Automated Sequential Analysis of Hydrophilic and Lipophilic Fractions of Biological Samples: Increasing Single-Injection Chemical Coverage in Untargeted Metabolomics
In order to increase metabolite coverage in LC–MS-based untargeted metabolomics, HILIC- and RPLC-mode separations are often combined. Unfortunately, these two techniques pose opposite requirements on sample composition, necessitating either dual sample preparations, increasing needed sample volume,...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-05-01
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| Series: | Metabolites |
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| Online Access: | https://www.mdpi.com/2218-1989/11/5/295 |
| _version_ | 1797535247993143296 |
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| author | Kristian Pirttilä Göran Laurell Curt Pettersson Mikael Hedeland |
| author_facet | Kristian Pirttilä Göran Laurell Curt Pettersson Mikael Hedeland |
| author_sort | Kristian Pirttilä |
| collection | DOAJ |
| description | In order to increase metabolite coverage in LC–MS-based untargeted metabolomics, HILIC- and RPLC-mode separations are often combined. Unfortunately, these two techniques pose opposite requirements on sample composition, necessitating either dual sample preparations, increasing needed sample volume, or manipulation of the samples after the first analysis, potentially leading to loss of analytes. When sample material is precious, the number of analyses that can be performed is limited. To that end, an automated single-injection LC–MS method for sequential analysis of both the hydrophilic and lipophilic fractions of biological samples is described. Early eluting compounds in a HILIC separation are collected on a trap column and subsequently analyzed in the RPLC mode. The instrument configuration, composed of commercially available components, allows easy modulation of the dilution ratio of the collected effluent, with sufficient dilution to obtain peak compression in the RPLC column. Furthermore, the method is validated and shown to be fit for purpose for application in untargeted metabolomics. Repeatability in both retention times and peak areas was excellent across over 140 injections of protein-precipitated blood plasma. Finally, the method has been applied to the analysis of real perilymph samples collected in a guinea pig model. The QC sample injections clustered tightly in the PCA scores plot and showed a high repeatability in both retention times and peak areas for selected compounds. |
| first_indexed | 2024-03-10T11:41:49Z |
| format | Article |
| id | doaj.art-dbce8175dce948a38afd1cbef1d9231c |
| institution | Directory Open Access Journal |
| issn | 2218-1989 |
| language | English |
| last_indexed | 2024-03-10T11:41:49Z |
| publishDate | 2021-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Metabolites |
| spelling | doaj.art-dbce8175dce948a38afd1cbef1d9231c2023-11-21T18:26:48ZengMDPI AGMetabolites2218-19892021-05-0111529510.3390/metabo11050295Automated Sequential Analysis of Hydrophilic and Lipophilic Fractions of Biological Samples: Increasing Single-Injection Chemical Coverage in Untargeted MetabolomicsKristian Pirttilä0Göran Laurell1Curt Pettersson2Mikael Hedeland3Department of Medicinal Chemistry, Uppsala University, SE-75123 Uppsala, SwedenDepartment of Surgical Science, Uppsala University, SE-75185 Uppsala, SwedenDepartment of Medicinal Chemistry, Uppsala University, SE-75123 Uppsala, SwedenDepartment of Medicinal Chemistry, Uppsala University, SE-75123 Uppsala, SwedenIn order to increase metabolite coverage in LC–MS-based untargeted metabolomics, HILIC- and RPLC-mode separations are often combined. Unfortunately, these two techniques pose opposite requirements on sample composition, necessitating either dual sample preparations, increasing needed sample volume, or manipulation of the samples after the first analysis, potentially leading to loss of analytes. When sample material is precious, the number of analyses that can be performed is limited. To that end, an automated single-injection LC–MS method for sequential analysis of both the hydrophilic and lipophilic fractions of biological samples is described. Early eluting compounds in a HILIC separation are collected on a trap column and subsequently analyzed in the RPLC mode. The instrument configuration, composed of commercially available components, allows easy modulation of the dilution ratio of the collected effluent, with sufficient dilution to obtain peak compression in the RPLC column. Furthermore, the method is validated and shown to be fit for purpose for application in untargeted metabolomics. Repeatability in both retention times and peak areas was excellent across over 140 injections of protein-precipitated blood plasma. Finally, the method has been applied to the analysis of real perilymph samples collected in a guinea pig model. The QC sample injections clustered tightly in the PCA scores plot and showed a high repeatability in both retention times and peak areas for selected compounds.https://www.mdpi.com/2218-1989/11/5/295LC–MSsequential columnsautomatedchemical coverageuntargeted metabolomics |
| spellingShingle | Kristian Pirttilä Göran Laurell Curt Pettersson Mikael Hedeland Automated Sequential Analysis of Hydrophilic and Lipophilic Fractions of Biological Samples: Increasing Single-Injection Chemical Coverage in Untargeted Metabolomics Metabolites LC–MS sequential columns automated chemical coverage untargeted metabolomics |
| title | Automated Sequential Analysis of Hydrophilic and Lipophilic Fractions of Biological Samples: Increasing Single-Injection Chemical Coverage in Untargeted Metabolomics |
| title_full | Automated Sequential Analysis of Hydrophilic and Lipophilic Fractions of Biological Samples: Increasing Single-Injection Chemical Coverage in Untargeted Metabolomics |
| title_fullStr | Automated Sequential Analysis of Hydrophilic and Lipophilic Fractions of Biological Samples: Increasing Single-Injection Chemical Coverage in Untargeted Metabolomics |
| title_full_unstemmed | Automated Sequential Analysis of Hydrophilic and Lipophilic Fractions of Biological Samples: Increasing Single-Injection Chemical Coverage in Untargeted Metabolomics |
| title_short | Automated Sequential Analysis of Hydrophilic and Lipophilic Fractions of Biological Samples: Increasing Single-Injection Chemical Coverage in Untargeted Metabolomics |
| title_sort | automated sequential analysis of hydrophilic and lipophilic fractions of biological samples increasing single injection chemical coverage in untargeted metabolomics |
| topic | LC–MS sequential columns automated chemical coverage untargeted metabolomics |
| url | https://www.mdpi.com/2218-1989/11/5/295 |
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