(M1430-06-40) Determination of Chemical Species Compound Concentrations (Free Form, Ion) during a Membrane Permeation Test Using Simulated Intestinal Fluid

Purpose: The flux test is a popular in vitro method for evaluating the solubility and absorption of oral pharmaceuticals in the gastrointestinal tract. The compound concentration in the test medium was measured at constant time intervals during the flux tests. However, in reality, the compound exists in equilibrium in a solution containing ions with high saturated solubility and free acids/bases with high membrane permeability. Although it is possible to predict the free form and ion concentrations in the test medium based on the p<em>K_a of the compound, it is known that p<em>K_a is affected by temperature, ionic strength, and surfactants in solution. This makes an offline analysis not suitable to determine. Furthermore, when a solution containing surfactants, such as artificial intestinal fluid, is used, the situation becomes more complicated due to the formation of micelles. In this study, we used a Fiber Optics UV Spectrometer for in-situ absorbance measurement and analysis software to quantify the concentrations of free (neutral) species and ions present in the test medium during the flux test. In addition, the effects of their ratio and the presence of bile micelles on membrane permeability are discussed.

Methods: Small volume flux test Warfarine sodium (pK_a 5.0), papaverine hydrochloride (pK_b 6.5), and propranolol hydrochloride (pK_b 9.5) were used as model drug compounds. Each compound was dissolved in dimethyl sulfoxide to make a high-concentration solution and added to the small-volume flux tester to reach a concentration of approximately 30 µg/mL in the Donor chamber for the flux test. Four types of media, FaSSIF (pH 6.5), FeSSIF (pH 5.0), plane FaSSIF, and plane FeSSIF, were used in the Donor Chamber, and Acceptor Sink Buffer (pH 7.4) was used in the Acceptor Chamber. (Fig. 1) Quantification of individual chemical species in the flux test - If there is a difference between the pH of the solution and the p<em>K_a of the compound is larger than three, the abundance ratio of one chemical species is theoretically less than 0.1% and, UV absorption can be ignored. Therefore, UV absorption spectra were obtained for warfarine and papaverine in the concentration range of 0–35 µg/mL in solutions adjusted to the following pH to obtain calibration curves for the free or ionized species: Warfarin: pH 2 (free acid) and pH 9 (ionized) - Papaverine: pH 3 (ionized) and pH 10 (free base) - From the obtained UV spectra of individual chemical species, the concentrations of the chemical species present in the test medium during the flux experiment were analyzed using AuPRO software.

Results: The concentration of each compound that permeated the Acceptor Chamber is shown in Figure 2. For the acidic compound warfarin, the membrane permeation rate followed the order: plane FeSSIF >FeSSIF >plane FaSSIF≒. In contrast, for the basic compounds papaverine and propranolol, the order was: plane FaSSIF >FaSSIF >plane FeSSIF >FaSSIF. To examine this result, the abundance ratio of warfarin species in the donor chamber during the flux experiments was analyzed. For the acidic compound warfarin, 41% was present as a free acid with high membrane permeability in the FeSSIF plane, whereas almost the entire amount was present as ions in the FeSSIF plane. For warfarin, there was no difference in the amount of membrane permeation between plain FaSSIF and FaSSIF; however, in the case of papaverine, the amount of membrane permeation was reduced by 28% in the presence of bile salts and lecithin. These results suggest that the presence of bile salts and lecithin does not affect chemical species concentration in FaSSIF, meaning that bile micelles were not formed. In contrast, 67% of papaverine was present in FaSSIF as a free form, indicating that the formation of bile micelles reduced the amount of papaverine available for membrane permeability, leading to an observed inhibition of membrane transport. The UV absorption spectrum of warfarin in FaSSIF is shown in Figure 3.

Conclusion: In this study, we successfully quantified the chemical species of compounds present in donor solutions during flux experiments for both acidic and basic compounds in situ, and simultaneously evaluated bile micelle formation. Membrane permeation of the acidic compound warfarin was not affected by bile salts or lecithin in FaSSIF (pH 6.5) but in FeSSIF (pH 5.0), where the proportion of free acid increased, membrane permeation was thought to decrease due to bile micelle formation. Thus, the presence of chemical species in the gastrointestinal tract is important for understanding the solubility and absorption of formulations and food effects. The purpose of this study was to develop an experimental model, and thus, only flux tests were performed in the low concentration range, where the compound was sufficiently soluble. No solubility enhancement by bile salts and lecithin was observed, and only a decrease in membrane permeation was noted. In the future, this analytical method will be applied to a wider concentration range and various compounds for further evaluation.

References: S. Kadar, A. Kennedy, S. Lee, R. Ruiz, A. Farkas, P. Tozser, D. Csicsak, G. Toth, B. Sinko, E. Borbas, 2024. Bioequivalence prediction with small-scale biphasic dissolution and simultaneous dissolution-permeation apparatus-An aripiprazole case study. European Journal of Pharmaceutical Sciences 198.


Images of a small-volume flux system. UV probes were inserted into the donor and acceptor chambers.


Concentrations of compounds that permeated into the acceptor chamber. Top: warfarin; middle: papaverine; and bottom: propranolol. Concentrations were determined by UV absorption using dip probes inserted into the chamber. In this figure, total compound concentration permeating the membrane is plotted without distinguishing between the free or ion forms.


UV absorption spectrum of warfarin acquired in FaSSIF, analyzed using the Multiple-Component analysis function. Black curve is the acquired spectrum, and purple dotted curve is the spectrum predicted from the sum of the calculated concentrations of the free and ionic forms. The calculated spectra overlap within the calculated range, indicated by the green lines.