Spectrophotometric Investigations of Charge Transfer Complexes of Tyrosine Kinase Inhibitors with Iodine as a σ-Electron Acceptor: Application to Development of Universal High-Throughput Microwell Assay for Their Determination in Pharmaceutical Formulations

Abstract

Background and Objective: Tyrosine kinase inhibitors (TKIs) are used for the treatment of different types of cancers. The current study describes, for the first time, the ultraviolet-visible spectrophotometric investigation of charge transfer complexes (CTCs) of seven TKIs, as electron donors, and iodine, as σ-electron. Materials and Methods: The formation of CTCs was promoted in dichloromethane, among the other solvents used in the investigation. The molar absorptivity values, association constants, and free energy changes of the CTCs were determined. Stoichiometric ratio of TKI: iodine as well as TKIs site(s) of interaction were addressed. Reaction was the basis for constructing a novel simple and accurate 96-microwell spectrophotometric assay (MW-SPA) with high-throughput property for the quantitative determination of TKIs in their pharmaceutical formulations. Results: Beer's law, which relates CTC absorbances to TKI concentrations, was followed within the optimal range of 2 to 100 µg/well (r ranged from 0.9991 to 0.9998). Detection and quantification limits ranged from 0.91 to 3.60 and 2.76 to 10.92 g µmL^-1, respectively. Relative standard deviations values for the intra- and inter-assay precisions of the proposed MW-SPA did not exceed 2.13 and 2.34%, respectively. Studies of recovery demonstrated MW-SPA accuracy, with results ranging from 98.9% to 102.4%. All TKIs, both in bulk form and in pharmaceutical formulations (tablets), were effectively determined using the suggested MW-SPA. Conclusions: The current MW-SPA involved a simple procedure and it was convenient as it could analyse all proposed TKIs utilizing a single assay system at once measuring wavelengths for all TKIs. In addition, the proposed MW-SPA has high throughput which enables the processing of a batch of huge samples' number in very short reasonable time period. In conclusion, TKIs can be routinely analysed in their dosage forms in quality control laboratories, and the assay can be highly valuable and helpful in this regard.

Keywords: 96-Microwell spectrophotometric assay; Iodine; charge-transfer complex; high-throughput analysis; tyrosine kinase inhibitors.

Figure 1

The chemical structures of the investigated tyrosine kinase inhibitors (TKIs) with their abbreviations. Asterisk symbols (*) denote the electron-donating sites of interactions of TKIs with iodine and form CTC.

Figure 2

The absorption spectra of CAB (1.99 × 10⁻⁵ M), iodine (1.58 × 10⁻³ M), and a reaction mixture of CAB (3.99 × 10⁻⁵ M) with iodine (1.58 × 10⁻³ M). All solutions were in dichloromethane.

Figure 3

Tauc plot of energy (hυ, in eV) against (αhυ)² against for CT complex of iodine with CER and LIN.

Figure 4

Benesi-Hildebrand plot of the CT complex of iodine with AXT and the linear fitting equation with correlation coefficient (r). [A], A and [D] are the molar concentration of iodine, the absorbance of the reaction mixture of the complex, and the molar concentration of AXT, respectively.

Figure 5

The association constant (K) versus free energy change (ΔG) for the CTC of TKIs with iodine. K values were presented in linear and logarithmic scale (panels (A) and (B), respectively). A value of 30 was added to all ΔG values to convert their negative values to positive ones.

Figure 6

Plot of absorbance versus molar ratio of [Iodine]/[Drug] obtained from reaction mixtures containing a fixed concentration of drug and varying concentrations of iodine. The mole ratio corresponds to the point of intersection of the tangents of the straight-line portions of the plots. Results are given for CER and AFA as representative examples for the molar ratios of 1:1 and 1:2, respectively.

Figure 7

The effect of iodine concentration on its reaction with TKIs (25 µg mL⁻¹).

Figure 8

Calibration curves for the determination of TKIs by the proposed MW-SPA based on their reaction with iodine.