Determination of ceramide NP and AP content in liposomes by high performance liquid chromatography-evaporative light scattering
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.
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