高效液相色谱法测定静电层层自组装载药多层膜中葛根素的载药量

Determination of Puerarin Loading Content in Electrostatic Layer-by-layer Self-assembly Multilayer Films by HPLC

[目的]建立测定静电层层自组装载药多层膜内葛根素载药量的高效液相色谱(high performance liquid chromatography,HPLC)方法。[方法]多层膜浸没于十二烷基硫酸钠饱和溶液,超声崩解,崩解液冻干后甲醇定容,由HPLC法定量测定。葛根素的定量分析采用Hypersil BDS C18色谱柱(4.6mm×250mm,5μm),流动相为甲醇-0.2%甲酸(18/82),流速1.0mL·min-1,检测波长250nm,柱温30℃,进样量为10μL。[结果]在本研究确定的色谱条件下,葛根素浓度2.0~12.0μg·mL-1范围内,葛根素浓度与出峰面积线性关系良好(r=0.9994)。以HPLC法测定静电层层自组装多层膜内葛根素载药量精密度、重复性和稳定性良好;在低、中、高三个剂量水平下的空白回收率为96.98%~102.34%。利用该方法定量测定10、15和20双层载药多层膜中葛根素的载药量,随着多层膜组装层数增加,载药量显著增大,葛根素含量分别为(1.92±0.54)μg·cm-2、(2.58±0.52)μg·cm-2和(4.23±0.77)μg·cm-2,差异有统计学意义(P<0.05)。[结论]以HPLC法测定静电层层自组装多层膜内葛根素载药量,方法简便、条件温和、结果可靠,为静电组装多层膜的载药量测定提供了参考。

[Objective]To establish a method for determination of the puerarin loading content in electrostatic layer-by-layer self-assembly multilayer films by high performance liquid chromatography(HPLC).[Methods]Multilayer films were immersed in a saturated solution of sodium dodecyl sulfate and deconstructed after sonication.After lyophilization,the mixture was dissolved in methyl alcohol.The quantitative analysis of puerarin was carried out with Hypersil BDS C18 chromatographic column(4.6 mm×250 mm,5μm),the mobile phase was methanol-0.2%formic acid(18/82)at a flow rate of 1.0 mL·min-1,the detection wavelength was 250 nm,the column temperature was 30℃,and the injection volume was 10μL.[Results]Under the chromatographic conditions determined in this research,the linear relationship between puerarin concentration and peak area was good with puerarin concentration in the range of 2.0~12.0μg·m L-1(r=0.9994).The precision,repeatability and stability of determination of puerarin loading content in electrostatic layer-by-layer self-assembly multilayer films by HPLC were satisfying.And the blank recovery rate at low,medium and high dose levels were 96.98%~102.34%.The method was successfully applied for quantitative determination of puerarin loading content in 10,15 and 20 double-layer drug loaded multilayer films.With the increase of the layer number of multilayer films,the drug loading content increased significantly,puerarin content was(1.92±0.54)μg·cm-2,(2.58±0.52)μg·cm-2 and(4.23±0.77)μg·cm-2,respectively,and the difference was statistically significant(P<0.05).[Conclusion]HPLC method was successfully established for the determination of puerarin loading content in electrostatic layer-by-layer self-assembly multilayer films.With the advantages of simplicity,mild condition and reliability,this method can provide a good strategy for quantitative determination of drugs loaded in electrostatic layer-by-layer self-assembly multilayer films.