bims-metlip 2023-11-05 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023–11–05
twelve papers selected by
Sofia Costa, Matterworks

Quantitative analysis of the glutathione pathway cellular metabolites by targeted liquid chromatography - tandem mass spectrometry.
Simultaneous determination of ergothioneine, selenoneine, and their methylated metabolites in human blood using ID-LC-MS/MS.
Determination of well-being-related markers in nails by liquid chromatography tandem mass spectrometry.
Fast and sensitive LC-MS/MS method for quantification of cannabinoids and their metabolites in plasma of cattle fed hemp.
Development and validation of Empagliflozin and Linagliptin simultaneous estimation in rat plasma using freezing lipid precipitation and SCX-SPE assisted HPLC-MS/MS method and its application in pharmacokinetic studies.
Preclinical pharmacokinetic and in vitro metabolic stability study of lysosomotropic autophagy inhibitor, IITZ-01 in mice by using UPLC-MS/MS.
Mass Spectrometry Analysis for Clinical Applications: A Review.
DEEP Picker1D and Voigt Fitter1D: a versatile tool set for the automated quantitative spectral deconvolution of complex 1D-NMR spectra.
A benchmark study of data normalisation methods for PTR-TOF-MS exhaled breath metabolomics.
Matrix effects demystified: Strategies for resolving challenges in analytical separations of complex samples.
Impact assessment of metabolite instability in the development and validation of LC-MS/MS bioanalytical assays for measurement of rosuvastatin in human plasma and urine samples.
Using in silico tools to predict flame retardant metabolites for more informative exposomics-based approaches.

J Sep Sci. 2023 Oct 29. e2300780

Quantitative analysis of the glutathione pathway cellular metabolites by targeted liquid chromatography - tandem mass spectrometry.

Kristian Serafimov, Yüsra Aydin, Michael Lämmerhofer.

  Glutathione, its biosynthesis intermediates and other thiol metabolites are of central relevance for the redox homeostasis of cells. Their analysis is critical due to the facile interconversion of redox pairs during sampling, sample preparation, and data acquisition, in particular in the electrospray ionization interface. In this work we propose a fast targeted LC-MS/MS method to accurately analyze 14 metabolites from the glutathione pathway. N-Ethylmaleimide reagent is added with the extraction solvent and instantly stabilizes the thiol-redox state by derivatization. Liquid chromatographic separation of the analytes was performed on a sub-2μm superficially porous HILIC column with sulfobetaine chemistry. Tandem MS with triple-quadrupole mass spectrometry in multiple-reaction monitoring acquisition mode allowed sensitive detection of the targeted metabolites with LOQs in the range of 5-25 nM. Run times of 3 min enable a high throughput analysis of cellular samples. For calibration a 13 C-labelled cell extract was used as internal standard. The method was validated and the concentrations of glutathione and its biosynthesis intermediates determined in HeLa cells. This article is protected by copyright. All rights reserved.

Keywords:  Targeted metabolomics; bioanalytical; derivatization; glutathione; hydrophilic interaction chromatography; quantitative; redox; redox homeostasis; tandem mass spectrometry; thiol

DOI:  https://doi.org/10.1002/jssc.202300780
Anal Bioanal Chem. 2023 Nov 02.

Simultaneous determination of ergothioneine, selenoneine, and their methylated metabolites in human blood using ID-LC-MS/MS.

Adel Achouba, Pierre Dumas, Pierre Ayotte.

