Analysis of free drug fractions using near-infrared fluorescent labels and an ultrafast immunoextraction/displacement assay

A chromatographic method was developed for measuring free drug fractions based on the use of an ultrafast immunoextraction/displacement assay (UFIDA) with near-infrared (NIR) fluorescent labels. This approach was evaluated by using it to determine the free fraction of phenytoin in serum or samples containing the binding protein human serum albumin (HSA). Items considered in the design of this method included the dissociation rate of HSA-bound phenytoin, the rate of capture of free phenytoin by immunoextraction microcolumns, the behavior of NIR fluorescent labels in a displacement format, and the overall response and stability of the resulting assay. In the final UFIDA method, the free fraction of phenytoin was extracted in similar to 100 ms by a microcolumn containing a small layer of anti-phenytoin antibodies. This gave a displacement peak for a NIR-fluorescent-labeled analogue of phenytoin that appeared within 2-3 min of sample injection, creating a signal proportional to the amount of free phenytoin in the sample. The UFIDA method provided results within 1-5% of those determined by ultrafiltration for reference samples. The lower limit of detection was 570 pM, and the linear range extended up to 10 mu M. This approach is not limited to phenytoin but can be adapted for other analytes through the use of appropriate antibodies and labeled analogues.