Quantitative analysis of flavonoids inAnnona squamosaleaf extracts and its pellet formulation by validated high-performance thin-layer chromatographic technique

Annona squamosa, commonly known as custard apple, possesses various medicinal properties such as antimicrobial, insecticidal, cardiotonic, etc. Leaves of A. squamosa are known to contain various types of flavonoids which are responsible for these activities. The aim of this article was to develop and validate a high-performance thin-layer chromatography (HPTLC) method for the determination of rutin and isoquercitrin in the extracts of A. squamosa and in an in-house developed herbal pharmaceutical formulation. Analysis was performed on TLC aluminum plates precoated with silica gel 60 F254 as the stationary phase. Linear ascending development was carried out in a twin-trough glass chamber saturated with a mobile phase consisting of ethyl acetate-formic acid-glacial acetic acid-ethyl methyl ketone-water 50:7:3:30:10 (ν/ν/ν/ν/ν) at room temperature (25 ± 2°C). A CAMAG TLC scanner III was used for the spectrodensitometric scanning at 366 nm. The system was found to give compact bands for rutin and isoquercitrin (RF values of 0.32 ± 0.05 and 0.59 ± 0.03, respectively). The detector response was linear for concentrations ranging between 200 and 1600 ng per band (r2 = 0.9885 ± 0.0041 for rutin and 0.9828 ± 0.0013 for isoquercitrin). The limits of detection and quantitation were 75 and 100 ng per band for rutin, and 40 and 80 ng per band for isoquercitrin, respectively. The recovery study was carried out by the standard addition method and was found to be 96–107%. The present method was found to be simple, specific, sensitive, and accurate and was used successfully to quantitate rutin and isoquercitrin in the leaf extracts of A. squamosa collected from five different locations of India and in a pellet formulation of the extract.