Alzheimer’s disease; biomarker discovery; data acquisition; high-resolution mass spectrometry; untargeted metabolomics

DOI:  https://doi.org/10.1021/jasms.2c00294
Metabolites. 2023 Feb 21. pii: 314. [Epub ahead of print]13(3):

MAD HATTER Correctly Annotates 98% of Small Molecule Tandem Mass Spectra Searching in PubChem.

Martin A Hoffmann, Fleming Kretschmer, Marcus Ludwig, Sebastian Böcker.

  Metabolites provide a direct functional signature of cellular state. Untargeted metabolomics usually relies on mass spectrometry, a technology capable of detecting thousands of compounds in a biological sample. Metabolite annotation is executed using tandem mass spectrometry. Spectral library search is far from comprehensive, and numerous compounds remain unannotated. So-called in silico methods allow us to overcome the restrictions of spectral libraries, by searching in much larger molecular structure databases. Yet, after more than a decade of method development, in silico methods still do not reach the correct annotation rates that users would wish for. Here, we present a novel computational method called Mad Hatter for this task. Mad Hatter combines CSI:FingerID results with information from the searched structure database via a metascore. Compound information includes the melting point, and the number of words in the compound description starting with the letter 'u'. We then show that Mad Hatter reaches a stunning 97.6% correct annotations when searching PubChem, one of the largest and most comprehensive molecular structure databases. Unfortunately, Mad Hatter is not a real method. Rather, we developed Mad Hatter solely for the purpose of demonstrating common issues in computational method development and evaluation. We explain what evaluation glitches were necessary for Mad Hatter to reach this annotation level, what is wrong with similar metascores in general, and why metascores may screw up not only method evaluations but also the analysis of biological experiments. This paper may serve as an example of problems in the development and evaluation of machine learning models for metabolite annotation.

Keywords:  database search; in silico methods; metabolite annotation; metascores; molecular structure; parody paper; tandem mass spectrometry

DOI:  https://doi.org/10.3390/metabo13030314
Metabolomics. 2023 Mar 27. 19(4): 24

Systematic untargeted UHPLC-Q-TOF-MS based lipidomics workflow for improved detection and annotation of lipid sub-classes in serum.

Seema Dhariwal, Kiran Maan, Ruchi Baghel, Apoorva Sharma, Dipankar Malakar, Poonam Rana.

  INTRODUCTION AND OBJECTIVE: Taking into consideration the challenges of lipid analytics, present study aims to design the best high-throughput workflow for detection and annotation of lipids.MATERIAL AND METHODS: Serum lipid profiling was performed on CSH-C18 and EVO-C18 columns using UHPLC Q-TOF-MS and generated lipid features were annotated based on m/z and fragment ion using different software.
RESULT AND DISCUSSION: Better detection of features was observed in CSH-C18 than EVO-C18 with enhanced resolution except for Glycerolipids (triacylglycerols) and Sphingolipids (sphingomyelin).
CONCLUSION: The study revealed an optimized untargeted Lipidomics-workflow with comprehensive lipid profiling (CSH-C18 column) and confirmatory annotation (LipidBlast).

Keywords:  LIPID MAPS®; LipidBlast; LipidView™; Lipidomics; Q-TOF; UHPLC–MS

DOI:  https://doi.org/10.1007/s11306-023-01983-2
J Am Soc Mass Spectrom. 2023 Mar 29.

Lipidomic Analysis of Mouse Brain to Evaluate the Efficacy and Preservation of Different Tissue Preparatory Techniques by IR-MALDESI-MSI.

Mary F Wang, Alexandria L Sohn, Juhi Samal, Kevin Erning, Tatiana Segura, David C Muddiman.

  Numerous preparatory methods have been developed to preserve the cellular and structural integrity of various biological tissues for different -omics studies. Herein, two preparatory methods for mass spectrometry imaging (MSI) were evaluated, fresh-frozen and sucrose-embedded, paraformaldehyde (PFA) fixed, in terms of ion abundance, putative lipid identifications, and preservation of analyte spatial distributions. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI)-MSI was utilized to compare the preparatory methods of interest with and without the use of the conventional ice matrix. There were 2.5-fold and 1.6-fold more lipid species putatively identified in positive- and negative-ion modes, respectively, for sucrose-embedded, PFA-fixed tissues without an ice matrix relative to the current IR-MALDESI-MSI gold-standard, fresh-frozen tissue preparation with an exogenous ice matrix. Furthermore, sucrose-embedded tissues demonstrated improved spatial distribution of ions resulting from the cryo-protective property of sucrose and paraformaldehyde fixation. Evidence from these investigations supports sucrose-embedding without ice matrix as an alternative preparatory technique for IR-MALDESI-MSI.

Keywords:  IR-MALDESI; PFA; mass spectrometry imaging; sucrose

DOI:  https://doi.org/10.1021/jasms.2c00353
Open Life Sci. 2023 ;18(1): 20220556

Performance comparison of three scaling algorithms in NMR-based metabolomics analysis.

Xia Liu, Yiqun Fang, Haifeng Ma, Naixia Zhang, Ci Li.

  Unit variance (UV) scaling, mean centering (CTR) scaling, and Pareto (Par) scaling are three commonly used algorithms in the preprocessing of metabolomics data. Based on our NMR-based metabolomics studies, we found that the clustering identification performances of these three scaling methods were dramatically different as tested by the spectra data of 48 young athletes' urine samples, spleen tissue (from mice), serum (from mice), and cell (from Staphylococcus aureus) samples. Our data suggested that for the extraction of clustering information, UV scaling could serve as a robust approach for NMR metabolomics data for the identification of clustering analysis even with the existence of technical errors. However, for the purpose of discriminative metabolite identification, UV scaling, CTR scaling, and Par scaling could equally extract discriminative metabolites efficiently based on the coefficient values. Based on the data presented in this study, we propose an optimal working pipeline for the selection of scaling algorithms in NMR-based metabolomics analysis, which has the potential to serve as guidance for junior researchers working in the NMR-based metabolomics research field.

Keywords:  CTR scaling; Par scaling; UV scaling; discriminative metabolites; metabolomics; nuclear magnetic resonance

DOI:  https://doi.org/10.1515/biol-2022-0556
Metabolites. 2023 Feb 21. pii: 311. [Epub ahead of print]13(3):

Development of an Untargeted Metabolomics Strategy to Study the Metabolic Rewiring of Dendritic Cells upon Lipopolysaccharide Activation.

