A method for the separation and determination of lactose and sucrose using high performance liquid chromatography-refractive index (HPLC-RI) detection base on the prepared amino-bonded silica gel chromatographic stationary phase was established. The method was accorded to the content determination method in Pharmacopoeia of the People's Republic of China (2015). Then, a series of chromatographic behaviors of lactose and sucrose such as the retention times, resolutions and peak area stabilities were investigated on three different types of amino-bonded silica columns (300 mm×4.6 mm, 5 μm). The mobile phase was acetonitrile-water (70:30, v/v) with isocratic elution, the flow rate was 1.0 mL/min and the injection volume was 10 μL. The resolution of lactose and sucrose was 3.03 by isopropyl side chain protected amino column, and the peak shape of each target compound was good. The relative standard deviation (RSD) of peak area of lactose was 1.14%, less than 2.0% prescribed in the Pharmacopoeia of the People's Republic of China (2015). Therefore, this method meets the requirements of lactose content determination, and is suitable as the quality control method in the above mentioned Pharmacopoeia.
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