Determination of Steviol Glycosides in Fermented Milk by High Performance Liquid Chromatography

A method for the determination of stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside F, dulcoside A, rubusoside and steviolbioside in fermented milk by high performance liquid chromatography (HPLC) was developed and validated. The chromatographic conditions and sample pretreatment conditions were optimized. Samples were ultrasonically extracted with water for 20 min and for another 10 min after addition of 30% acetonitrile in water bath at 45 ℃. Potassium ferrocyanide and zinc acetate were added to precipitate proteins. The chromatographic separation was achieved on a C₁₈ with gradient elution using a mobile phase consisting of sodium phosphate buffer and acetonitrile, and the analytes were detected by an ultraviolet (UV) detector at 210 nm. The results showed that the method was linear in the concentration range of 1–50 μg/mL, the limit of detection (LOD, R_S/N = 3) was 3.0 mg/kg, and the limit of quantitation (LOQ, R_S/N = 10) was 10.0 mg/kg. The average recoveries of all analytes spiked at 15–200 mg/kg were between 92.0% and 103.1%, with relative standard deviation (RSDs) (n = 6) less than 3.0%. This method was rapid, accurate, stable, sensitive, and suitable for the analysis of the content of steviol glycosides in fermented milk.