  Ergothioneine and selenoneine are structurally related dietary antioxidants and cytoprotectants that may help prevent several chronic diseases associated with inflammation and aging. Both compounds share pharmacokinetic characteristics such as cellular uptake through the ergothioneine transporter, accumulation in red blood cells, and biotransformation to methylated metabolites. A rapid, sensitive, specific, precise, and cost-effective analytical method is required to further investigate the potential health benefits of these compounds, individually or combined, in large epidemiological studies. We developed and validated an isotope-dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous specific quantification of these analytes in human blood following a simple sample preparation consisting of dilution in aqueous dithiothreitol followed by centrifugal filtration. Chromatographic separation of the analytes is achieved using a reversed-phase chromatography within an 8-min run. Analyte detection is performed using triple quadrupole mass spectrometry in multiple reaction monitoring mode. Each analyte is quantified against its corresponding isotopically labeled internal standard either commercially available or synthesized in-house (77Se-labeled selenoneine compounds). The validated method demonstrates excellent linearity and very good precision (all CV < 10%). Matrix effects are minimal, suggesting that this method could easily be adapted to other matrices. Freeze/thaw cycles have little effect on methylated metabolites but significantly reduced concentrations of the parent compounds. The method was successfully applied to a small set of volunteer blood samples containing low levels of the analytes. The developed ID-LC-MS/MS method opens new avenues for exploring the roles of these bioactive compounds and their metabolites in human health and disease.

Keywords:  Ergothioneine; Human blood; Isotope-dilution LC–MS/MS; Selenoneine

DOI:  https://doi.org/10.1007/s00216-023-04994-z
Ecotoxicol Environ Saf. 2023 Oct 26. pii: S0147-6513(23)01090-4. [Epub ahead of print]267 115586

Determination of well-being-related markers in nails by liquid chromatography tandem mass spectrometry.

Alex Gomez-Gomez, Blanca Montero-San Martin, Noemí Haro, Oscar J Pozo.

  Well-being is a multifactorial positive state that is highly influenced by some endogenous molecules that control happiness and euphoric feelings. These molecules, e.g., neurotransmitters, hormones and their derivatives, play a crucial role in metabolism and may be referred to as "well-being-related markers". The deregulation of well-being-related markers can lead to organism malfunctions and life-threatening states. In this research, we aimed to evaluate the potential of nails for the chronic production of several well-being-related markers. For this purpose, we developed an LCMS /MS-based method for the determination of 10 well-being-related markers, including melatonin, serotonin, cortisol, kynurenine and several precursors and metabolites. The method was optimized regarding different analytical steps: required sample amount, extraction time, number of required extractions, preconcentration, injection volume and MS conditions. Method validation was performed by two different approaches: (i) using surrogate nail matrix and (ii) using authentic nail samples by standard additions. The method was found to be linear in the expected endogenous range and sensitive enough to determine the low endogenous concentration levels in nails. Accuracy and precision were appropriate in both validation approaches. As proof of concept, the method was used (i) to correlate fingernail and toenail levels for all metabolites in 22 volunteers, (ii) to establish the endogenous concentration range of all metabolites in females (n = 50) and males (n = 34) and (iii) to correlate the metabolite levels with age. For some metabolites, the calculated ranges have been reported for the first time. In summary, the present strategy to evaluate well-being-related markers in nails may be a useful tool for the evaluation of the production of these important compounds with high potential for a wide range of clinical purposes.

Keywords:  Cortisol; Mass spectrometry; Melatonin; Nails; Sleep-wake cycle

DOI:  https://doi.org/10.1016/j.ecoenv.2023.115586
J Sep Sci. 2023 Oct 30. e2300630

Fast and sensitive LC-MS/MS method for quantification of cannabinoids and their metabolites in plasma of cattle fed hemp.

Xin Xu, Lisa A Murphy.

  Hemp-based materials have gained interest as alternative feed ingredients for livestock. However, safety concerns arise regarding the transfer of cannabinoids from the plant to the animals. Addressing these concerns requires the use of methods capable of detecting and quantifying cannabinoids in livestock. In this study, a fast and sensitive method was developed for quantification of cannabinoids and cannabinoid metabolites in cattle plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The extraction of cannabinoids from the plasma matrix was achieved by combining the Captiva Enhanced Matrix Removal-Lipid clean-up and salting-out assisted liquid-liquid extraction procedure. The developed method underwent validation using various analytical parameters, and the results demonstrated good accuracy, precision, specificity, and high sensitivity. The method was applied to real plasma samples obtained from cattle fed hemp for 2 weeks, and successfully detected various cannabinoids, including delta-9-tetrahydrocannabinol. Furthermore, the study revealed that 7-carboxy cannabidiol, a metabolite of cannabidiol, was the predominant cannabinoid present in the cattle plasma throughout the feeding period, which could remain detectable for weeks after the hemp feeding had ended.

