A validated high-performance thin-layer chromatography method for the simultaneous quantification of amino acids and sugars in Eulophia nuda Lindl. corm

Abstract

Eulophia nuda Lindl. is an edible orchid having numerous folklore claims in India. The aim of the present study was the quantification of amino acids and sugars in this species by using a validated high-performance thin-layer chromatography method. Several sets of solvent systems were tried; based on the separation and peak area, a fingerprint of amino acids and sugars was developed in the tertiary solvent of n-butanol–acetic acid–water at variable concentrations. Densitometric quantification of amino acids and sugars was done at 500 nm and 450 nm. The results suggest the presence of l-arginine monohydrochloride, dl-aspartic acid, glutamic acid, and leucine amino acid in this species. Besides, the corm also contains sucrose and fructose. The retention factor (R_F) of amino acids, viz., l-arginine monohydrochloride, dl-aspartic acid, glutamic acid, and leucine, was 0.049 ± 0.001, 0.164 ± 0.01, 0.261 ± 0.02, and 0.572 ± 0.01, respectively, and of sugars (sucrose and fructose) was 0.35 ± 0.03 and 0.479 ± 0.02. The developed method was found to be linear in the concentration range of 50–250 ng spot⁻¹ for amino acids and 200–600 ng spot⁻¹ for sugars. Intraday (reproducibility) and interday (repeatability) precision were within the acceptance limits of International Council for Harmonisation (ICH) guidelines. The recovery of amino acids and sugars ranged from 99.49% to 100.87% and from 99.73% to 100.49% for the developed method. Among the quantified amino acids, dl-aspartic acid (10.46 ± 0.12 µg mg⁻¹) showed the highest content in this species, whereas the content of fructose (11.07 ± 0.26 µg mg⁻¹) was higher than that of sucrose.