Analysis of free drug fractions by ultrafast immunoaffinity chromatography.

A chromatographic method was developed for measuring the nonbound (or free) fraction of drugs by using millisecond-scale extractions on small immunoaffinity columns. The design of this system was developed by considering the dissociation rates of (R)- and (S)-warfarin from the binding protein human serum albumin (HSA) and by performing computer simulations of the immunoaffinity extraction of these drugs. The final system was tested by using it to measure the free fractions of (R)- or (S)-warfarin in samples with known concentrations of these agents and HSA. The free warfarin fraction was extracted in 180 ms by a 2.1-mm-i.d. sandwich microcolumn that contained a 1.1-mm layer of an anti-warfarin antibody support. The nonretained peaks from this immunoaffinity column were passed through a series internal surface reversed-phase columns and a fluorescence detector for the analysis of any protein-bound warfarin that remained in the sample. The experimental results were found to have good agreement with those predicted from the known equilibrium constants for the binding of (R)- and (S)-warfarin with HSA. This approach can be modified for other analytes by changing the types of antibodies that are used in the immunoaffinity column and by using an appropriate detector for the nonretained drug fraction.