Keywords:  animal feed; cannabinoid; cattle plasma; hemp; liquid chromatography-tandem mass spectrometry

DOI:  https://doi.org/10.1002/jssc.202300630
Anal Sci. 2023 Nov 03.

Development and validation of Empagliflozin and Linagliptin simultaneous estimation in rat plasma using freezing lipid precipitation and SCX-SPE assisted HPLC-MS/MS method and its application in pharmacokinetic studies.

Anil Kumar Goud Thallapalli, Ram Mohan Manda.

  A quick and sensitive liquid chromatography-mass spectrometry technique was designed, improved, and validated for simultaneous determination of Empagliflozin (EPG) and Linagliptin (LNG) using Empagliflozin-d4 (EPG-d4) and linagliptin-d4 (LNG-d4) as internal standards (IS) in rat plasma. Target analytes and the IS were extracted using freezing lipid precipitation (FLP) and optimized using the strong cation exchange solid phase extraction (SCX-SPE) method to achieve the maximum sample clean-up. In particular, when combined with SPE clean-up, FLP can efficiently eliminate the plasma sample's high lipid content. More than 84.14% of plasma lipids were rapidly removed during the FLP procedure, with minimal loss of EPG and LNG. We used LC-atmospheric chemical ionization (APCI)-mass spectrometry was employed to assess the efficiency of FLP in lipid removal. The SCX-SPE cartridges removed the remaining impurities from EPG and LNG, allowing for further purification. The samples were chromatographically separated using a Spherisorb RP/Cyano column by pumping a gradient mobile phase comprised of acetonitrile and 25 mM ammonium acetate buffer (pH 8.1) in positive ion mode at a flow rate of 0.8 mL/min. The selected reaction monitoring technique was performed using a Waters triple-stage quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source. The chromatographic separation was accomplished using a Waters Acquity® high-performance liquid chromatography (HPLC) system. Mass transition (m/z) of 451.15/71.12 for EPG, m/z 473.27/419.94 for LNG; m/z 455.19/71.12 for EPG-d4, and 477.27/423.94 for LNG-d4 was successfully achieved. This study successfully examined the concentration ranges of 25-1050 ng/mL for EPG and 0.35-15 ng/mL for LNG. The results showed that the linearity of EPG ranged from 25.14 to 985.26 ng/mL, while the linearity of LNG ranged from 0.59 to 14.86 ng/mL. The relative standard deviation (RSD) for both EPG and LNG, within and between days, were below 3.83%, indicating that they fall within acceptable limits. This novel approach demonstrated favourable outcomes in a pharmacokinetic study involving healthy rats, where EPG and LNG were co-administered. This study found that the co-administration of both drugs did not have a significant impact on their pharmacokinetic behavior, suggesting the absence of any drug-drug interactions.

Keywords:  Empagliflozin; Freezing lipid precipitation; LC–MS; Linagliptin; MS; SCX-SPE etc.; Validation

DOI:  https://doi.org/10.1007/s44211-023-00444-z
Anal Biochem. 2023 Oct 30. pii: S0003-2697(23)00298-1. [Epub ahead of print]683 115333

Preclinical pharmacokinetic and in vitro metabolic stability study of lysosomotropic autophagy inhibitor, IITZ-01 in mice by using UPLC-MS/MS.

Basveshwar Gawali, Aishwarya Jala, Jagadeesh Kumar Gangasani, Eswara Rao Puppala, Roshan M Borkar, V G M Naidu.

