Quantitative determination of diosgenin in Dioscorea zingiberensis cell cultures by microplate-spectrophotometry and high-performance liquid chromatography

Abstract

Quantitative determination of diosgenin was carried out by multi-well microplate-spectrophotometry and high-performance liquid chromatography (HPLC) in this study. Diosgenin detection by microplate-spectrophotometry depended on the formed yellow substance developed by perchloric acid, and measured at 410 nm. The calibration graph was linear over the range of 2 to 10 μg diosgenin in each well with correlation coefficient (R) as 0.9988, detection limit as 0.6111 μg, and quantification limit as 1.8518 μg. For HPLC analysis, diosgenin detection was conducted at 203 nm on an Agilent TC-C18 column with a mobile phase of acetonitrile-water (90:10, v/v). The calibration graph was linear over the range of 0.0625 to 1.000 µg diosgenin with correlation coefficient (R) as 0.9995, detection limit as 0.0372 μg, and quantification limit as 0.1127 μg of diosgenin. Both methods were characterized by satisfactory precision and accuracy, which were then employed to detect diosgenin content in cell cultures of Dioscorea zingiberensis. No statistical significant differences were observed between the results obtained by microplate-spectrophotometry and HPLC (ANOVA, p < 0.05). Quantitative determination of diosgenin by microplate-spectrophotometry should be a rapid, accurate, simple and economic alternative method, though HPLC is more sensitive for diosgenin analysis. Key words: Diosgenin, Dioscorea zingiberensis, cell cultures, microplate-spectrophotometry, high-performance liquid chromatography, quantitative determination.