Acid–base equilibrium of drugs in time-resolved fluorescence measurements: Theoretical aspects and expressions for apparent p Ka shifts

Abstract

Spectroscopic properties of molecules which undergo acid–base equilibrium are greatly influenced by the local pH. Their acid–base equilibrium constants can be experimentally obtained under different environments by measuring spectroscopic parameters such as absorbance, fluorescence intensity and fluorescence lifetimes as a function of pH. The p K a values are obtained fitting the data with the well known Henderson–Hasselbalch equation. Decay curves obtained with time-resolved pulse fluorometry are usually analyzed in terms of multi-exponential functions characterized by the pre-exponential factors or by the integrated fractional fluorescence intensities associated with each lifetime, this last being analogous to the steady state fluorescence contributions. In this paper we show that under normal conditions for many fluorescent drugs, a two-exponential analysis is adequate. We develop expressions for the pH dependence of pre-exponential factors and fractional fluorescence intensities in time-resolved pulse fluorometry of a fluorophore undergoing acid–base transition. We show that the obtained p K a values are apparently shifted, and give the expressions for the pK-shifts. The expressions were tested using the fluorescence decay properties of two compounds: the well characterized local anesthetic dibucaine and the antibiotic levofloxacin.