  The present study evaluates the pharmacokinetics and metabolic stability of a novel lysosomotropic autophagy inhibitor, IITZ-01 using an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). It is required as this lead molecule awaits pre-clinical studies for development because of significant therapeutic outcomes in triple-negative breast cancer and renal cancer. A bioanalytical method for the quantitative determination of IITZ-01 in the plasma of mice was developed using the UPLC-MS/MS technique. The UPLC-MS/MS method was validated according to US-FDA bioanalytical guidance and successfully applied to study the pharmacokinetics and metabolic stability. Separation of IITZ- 01 and ZSTK474 (IS) from endogenous components with high selectivity and sensitivity (0.5 ng/mL) was achieved using Waters Acquity BEH C-18 column (50 mm × 2.1 mm, 1.7 μm). A gradient mobile phase consisting of 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile was applied at a flow rate of 0.2 mL/min. Electrospray ionization was employed in positive ion mode for detection, while quantification utilized the multiple reaction monitoring (MRM) mode. This involved using [M+H]+fragment ions at m/z 483.19 → 235.09 for IITZ-01 and m/z 418 → 138 for the internal standard (IS). The method was validated over the calibration range of 0.5-800 ng/mL. The LLOQ of IITZ-01 was 0.5 ng/mL in mice plasma. The method demonstrated good in terms of intra- and inter-day precision and accuracy. The matrix effect was found to be negligible, and the stability data were within acceptable limits. The validated technique supports suitability, reliability, reproducibility, and sensitivity for the pre-clinical investigation of IITZ-01 pharmacokinetics in mice and metabolic stability in human liver microsomes.

Keywords:  Bioanalytical method development and validation; IITZ-01; LC-MS/MS; Lysosomotropic autophagy inhibitor

DOI:  https://doi.org/10.1016/j.ab.2023.115333
Crit Rev Anal Chem. 2023 Nov 01. 1-20

Mass Spectrometry Analysis for Clinical Applications: A Review.

Hong Zhang, Yali Yang, Yanxiao Jiang, Meng Zhang, Zhilong Xu, Xiaofei Wang, Jie Jiang.

  Mass spectrometry (MS) has become an attractive analytical method in clinical analysis due to its comprehensive advantages of high sensitivity, high specificity and high throughput. Separation techniques coupled MS detection (e.g., LC-MS/MS) have shown unique advantages over immunoassay and have developed as golden criterion for many clinical applications. This review summarizes the characteristics and applications of MS, and emphasizes the high efficiency of MS in clinical research. In addition, this review also put forward further prospects for the future of mass spectrometry technology, including the introduction of miniature MS instruments, point-of-care detection and high-throughput analysis, to achieve better development of MS technology in various fields of clinical application. Moreover, as ambient ionization mass spectrometry (AIMS) requires little or no sample pretreatment and improves the flux of MS, this review also summarizes its potential applications in clinic.

Keywords:  Mass spectrometry; ambient ionization mass spectrometry; application; clinical research

DOI:  https://doi.org/10.1080/10408347.2023.2274039
Magn Reson (Gott). 2023 ;4(1): 19-26

DEEP Picker1D and Voigt Fitter1D: a versatile tool set for the automated quantitative spectral deconvolution of complex 1D-NMR spectra.

Da-Wei Li, Lei Bruschweiler-Li, Alexandar L Hansen, Rafael Brüschweiler.

The quantitative deconvolution of 1D-NMR spectra into individual resonances or peaks is a key step in many modern NMR workflows as it critically affects downstream analysis and interpretation. Depending on the complexity of the NMR spectrum, spectral deconvolution can be a notable challenge. Based on the recent deep neural network DEEP Picker and Voigt Fitter for 2D NMR spectral deconvolution, we present here an accurate, fully automated solution for 1D-NMR spectral analysis, including peak picking, fitting, and reconstruction. The method is demonstrated for complex 1D solution NMR spectra showing excellent performance also for spectral regions with multiple strong overlaps and a large dynamic range whose analysis is challenging for current computational methods. The new tool will help streamline 1D-NMR spectral analysis for a wide range of applications and expand their reach toward ever more complex molecular systems and their mixtures.

DOI: https://doi.org/10.5194/mr-4-19-2023
J Breath Res. 2023 Nov 02.

