Developing an SPE protocol for the analysis of biological samples

Many bioanalytical LC-MS assays require a number of injections to condition the analytical column to ensure stable and reproducible quantitative data is obtained. This work aims to assess these instabilities and specifically investigate the impact of sample preparation techniques and look at methods to enable the reduction of sample and solvent use in the workflow.

A different approach will be taken to share the information, using an interactive discussion between leading experts in the field, who will look at how to optimize an SPE method for the analysis of small and large molecules. The five steps to the SPE methodology will be investigated to determine the impact that each of these steps can have on the assay performance. Data will be shared looking at the impact of some standard variables at each of the five critical stages of an SPE method, namely;

• Condition
• Re-equilibration
• Load
• Wash
• Elute

There are many different parameters that impact the performance of the extraction, including phase selection, solvent and pH selection as well as bed weight. Even the flow rate can affect analyte retention and elution. Data will be presented which highlights the impact of these important parameters along with a methodology for determining the loading capacity of an SPE cartridge, critical for ensuring that the correct bed weight is chosen. This selection of experimental parameters is particularly important when dealing with more complex molecules. Examples will be given of the impact of loading and elution solvents on the retention and elution of oligonucleotides, and an approach to method optimisation will be presented for these popular therapeutic compounds.

The discussion will move on to the impact of matrix removal and how different approaches to sample preparation can have an impact on specifically phospholipid removal. Data will be presented and reviewed which shows the impact that the matrix has on the validation of the assay and the implications of enhanced matrix will be reviewed in the context of the validation process and also the stability of the assay and sample analysis.

Finally, the work is further developed to look at the impact that the bed weight and nature of the SPE material can have on the use of reduced eluting volumes. A novel composite silica based SPE was used to investigate the use of reducing the eluting volume and data will be presented that compares the 2 mg composite bed mass with a 10 mg bed loose packed mass which demonstrates superior recovery and precision that a 2 mg composite bed has over a 10 mg loose packed SPE material.

For more information, please contact us at webinar@avantorsciences.com