Development and Validation of HPLC and HPTLC Methods for Estimation of Glabridin in Extracts of Glycyrrhiza glabra

Abstract

Glabridin is a major bioactive phytoconstituent of licorice. This work discusses the development and validation of HPLC and HPTLC methods for analysis of glabridin in licorice. The HPLC separation was performed using a Purospher STAR RP-18e column (5 μm silica particle size, 250 mm × 4.6 mm inner diameter) with gradient elution of 0.2% acetic acid in water-acetonitrile. The flow rate was 1 mL/min. Quantification was performed at a detection wavelength of 280 nm. HTPLC separation was performed on precoated silica gel 60 F254 aluminum plate (10 × 10 cm, 250 μm thickness). A linear ascending development was done using a mobile phase of hexane-ethyl acetate-chloroform (5 + 4 + 3, v/v/v). After development, the plates were scanned at 285 nm. Both of the methods provided good separation of glabridin from other constituents of licorice extract. The methods were validated as per ICH guidelines. Comparison by Student t-test showed that there was a statistically insignificant difference between the mean glabridin content estimated by both methods at 95% confidence interval. The glabridin content in licorice extract was 3.90% by HPLC and 3.79% by HPTLC.

Construction of a monoclonal antibody against glabridin (2G4) and development of an enzyme‐linked immunosorbent assay

“…The HPLC analysis was performed according to a previously published protocol. 9 The UV-visible detector was operated on a Shimadzu (i-series; Kyoto, Japan) at a detection wavelength of 254 nm. The Purospher STAR (RP-18 endcapped, 250 mm  4 mm, 5 μm) used a gradient mobile phase between acetonitrile (A) and 1.5% acetic acid (B), with a constant flow rate of 1 ml/min.…”

“…When comparing the sensitivity of the two methods, HPLC 9 and ELISA had LODs equal to 0.5 and 0.16 μg/ml, and limit of quantification (LOQ) equal to 2 and 0.28 μg/ml, respectively. ELISA was more sensitive for glabridin analysis.…”

“…The HPLC analysis was performed according to a previously published protocol 9 . The UV‐visible detector was operated on a Shimadzu (i‐series; Kyoto, Japan) at a detection wavelength of 254 nm.…”

“…However, there is a gap in the identification of other active compounds, including glabridin, which are important pharmacological components of this plant. Previous reports on the development of glabridin analysis involved the use of HPLC with a UV detector 9,10 . However, as glabridin has limited UV absorption, the detection sensitivity of this method is inadequate.…”

“…Previous reports on the development of glabridin analysis involved the use of HPLC with a UV detector. 9,10 However, as glabridin has limited UV absorption, the detection sensitivity of this method is inadequate. A literature review indicated that an immunological method using antibodies to develop an enzyme-linked immunosorbent assay (ELISA) method for licorice active compounds can enhance the sensitivity of the analysis compared to the conventional HPLC method.…”