Jessica Michieletto, Aurélie Delvaux, Emeline Chu-Van, Christophe Junot, François Fenaille, Florence A Castelli.

  Dendritic cells (DCs) are essential immune cells for defense against external pathogens. Upon activation, DCs undergo profound metabolic alterations whose precise nature remains poorly studied at a large scale and is thus far from being fully understood. The goal of the present work was to develop a reliable and accurate untargeted metabolomics workflow to get a deeper insight into the metabolism of DCs when exposed to an infectious agent (lipopolysaccharide, LPS, was used to mimic bacterial infection). As DCs transition rapidly from a non-adherent to an adherent state upon LPS exposure, one of the leading analytical challenges was to implement a single protocol suitable for getting comparable metabolomic snapshots of those two cellular states. Thus, a thoroughly optimized and robust sample preparation method consisting of a one-pot solvent-assisted method for the simultaneous cell lysis/metabolism quenching and metabolite extraction was first implemented to measure intracellular DC metabolites in an unbiased manner. We also placed special emphasis on metabolome coverage and annotation by using a combination of hydrophilic interaction liquid chromatography and reverse phase columns coupled to high-resolution mass spectrometry in conjunction with an in-house developed spectral database to identify metabolites at a high confidence level. Overall, we were able to characterize up to 171 unique meaningful metabolites in DCs. We then preliminarily compared the metabolic profiles of DCs derived from monocytes of 12 healthy donors upon in vitro LPS activation in a time-course experiment. Interestingly, the resulting data revealed differential and time-dependent activation of some particular metabolic pathways, the most impacted being nucleotides, nucleotide sugars, polyamines pathways, the TCA cycle, and to a lesser extent, the arginine pathway.

Keywords:  adherent cells; dendritic cells; high-resolution mass spectrometry; immunometabolomics; liquid chromatography; metabolomics; sample preparation

DOI:  https://doi.org/10.3390/metabo13030311
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Mar 20. pii: S1570-0232(23)00091-0. [Epub ahead of print]1221 123681

A simple and sensitive LC-MS/MS method for therapeutic drug monitoring of digoxin in children.

Zhi-Mei Yang, Ya-Bin Qin, De-Yun Zhao, Lan-Tao Wang, Ying-Ping Tian, Jian-Fang Liu.

  This short communication introduced a simple and sensitive LC-MS/MS method for therapeutic drug monitoring of digoxin in children with the lower limit of quantitation of 0.2 ng/mL based on 30 μL of plasma. The plasma sample was pretreated by one-step protein precipitation. Then the chromatographic separation was performed on a short C-18 column with a total run time of 2.4 min. The detection was achieved through multiple reaction monitoring using positive ionization mode on a triple quadrupole mass spectrometer. The linear range of digoxin in human plasma was among 0.2-6.4 ng/mL. The intra-day and inter-day accuracies of digoxin ranged from -6.0 % to 10.1 % and imprecisions were less than 8.8 %. The extraction recovery rate of digoxin in plasma samples was above 90 %. Matrix factor normalized by internal standard was within acceptance criteria. This method was fully verified and applied to determine the plasma digoxin concentrations of 43 pediatric patients. It is approved appropriate and practical for the therapeutic drug monitoring of digoxin in routine clinical laboratory practice, especially for children.

Keywords:  Children; Digoxin; LC-MS/MS; Protein precipitation; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.jchromb.2023.123681
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jan 27. pii: S1570-0232(23)00024-7. [Epub ahead of print]1220 123614

High throughput FAMS - A fatty acid mass spectrometry method for monitoring polysorbate hydrolysis in QC.

Sina Hoelterhoff, Jan Wendler, Liya Arackal, Benjamin Felkel, Christian H Bell, Anja Bathke.

  Surfactant degradation in biopharmaceuticals has recently gained significant attention in the pharmaceutical industry. Specifically, hydrolytic degradation of polysorbates, leading to the release of free fatty acids potentially forming visible particles, is a key theme in technical development. To address this emerging topic, we present the development of a fully automated liquid-chromatography single quad mass detector method for the quantification of free fatty acids in biopharmaceuticals. For the first time, we have quantified the longer chain fatty acid degradation products of polysorbate, palmitic and stearic acid, allowing reliable detection and early critical insights for process improvements. This high-throughput method was validated underlining its robust performance in an interlaboratory trial as well as high flexibility allowing different robotic platforms and preparation techniques. The combination of automated sample preparation, separation by liquid chromatography and single quad mass detection makes the validated fatty acid mass spectrometry assay ready for routine use in a regulated environment.

Keywords:  automation; biopharmaceuticals; free fatty acid; high-throughput; liquid chromatography; mass spectrometry; method validation; polysorbate degradation; single quad

DOI:  https://doi.org/10.1016/j.jchromb.2023.123614
Talanta. 2023 Mar 23. pii: S0039-9140(23)00228-X. [Epub ahead of print]259 124477

Reliable quantification of citrate isomers and isobars with direct-infusion tandem mass spectrometry.

Qingyu Hu, Yuting Sun, Xiyan Mu, Yulan Wang, Huiru Tang.

  Direct-infusion tandem mass spectrometry (DI-MS/MS) is an excellent tool for large cohort high-throughput quantitative metabolomics, MS imaging and single cell studies but incapable of discriminating isomers/isobars with similar MS spectral features. With experimental and density-functional theory (DFT) approaches, here, we comprehensively investigated the fragmentation pathways and characteristics of differential ion-mobility spectrometry (DMS) for three citrate isomers (citrate, isocitrate, glucaro-1,4-lactone) and an isobar (quinate) co-existing in biological sample such as urine. Results showed that all these compounds gave better MS spectra in negative-ion mode than positive-ion one and had numerous fragment ions under collision-induced dissociation (CID) with sequential losses of H2O and CO2. All observed fragment ions were assignable by combining experimental with DFT calculation results. A DI-DMS-MS/MS method was then developed to simultaneously quantify these four isomers/isobars with m/z 191-87 (CoV, -5.5 V), 191-73 (CoV, -3.5 V), 191-85 (CoV, -29.5 V) and m/z 191-93 (CoV, -41.5 V) for citrate, isocitrate, glucaro-1,4-lactone and quinate, respectively. The low limit-of-quantification was below 5.5 nM whilst accuracy was above 94% for all above compounds. The urinary concentrations of them in human and C57BL/6 mouse samples were further quantified showing clear inter-individual and inter-species level differences with significantly higher levels of isocitrate, glucaro-1,4-lactone and quinate in human urine samples than mouse ones. This provides an approach to understand the detailed fragmentation pathways for organic isomers/isobars and a high-throughput MS strategy to quantify them in complex mixtures for metabolomics, lipidomics, foodomics and exposomics especially when chromatographic separations are not useable.

