Ultratrace analysis of drugs in biological fluids using affinity probe capillary electrophoresis: analysis of dorzolamide with fluorescently labeled carbonic anhydrase.

This work demonstrates the use of affinity probe capillary electrophoresis (APCE) in the quantitative analysis of drugs in biological fluids at the low pM level. The interaction of human carbonic anhydrase II (HCAII) with the glaucoma drug dorzolamide (Dz) was chosen as a model system. HCAII was labeled at its single cysteine residue using a thiol-specific fluorescein reagent. The peak area of HCAII complexed with the tight-binding drug Dz provided a direct assay of the drug concentration in solution. A charged competitive ligand added to the running buffer was employed in APCE to distinguish Dz-bound from free forms of the HCAII. Using laser-induced fluorescence (LIF), the Dz detection limit was 16.5 pM in aqueous solution and 62.5 pM in both urine and plasma. Normalized peak area reproducibility of the drug was within 3.4% RSD. Each analysis was completed within 10 min, including incubation, and consumed only 0.3 pmol of labeled protein. The APCE approach provides an effective method for trace level detection of drugs in biological matrices.