Optimization of extraction methods with alkali and determination of stachyose, sucrose, and raffinose in fresh rehmannia (Rehmannia glutinosa Libosch) using high-performance liquid chromatography with evaporative light scattering detection

A high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was used to determine the stachyose, sucrose, and raffinose in Rehmannia glutinosa Libosch simultaneously. Carbohydrates were separated on an XBridgeTM Amide column using a mixture solvent of acetonitrile: water (70:30, v/v) as mobile phase. Four different methods for the extraction and purification of stachyose in fresh rehmannia (FR) tubers were detailed and evaluated. According to the analysis of the three factors in orthogonal array design (OAD), the optimal combination parameters of the processing technology were as follows: pH value of 12.0, extraction time of 2 h and ethanol concentration of 5%. The extraction method with alkali can be used to extract the carbohydrates completely from the FR. The stachyose content (652.01 mg/g) obtained using the extraction method with alkali was obviously higher than that using other methods. Interestingly, the results show that the stachyose content decreased by approximately 30 to 40% when precipitated with the addition of ethanol. Therefore, the current method is suitable for the determination of sugars in FR and provides the basis for industrial production and purification in future studies.   Key words: Fresh rehmannia, alkali, oligosaccharide, evaporative light scattering detection, orthogonal array design.