Keywords:  Citrate isomers; Collision-induced dissociation; Differential ion-mobility spectrometry; Direct-infusion tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.talanta.2023.124477
Molecules. 2023 Mar 07. pii: 2439. [Epub ahead of print]28(6):

A High-Performance Liquid Chromatography-Mass Spectrometry Method for Simultaneous Determination of Vancomycin, Meropenem, and Valproate in Patients with Post-Craniotomy Infection.

Yuting Jin, Qiang Sun, Yumei Pei, Jing Huang.

  Vancomycin (VAN), meropenem (MER), and valproate (VPA) are commonly used to treat intracranial infection post-craniotomy and prevent associated epilepsy. To monitor their levels, we developed a novel bioassay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneous determination of these three drugs in human serum and cerebrospinal fluid (CSF). Sample preparation by protein precipitation using acetonitrile was followed by HPLC on a Zorbax 300SB-C8 column (150 mm × 4.6 mm, 5 μm) maintained at 40 °C. The lower limit of quantification (LLOQ) was 5 ng/mL for MER, 0.1 μg/mL for VAN, and 1 μg/mL for VPA in serum and 50 ng/mL for MER, 1 μg/mL for VAN, and 2 μg/mL for VPA in CSF. This method was validated with satisfactory linearity, sensitivity, precision, accuracy, recovery, matrix effects, and stability for all analytes. The assay was then successfully applied to evaluate VPA, MER, and VAN levels in serum and CSF from patients with intracranial infection administrated by intrathecal injection. Compared with intravenous injections, an intrathecal injection can provide sufficient therapeutic effects even if the CSF levels did not reach the effective concentration reported. Our method provided a detection tool to study the effective concentrations of these three drugs in CSF from patients administered via intrathecal injection.

Keywords:  LC–MS/MS; cranial infection; meropenem; valproate; vancomycin

DOI:  https://doi.org/10.3390/molecules28062439
Molecules. 2023 Mar 15. pii: 2653. [Epub ahead of print]28(6):

Matrix Effects in GC-MS Profiling of Common Metabolites after Trimethylsilyl Derivatization.

Elena Tarakhovskaya, Andrea Marcillo, Caroline Davis, Sanja Milkovska-Stamenova, Antje Hutschenreuther, Claudia Birkemeyer.

  Metabolite profiling using gas chromatography coupled to mass spectrometry (GC-MS) is one of the most frequently applied and standardized methods in research projects using metabolomics to analyze complex samples. However, more than 20 years after the introduction of non-targeted approaches using GC-MS, there are still unsolved challenges to accurate quantification in such investigations. One particularly difficult aspect in this respect is the occurrence of sample-dependent matrix effects. In this project, we used model compound mixtures of different compositions to simplify the study of the complex interactions between common constituents of biological samples in more detail and subjected those to a frequently applied derivatization protocol for GC-MS analysis, namely trimethylsilylation. We found matrix effects as signal suppression and enhancement of carbohydrates and organic acids not to exceed a factor of ~2, while amino acids can be more affected. Our results suggest that the main reason for our observations may be an incomplete transfer of carbohydrate and organic acid derivatives during the injection process and compound interaction at the start of the separation process. The observed effects were reduced at higher target compound concentrations and by using a more suitable injection-liner geometry.

Keywords:  compound saturation; gas chromatography–mass spectrometry; metabolomics; quantification; signal enhancement; signal suppression

DOI:  https://doi.org/10.3390/molecules28062653
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Mar 20. pii: S1570-0232(23)00090-9. [Epub ahead of print]1221 123680

Quantification of orelabrutinib in human plasma and cerebrospinal fluid by liquid chromatography tandem mass spectrometry.

Shengnan Ji, Xiaomeng Liu, Jing Ha, Lianfeng Ai, Zheng Li.

  A sensitive and simple method was developed to determine orelabrutinib in human plasma and cerebrospinal fluid by liquid chromatography tandem mass spectrometry (LC-MS/MS). The samples were prepared by simple protein precipitation with by 0.1% formic acid acetonitrile solution and efficient separations were performed on the Thermo Hypersll GOLD C18 column (2.1 mm × 150 mm, 5 μm) under a gradient program in a total run time of 9 min. The orelabrutinib was detected by electrospray ionization in positive ion mode with selective reaction monitoring (SRM) and mass spectrometric conditions were optimized in order to increase selectivity and sensitivity. The developed method was validated in terms of its accuracy, precision, selectivity, linearity, recovery, matrix effect, stability, and limits of quantification (LOQ). The lower limit of quantification is 0.50 ng/mL, the intraday and interday precision RSD are both less than 15%, and the recovery rate is 85.7%-92.9%. Finally, the method was successfully applied for the quantitation of orelabrutinib in human plasma and cerebrospinal fluid of clinical patients treated with orelabrutinib.

Keywords:  Cerebrospinal fluid; Liquid chromatography-mass spectrometry; Orelabrutinib; Plasma

DOI:  https://doi.org/10.1016/j.jchromb.2023.123680
Molecules. 2023 Mar 14. pii: 2618. [Epub ahead of print]28(6):

Assessment of In Silico and In Vitro Selpercatinib Metabolic Stability in Human Liver Microsomes Using a Validated LC-MS/MS Method.

Mohamed W Attwa, Haitham AlRabiah, Gamal A E Mostafa, Ahmed H Bakheit, Adnan A Kadi.

