Spectrophotometric and computational investigations of charge transfer complexes of chloranilic acid with tyrosine kinase inhibitors and application to development of novel universal 96-microwell assay for their determination in pharmaceutical formulations.

The tyrosine kinase inhibitors (TKIs) are chemotherapeutic drugs used for targeted therapy of various types of cancer. In literature, there is no existing universal chromogenic reagent used for development of spectrophotometric assay for all TKIs regardless the diversity of their chemical structures. This work discusses, for the first time, the experimental and computational evaluation of chloranilic acid (CLA) as a universal chromogenic reagent for developing a novel 96-microwell spectrophotometric assay (MW-SPA) for TKIs. The reaction of CLA with seven TKIs was examined in different organic solvents of various dielectric constants and polarity indexes. The reaction resulted in an instantaneous formation of intensely purple coloured products with all the investigated TKIs. Spectrophotometric investigations confirmed that the reactions proceeded via the formation of charge-transfer complexes (CTC). The physical parameters (molar absorptivity, molar ratio, association constant and standard free energy) were determined for the CTC of all TKIs. Computational calculations for the relative electron densities on each atom of the TKI molecule and molecular modelling for the CTC were conducted, and the site(s) of interaction on each TKI molecule were determined. Under the optimized conditions, Beer's law correlating the absorbances of the CTC with the concentrations of TKIs were obeyed in the range of 5-500 µg/well with good correlation coefficients (0.9991-0.9998). The limits of detection and quantitation were in the ranges of 1.89-5.09 and 5.74-15.42 µg/well, respectively. The proposed MW-SPA showed high precisions as the values of the relative standard deviations did not exceed 2.01 and 2.45% for the intra- and inter-assay precision, respectively. The accuracy of MW-SPA was proved by recovery studies as the recovery values were in the range of 98.8-103.7%. The proposed MW-SPA was successfully applied for the determination of all TKIs in their bulk forms and pharmaceutical formulations (tablets) with good accuracy and precisions. The proposed MW-SPA is the first assay that can analyse all the TKIs on a single assay system without modifications in the detection wavelength. Additional advantages of the proposed MW-SPA are simple, economic, and more importantly have high throughput. Therefore, the assay can be helpful and beneficial for routine analysis of TKIs in their pharmaceutical formulations in quality control laboratories.