Second Derivative Spectrophotometric Determination of the Binding Constant Between Codeine Phosphate and Bovine Serum Albumin

Second derivative spectrophotometry was applied to determine the binding constant (K) between codeine phosphate (COD) and bovine serum albumin (BSA) at simulated physiological conditions (37.00 °C and pH = 7.4). The second derivative spectra of COD in buffer solutions containing various amounts of BSA showed derivative isosbestic points. The residual background signals derived from incomplete suppression of BSA signals can be entirely eliminated in the second derivative spectra indicating that BSA has spectrophotometrically one kind of binding site for COD. The fractions of COD bound to BSA were calculated from the derivative intensity differences (ΔD values) of COD before and after the addition of BSA. Scatchard plot calculation suggested that the binding of COD to BSA can be explained by a partition-like non-specific binding model. The binding constant (K) was calculated from ΔD values according to the non-specific binding model by a nonlinear least-squares method. K values were almost constant for all of the COD concentrations studied with good reproducibility. The fractions predicted by the K values were in good agreement with the observed values. The results indicate the usefulness of the derivative method in drug–albumin binding studies without the need for prior separation procedures which may disturb the equilibrium states of the samples solutions.