Erik Ruijters (Presenter)
MagnaMedics Diagnostics BV
Authorship: Erik Ruijters, Leon de Bruin
MagnaMedics Diagnostics BV, Geleen, The Netherlands
| Short Abstract MagnaMedics developed the MagSiMUS sample preparation method for LC-MS/MS analysis. Proteins are precipitated on a magnetic silica based solid support and removed from the sample by magnetic separation. The levels of immunosuppressants, benzodiazepines, vitamin D, and steroids could be accurately determined in clinical samples using this process. MagSiMUS is a reliable sample preparation methods which can be applied flexibly in a manual or medium to high throughput automated work-flow. The absence of a centrifugation step improves the total sample throughput and reduces the preparation time. |
Long Abstract
INTRODUCTION
Next to medication that require Therapeutic Drug Monitoring (TDM), e.g. immunosuppressants and benzodiazepines, endogenous biomolecules like vitamins and steroids are also routinely measured by various assays, including LC-MS/MS. The advantage of LC-MS/MS is the simultaneous measurement of multiple analytes and the high sensitivity. This also makes it very susceptible for impurities which cause matrix effects, emphasizing the need for a good sample preparation process. Generally, sample preparation consists of a protein precipitation with either organic solutions or water solutions containing acid, base or salts and the protein precipitate is removed by centrifugation. MagnaMedics developed the MagSiMUS sample preparation process based on magnetic beads. Proteins are precipitated on a magnetic silica based solid support and removed from the sample by magnetic separation. This makes the use of a centrifuge obsolete and opens up high throughput automation possibilities and fast preparation of clinical samples.
METHODS
Samples, either whole blood or serum, were prepared on a modified Bioanalytical STARlet (Hamilton). The automated process reduces hands-on time, is fully flexible (i.e. multiple analytes and sample types can be processed in one batch) and includes sample tracking. From a standard collection tube, 25 µl whole blood or 50 µl serum was transferred to a tube or microtiter plate. For whole blood, 60 µl lysis buffer was added. Next, 20 µl deuterated internal standard and 40 µl MagSiMUS particle mix was added. Proteins were precipitated by the addition of precipitation reagent and mixing. After magnetic separation of the precipitate, 5 to 10 µl clean supernatant was injected into an LC-MS/MS system.
RESULTS
The automated MagSiMUS sample preparation, combined with LC-MS/MS analysis, was performed to determine the levels of immunosuppressants, benzodiazepines, 25-OH vitamin D2/D3, and steroids. Samples were completely free of any particles and protein removal was >98%. Detection limits and calibration curves covered the diagnostic range of the analytes with RSD < 15% and accuracy between 80-120% compared to conventional protein crash protocols. Total processing time for 96 samples was 26 minutes for serum samples and 46 minutes for whole blood samples.
CONCLUSIONS
MagSiMUS is a reliable sample preparation methods which can be applied flexibly in a manual or medium to high throughput automated work-flow. The absence of a centrifugation step, which can take up to 10 minutes and limits the number of samples that can be treated simultaneously, improves the total sample throughput and reduces the preparation time. Since the prepared sample has properties that are at least as good as a conventionally prepared sample using protein crash and centrifugation, it is very easy to implement in the current work-flow.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | MagnaMedics Diagnostics BV |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |