High throughput determination of relevant physicochemical parameters in the drug discovery and HPLC processes. Microfluidic devices

Abstract

Determining the acidity (pKa) and lipophilicity (log Po/w) of organic compounds is fundamental in analytical chemistry fields, with potential relevance in drug development, material science, analytical separation, and environmental research. A precise estimation of these physicochemical parameters is important for estimating compound behavior and distribution in different biological and chemical systems. Fast determination of pKa of very insoluble drugs has become an essential tool in drug development as it frequently creates compounds that are highly lipophilic and sparingly soluble in water. Also, studying the pharmacokinetics and pharmacodynamics of a proposed drug involves a thorough knowledge of its ionization state and hydrophobicity. In the first part of the thesis, a high-throughput internal standard capillary electrophoresis (IS-CE) method was established to determine the pKa of ISs at different concentrations of methanol and acetonitrile from 0 to 90% (v/v). IS-CE is a pKa determination method based on the use of a known pKa reference compound as an internal standard (IS), whose nature and pKa value are similar to those one of the analytes. Internal standard mobility is measured under the same conditions as analyte mobility when injected at the same time, hence any change in experimental conditions influences both analyte and IS equally. Whereas the traditional CE approach needs potentiometric measurement of the pH of the buffers used, IS-CE uses IS to calculate the true pH in our electrophoretic system and reduces measurement mistakes. Herein, the acid and base scales of methanol-water mixtures and acetonitrile-water mixtures were properly anchored to the potentiometrically obtained pKa values of reference compounds to get absolute pKa scales. As a consequence, a set of 46 acid-base compounds with changing structures were proposed as internal standards for consistent pKa measurements in methanol-water and acetonitrile-water mixtures buffers using capillary electrophoresis. The determined ISs reference set facilitates the determination of analytes pKa and measurement of buffer pH in the range 4-11.5 (in water) for any methanol-water and acetonitrile-water composition. Secondly, to prove its feasibility, the IS-CE approach was successfully used to determine the aqueous pKa in methanol-aqueous buffer compositions up to 40% of methanol in volume. The Yasuda-Shedlovsky extrapolation method was utilized to determine seven drugs of different chemical nature with intrinsic water solubilities lower than 10−6 M. The results were successfully compared to literature ones obtained by other approaches. It is concluded then that the IS-CE methodolgy permits the measurement of aqueous pKa values using lower ratios of methanol than the classical method, becoming then more accurate in the extrapolation procedure than other reference methods. Finally, since methanol-water and acetonitrile-water mixtures are solvents of interest in liquid chromatographic separations because of their use as the mobile phase, the IS-CE method was also applied to measure the pKa of eight organic bases in methanol-water and acetonitrile-water mixtures (0-90%,v/v), which are usually used as test compounds in HPLC column evaluation. As a result of this work, the IS-CE method was proven to be a fast and simple approach for determining the pH of the buffer and the pKa of analytes in typical HPLC systems (RPLC, HILIC) in both methanol-water mixtures and acetonitrile-water mixtures. The degree of ionization of the analytes may be easily determined using them, making it easier to choose the mobile phase composition and thus enhance analytical separations. In the second part of the thesis, a new approach based on microfluidics was developed to determine the octanol-water partition. The octanol-water partition coefficient is crucial in pharmaceutical and biological sciences as it is a vital metric in predicting chemical distribution and behavior in biological systems. However, the current techniques are time consuming and requires high amounts of solvents. From the need to develop a quicker, more cost-effective, and more sustainable method, microfluidics has raised as a powerful miniaturized analytical tool. As a first step, a design with a perpendicular configuration of the channels was developed using direct 3D printed microfluidics. A gravitational perfusion system was implemented to create a spontaneous flow within the octanol and water channels without the need for external pump. The movement of octanol and water phases was successfully validated using fluorescent dyes. After that, the intensity of the fluorescent dye was used to evaluate the partition dynamics in static and dynamic conditions. The results prove that the proposed design with this microfluidic methodology allows the evaluation of molecule partition, achieving high efficiency partition and reaching the equilibrium of O/W partition faster than conventional techniques. Later, the design was adapted to a parallel configuration of the channels to be compatible with up-scalable manufacturing techniques and parallelize it for up to 56 simultaneous determinations in a single platform. Finally, both the perpendicular and parallel designs were validated using several drugs with well standardize log Po/w values that cover a wide range of lipophilicity. The microfluidic device was coupled with HPLC to determine their partition coefficients from the peak areas of the compounds in octanol and in water after partition. Good agreement with the literature values was achieved, showing the capability of microfluidic chips for precise and accurate prediction of the partition coefficient. Finally, the progress of a cost-effective and consistent method for predicting partition coefficient via microfluidic chips demonstrated a great advancement in the field of analytical chemistry, with powerful applications in drug discovery and other related fields. The results gotten from this investigation offer an establishment for additional research and advance of this approach. To summarize, estimating pKa and log Po/w values is vital in analytical chemistry, with capacity applications in drug development, material science, and environmental research. A precise estimation of these physicochemical parameters is important for predicting compound performance and distribution in diverse biological and chemical systems. The use of innovative analytical techniques for verifying pKa values, like the IS-CE method for pKa and microfluidics for measuring log Po/w, represents valuable advances in the field of analytical chemistry. These methods provide low-reagent-consumption, cost-effective, and reliable determination for evaluating these parameters, for high-throughput analysis.