Joseph Takarewski (Presenter)
Thermo Fisher Scientific
Authorship: Joseph Takarewski
Thermo Fisher Scientific; Franklin, MA
| Short Abstract In an online SPE-LC-MS/MS method for the analysis of immunosuppressant drugs in whole blood, we observed differences in response between injections of standards prepared in mobile phase and standards prepared in matrix. We configured the system and method to permit concomitant injection within the same method cycle to both first and second dimension columns. We injected standard solutions to the second dimension while injecting either mobile phase or matrix blanks to the first dimension. This approach has allowed us to directly assess the influence of residual matrix on signal. Having indirectly established that the signal differences were related to matrix and not to recovery in the first dimension of the separation, we evaluated changes to the analytical gradient to correct the problem. |
Long Abstract
1. Problem
We observed differences in response between injections of standards prepared in mobile phase and standards prepared in matrix. It has become common practice to evaluate matrix effects during the development of methods for the analysis of small molecules from biological fluids. In a canonical HPLC-MS hardware setting, one could compare (a) standard in mobile phase, (b) pre-extracted blank matrix spiked with standard and (c) a matrix sample spiked prior to sample preparation to establish whether observed differences during sample analysis are attributable to ion suppression alone. Where some LC-MS/MS methods combine on-line solid phase extraction with an HPLC separation, distinguishing ion suppression from incomplete recovery in the first dimension separation is more problematic. In these integrated configurations, matrix extract is not isolated after online SPE in advance of the HPLC separation. An instrument method based approach is needed to emulate the spiking of a pre-extracted blank.
2. Method Information
* Immunosuppressant drugs in human blood
* Whole blood or extracts of blood as samples
* Samples directly injected into a two-dimensional LC system
* Thermo Scientific Transcend II LC system
* Thermo Scientific TSQ Quantum Ultra mass spectrometer
* Sample extraction on a Thermo Scientific 0.5 x 50 mm Cyclone-P column
* HPLC separation on a Thermo Scientific 3 x 30 mm Accucore C8 column
3. Troubleshooting Steps
We configured the system and method to permit concomitant injection within the same method cycle to both first and second dimension columns. We injected standard solutions to the second dimension while injecting either mobile phase or matrix blanks to the first dimension. This approach has allowed us to directly assess the influence of residual matrix on signal.
4. Outcome
Having indirectly established that signal differences were related to matrix and not to recovery in the first dimension of the separation, we evaluated changes to the analytical gradient to correct the problem.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Thermo Fisher Scientific |
| Board Member | no | |
| Stock | yes | Thermo Fisher Scientific |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |