Collision-energy breakdown curves – an additional tool to characterize MS/MS methods
Sophie Mörlein (1), Michael Paal (2), Michael Vogeser (3) Institute of Laboratory Medicine, University Hospital, LMU Munich
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Sophie Mörlein (Presenter) University Hospital, LMU Munich
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
INTRODUCTION: A fundamental assumption in quantitative mass spectrometry is that the internal standard compound behaves identical on all stages of the analytic process chain (ionization-fragmentation-detection) in relation to the target compound and is so used to compensate any inconsistency during sample preparation and analysis. Differential impact of matrix effects on these processes can potentially affect accuracy and sensitivity. Consequently, it can be of significance to investigate the fragmentation characteristics of compounds comparatively (analyte vs. internal standard compound) and under various matrix conditions (reflecting different approaches to sample preparation) in addition to the usual investigated ion source phenomena. The evaluation of such experiments can be performed by breakdown curves, which can be described as a function of the collision energy.
OBJECTIVES: The objective of this approach was to study the ionization yield for various collision energy settings using two immunosuppressant compounds and visualizing the results as breakdown curves in plots.
METHODS: To study this approach, we investigated the immunosuppresants cyclosporine A and tacrolimus and the corresponding internal standards, 4-fold deuterated cyclosporine A and ascomycin as exemplary analytes, both on the background of biological sample matrix and in matrix free solutions for comparison. For this aim, a routine quantitative method used in the therapeutic drug monitoring, including protein precipitation followed by online solid phase extraction for these two immunosuppressants, was modified. Analyses were performed for two different concentrations and in duplicate and a mean of responses was calculated to generate the breakdown curve plots.
RESULTS: We found consistent breakdown curves for all four compounds studied, whereby the curves showed different patterns that were independent from the concentration. Slight differences were observed for the compounds in biological matrix-based samples compared to matrix free solutions, indicating potential matrix effects on fragmentation. Additionally, stable isotope-labelled 4-fold deuterated cyclosporine A showed an identical breakdown curve to native cyclosporine A, while ascomycin, used as an analogous molecule for internal standardization, showed a shifted breakdown curve of 5eV compared to tacrolimus.
CONCLUSION: We conclude that collision-energy breakdown curves may be an interesting additional tool to characterize MS/MS methods with respect to matrix effects and similarity of target analyte and corresponding internal standard compound.
= Discovery stage.
= Translation stage.
= Clinically available.