  Selpercatinib (SLP; brand name Retevmo®) is a selective and potent RE arranged during transfection (RET) inhibitor. On 21 September 2022, the FDA granted regular approval to SLP (Retevmo, Eli Lilly, and Company). It is considered the only and first RET inhibitor for adults with metastatic or locally advanced solid tumors with RET gene fusion. In the current experiment, a highly specific, sensitive, and fast liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantifying SLP in human liver microsomes (HLMs) was developed and applied to the metabolic stability evaluation of SLP. The LC-MS/MS method was validated following the bioanalytical methodology validation guidelines outlined by the FDA (linearity, selectivity, matrix effect, accuracy, precision, carryover, and extraction recovery). SLP was detected by a triple quadrupole detector (TQD) using a positive ESI source and multiple reaction monitoring (MRM) mode for mass spectrometric analysis and estimation of analytes ions. The IS-normalized matrix effect and extraction recovery were acceptable according to the FDA guidelines for the bioanalysis of SLP. The SLP calibration standards were linear from 1 to 3000 ng/mL HLMs matrix, with a regression equation (y = 1.7298x + 3.62941) and coefficient of variation (r2 = 0.9949). The intra-batch and inter-batch precision and accuracy of the developed LC-MS/MS method were -6.56-5.22% and 5.08-3.15%, respectively. SLP and filgotinib (FLG) (internal standard; IS) were chromatographically separated using a Luna 3 µm PFP (2) stationary phase (150 × 4.6 mm) with an isocratic mobile phase at 23 ± 1 °C. The limit of quantification (LOQ) was 0.78 ng/mL, revealing the LC-MS/MS method sensitivity. The intrinsic clearance and in vitro t1/2 (metabolic stability) of SLP in the HLMs matrix were 34 mL/min/kg and 23.82 min, respectively, which proposed an intermediate metabolic clearance rate of SLP, confirming the great value of this type of kinetic experiment for more accurate metabolic stability predictions. The literature review approved that the established LC-MS/MS method is the first developed and reported method for quantifying SLP metabolic stability.

Keywords:  LC-MS/MS; WhichP450 module; in vitro half-life; intrinsic clearance; metabolic stability; selpercatinib

DOI:  https://doi.org/10.3390/molecules28062618
Metabolites. 2023 Mar 22. pii: 460. [Epub ahead of print]13(3):

A Strategy for Uncovering the Serum Metabolome by Direct-Infusion High-Resolution Mass Spectrometry.

Xiaoshan Sun, Zhen Jia, Yuqing Zhang, Xinjie Zhao, Chunxia Zhao, Xin Lu, Guowang Xu.

  Direct infusion nanoelectrospray high-resolution mass spectrometry (DI-nESI-HRMS) is a promising tool for high-throughput metabolomics analysis. However, metabolite assignment is limited by the inadequate mass accuracy and chemical space of the metabolome database. Here, a serum metabolome characterization method was proposed to make full use of the potential of DI-nESI-HRMS. Different from the widely used database search approach, unambiguous formula assignments were achieved by a reaction network combined with mass accuracy and isotopic patterns filter. To provide enough initial known nodes, an initial network was directly constructed by known metabolite formulas. Then experimental formula candidates were screened by the predefined reaction with the network. The effects of sources and scales of networks on assignment performance were investigated. Further, a scoring rule for filtering unambiguous formula candidates was proposed. The developed approach was validated by a pooled serum sample spiked with reference standards. The coverage and accuracy rates for the spiked standards were 98.9% and 93.6%, respectively. A total of 1958 monoisotopic features were assigned with unique formula candidates for the pooled serum, which is twice more than the database search. Finally, a case study of serum metabolomics in diabetes was carried out using the developed method.

Keywords:  direct-infusion; formula assignment; high-resolution mass spectrometry; metabolomics; reaction network

DOI:  https://doi.org/10.3390/metabo13030460
Curr Opin Chem Biol. 2023 Mar 24. pii: S1367-5931(23)00026-1. [Epub ahead of print]74 102288

Recent advances in mass spectrometry-based computational metabolomics.

Timothy M D Ebbels, Justin J J van der Hooft, Haley Chatelaine, Corey Broeckling, Nicola Zamboni, Soha Hassoun, Ewy A Mathé.

  The computational metabolomics field brings together computer scientists, bioinformaticians, chemists, clinicians, and biologists to maximize the impact of metabolomics across a wide array of scientific and medical disciplines. The field continues to expand as modern instrumentation produces datasets with increasing complexity, resolution, and sensitivity. These datasets must be processed, annotated, modeled, and interpreted to enable biological insight. Techniques for visualization, integration (within or between omics), and interpretation of metabolomics data have evolved along with innovation in the databases and knowledge resources required to aid understanding. In this review, we highlight recent advances in the field and reflect on opportunities and innovations in response to the most pressing challenges. This review was compiled from discussions from the 2022 Dagstuhl seminar entitled "Computational Metabolomics: From Spectra to Knowledge".

Keywords:  Benchmarking; Cheminformatics; Chemometrics; Machine learning; Metabolite identification; Metabolomics; Multi-omics; Small molecules; Visualization

DOI:  https://doi.org/10.1016/j.cbpa.2023.102288
Metabolites. 2023 Mar 08. pii: 402. [Epub ahead of print]13(3):

Cross-Platform Comparison of Amino Acid Metabolic Profiling in Three Model Organisms Used in Environmental Metabolomics.

Jessica C D'eon, Brian P Lankadurai, André J Simpson, Eric J Reiner, David G Poirier, Greg C Vanlerberghe, Myrna J Simpson.

  Environmental metabolomics is a promising approach to study pollutant impacts to target organisms in both terrestrial and aquatic environments. To this end, both nuclear magnetic resonance (NMR)- and mass spectrometry (MS)-based methods are used to profile amino acids in different environmental metabolomic studies. However, these two methods have not been compared directly which is an important consideration for broader comparisons in the environmental metabolomics field. We compared the quantification of 18 amino acids in the tissue extracts of Daphnia magna, a common model organism used in both ecotoxicology and ecology, using both 1H NMR spectroscopy and liquid chromatography with tandem MS (LC-MS/MS). 1H NMR quantification of amino acids agreed with the LC-MS/MS quantification for 17 of 18 amino acids measured. We also tested both quantitative methods in a D. magna sub-lethal exposure study to copper and lithium. Again, both NMR and LC-MS/MS measurements showed agreement. We extended our analyses with extracts from the earthworm Eisenia fetida and the plant model Nicotiana tabacum. The concentrations of amino acids by both 1H NMR and LC-MS/MS, agreed and demonstrated the robustness of both techniques for quantitative metabolomics. These findings demonstrate the compatibility of these two analytical platforms for amino acid profiling in environmentally relevant model organisms and emphasizes that data from either method is robust for comparisons across studies to further build the knowledge base related to pollutant exposure impacts and toxic responses of diverse environmental organisms.

