Identification, Determination and Transdermal Behavior Characterization of Nine Components in WenTong HuoXue Cream by UPLC-MS/MS

Background/Objectives: WenTong HuoXue Cream (WTHXC) plays a significant role in the treatment of diabetic peripheral neuropathy (DPN). However, the material basis and quality control methods for this formulation remain largely unexplored. Methods: In this study, UPLC-HRMS/MS combined with standard reference substances was employed to comprehensively identify and confirm the chemical constituents of WTHXC. Furthermore, a rapid and sensitive UPLC-MS/MS method based on multiple reaction monitoring (MRM) mode was developed and validated for the simultaneous quantification of the marker components. Results: Nine compounds were unambiguous characterized, including Di-hydrocapsaicin (DHC), Oxypeucedanin hydrate (OPH), Imperatorin (IMP), Isoimperatorin (IIMP), Xanthotoxin (XAN), Hydroxysafflor yellow A (HSYA), Chlorogenic acid (CGA), Ferulic Acid (FA) and Ligustilide (LIG). The results of method validation denotes that all the analytes showed good linearity between concentration and peak area in the tested ranges, with correlation coefficients (r) not less than 0.9990. The relative standard deviation (RSD) of precision was in the range of 0.57–7.11%. The accuracy of the method, verified by recovery experiments at three concentration levels, ranged from 96.51% to 101.04% for all analytes. Transdermal behavior determination results demonstrate that OPH, HYSA, CGA, FA and LIG exhibited favorable skin permeability and may serve as the key active components of WTHXC. Conclusions: This study elucidates the material basis of WTHXC, providing a scientific foundation for the development of quality control methods and facilitating its broader clinical application.