Publicação
Identification of metabolites by liquid chromatography quadrupole time-of-flight mass spectrometric technique
| Resumo: | Analytical methods based on mass spectrometry and coupled to a chromatographic separation technique such as HPLC or GC, are crucial for investigating human metabolome, UPLC coupled with ESI-MS is a recent methodology representing an effective tool to separate and analyze molecules. The mains focus of this experimental work was to elucidate the influence of HILIC and Reversed-Phase chromatography on the analysis of compounds through ESI-MS. One additional aim was also to collect the identification data of selected compounds on a database for the building of a metabolomics library. This tool would allow to establish a match with results of subsequent analyses, minimizing the time spent on the identification of future analytes. Herein a UHPLC–MS method was applied for the first time to detect and identify a series of human metabolites and compounds found naturally on the human body. The procedures and molecules subjected to analysis are based on the protocol “Mass Spectrometry Metabolite Library of Standards”, commercially available by IROA Technologies. The kit contains a set of 619 standards. This work covers the first 253 compounds of the list. The analysis of compounds previously prepared as mixtures consisted on the chromatographic separation by HILIC or RP followed by positive or negative ESI-MS/MS. All the compounds were analyzed by the four possible combinations (HILIC, positive ESI; HILIC, negative ESI; RP, positive ESI; RP, negative ESI) as mixes of 12 standards distributed by four vials, one for each type of analysis. The results obtained were compiled and it was possible to conclude that HILIC is suitable to polar analytes whereas RP is preferable to non-polar compounds. Relatively to ESI, the positive ionization mode is applied with advantage to compounds that are able to accept a proton. Conversely, negative ESI is more effective to molecules which contain functional groups that readily lose a proton. Zwitterionic molecules are suitable to be analyzed by both modes. Some examples and exceptions to these general statements are presented. |
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| Autores principais: | Santos, David Jorge Pires dos |
| Assunto: | Hydrophilic interaction liquid chromatography Electrospray ionization Mass spectrometry Reversed phase Library Metabolites Mestrado Integrado - 2016 |
| Ano: | 2016 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Analytical methods based on mass spectrometry and coupled to a chromatographic separation technique such as HPLC or GC, are crucial for investigating human metabolome, UPLC coupled with ESI-MS is a recent methodology representing an effective tool to separate and analyze molecules. The mains focus of this experimental work was to elucidate the influence of HILIC and Reversed-Phase chromatography on the analysis of compounds through ESI-MS. One additional aim was also to collect the identification data of selected compounds on a database for the building of a metabolomics library. This tool would allow to establish a match with results of subsequent analyses, minimizing the time spent on the identification of future analytes. Herein a UHPLC–MS method was applied for the first time to detect and identify a series of human metabolites and compounds found naturally on the human body. The procedures and molecules subjected to analysis are based on the protocol “Mass Spectrometry Metabolite Library of Standards”, commercially available by IROA Technologies. The kit contains a set of 619 standards. This work covers the first 253 compounds of the list. The analysis of compounds previously prepared as mixtures consisted on the chromatographic separation by HILIC or RP followed by positive or negative ESI-MS/MS. All the compounds were analyzed by the four possible combinations (HILIC, positive ESI; HILIC, negative ESI; RP, positive ESI; RP, negative ESI) as mixes of 12 standards distributed by four vials, one for each type of analysis. The results obtained were compiled and it was possible to conclude that HILIC is suitable to polar analytes whereas RP is preferable to non-polar compounds. Relatively to ESI, the positive ionization mode is applied with advantage to compounds that are able to accept a proton. Conversely, negative ESI is more effective to molecules which contain functional groups that readily lose a proton. Zwitterionic molecules are suitable to be analyzed by both modes. Some examples and exceptions to these general statements are presented. |
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