Keywords:  Daphnia magna; Eisenia fetida; LC-MS/MS; NMR spectroscopy; Nicotiana tabacum; metabolite profiling

DOI:  https://doi.org/10.3390/metabo13030402
Anal Chem. 2023 Mar 31.

Separation and Simultaneous Trapping of Multiply Charged and Singly Charged Ions for Mass Spectrometry: Application to Lipid Mixtures.

Kimberly C Fabijanczuk, James W Hager, Scott A McLuckey.

Conventional electrospray ionization (ESI) of mixtures can give rise to singly and multiply charged analyte species that overlap in mass-to-charge (m/z) ratios, which can complicate the analysis of individual components. The overlap in m/z for ions of different mass and charge is particularly problematic when ions of low relative abundance are of interest. For example, cardiolipins (CLs) are structurally complex phospholipids present in low relative abundance in the lipidome but play crucial roles in mitochondrial metabolism and various regulatory processes. ESI of CLs in negative ion mode shows abundant doubly deprotonated ions and minor singly deprotonated ions. In the ESI of lipid extracts, highly abundant singly charged phospholipids extensively overlap in m/z space with CL dianions of much lesser abundance, thereby complicating the study of the CLs. To address this challenge, we employed a gas-phase approach to separate singly charged ions from a population of ions of mixed charge states while allowing for the storage of one or both of the separated ion populations. Herein, we describe the considerations for applying enhanced singly charged (ESC) and enhanced multiply charged (EMC) scans to perform a gas-phase separation of singly charged lipids from doubly charged lipids in an Escherichia coli extract. This method allows for improved signal-to-noise (S/N) ratio of low abundance ions with minimal overall signal loss, removal of "chemical noise" arising from singly charged ions, and allows for retention of spatially separated ions within a mass spectrometer.

DOI: https://doi.org/10.1021/acs.analchem.3c00420
Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2023 Mar 27. 1-10

Analysis of N-nitrosamines and N-nitrosatable substances from baby bottle rubber teats by liquid chromatography tandem mass spectrometry.

Joung Boon Hwang, Jung Eun Lee, Eunbi Kim, Kwon Yong Eom, Hyun-Ah Kim, Soonho Lee.

  A simple and sensitive method based on liquid chromatography-atmospheric pressure chemical ionisation-tandem mass spectrometry (LC-APCI-MS/MS) was developed and validated to determine the levels of 13N-nitrosamines and N-nitrosatable substances migrated from rubber teats into artificial saliva. The migration test from rubber teats was conducted at 40 °C and for 24 h in artificial saliva, and the migrated artificial saliva solution was analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) without further extracting steps. The sensitivity of N-nitrosamines was examined by applying atmospheric chemical ionisation and electrospray ionisation to optimise the mass spectrometric conditions, and the atmospheric chemical ionisation (APCI) mode exhibited 1.6-19 times higher sensitivity. Method validation showed acceptable linearity, precision, and accuracy, and the detection and quantification limits were 0.07-0.35 and 0.24-1.1 μg kg-1, respectively. The developed liquid chromatography-atmospheric chemical ionisation-tandem mass spectrometry method was applied to 39 domestic and imported rubber teats. From 39 samples, N-nitrosamines [N-nitrosodimethylamine (NDMA), N-nitrosomorpholine (NMOR), and N-nitroso n-methyl N-phenylamine (NMPhA)] were detected in 30 samples, with N-nitrosatable substances in 17 samples give rise to NDMA, NMOR, and N-nitrosodiethylamine. However, the levels were below the specific migration limit of Korean Standards and Specifications for Food Containers, Utensils, and Packages and EC Directive 93/11/EEC.

Keywords:  N-nitrosamines; N-nitrosatable substances; high-performance liquid chromatography-atmospheric pressure-tandem mass spectrometry; high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry; rubber teat

DOI:  https://doi.org/10.1080/19440049.2023.2193276
Bioanalysis. 2023 Mar 28.

A sensitive, robust and high-throughput isotope dilution LC-MS/MS method for quantifying three folate forms in serum.

Yunfeng Cao, Jia Zeng, Ting Huang, Jianxing Chen.

  This study aimed to establish an isotope dilution LC-MS/MS method for the determination of folic acid, 5-formyltetrahydrofolate and 5-methyltetrahydrofolate in human serum. This method was then used to quantify these three folate forms in the healthy adult population and supplement users. A stable 96-well solid-phase extraction system was used to prepare serum samples. The highly sensitive method was established using a Shimadzu LCMS-8060NX. The linearity was good in the range of 0.1-10 nmol/l for folic acid and 5-formyltetrahydrofolate and 1.0-100 nmol/l for 5-methyltetrahydrofolate. The accuracy and precision were good. The method was sensitive, robust and high-throughput and could be used for the routine clinical monitoring of these three folate forms in the Chinese population.

Keywords:  5-formyltetrahydrofolate; 5-methyltetrahydrofolate; 96-well solid phase extraction; ID-LC–MS/MS; folic acid; sensitivity; throughput

DOI:  https://doi.org/10.4155/bio-2023-0007
Int J Mol Sci. 2023 Mar 13. pii: 5489. [Epub ahead of print]24(6):

A Simultaneous Extraction/Derivatization Strategy for Quantitation of Vitamin D in Dried Blood Spots Using LC-MS/MS: Application to Biomarker Study in Subjects Tested for SARS-CoV-2.

Yashpal S Chhonker, Nusrat Ahmed, Christine M Johnston, Ruanne V Barnabas, Daryl J Murry.

