Development and Validation of a HPLC-UV Method for Simultaneous Determination of Lutein and β -Carotene in Food Formulated with Lutein Diester and β-carotene

A simple and rapid method has been developed for determination of lutein and β-carotene in food formulated with lutein diester beadlet, microencapsulated β-carotene as ingredients by reverse-phase high performance liquid chromatograph. This method combines extraction and saponification to one step reaction and lack of evaporation. Dimethyl sulfoxide (DMSO) was selected as solvent to release lutein diester, β-carotene from lutein diester beadlet and microencapsulated β-carotene, then add acetone-ethanol-20%KOH/methanol (10:5:4, v/v/v) to saponify lutein diester as well as extract lutein and β-carotene directly at the same time. The prepared sample solution was subjected to a HPLC analysis using Athena C30 column eluting with Methanol / methyl tert-butyl ether (MTBE) mobile phase. The precision, accuracy, linearity, specificity and ruggedness of the developed method were validated following AOAC guidance for single laboratory validation procedure. The recovery of lutein and β-carotene are 94.80%-104.76% and 98.94%-104.69% respectively. The injection repeatability: RSD(r) =1.8% for lutein, RSD(r) =5.5% for β-carotene. This method developed can be used for the quantitation of free lutein and β-carotene in products adding with lutein diester and β-carotene in quality control.