A benchmark study of data normalisation methods for PTR-TOF-MS exhaled breath metabolomics.

Camille Roquencourt, Elodie Lamy, Emmanuelle Bardin, Philippe Devillier, Stanislas Grassin-Delyle.

   BACKGROUND: Volatilomics is the branch of metabolomics dedicated to the analysis of volatile organic compounds (VOCs) in exhaled breath for medical diagnostic or therapeutic monitoring purposes. Real-time mass spectrometry technologies such as proton transfer reaction mass spectrometry (PTR-MS) are commonly used, and data normalisation is an important step to discard unwanted variation from non-biological sources, as batch effects and loss of sensitivity over time may be observed. As normalisation methods for real-time breath analysis have been poorly investigated, we aimed to benchmark known metabolomic data normalisation methods and apply them to PTR-MS data analysis.
METHODS: We compared seven normalisation methods, five statistically based and two using multiple standard metabolites, on two datasets from clinical trials for COVID-19 diagnosis in patients from the emergency department or intensive care unit. We evaluated different means of feature selection to select the standard metabolites, as well as the use of multiple repeat measurements of ambient air to train the normalisation methods.
RESULTS: We show that the normalisation tools can correct for time-dependent drift. The methods that provided the best corrections for both cohorts were Probabilistic Quotient Normalisation and Normalisation using Optimal Selection of Multiple Internal Standards. Normalisation also improved the diagnostic performance of the machine learning models, significantly increasing sensitivity, specificity and area under the ROC curve for the diagnosis of COVID-19.
CONCLUSIONS: Our results highlight the importance of adding an appropriate normalisation step during the processing of PTR-MS data, which allows significant improvements in the predictive performance of statistical models.Clinical trials: VOC-COVID-Diag (EudraCT 2020-A02682-37); RECORDS trial (EudraCT 2020-000296-21)Keywords: Data normalisation, PTR-TOF-MS, machine learning, exhaled breath&#xD.

Keywords:  PTR-TOF-MS; data normalisation; exhaled breath; machine learning

DOI:  https://doi.org/10.1088/1752-7163/ad08ce
J Sep Sci. 2023 Oct 28. e2300571

Matrix effects demystified: Strategies for resolving challenges in analytical separations of complex samples.

Madison L Williams, Aghogho Abigail Olomukoro, Ronald V Emmons, Nipunika H Goodage, Emanuela Gionfriddo.

  Matrix effects can significantly impede the accuracy, sensitivity, and reliability of separation techniques presenting a formidable challenge to the analytical process. It is crucial to address matrix effects to achieve accurate and precise measurements in complex matrices. The multifaceted nature of matrix effects which can be influenced by factors such as target analyte, sample preparation protocol, composition, and choice of instrument necessitates a pragmatic approach when analyzing complex matrices. This review aims to highlight common challenges associated with matrix effects throughout the entire analytical process with emphasis on gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and sample preparation techniques. These techniques are susceptible to matrix effects that could lead to ion suppression/enhancement or impact the analyte signal at various stages of the analytical workflow. The assessment, quantification, and mitigation of matrix effects are necessary in developing any analytical method. Strategies can be implemented to reduce or eliminate the matrix effect by changing the type of ionization, improving extraction and clean-up methods, optimization of chromatography conditions, and corrective calibration methods. While development of an effective strategy to completely mitigate matrix effects remains elusive, an integrated approach that combines sample preparation, analytical extraction, and effective instrumental analysis remains the most promising avenue for identifying and resolving matrix effects.

Keywords:  analytical method development; biological analysis; complex samples; matrix effects; sample preparation

DOI:  https://doi.org/10.1002/jssc.202300571
Biomed Chromatogr. 2023 Nov 02. e5766

Impact assessment of metabolite instability in the development and validation of LC-MS/MS bioanalytical assays for measurement of rosuvastatin in human plasma and urine samples.

Yuxiang Cui, Kevin Kowalski, Michael Van Parys, Dennis Miller, Patricia Hansen, Xiaorong Liang, Brian Dean, Liuxi Chen.