  Vitamin D plays a critical role in bone development and maintenance, and in other physiological functions. The quantitation of endogenous levels of individual vitamin D and its metabolites is crucial for assessing several disease state conditions. With cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to the coronavirus disease 2019 (COVID-19) pandemic, there are several studies that have associated lower levels of serum vitamin D with severity of infection in COVID-19 patients. In this context, we have developed and validated a robust LC-MS/MS method for simultaneous quantitation of vitamin D and its metabolites in human dried blood spot (DBS) obtained from participants tested for COVID-19. The chromatographic separation for vitamin D and metabolites was performed using an ACE Excel C18 PFP column protected with a C18 guard column (Phenomenex, Torrance, CA, USA). The mobile phase consisted of formic acid in water (0.1% v/v) as mobile phase A and formic acid in methanol (0.1% v/v) as mobile phase B, operated at a flow rate of 0.5 mL/min. Analysis was performed utilizing the LC-MS/MS technique. The method was sensitive with a limit of quantification of 0.78 ng/mL for all analytes, and had a large dynamic range (200 ng/mL) with a total run time of 11 min. The inter- and intraday accuracy and precision values met the acceptance criteria per the US Food and Drug Administration guidelines. Blood concentrations of 25(OH)D3, vitamin D3, 25(OH)D2, and vitamin D2 over a range of 2-195.6, 0.5-121.5, 0.6-54.9, and 0.5-23.9 ng/mL, respectively, were quantified in 909 DBS samples. In summary, our developed LC-MS/MS method may be used for quantification of vitamin D and its metabolites in DBS, and may be applied to investigations of the emerging role of these compounds in various physiological processes.

Keywords:  LC–MS/MS; SARS-CoV-2(+); biomarker; vitamin D

DOI:  https://doi.org/10.3390/ijms24065489
Foods. 2023 Mar 13. pii: 1226. [Epub ahead of print]12(6):

Impact of Matrix Species and Mass Spectrometry on Matrix Effects in Multi-Residue Pesticide Analysis Based on QuEChERS-LC-MS.

Shuang Zhang, Zhiyong He, Maomao Zeng, Jie Chen.

  With the popularity of multi-residue pesticide analysis based on quick, easy, cheap, effective, rugged, and safe (QuEChERS) cleanup and liquid chromatography-mass spectrometry (LC-MS), matching optimal matrix-matched calibration protocols and LC-MS conditions to reduce matrix effects (MEs) has become a crucial task for analysts in their routines. However, dozens to hundreds of pesticide analytes in a single run generate increasingly multi-dimensional ME data, requiring appropriate tools to handle these data sets. Therefore, we established an ME analysis strategy by drawing on analytical thinking and tools from metabolomics analysis. Using this, matrix species-induced and mass spectrometry-induced systematic ME variations were distinguished, and pesticides contributed to the variations were scanned out. A simultaneous weakening of MEs on 24 pesticides in 32 different matrices was achieved using the time-of-flight-mass spectrometry (TOF-MS) scan under the information-dependent acquisition (IDA) mode of high-resolution mass spectrometry (HR-MS), compared to multiple reaction monitoring (MRM) scanning by tandem mass spectrometry (MS/MS). Bay leaf, ginger, rosemary, Amomum tsao-ko, Sichuan pepper, cilantro, Houttuynia cordata, and garlic sprout showed enhanced signal suppression in the MRM scan for 105 differential MRM transitions for 42 pesticides and in IDA mode for 33 pesticides, respectively. This study revealed the interference of matrix species and mass spectrometry on MEs and provided a novel strategy for ME analysis.

Keywords:  IDA; MRM; OPLS-DA; UPLC MS/MS; UPLC QTOF-MS; matrix effects; matrix species; multi-residue pesticide analysis

DOI:  https://doi.org/10.3390/foods12061226
MethodsX. 2023 ;10 102129

A simultaneous, high-throughput and sensitive method for analysing 13 neonicotinoids and metabolites in urine using a liquid chromatography-tandem mass spectrometry.

Yukiko Nishihama, Shoji F Nakayama, Tomohiko Isobe.

  A simultaneous, high-throughput and sensitive method for analysing nine neonicotinoid pesticides (NEOs) and four metabolites (NEOms) in urine using a liquid chromatography-tandem mass spectrometry (LC-MSMS) was developed. The method detection limit (MDL) and lowest concentration minimum reporting limit (LCMRL) of the nine NEOs were 0.0013-0.048 ng/ml and 0.0050-0.17 ng/ml, respectively. The MDL and LCMRL of the four NEOms were 0.0052-0.52 ng/ml and 0.011-1.6 ng/ml, respectively. Intermediate precision for the nine NEOs and four NEOms was 7.5-12.5% and 7.4-10.9%, respectively. Accuracy for the nine NEOs and four NEOms was 3.83-5.60% and 3.01-29.2%, respectively. The developed method was applied to analyse urine samples collected from participants of a large-scale birth cohort study, namely, the Japan Environment and Children's Study (JECS). •The NEO and NEOm concentrations in 100 µl urine samples were analysed using a highly sensitive LC-MSMS.•Automated solid phase extraction in a 96-well plate was utilised to achieve high-throughput analysis.•Intermediate precision and accuracy were less than 12.5% and 94.8-99.1%, respectively.

Keywords:  Neonicotinoid; Simultaneous and high-throughput determination of 13 urinary neonicotinoid and metabolite concentrations; high-throughput analysis; urine

DOI:  https://doi.org/10.1016/j.mex.2023.102129
Metabolites. 2023 Mar 03. pii: 377. [Epub ahead of print]13(3):

Targeted Desorption Electrospray Ionization Mass Spectrometry Imaging for Drug Distribution, Toxicity, and Tissue Classification Studies.

Andreas Dannhorn, Maria Luisa Doria, James McKenzie, Paolo Inglese, John G Swales, Gregory Hamm, Nicole Strittmatter, Gareth Maglennon, Sadaf Ghaem-Maghami, Richard J A Goodwin, Zoltan Takats.

  With increased use of mass spectrometry imaging (MSI) in support of pharmaceutical research and development, there are opportunities to develop analytical pipelines that incorporate exploratory high-performance analysis with higher capacity and faster targeted MSI. Therefore, to enable faster MSI data acquisition we present analyte-targeted desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) utilizing a triple-quadrupole (TQ) mass analyzer. The evaluated platform configuration provided superior sensitivity compared to a conventional time-of-flight (TOF) mass analyzer and thus holds the potential to generate data applicable to pharmaceutical research and development. The platform was successfully operated with sampling rates up to 10 scans/s, comparing positively to the 1 scan/s commonly used on comparable DESI-TOF setups. The higher scan rate enabled investigation of the desorption/ionization processes of endogenous lipid species such as phosphatidylcholines and a co-administered cassette of four orally dosed drugs-erlotininb, moxifloxacin, olanzapine, and terfenadine. This was used to enable understanding of the impact of the desorption/ionization processes in order to optimize the operational parameters, resulting in improved compound coverage for olanzapine and the main olanzapine metabolite, hydroxy-olanzapine, in brain tissue sections compared to DESI-TOF analysis or matrix-assisted laser desorption/ionization (MALDI) platforms. The approach allowed reducing the amount of recorded information, thus reducing the size of datasets from up to 150 GB per experiment down to several hundred MB. The improved performance was demonstrated in case studies investigating the suitability of this approach for mapping drug distribution, spatially resolved profiling of drug-induced nephrotoxicity, and molecular-histological tissue classification of ovarian tumors specimens.

