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Title: Development and Validation of a Decigram-Scale Method for the Separation of Limonin from Limonin Glucoside by C-18 Flash Chromatography

item Breksa, Andrew
item Dragull, Klaus

Submitted to: Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/18/2008
Publication Date: 4/15/2009
Citation: Breksa III, A.P., Dragull, K.D. 2009. Development and Validation of a Decigram-Scale Method for the Separation of Limonin from Limonin Glucoside by C-18 Flash Chromatography. Food Chemistry. 113:1308-1311.

Interpretive Summary: After learning that the limonin glucoside containing beverages to be used for the human study were too bitter for consumption due to the presence of limonin (less than 1%), a compound in the same family as limonin glucoside, but structurally different, researchers in the Processed Foods Research Unit, Albany, CA, quickly developed a pilot-scale chromatographic method for removing residual limonin. Purity of the material treated by this polishing method increased to 99.9% with only a minimal loss of the starting material. The two-step process uses only ethanol and water as the solvents and is readily scalable to a production scale method that may be used by producers whose customer requirements include increased purity.

Technical Abstract: A preparative method for decigram-scale polishing of limonin glucoside with regard to removing traces of limonin, the main bitter principle in Citrus, is reported. During the method development and up-scaling stages, in which sample purity, sample size, solvent amounts, and drying conditions were varied, a rapid isocratic HPLC method with UV absorbance detection was used to monitor the elution behavior of limonin and subsequently, fractions obtained using the finalized method were analyzed for their limonoid glucoside and aglycone content in a single HPLC run using a binary gradient HPLC method and an evaporative light scattering detector. The resulting polishing method consists of a simple step gradient that utilizes food-grade solvents, ethanol and water at 15% EtOH and 50% EtOH steps. Using a 75 x 300 mm C-18 column, this method is capable of processing twenty-grams of material per run in less than 3 h. Recovery of the purified limonin glucoside following evaporation of the solvent was 93.5% (+/-2.8, n=6) and the limonin concentrations in the resulting materials were found to be reduced 10- to 15-fold.