Determination of Rebaudioside A and Stevioside in Beverage and Stevia Samples by High-Performance Liquid Chromatography

Khajadpai Thipyapong, Kittiya Deelert, Somsak Sirichai


The optimized conditions for the determination of two steviol glycosides, namely rebaudioside A and stevioside in beverage and stevia samples by using high-performance liquid chromatography coupled with diode array detector was studied. Analytical column was Luna C18 (4.6 mm x 250 mm, 5 micrometer). Both analytes were detected at 210 nm. It was found that the optimized mobile phase comprised acetonitrile and water (pH 3.5) in ratio of 35:65 (%v/v). The flow rate of mobile phase was 0.60 mL/min. Under these optimized conditions, analysis time was about 8 min. The limits of detection were 0.03 mg/L for rebaudioside A and stevioside, and the limits of quantitation were 0.10 mg/L for rebaudioside A and stevioside. Correlation coefficients of calibration curves from 0.01 to 5.00 mg/L were 0.9990 for rebaudioside A, and 0.9998 for stevioside. Intra-day precisions in terms of percent relative standard deviation of retention times and peak areas were 0.17-2.36 for rebaudioside A, and 00.10-2.04 for stevioside. Inter-day precisions in terms of percent relative standard deviation of retention times and peak areas were 0.07-3.99 for rebaudioside A, and 0.11-6.94 for stevioside. Accuracy of the purposed methods for rebaudioside A and stevioside in terms of percent recovery was in the range 92.0-101.3. The proposed method could be applied to determine rebaudioside A and stevioside in beverage and stevia samples.


Keywords :  rebaudioside  A, stevioside, stevia, high-performance liquid chromatography

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