Keywords:  DESI; MALDI; MSI; TQ; mass spectrometry imaging

DOI:  https://doi.org/10.3390/metabo13030377
J Chromatogr A. 2023 Mar 12. pii: S0021-9673(23)00151-6. [Epub ahead of print]1695 463925

Development of tandem-column liquid chromatographic methods for pharmaceutical compounds using simulations based on hydrophobic subtraction model parameters.

Zhiyang Liu, Yiyang Zhou, Qinggang Wang, Joe P Foley, Dwight R Stoll, Jonathan G Shackman.

  The liquid chromatography (LC) analysis of small molecule pharmaceutical compounds and related impurities is crucial in the development of new drug substances, but developing these separations is usually challenging due to analyte structural similarities. Tandem-column LC (TC-LC) has emerged as a powerful approach to achieve alternative separation selectivity compared to conventional single column separations. However, one of the bottlenecks associated with use of tandem column approaches is time-consuming column pair screening and selection. Herein, we compared critical resolution (Rc) in single column vs. TC-LC separations for a given set of small molecule pharmaceutical compounds and developed a column selection workflow that uses separation simulations based on parameters from the hydrophobic subtraction model (HSM) of reversed-phase selectivity. In this study, HSM solute parameters were experimentally determined for a small molecule pharmaceutical (Linrodostat) and ten of its related impurities using multiple linear regression of their retentions on 16 selected RPLC columns against in-house determined HSM column parameters. Rc values were calculated based on HSM database column parameters for a pool of about 200 available stationary phases in both single-phase column (2.1 mm i.d. × 100 mm) or tandem column paired (two 2.1 mm i.d. × 50 mm) formats. Four column configurations (two single and two tandem) were predicted to achieve successful separations under isocratic HSM separation conditions, with a fifth tandem pair predicted to have a single co-elution. Of these five potential candidates, one tandem pair yielded compete baseline resolution of the 11-component mixture in an experimental separation. In this specific case, the tandem column pairs outperformed single-phase columns, with better predicted and experimental Rc values for the Linrodostat mixture under the HSM separation conditions. The results reported in this study demonstrated the enormous selectivity potential of TC-LC in pharmaceutical compound separations and are consistent with our previous study that examined the potential of tandem column approaches using purely computational means, though there is room for substantial improvement in the prediction accuracy. The proposed workflow can be used to prioritize a small number of column combinations by computational means before any experiments are conducted. This is highly attractive from the point of view of time and resource savings considering over 200,000 different tandem column pairings are possible using columns for which there are data in the HSM database.

Keywords:  Hydrophobic Subtraction Model; Method development; Pharmaceuticals; Simulated separations; Tandem-column liquid chromatography

DOI:  https://doi.org/10.1016/j.chroma.2023.463925
Foods. 2023 Mar 09. pii: 1152. [Epub ahead of print]12(6):

Metabolite Fingerprinting for Identification of Panax ginseng Metabolites Using Internal Extractive Electrospray Ionization Mass Spectrometry.

Xueyan Yuan, Xiaoping Zhang, Jiaquan Xu, Jianhua Ye, Zhendong Yu, Xinglei Zhang.

  Ginseng, a kind of functional food and medicine with high nutritional value, contains various pharmacological metabolites that influence human metabolic functions. Therefore, it is very important to analyze the composition and metabolites of ginseng. However, the analysis of active metabolites in ginseng samples usually involves various experimental steps, such as extraction, chromatographic separation, and characterization, which may be time-consuming and laborious. In this study, an internal extractive electrospray ionization mass spectrometry (iEESI-MS) method was developed to analyze active metabolites in ginseng samples with sequential sampling and no pretreatment. A total of 44 metabolites, with 32 ginsenosides, 6 sugars, and 6 organic acids, were identified in the ginseng samples. The orthogonal partial least-squares discriminant analysis (OPLS-DA) score plot showed a clear separation of ginseng samples from different origins, indicating that metabolic changes occurred under different growing conditions. This study demonstrated that different cultivation conditions of ginseng can be successfully discriminated when using iEESI-MS-based metabolite fingerprints, which provide an alternative solution for the quality identification of plant drugs.

Keywords:  ginseng; internal extractive electrospray ionization mass spectrometry; metabolite fingerprinting; metabolites; sequential sampling

DOI:  https://doi.org/10.3390/foods12061152
J Hazard Mater. 2023 Mar 15. pii: S0304-3894(23)00472-7. [Epub ahead of print]452 131190

In-depth profiling of di(2-ethylhexyl) phthalate metabolic footprints in rats using click chemistry-mass spectrometry probes.

Yu-Ning Hu, Jin-Tao Zhan, Pei-Rong Bai, Na An, Jun-Jie Tan, Yan-Zhen Wang, Quan-Fei Zhu, Yu-Qi Feng.

  Di(2-ethylhexyl) phthalate (DEHP), the most widely used plasticizers in the world, has been regarded as an endocrine disrupting chemical with serious adverse health outcomes. Accumulating evidence strongly suggests that the undesirable biological effects of DEHP are meditated by its metabolites rather than itself. However, the metabolic footprints of DEHP in vivo are still unclear. Here we developed a click chemistry-assisted mass spectrometry (CC-MS) strategy for in-depth profiling DEHP metabolites in rats. An alkyne-modified DEHP analogue (alkyne-DEHP) was synthesized as a tracer for in vivo tracing, and a pair of MS probes (4-azido-nphenylbenzamide, 4-ANPA, and its deuterated reagent d5-4-ANPA) were prepared to specifically label the alkyne-DEHP metabolites, and prominently improve their detection sensitivity and selectivity. Using the CC-MS strategy, we successfully screened 247 alkyne-DEHP metabolites from rat urine, feces, and serum, including many unrevealed metabolites, such as oxidized phthalate diester metabolites and glucuronides of phthalate monoester metabolites. The discovery of new DEHP metabolites provides additional insights for understanding the metabolism of DEHP, which may be beneficial in exploring the mechanism underlying DEHP induced-toxicity in the future.

