CPSA Digest 2003

Day 2

Vendor Session - Drug Discovery Technologies & Applications

Plasma Protein Binding Determination By Equilibrium Dialysis Based On LC/MS/MS Peak Area Ratios In Support Of Drug Discovery

Plasma protein binding information is often required during the discovery phase in selecting drug candidates: projecting human therapeutic dose and calculating safety margin. Ultrafiltration method is less time consuming but limited to compounds without non-specific binding. For compounds that may have non-specific binding issues, equilibrium dialysis is the method of choice. Protein binding studies are often conducted in human plasma, plasma of efficacy model, and/or plasma of toxicity species. After equilibrium dialysis, sample analysis often requires a number of calibration curves prepared in different matrix and run separately. This presentation focuses on streamlining the sample analysis process by reducing the LC/MS and sample preparation time. Plasma and buffer samples collected from the dialysis cells were diluted with either buffer or plasma to become the same matrix of approximately 1:1 plasma and buffer mix. Samples were analyzed by LC/MS/MS without calibration curves. LC/MS peak area ratios were used to calculate the free fraction. Compounds with a wide range of binding properties were tested. Protein binding results using the peak area ratios showed excellent agreement with the results using the appropriate calibration curves. In addition to the advantage of eliminating the need of preparing standards, this method allows the analysis of protein binding samples from multiple species in the same LC/MS run, thus further streamline the protein binding determination process.

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