A dual-detection HPTLC platform: combining smartphone-based imaging and densitometry for the analysis of diazepam, its metabolite and degradation product

In recent years, high-performance thin-layer chromatography (HPTLC) has gained prominence as a cost-effective, straightforward, and dependable analytical technique, particularly in forensic and pharmaceutical laboratories. With the rapid evolution of smartphone technologies, a novel dimension in analytical detection has emerged. Advent of smartphones with superior imaging modalities combined with simplicity of use and easy transition into the healthcare ecosystem has made the existing benchtop-based techniques much sleeker, cost-effective and rapid screening approaches. The developed and validated HPTLC method employing smartphone camera detection for simultaneous determination of Diazepam (DZP), its metabolite Oxazepam (OXP) and its degradation product 2-methylamino-5-chlorobenzophenone (ACB) was intended to meet this requirement and provide a useful replacement for the usual densitometric analysis. The chromatographic separation was performed on silica gel HPTLC plates with green mobile phase of heptane: ethyl acetate (7.0:3.0, v/v). After chromatographic development, the Dragendorff's reagent was applied for visualization, and the plates were photographed with smartphone camera. Spot intensities were quantitatively analyzed using ImageJ software in the concentration range 3.0-35.0 µg/spot for both DZP and ACB, and 5.0-35.0 µg/spot for OXP. The data developed by the proposed method were compared with benchtop densitometric method. Linearity was established from 0.2 to 1.0 µg/spot for the three analytes detected at 230.0 nm using HPTLC/densitometry while the HPTLC/smartphone method exhibited linearity from 3.0 to 35.0 µg/spot for both DZP and ACB, and 5.0-35.0 µg/spot for OXP. The developed HPTLC/smartphone method showed that it could successfully be employed to quantify DZP in pharmaceutically marketed formulations in terms of speed, eco-friendly and simplicity. Additionally, the enhanced sensitivity of the densitometric technique enabled successful determination of DZP and OXP in spiked human plasma samples using ACB as an internal standard. To assess the environmental and practical merit of the methods, greenness and sustainability were evaluated using the Green Analytical Procedure Index (GAPI), Analytical Greenness Metric (AGREE), White Analytical Chemistry (WAC), and Blue Applicability Grade Index (BAGI). Results confirmed the developed methods' analytical efficiency, environmental compatibility, and alignment with the principles of green and white analytical chemistry.