  During bioanalytical assay development and validation, maintaining the stability of the parent drug and metabolites of interest is critical. While stability of the parent drug has been thoroughly investigated, the stability of unanalyzed metabolites is often overlooked. When an unstable metabolite is known or suspected to interfere with measurement of the parent drug or other metabolites of interest through back-conversion or other routes, additional tests with these unstable metabolites should be conducted. Here, the development and validation of two assays for quantification of rosuvastatin, one in human plasma and one in human urine, was reported. To this end, additional sets of quality control samples were added during assay validation to ensure the reliability of the assays. Acid treatment of samples is shown to be necessary for rosuvastatin quantification. In this regard, stability issues caused by the metabolite, rosuvastatin lactone, may have been overlooked if assay development and validation had only considered the parent drug, rosuvastatin. These assays represent a case study for how to develop and validate assays with unstable metabolites. Taken together, unstable metabolites should be included in all applicable stability tests.

Keywords:  bioanalytical assay validation; liquid chromatography-tandem mass spectrometry; rosuvastatin; rosuvastatin lactone; unstable drug metabolite

DOI:  https://doi.org/10.1002/bmc.5766
Front Toxicol. 2023 ;5 1216802

Using in silico tools to predict flame retardant metabolites for more informative exposomics-based approaches.

Breanne Kincaid, Przemyslaw Piechota, Emily Golden, Mikhail Maertens, Thomas Hartung, Alexandra Maertens.

  Introduction: The positive identification of xenobiotics and their metabolites in human biosamples is an integral aspect of exposomics research, yet challenges in compound annotation and identification continue to limit the feasibility of comprehensive identification of total chemical exposure. Nonetheless, the adoption of in silico tools such as metabolite prediction software, QSAR-ready structural conversion workflows, and molecular standards databases can aid in identifying novel compounds in untargeted mass spectral investigations, permitting the assessment of a more expansive pool of compounds for human health hazard. This strategy is particularly applicable when it comes to flame retardant chemicals. The population is ubiquitously exposed to flame retardants, and evidence implicates some of these compounds as developmental neurotoxicants, endocrine disruptors, reproductive toxicants, immunotoxicants, and carcinogens. However, many flame retardants are poorly characterized, have not been linked to a definitive mode of toxic action, and are known to share metabolic breakdown products which may themselves harbor toxicity. As U.S. regulatory bodies begin to pursue a subclass- based risk assessment of organohalogen flame retardants, little consideration has been paid to the role of potentially toxic metabolites, or to expanding the identification of parent flame retardants and their metabolic breakdown products in human biosamples to better inform the human health hazards imposed by these compounds. Methods: The purpose of this study is to utilize publicly available in silico tools to 1) characterize the structural and metabolic fates of proposed flame retardant classes, 2) predict first pass metabolites, 3) ascertain whether metabolic products segregate among parent flame retardant classification patterns, and 4) assess the existing coverage in of these compounds in mass spectral database. Results: We found that flame retardant classes as currently defined by the National Academies of Science, Engineering and Medicine (NASEM) are structurally diverse, with highly variable predicted pharmacokinetic properties and metabolic fates among member compounds. The vast majority of flame retardants (96%) and their predicted metabolites (99%) are not present in spectral databases, posing a challenge for identifying these compounds in human biosamples. However, we also demonstrate the utility of publicly available in silico methods in generating a fit for purpose synthetic spectral library for flame retardants and their metabolites that have yet to be identified in human biosamples. Discussion: In conclusion, exposomics studies making use of fit-for-purpose synthetic spectral databases will better resolve internal exposure and windows of vulnerability associated with complex exposures to flame retardant chemicals and perturbed neurodevelopmental, reproductive, and other associated apical human health impacts.

Keywords:  chemical classification; exposomics; flame retardant (FR); hazard assessment; in silico spectra; metabolite prediction; metabolomics; risk assessment

DOI:  https://doi.org/10.3389/ftox.2023.1216802