Sarah Aijaz (Presenter)
Cerilliant Corporation
Authorship: Sarah M. Aijaz, Isil Dilek, and Uma Sreenivasan
Cerilliant Corporation
| Short Abstract LC/MS/MS assays of stable isotope labeled compounds and their native counterparts were compared to investigate relative ionization responses. Native, deuterated, and 13C analogs of Testosterone, Pregabalin, and L-Thyroxine were chosen as representative small molecules. Ionization responses were not equivalent between native and labeled compounds analyzed under identical conditions and concentrations. The magnitude of ionization response differences is affected by experimental parameters such as collision energy. Assays performed at multiple collision energies demonstrated the relative ionization responses can be varied by greater than 20%. These ionization response differences must be evaluated, understood, and optimized when considering the use of stable labeled standards for quantitation. |
Long Abstract
INTRODUCTION:
Stable isotope labeled (SIL) compounds such as deuterium and 13C analogs are widely used as internal standards for clinical LC/MS/MS analyses. Internal standards are used to improve the accuracy of analyte quantitation by correcting for matrix effects and analytical variability. The suitability of an internal standard is dependent upon how closely it behaves to the analyte of interest throughout sample preparation and LC/MS/MS analysis. This is a strength of SIL compounds over structural analogs, especially 13C SIL compounds which co-elute with their native counterparts under UHPLC methods. When used as an internal standard, it is more critical for the relative response of an SIL compound to mimic the analyte rather than absolute response. SIL compounds may also be used as calibrators in assays of native analytes. In this case, the absolute response comparability between the native and labeled compounds is critical for accurate concentration assignment. Comprehensive studies comparing ionization responses between SIL and native compounds are needed to fully understand how the use of SIL compounds in roles other than those of internal standards may affect the accuracy of LC/MS/MS assays.
METHODS:
LC/MS/MS assays were performed on a Shimadzu Nexera X2 UHPLC coupled to an AB Sciex 5500 QTRAP system. UHPLC methods were optimized for each compound. All methods used a Supelco Ascentis Express C18, 2.7 micron, 3.0 x 50 mm column and 0.1% Formic Acid in Water and 0.1% Formic Acid in Acetonitrile as mobile phases. The LC/MS parameters of Declustering Potential, Entrance Potential, Collision Cell Exit Potential, and Collision Energy were optimized for all analyzed compounds. The UHPLC method runtimes were minimized to allow for omission of internal standards. All assays used six replicate injections of each analyte and bracketing injections were used as a system suitability measure.
PRELIMINARY RESULTS:
Native, deuterated, and 13C analogs of Testosterone, Pregabalin, and L-Thyroxine were chosen as representative small molecules. The stable isotope labeled compounds and their native counterparts were compared by LC/MS/MS assay. Ionization responses were not equivalent between native and labeled compounds analyzed under identical conditions and concentrations. The magnitude of ionization response differences is affected by experimental parameters such as collision energy. Assays performed at multiple collision energies demonstrated the relative ionization responses can be varied by greater than 20%. These ionization response differences must be evaluated, understood, and optimized when considering the use of stable labeled standards for quantitation.
References & Acknowledgements:
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| Salary | no | |
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IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |