兔皮肤中全缘千里光碱的HPLC测定

目的：建立一种兔皮肤中全缘千里光碱测定的HPLC方法。方法：采用X-Terra RP18色谱柱（250 mm×4.6 mm，5 μm），流动相为乙腈-水（23:77，以冰醋酸、氨水调节pH至7.1），流速0.8 mL·min^-1，柱温为室温，检测波长213 nm，进样量20 μL。样品处理过程包括皮肤匀浆、液- 液萃取、氮气吹干有机层、残留物复溶及进样分析。结果：皮肤中的内源性物质对测定无干扰；样品皮肤（1 cm²）中药物含量在0.525~31.5 μg范围内与其峰面积的线性关系良好（r=0.999 8，n=7）；日内、日间精密度良好（RSD ≤ 12.2%）；准确度（相对回收率）为96.2%~105.0%，提取回收率为79.4%~83.7%；复溶的供试品溶液在24 h内稳定（相对回收率为95.2%~100.3%），皮肤样品冷冻- 解冻循环3次测定结果稳定（相对回收率为96.2%~101.8%）；皮肤中全缘千里光碱含量测定结果表明，应用12 h以后全皮及角质层下皮肤中药物滞留量的大小关系为：全缘千里光碱脂质体混悬液> 全缘千里光碱脂质体凝胶剂> 全缘千里光碱普通凝胶剂。结论：该方法的精密度、准确度、稳定性均符合生物样本测定要求，可用于兔皮肤中全缘千里光碱的测定。

Objective:To develop an HPLC method for determination of integerrimine in rabbit skin.Methods: A Terra RP18 column(250 mm×4.6 mm,5 μm)was used with an isocratic elution composed of acetonitrile and water in the ratio of 23:77(adjust pH to 7.1 with acetic acid and ammonia)at a flow rate of 0.8 mL·min^-1.The column was maintained at room temperature.The UV detector was set at 213 nm.The injected volume was 20 μL. The preparation of test solutions involved homogenization of skin samples,liquid-liquid extraction,evaporation of the organic phase to dryness under nitrogen flow,re-dissolution of the residue and sample analysis.Results:The method validation tests showed that endogenous substances of rabbit skin had no interference with the assay of integerrimine.A good linear relation was observed between the amount(0.525-31.5 μg)and the peak area of integerrimine in one square centimeter of skin assayed(r=0.999 8,n=7).The intra-day and inter-day precisions were qualified with RSDs of less than 12.2%,the relative recoveries were between 96.2%-105.0%,the extraction recoveries were ranged at 79.4%-83.7%,the test solutions were stable within 24 h(relative recoveries were between 95.2%-100.3%),and the analyte was stable after three freeze-thaw cycles in skin with recoveries calculated from quality control samples ranging from 96.2%-101.8%.The determination result of integerrimine in the skin showed that the skin deposition amount of integerrimine liposomes,integerrimine liposomes gel and integerrimine ordinary gel in whole skin and the skin beneath stratum after 12 h was as follows:integerrimine liposomes > gerrimine liposomes gel > integerrimine ordinary gel.Conclusion:The established method which is proved to be accurate,precise,and stable,is suitable for the determination of integerrimine in rabbit skin.