Validation and greenness assessment of HPTLC-densitometric method for simultaneous estimation of six bioactive alkamides in Piper longum Linn. root

The substantial structural similarity of alkamides poses a challenge in the chromatographic separation for simultaneous quantification of bioactive alkaloids in agro-waste of Piper longum. Here, we report a novel HPTLC method for the simultaneous quantitation of six alkamides, viz., pellitorine (1), piperanine (2), piperine (3), piperdardine (4), piplartine (5), and piperlonguminine (6) in Piper longum. PRISMA approach was applied for optimum separation on silica gel 60 F254 plates using n-hexane-dichloroethane-diethylamine (7.8:1.2:0.5, v/v/v) as the solvent system. In addition to Rf and colour visualization, the specificity of determination was demon-strated by UV, and mass spectral matching. The method was consistent with the Beer's law over the 1-5 mu g/band range. The detection limits and the limits of quantification are in the range of 0.073-0.531, and 0.243-0.769 mu g for pellitorine (1), piperanine (2), piperine (3), piperdardine (4), piplartine (5), and piperlonguminine (6); respectively. The proposed method was successfully applied to determine the six cited alkamides with mean recoveries ranging from 98.81 to 100.05 %, and intra-and inter-day precisions with relative standard deviations below 2 %. Moreover, the greenness was investigated using National Environmental Methods Index (NEMI), Eco-scale Assessment (ESA), Green Analytical Procedure Index (GAPI), and Analytical GREEness metric (AGREE) metrics revealing the current HPTLC method environmentally benign and energy efficient. It is the first report of an analytical method that supports precise, high-throughput, and simultaneous quantification of six alkamides (1-6) in Piper longum roots with application in food and pharmaceutical.

Automated summary

A novel HPTLC method was developed for the simultaneous quantification of six bioactive alkaloids in agro-waste of Piper longum. The method demonstrated excellent separation and specificity, and was successfully applied for accurate quantification in Piper longum roots. Additionally, the method was found to be environmentally friendly and energy efficient.