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Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry

Using manual derivatization in gas chromatography-mass spectrometry samples have varying equilibration times before analysis which increases technical variability and limits the number of potential samples analyzed. By contrast, automated derivatization methods can derivatize and inject each sample...

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Autores principales: Fritsche-Guenther, Raphaela, Gloaguen, Yoann, Bauer, Anna, Opialla, Tobias, Kempa, Stefan, Fleming, Christina A., Redmond, Henry Paul, Kirwan, Jennifer A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8703763/
https://www.ncbi.nlm.nih.gov/pubmed/34940646
http://dx.doi.org/10.3390/metabo11120888
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author Fritsche-Guenther, Raphaela
Gloaguen, Yoann
Bauer, Anna
Opialla, Tobias
Kempa, Stefan
Fleming, Christina A.
Redmond, Henry Paul
Kirwan, Jennifer A.
author_facet Fritsche-Guenther, Raphaela
Gloaguen, Yoann
Bauer, Anna
Opialla, Tobias
Kempa, Stefan
Fleming, Christina A.
Redmond, Henry Paul
Kirwan, Jennifer A.
author_sort Fritsche-Guenther, Raphaela
collection PubMed
description Using manual derivatization in gas chromatography-mass spectrometry samples have varying equilibration times before analysis which increases technical variability and limits the number of potential samples analyzed. By contrast, automated derivatization methods can derivatize and inject each sample in an identical manner. We present a fully automated (on-line) derivatization method used for targeted analysis of different matrices. We describe method optimization and compare results from using off-line and on-line derivatization protocols, including the robustness and reproducibility of the methods. Our final parameters for the derivatization process were 20 µL of methoxyamine (MeOx) in pyridine for 60 min at 30 °C followed by 80 µL N-Methyl-N-trimethylsilyltrifluoracetamide (MSTFA) for 30 min at 30 °C combined with 4 h of equilibration time. The repeatability test in plasma and liver revealed a median relative standard deviation (RSD) of 16% and 10%, respectively. Serum samples showed a consistent intra-batch median RSD of 20% with an inter-batch variability of 27% across three batches. The direct comparison of on-line versus off-line demonstrated that on-line was fit for purpose and improves repeatability with a measured median RSD of 11% compared to 17% using the same method off-line. In summary, we recommend that optimized on-line methods may improve results for metabolomics and should be used where available.
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spelling pubmed-87037632021-12-25 Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry Fritsche-Guenther, Raphaela Gloaguen, Yoann Bauer, Anna Opialla, Tobias Kempa, Stefan Fleming, Christina A. Redmond, Henry Paul Kirwan, Jennifer A. Metabolites Article Using manual derivatization in gas chromatography-mass spectrometry samples have varying equilibration times before analysis which increases technical variability and limits the number of potential samples analyzed. By contrast, automated derivatization methods can derivatize and inject each sample in an identical manner. We present a fully automated (on-line) derivatization method used for targeted analysis of different matrices. We describe method optimization and compare results from using off-line and on-line derivatization protocols, including the robustness and reproducibility of the methods. Our final parameters for the derivatization process were 20 µL of methoxyamine (MeOx) in pyridine for 60 min at 30 °C followed by 80 µL N-Methyl-N-trimethylsilyltrifluoracetamide (MSTFA) for 30 min at 30 °C combined with 4 h of equilibration time. The repeatability test in plasma and liver revealed a median relative standard deviation (RSD) of 16% and 10%, respectively. Serum samples showed a consistent intra-batch median RSD of 20% with an inter-batch variability of 27% across three batches. The direct comparison of on-line versus off-line demonstrated that on-line was fit for purpose and improves repeatability with a measured median RSD of 11% compared to 17% using the same method off-line. In summary, we recommend that optimized on-line methods may improve results for metabolomics and should be used where available. MDPI 2021-12-18 /pmc/articles/PMC8703763/ /pubmed/34940646 http://dx.doi.org/10.3390/metabo11120888 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Fritsche-Guenther, Raphaela
Gloaguen, Yoann
Bauer, Anna
Opialla, Tobias
Kempa, Stefan
Fleming, Christina A.
Redmond, Henry Paul
Kirwan, Jennifer A.
Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry
title Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry
title_full Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry
title_fullStr Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry
title_full_unstemmed Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry
title_short Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry
title_sort optimized workflow for on-line derivatization for targeted metabolomics approach by gas chromatography-mass spectrometry
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8703763/
https://www.ncbi.nlm.nih.gov/pubmed/34940646
http://dx.doi.org/10.3390/metabo11120888
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