Microscale titrimetric and spectrophotometric methods for determination of ionization constants and partition coefficients of new drug candidates

This study describes the adaptation of conventional titrimetric and spectrophotometric techniques to a microscale for the determination of drug ionization constants (pK(a)) and partition coefficients (log P). The apparatus for determining pK(a) and compound purity (or equivalent weight) consists of a three-port conical glass microvial maintained at 25 degrees C, a pH microelectrode, and a microinjection pump equipped with a 10 mu L gastight syringe for titrant delivery. Sample mixing and protection from atmospheric CO2, which is particularly important at the microscale, is accomplished using a fine stream of water-saturated N-2 bubbles. Simple titrimetric procedures combined with ionic equilibria models which allow the accurate determination of pK(a) and purity (or equivalent weight) using sample sizes in the microgram range and solution volumes of 10-100 mu L were developed and validated using acetic acid and tromethamine. Simultaneous determinations of pK(a), purity or equivalent weight, and octanol/water partition coefficient were shown to be possible from a single sample of a test solute by adapting the pH-metric technique to a microscale. Using benzoic acid as a model compound, a pK(a) of 4.24 and octanol/water partition coefficient of 64 were obtained, in close agreement with the literature values. The principles employed in titrimetric analysis were also applied to demonstrate the spectrophotometric determination of benzoic acid's pK(a) and partition coefficient using only 6 mu g of compound. The microscale titration method was then used to determine the two pK(a) values of an "unknown" diprotic acid containing a carboxyl and an aromatic SH group. The phenyl thiol pK(a) was confirmed using the microscale spectrophotometric procedure.