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 sp ectrophotometric techniques to a microscale for the determination of d rug ionization constants (pK(a)) and partition coefficients (log P). T he 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 a nd protection from atmospheric CO2, which is particularly important at the microscale, is accomplished using a fine stream of water-saturate d N-2 bubbles. Simple titrimetric procedures combined with ionic equil ibria models which allow the accurate determination of pK(a) and purit y (or equivalent weight) using sample sizes in the microgram range and solution volumes of 10-100 mu L were developed and validated using ac etic acid and tromethamine. Simultaneous determinations of pK(a), puri ty 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 mode l 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 demon strate the spectrophotometric determination of benzoic acid's pK(a) an d 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 gr oup. The phenyl thiol pK(a) was confirmed using the microscale spectro photometric procedure.