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Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry
| Content Provider | MDPI |
|---|---|
| Author | Fritsche-Guenther, Raphaela Gloaguen, Yoann Bauer, Anna Opialla, Tobias Kempa, Stefan Fleming, Christina A. Redmond, Henry Paul Kirwan, Jennifer A. |
| Copyright Year | 2021 |
| 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. |
| Starting Page | 888 |
| e-ISSN | 22181989 |
| DOI | 10.3390/metabo11120888 |
| Journal | Metabolites |
| Issue Number | 12 |
| Volume Number | 11 |
| Language | English |
| Publisher | MDPI |
| Publisher Date | 2021-12-18 |
| Access Restriction | Open |
| Subject Keyword | Metabolites Biochemical Research Automated Derivatization On-line Derivatization Optimization Gas-chromatography Mass Spectrometry Validation Metabolomics Quality Assurance (qa) Quality Control (qc) |
| Content Type | Text |
| Resource Type | Article |
