Dawid Wardecki, Małgorzata Dołowy

Detection and quantification of bicalutamide and febuxostat by TLC in combination with densitometry

 

2025-03-20

The Subject of the Study. The subject of the study focuses on two active substances:bicalutamide, which is used in the treatment of prostate cancer, and febuxostat used in the treatment of gout. These diseases often co-occur, and the drugs studied are one of their primary treatment methods.

Aim of Study. The main objective of the study was to develop the previously undescribed chromatographic conditions necessary for the separation of the two studied medicinal substances, i.e., bicalutamide and febuxostat, and then, using Thin-layer Chromatography (TLC) in combination with densitometry, to determine their Limit of Detection (LOD) and Limit of Quantification (LOQ). The TLC technique is a popular tool that enables the separation and analysis of two or more compounds simultaneously in different samples, including biological material. Developing conditions for efficient separation of bicalutamide and febuxostat by TLC coupled with densitometry and estimating the LOD and LOQ values of the two substances may be useful in selecting a specific TLC procedure in biomedical analysis, i.e., to monitor drug concentration during pharmacotherapy and therapy personalization.

Material and Methods. The TLC technique in combination with densitometry was used to determine the LOD and LOQ values of bicalutamide and febuxostat, as well as to determine the separation conditions of the tested drugs. Mixtures of different composition and suitable volume ratio of various organic solvents, such as acetonitrile, chloroform, n-hexane, propan-2-ol, methanol, acetic acid and ethyl acetate were used as mobile phases. On the other hand, glass and aluminum chromatographic plates coated with silica gel 60F₂₅₄, a mixture of silica gel 60 and kieselguhr F_254, as well as plates coated with silica gel 60 RP-2F₂₅₄, RP-8F₂₅₄ and RP-18F₂₅₄ were used as stationary phases. The resulting chromatograms were subjected to densitometric analysis in the UV light. In addition, in order to develop new ways of detecting the investigated compounds in daylight, a number of visualizing reagents were tested, such as aniline blue, red neutral, brilliant green, brilliant-cresyl blue, bromophenol blue, phenol red, bromocresyl green, eosin yellow, titanium yellow, copper (II) sulfate, nickel (II) sulfate and iron (III) chloride.

The obtained results were used to evaluate the suitability of newly developed TLC procedures for simultaneous analysis of bicalutamide and febuxostat.

Results. The obtained values of the separation and selectivity parameters for the pair of studied compounds, i.e., bicalutamide and febuxostat such as ΔRf, Rs, Rf, and , made it possible to determine the optimal chromatographic systems for fast and efficient separation of these compounds. The effects of visualization of the bands of the tested compounds obtained after the use of color solutions of different organic reagents and, respectively, inorganic salts were evaluated for their usefulness as new and fast ways of detecting both of these compounds analyzed simultaneously.

The lowest bicalutamide LOD and LOQ values of 0.075 and 0.226 μg/spot, respectively, were achieved by a system composed of silica gel 60F₂₅₄ coated plates and a mixture of n-hexane-propan-2-ol (28 : 22, v/v) as the mobile phase. On the other hand, for febuxostat, the lowest results, i.e., LOD = 0.055 μg/spot and LOQ = 0.165 μg/spot, were achieved in a reverse phase system, i.e., on RP-8F₂₅₄ and RP-2F₂₅₄ plates using an acetonitrile-water mobile phase (35 : 15 and 20 : 30, v/v). It was observed that plates coated with silica gel RP-18F₂₅₄ and the mobile phase composed of acetonitrile and water (35 : 15, v/v) and the silica gel 60F₂₅₄ plates, respectively, with a mixture of n-hexane-propan- 2-ol (28 : 22) allowed values for each other to be achieved; LOD_B = 0.092 and LOD_FB = 0.102 μg/spot and LOQ_B = 0.280 and LOQ_FB = 0.310 μg/spot.

Conclusions. The analysis of the obtained results, including the LOD and LOQ values of febuxostat and bicalutamide, indicates that the selection of the appropriate chromatographic conditions can favorably influence the separation and the detection and quantification of the two compounds using the TLC method in combination with densitometry. In the course of numerous chromatographic experiments conducted under different conditions, the suitability for separation and analysis of the two compounds of stationary phase as yet unexplored for this purpose coated with a mixture of silica gel 60 and silica F₂₅₄ was confirmed. In addition, the visualizing reagents tested in the present work enabled the identification of those that can be successfully used as new ways to rapidly detect bicalutamide and febuxostat in daylight.

The LOD range of 0.075 to 0.767 μg/spot for bicalutamide and 0.055–0.165 μg/spot for febuxostat and the LOQ range of 0.226–2.324 μg/spot for bicalutamide, and from 0.165 to 0.509 μg/spot for febuxostat, indicates the possibility of performing an effective qualitative and quantitative analysis of these compounds by the TLC method coupled with the densitometer in biological samples when monitoring therapy with these active substances, which in turn may have a beneficial effect on the optimization of pharmacotherapy of patients affected by diseases such as prostate cancer and gout in which these drugs are used.

Keywords: bicalutamide, febuxostat; thin-layer chromatography, limit of detection, limit of quantification.

© Farm Pol, 2024, 80(10): 659–670