High Performance Thin Layer Chromatography (HPTLC) Methods for Simultaneous Estimation and Quantification of Vasicine, Gallic acid, Piperine and Pterostilbene from Two Ayurvedic Formulations and Herbal Extracts

Background

High Performance Thin Layer Chromatography (HPTLC) is an advanced analytical technique used for the identification and quantification of marker compounds in Ayurvedic formulations.

Aims and Objectives

The purpose of this study is to create and validate an HPTLC method for the simultaneous estimation and quantification of four marker compounds: Vasicine (VS), Gallic Acid (GA), Piperine (PP), and Pterostilbene (PTS) from two formulations: Amarantha Gynorite syrup and Amarantha Gynorite capsule.

Materials and Methods

A precoated silica gel aluminum plate (G60F254) was selected as the stationary phase to separate the four markers. The samples were developed using methanol. The mobile phase was composed of a mixture of Toluene, Ethyl acetate, Formic acid, and Glacial Acetic Acid (GAA) in a 2:1:1:0.75 v/v ratio. Quantification was performed at the isosbestic point of 280 nm utilizing the absorbance reflectance mode.

Results

The Retention factor (R_f) values for VS, GA, PP, and PTS were determined to be 0.11, 0.34, 0.68, and 0.71, respectively. Linearity was achieved within the range of 5-50 μg/mL for all four compounds, with correlation coefficients falling within acceptable limits. Satisfactory recovery rates of 100.21%, 99.96%, 100.66%, and 99.28% were recorded for VS, GA, PP, and PTS, respectively. The analysis results were validated concerning accuracy and precision. The Limits of Detection (LOD) were found to be 0.662 ng, 1.573 ng, 1.152 ng, and 1.688 ng for VS, GA, PP, and PTS, while the Limits of Quantification (LOQ) were 2.018 ng, 4.769 ng, 3.492 ng, and 5.115 ng for the same compounds, respectively. The results obtained from the validation tests indicate that the developed simultaneous method is both accurate and reliable.

Conclusion

The HPTLC method established in this study offers a quicker and more cost-effective approach for the simultaneous analysis of VS, GA, PP, and PTS from Amarantha Gynorite syrup and Amarantha Gynorite capsule.