THIN-LAYER AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ANALYSES OF LIMONOIDS AND LIMONOID GLUCOSIDES IN CITRUS SEEDS

A routine method for the analysis of limonoids and limonoid glucosides in citrus seeds, which utilizes thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), is described. Seed meals were washed with n-hexane to remove oily materials, after which limonoids and limonoid glucosides were extracted with acetone and then methanol. The methanol extract contains the remainder of the limonoid aglycones and all limonoid glucosides. After the methanol was evaporated off, the residue was extracted with methylene chloride-water (1:1). The limonoids of both the acetone and methylene chloride fractions were separated and quantified by HPLC. The total limonoid glucoside content of the water fraction was determined by silica gel TLC. Each of the limonoid glucosides was then eluted from the reversed-phase HPLC column by a linear gradient system starting at 15% acetonitrile in 3 mM phosphoric acid and ending with 26% acetonitrile after 35 min, and quantitated by spectrophotometric detection at 210 nm. The limonoid aglycones and limonoid glucosides in two kinds of citrus seeds, Shiikuwasha (Citrus depressa) and Iyo (Citrus iyo), were determined. The content of limonoid glucosides in Shiikuwasha was found to be approximately two-fold higher than that of Iyo.