The analysis of isobaric molecules by tandem mass spectrometry, especially if they are isomers, is often complicated by the similarity between their fragmentation patterns. In fact, it is common that the same MS/MS product ions are present in the spectra of all isomers. In this case, an adequate chromatographic separation between compounds should be developed in order to eliminate mutual interferences. However, the optimization of chromatographic separation of the isomers mixtures requires a lot of time and often involves the use of different instrument set up (other columns, solvents, etc..). On the other hand, several tandem mass spectrometric techniques were proposed to overcome these issues and allowing the isomers distinction also in the case of chromatographically unresolved peaks. Unfortunately, none of these MS/MS methods were useful to solve the isomers pairs in the proposed experimental conditions. In our work, a series of drug plasma stability experiments were carried out to evaluate the bioavailability of several isomers and, despite the attempts for optimizing the chromatographic separation, an unique LC-MS/MS method suitable to separate all compounds was not achieved. Therefore, to ensure the specificity of the LC-MS/MS methods, a different approach based on a series of energy resolved MS/MS experiments were carried out. By this approach a clear differentiation among each isomer was obtained but, in order to emphasize such differences, it was necessary to develop a mathematical algorithm that resolved the MS/MS spectra of the components of mixture. This algorithm consists in the application of a matrix of linear regression equations to different experimental data. In our case, the experimental data were the abundance ratios of product and precursor ions selected during MS/MS method set-up. In this way, it was possible to resolve the MS/MS spectra assigning the correct signal to the isomer also for chromatographically unresolved peaks. Considering the pharmaceutical interest on the compounds under investigation, the LC-MS/MS method developed was tested to be effective at the pharmacological active concentration levels of studied compounds, corresponding to a range between nM to M (i.e. ng mL-1 on processed sample). The performances of the proposed algorithm (LEDA) was checked proving its effectiveness both in the correct assignment of the isomer in the processed sample and the relative composition of isomers in mixtures. Therefore, because the isomers do not need to be resolved by LC, this approach has the other advantage that faster LC separation conditions can be used to quantify the analytes than are typically required.

Energy Resolved Tandem Mass Spectrometry experiments to resolve coeluting isomers / Menicatti Marta; Bartolucci Gian Luca. - STAMPA. - (2018), pp. 52-52. (Intervento presentato al convegno 36th Informal Meeting on Mass Spectrometry tenutosi a Koszeg (Hungary) nel 6-9 May 2018).

Energy Resolved Tandem Mass Spectrometry experiments to resolve coeluting isomers

Menicatti Marta;Bartolucci Gian Luca
2018

Abstract

The analysis of isobaric molecules by tandem mass spectrometry, especially if they are isomers, is often complicated by the similarity between their fragmentation patterns. In fact, it is common that the same MS/MS product ions are present in the spectra of all isomers. In this case, an adequate chromatographic separation between compounds should be developed in order to eliminate mutual interferences. However, the optimization of chromatographic separation of the isomers mixtures requires a lot of time and often involves the use of different instrument set up (other columns, solvents, etc..). On the other hand, several tandem mass spectrometric techniques were proposed to overcome these issues and allowing the isomers distinction also in the case of chromatographically unresolved peaks. Unfortunately, none of these MS/MS methods were useful to solve the isomers pairs in the proposed experimental conditions. In our work, a series of drug plasma stability experiments were carried out to evaluate the bioavailability of several isomers and, despite the attempts for optimizing the chromatographic separation, an unique LC-MS/MS method suitable to separate all compounds was not achieved. Therefore, to ensure the specificity of the LC-MS/MS methods, a different approach based on a series of energy resolved MS/MS experiments were carried out. By this approach a clear differentiation among each isomer was obtained but, in order to emphasize such differences, it was necessary to develop a mathematical algorithm that resolved the MS/MS spectra of the components of mixture. This algorithm consists in the application of a matrix of linear regression equations to different experimental data. In our case, the experimental data were the abundance ratios of product and precursor ions selected during MS/MS method set-up. In this way, it was possible to resolve the MS/MS spectra assigning the correct signal to the isomer also for chromatographically unresolved peaks. Considering the pharmaceutical interest on the compounds under investigation, the LC-MS/MS method developed was tested to be effective at the pharmacological active concentration levels of studied compounds, corresponding to a range between nM to M (i.e. ng mL-1 on processed sample). The performances of the proposed algorithm (LEDA) was checked proving its effectiveness both in the correct assignment of the isomer in the processed sample and the relative composition of isomers in mixtures. Therefore, because the isomers do not need to be resolved by LC, this approach has the other advantage that faster LC separation conditions can be used to quantify the analytes than are typically required.
2018
36th Informal Meeting on Mass Spectrometry
36th Informal Meeting on Mass Spectrometry
Koszeg (Hungary)
Menicatti Marta; Bartolucci Gian Luca
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/1153259
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