Determination of Citrus Limonoid Glucosides by High Performance Liquid Chromatography Coupled to Post-Column Reaction with Ehrlich’s Reagent

A method for the identification and quantification of citrus limonoid glucosides in juices, based upon high performance liquid chromatography (HPLC) separation coupled to post-column reaction with Ehrlich’s reagent, has been developed. This method utilizes a phenyl stationary phase and an isocratic mobile phase. Limit of detection for the method was determined to be 50 ng of limonin glucoside. Limonin glucoside and the other limonoid glucosides tested responded equivalently to the post-column reaction with Ehrlich’s reagent suggesting that limonin glucoside may be used as a surrogate for the quantification of other limonoid glucosides, including nomilin, deacetyl nomilin, nomilinic acid, deacetyl nomilinic acid, and obacunone glucosides. For most juice samples, no sample treatment is required to determine limonin glucoside content. Concentration by solid phase extraction is recommended for juices with low levels of limonin glucoside and to facilitate the detection and quantification of other limonoid glucosides present at low concentrations. With the exception of blood oranges, the method was successfully applied to the analysis of juices from a variety of citrus varieties.