Bioequivalence and Bioavailability Forum

Dear Ohlbe!

❝ That's indeed the easiest solution, but not always applicable. If your molecule has a high distribution or binding to the red blood cells, it may take some time after spiking to reach an equilibrium. In that case you will see a decrease of the plasma concentration over time, which will not be due to instability but to this partitioning or binding.

You are right!

❝ That's one of the reasons why the EBF recommended to use a "qualified" whole blood method. However this recommendation is discussed. In the same issue of Bioanalysis journal the GCC recommended your approach, and to only go for a direct evaluation in whole blood if a partitioning or binding problem is expected or suspected.

Thanks for the article – quite interesting! The latter case will be challenging, though. Imagine a method validated for plasma and applied to whole blood

• Direct injection of matrix, online trace-enrichment (column switching): after the first attempt with whole blood your pre-column is blocked and the pump will shut down.
  
• Protein precipitation (the mostly applied method): inclusion of the analyte(s) in denatured proteins / red blood cells / platelets likely.