Abstract:

Simultaneous chromatographic separations of ceramide NP and ceramide AP in ceramide liposomes were carried out by high-performance liquid chromatography （HPLC） coupled to evaporative light scattering detector （ELSD）. The ceramide liposomes samples were treated with methanol-isopropanol （volume ratio 7:3） and analyzed by Agilent ZORBAX StableBond C8 （4.6×150 mm, 3.5 μm） column. The simultaneous separation of ceramide NP and AP was achieved by gradient elution with 0.1% formic acid in methanol-isopropanol （volume ratio 7:3） and acetonitrile as the mobile phases. The flow rate was 1.0 mL/min and the column temperature was 40 ℃. Evaporator and nebulizer temperature were set at 90 ℃ and 70 ℃, and the gas flow rate was 1.0 L/min. The result shows the detection limits of ceramide NP and AP in ceramide liposomes are 3 and 6 μg/mL, respectively, and the quantitative limits are 1 and 2 μg/mL, respectively. The RSD of test on ceramide NP and AP repeatability and precision are all less than 2%, and the linear relationship of calibration curves for ceramide NP and AP is good （R²>0.999） in the mass concentration range of 50-500 μg/mL and 25-250 μg/mL,respectively. Moreover, the recovery rates range from 96.2% to 105.1% at different concentration levels. A simple, reproducible and reliable method is provided for the determination of ceramide NP and AP contents in different ceramide liposomes.

Key words: ceramide NP, ceramide AP, liposomes, high-performance liquid chromatography, evaporative light scattering detector