Keywords:  Alkyne analogue; DEHP; Derivatization; LC-MS; Metabolic tracing

DOI:  https://doi.org/10.1016/j.jhazmat.2023.131190
PLoS One. 2023 ;18(3): e0283783

Quantitative determination of lurbinectedin, its unbound fraction and its metabolites in human plasma utilizing ultra-performance LC-MS/MS.

Nicholas King, Soledad Garcia-Martinez, Eider Alcaraz, Alba Grisalena, Rubin Lubomirov, Raquel Altares, Carlos Fernandez-Teruel, Andrés M Francesch, Pablo M Avilés, Salvador Fudio.

AIMS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods to quantify total lurbinectedin, its plasma protein binding to derive the unbound fraction and its main metabolites 1',3'-dihydroxy-lurbinectedin (M4) and N-desmethyl-lurbinectedin (M6) in human plasma, were developed and validated.MATERIALS & METHODS: For lurbinectedin, sample extraction was performed using supported liquid extraction. For metabolites, liquid-liquid extraction with stable isotope-labeled analogue internal standards was used. Plasma protein binding was evaluated using rapid equilibrium dialysis. In vitro investigations at different plasma protein concentrations were carried out to estimate dissociation rate constants to albumin and alpha-1-acid glycoprotein (AAG).
RESULTS: Calibration curves displayed good linearity over 0.1 to 50 ng/mL for lurbinectedin and 0.5 to 20 ng/mL for the metabolites. Methods were validated in accordance with established guidance. The inter-day precision and accuracy ranged from 5.1% to 10.7%, and from -5% to 6% (lurbinectedin in plasma); from 3.1% to 6.6%, and from 4% to 6% (lurbinectedin in plasma:PBS); from 4.5% to 12.9%, and from 4% to 9% (M4); and from 7.5% to 10.5%, and from 6% to 12% (M6). All methods displayed good linearity (r2 >0.99). Recovery was evaluated for lurbinectedin in plasma:PBS (66.4% to 86.6%), M4 (7.82% to 13.4%) and M6 (22.2% to 34.3%). The method for lurbinectedin in plasma has been applied in most clinical studies, while the plasma:PBS and metabolites methods were used to evaluate the impact of special conditions on lurbinectedin PK. Lurbinectedin plasma protein binding was 99.6% and highly affected by AAG concentration.
CONCLUSIONS: These UPLC-MS/MS methods enable the rapid and sensitive quantification of lurbinectedin and its main metabolites in clinical samples.

DOI: https://doi.org/10.1371/journal.pone.0283783
Metabolites. 2023 Mar 01. pii: 367. [Epub ahead of print]13(3):

Assessing the Effectiveness of Chemical Marker Extraction from Amazonian Plant Cupuassu (Theobroma grandiflorum) by PSI-HRMS/MS and LC-HRMS/MS.

Nerilson M Lima, Gesiane S Lima, Gabriel F Dos Santos, Gagan Preet, Lanaia I L Maciel, Teresinha de Jesus A S Andrade, Marcel Jaspars, Andrea R Chaves, Boniek G Vaz.

  Employing a combination of liquid chromatography electrospray ionization and paper spray ionization high-resolution tandem mass spectrometry, extracts from cupuassu (Theobroma grandiflorum) pulp prepared with either water, methanol, acetonitrile or combinations thereof were subjected to metabolite fingerprinting. Among the tested extractors, 100% methanol extracted preferentially phenols and cinnamic acids derivatives, whereas acetonitrile and acetonitrile/methanol were more effective in extracting terpenoids and flavonoids, respectively. And while liquid chromatography- mass spectrometry detected twice as many metabolites as paper spray ionization tandem mass spectrometry, the latter proved its potential as a screening technique. Comprehensive structural annotation showed a high production of terpenes, mainly oleanane triterpene derivatives. of the mass spectra Further, five major metabolites with known antioxidant activity, namely catechin, citric acid, epigallocatechin-3'-glucuronide, 5,7,8-trihydroxyflavanone, and asiatic acid, were subjected to molecular docking analysis using the antioxidative enzyme peroxiredoxin 5 (PRDX5) as a model receptor. Based on its excellent docking score, a pharmacophore model of 5,7,8-trihydroxyflavanone was generated, which may help the design of new antioxidants.

Keywords:  Theobroma grandiflorum; ambient mass spectrometry ionization; cupuassu; liquid chromatography–mass spectrometry; paper spray ionization mass spectrometry; untargeted metabolomics

DOI:  https://doi.org/10.3390/metabo13030367
Biomed Chromatogr. 2023 Mar 28. e5632

A review on various analytical methods for the estimation of olanzapine - An antipsychotic drug.

S Reka, Afsana Sheikh, Waleed H Almalki, Prashant Kesharwani, Punniyakoti Veeraveedu Thanikachalam.

  Olanzapine (OLZ) is an antipsychotic agent which is thienobenzodiazepine derivative. It is used either in combination with other drugs like Carbamazepine, Simvastatin, Clozapine etc. or as a single drug. The present work is mainly focused on various approaches for the analysis of OLZ in bulk drugs as well as their pharmaceutical formulations. It is also focused on various bioanalytical methods used for the analysis. Based on our survey, it is observed that many analytical techniques were carried out using UV-Spectrophotometry, Mass spectrophotometry, LC-MS/MS techniques, and chromatographic techniques like HPLC, and HPTLC in both bulk and solid dosage forms. Bioanalytical techniques are also performed using human plasma or serum etc., The analysis was carried out either for the single drug or for a combination of drugs. The review depicts the rate usage of the different methodologies for the analysis of OLZ. A measurable amount of information is gathered and utilized for the strategies.

Keywords:  Analytical methods; Antipsychotic drug; Bipolar disorder; Olanzapine; Schizophrenia

DOI:  https://doi.org/10.1002/bmc.5632