Development and validation of a HPTLC method for simultaneous estimation of phytoconstituents in Amalakyadi churna- a classical Ayurvedic formulation

Abstract Amalakyadi churna is a well-known Ayurvedic formulation; contains four main Plants: Amalaki (Emblica officinalis), Chitrak (Plumbago zeylanica), Haritaki (Terminalia chebula) and Pippali (Piper longum), which are rich sources of Gallic acid(GA), Plumbagin(PB), Ellagic acid(EA) and Piperine(PR). The present study aimed to develop and validate an HPTLC method for the simultaneous estimation of these phytoconstituents in Amlakyadi churna. Optimal separation was done with a mobile phase of Toluene, Ethyl acetate, Formic acid (5:4:1; v/v/v) that ensured efficient separation of all selected phytoconstituents. The developed method was validated according to ICH Q2(R2) guidelines.GA, PB, EA and PR showed good linearity in the range of 2000–10000 ng band−1 with Regression coefficient (R2) value 0.9989,0.998,0.9984 & 0.9985. The corresponding Rf values were 0.23, 0.29, 0.62 and 0.91. Recovery studies established accuracy with mean recoveries between 99.66% and 100.1% with %RSD <2. The developed method was found to be precise with %RSD 1.26(GA), 1.12 (PB),0.88 (EA), 0.90(PR) for Interday while 1.07(GA),1.06(PB),1.22(EA),1.12(PR) for Intraday study. LOD values for GA,PB,EA,PR were 401.94,531,483.63,483.16 ng/band while LOQ values of 1218,1609.90,1465.55,1464.12 ng/band were found. The method was found to be simple, sensitive, accurate, precise and robust for routine qualitative and quantitative analysis of Gallic acid, Plumbagin, Ellagic acid and Piperine in Amalakyadi churna and related herbal formulation. Graphical Abstract