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The objective of the present study was to develop an effective method of High Performance Liquid Chromatography-Refractive Index Detector (HPLC-RID) for the simultaneous quantification of stachyose, raffinose and galactose in enzymatic degradation of stachyose. The three sugars were separated on an Agilent zorbax carbohydrate column with acetonitrile water (75:25 v/v). The calibration curves of the three standards exhibited good linearity (R 2 >0.9989). The Limits of Detection (LOD) values of the stachyose, raffinose and galactose were 0.137, 0.301 and 0.386 μg/mL, respectively and their Limits of Quantification (LOQ) values were 0.452, 0.993 and 1.25 μg/mL, respectively. The Relative Standard Deviations (RSDs) of the data of the intra- and inter-day experiments were less than 2.68, 2.42 and 3.70%, respectively. The results of the recovery test were 97.71-102.61% with an RSD value of 0.92-2.41%. In conclusion, the results of validation showed that the proposed HPLC-RID method was useful for the quantification of the three sugars in enzymatic degradation of stachyose and similar reaction.

Rapid Simultaneous Analysis for Three Sugars Fraction in Enzymatic Degradation of Stachyose