LC-MS/MS方法研究新型酪氨酸酶抑制剂UP302的大鼠皮肤吸收量和皮肤代谢稳定性

LC-MS/MS Determination of a Natural Novel Tyrosinase Inhibitor UP302 in Rat Skin Homogenate and Its Application to Evaluate Skin Absorption Amount and Skin Metabolic Stability

目的建立大鼠皮肤中UP302定量的LC-MS/MS方法,并应用于研究UP302乳膏在大鼠皮肤局部给药24h后的皮肤吸收量,以及离体大鼠皮肤中UP302的代谢稳定性。方法 UP302用甲醇溶解稀释,皮肤样品用2倍体积甲醇沉淀处理,内标法进行定量。采用Hypersil Gold C18色谱柱,柱温30℃;甲醇为流动相A,5mmol.L-1甲酸铵水溶液为流动相B,以0.2mL.min-1梯度洗脱,进行色谱分离,运行时间6min。采用负离子电喷雾离子化电离源和选择反应监测(SRM)模式进行串联质谱分析,用于定量分析的离子反应分别为m/z 301.1→135.2(UP302)和m/z 252.9→132.0(内标大豆苷元)。结果 UP302皮肤标准样品在5～2000ng.mL-1的浓度范围内线性关系良好(r=0.9998)。UP302在大鼠皮肤匀浆中的最低定量限是5ng.mL-1。本方法日内准确度在100.00%～105.23%,日内精密度小于5.82%。结论本LC-MS/MS方法专属性强、灵敏度高、重现性好、操作简便、结果准确,可用于UP302在皮肤组织中含量的测定,以及UP302在大鼠皮肤组织中的代谢稳定性研究。

Objective To establish an LC-tandem mass spectrometric method for determination of UP302 in rat skin,quantify skin absorption amount of UP302 after cream applied to rat skin,and evaluate metabolic stability incubated in rat skin.Methods The methodology involved extraction of the analyze from rat skin samples by methanol and daidzein as an internal standard.The chromatographic separation was achieved within 6 min on Hypersil Gold C18 column with gradient mobile phase containing a mixture of methanol and 5 mmol·L-1 ammonium formate in water at the flow rate of 0.2mL·min-1 at 30℃.Selective reaction monitoring transitions of m/z 301.1→135.2 and 252.9→132.0 were measured in negative electrospray ionization mode for UP302 and daidzein,respectively.Results The calibration curves were well linear in the range of 5-2000ng·min-1 with the correlation coefficient of 0.9998.The lower limit of quantitation was 5ng·mL-1.The intra-day accuracy was between 100.0% and 105.23%,and precision was less than 5.82%.Conclusion The LC-MS/MS method is simple,sensitive,precise,accurate and suitable for the quantification of UP302 in rat skin.The method is applied to quantify the absorption amount of UP302 in rat skin after UP302 cream topical treatment,and evaluate the metabolic stability of UP302 in rat skin homogenate.