Box‒Behnken design-assisted development of an eco-friendly thin-layer chromatography‒densitometry method for the quantification of amlodipine besylate, metoprolol succinate, and telmisartan in synthetic mixture using AGREE tool

This study describes the systematic development of a high-performance thin-layer chromatography (HPTLC) method for the quantification of amlodipine besylate (AML), metoprolol succinate (MET), and telmisartan (TEL) in synthetic mixture within the quality by design framework. A mixture of toluene‒methanol‒isopropanol‒triethylamine (6:0.8:2:0.2, V/V) was used as the mobile phase for chromatographic separation on aluminum-backed silica gel 60 F254 plates with densitometric scanning at 233 nm. The effects of identified critical method parameters, volume of methanol, chamber saturation duration, and solvent front on the RF value of all three drugs and resolution between two drugs were investigated using Box‒Behnken experimental design. To understand potential interactions among critical method parameters, statistical modelization was used, followed by response surface analysis. By using numerical and graphical optimization to define the design space, the search for the best solution was carried out. The RF values discovered after optimization for AML, MET, and TEL were 0.25, 0.39, and 0.48, respectively. The calibration plots’ data from the linear regression analysis indicated a regression coefficient more than 0.99 for a concentration range of 500–2500 ng/band for AML, 400–2000 ng/band for MET, and 100–500 ng/band for TEL. The optimized HPTLC method was validated according to the International Council for Harmonisation (ICH) guideline Q2 (R1). The %RSD for accuracy, precision, and robustness were all within the acceptable range, less than 2. Additionally, the AGREE greenness assessment tool was used to analyze the greenness profile of the developed method, the score of 0.79 proved that the technique is environmentally friendly and can be regularly used for the analysis of all three drugs in combination. The investigations effectively show the applicability of the quality by design (QbD) strategy in the development of the highly sensitive HPTLC method with improved method performance.