Analytica Chimica Acta 583 (2007) 418–428 Potentiometric and spectrophotometric pK a determination of water-insoluble compounds: Validation study in a new cosolvent system Gergely V¨ olgyi a , Rebeca Ruiz b , Karl Box c , John Comer c , Elisabeth Bosch b , Krisztina Tak´ acs-Nov´ ak a,∗ a Semmelweis University, Department of Pharmaceutical Chemistry, H¨ ogyes E. u. 9, H-1092 Budapest, Hungary b Departament de Quimica Analitica, Universitat de Barcelona, Diagonal 647, 08028 Barcelona, Spain c Sirius Analytical Instruments Ltd., Riverside, Forest Row Business Park, Forest Row, East Sussex RH18 5DW, United Kingdom Received 24 August 2006; received in revised form 2 October 2006; accepted 10 October 2006 Available online 18 October 2006 Abstract In this paper the validation of pK a determination in MDM–water mixtures is presented. The MDM–water mixture is a new multicomponent cosolvent mixture (consisting of equal volumes of methanol, dioxane and acetonitrile, as organic solvents) that dissolves a wide range of poorly water- soluble compounds. The cosolvent dissociation constants (p s K a ) of 50 chemically diverse compounds (acids, bases and ampholytes) were measured in 15–56 wt% MDM–water mixtures by potentiometric or spectrophotometric titration and the aqueous pK a values obtained by extrapolation. Three different extrapolation procedures were compared in order to choose the best extrapolation in MDM–water mixture using a sub-set of 30 water- soluble compounds. The extrapolated results are in good agreement with pK a values measured in aqueous medium. No significant difference was found among these extrapolation procedures thus the widely used Yasuda–Shedlovsky plot was proposed for MDM cosolvent also. Further we also present that the single point estimation based on measurement in 20%/v MDM-mixture using a general calibration equation may be suitable for rapid pK a determination in the early phase of drug research. © 2006 Elsevier B.V. All rights reserved. Keywords: pK a ; Cosolvent procedure; Methanol/dioxane/acetonitrile–water (MDM); Validation 1. Introduction The role and timing of physico-chemical profiling in drug research has significantly changed during the last decade. Deter- mination of fundamentally important physico-chemical proper- ties such as ionization (pK a ), solubility (log S) and lipophilicity (log P) has been shifted to the early stage of drug discovery in order to facilitate the screening of drug-like candidates [1–3]. The pK a value is a key parameter to predict the ionization state of a molecule with respect to pH. Knowledge of this param- eter is essential in the estimation of ADME properties since absorption and distribution are highly affected by the ionization of the compound. It is also necessary for the measurements of pH-dependent molecular properties, for example solubility and lipophilicity. ∗ Corresponding author. Tel.: +36 1 215 5241. E-mail address: novkri@hogyes.sote.hu (K. Tak´ acs-Nov´ ak). Remarkable developments have been observed in the automa- tion of standard pK a determination methods using both poten- tiometry (GLpK a ) and spectroscopy (GLpK a + D-PAS) [4–7]. Recently a new method and instrument (ProfilerSGA) has been developed to address the need for high throughput measurements of pK a . Spectral gradient analysis (SGA) is suitable for mea- surements of large number of compounds using small amounts of samples, a typical requirement of early phase drug discov- ery [8]. However, the application of these highly sophisticated methods in aqueous solutions is often hindered by the low water solubility of samples. This is a frequent problem today since the new molecules in drug research are less water-soluble and more lipophilic [9]. The mixed-solvent procedure mainly using methanol–water mixtures provides a good alternative for spar- ingly soluble compounds [10–12]. Here, the cosolvent ionization constants (p s K a ) in different ratios of MeOH–water mixtures are measured and the aqueous pK a is obtained by extrapolation. But experience shows that not all compounds dissolve in single component organic solvent-water mixture. Thus recently a new, 0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.10.015