Development of a MD-LC-MS/MS Method to Analyze 3 Bioactive Compounds in Huoxuezhitong Rubber Patch and Application to a Pharmacokinetic Study in Rats

Huoxuezhitong rubber patch, a well-known traditional Chinese medicine (TCM) prescription, is utilized to treat pain and inflammation. In this study, a microdialysis-ultra-high-performance liquid chromatography-tandem mass spectrometry (MD-LC-MS/MS) method was designed for the simultaneous determination of active constituents in the rubber patch, such as paeonol (Pae), eugenol (Eug), and piperine (Pip). A microdialysis probe was implanted in the subcutaneous tissue of a rat, which is intended to detect the subcutaneous concentrations of target components. Saline containing 30% ethanol acted as perfusion fluid. Analytes in the microdialysate were completely separated over an ACQITY UPLC RBEH C(18) column (2.1mm×100mm, 1.7μm). The mobile phase was composed of 0.01% ammonia aqueous and acetonitrile-0.01% ammonia with gradient elution. The single-run analysis time was 10.0 minutes. The linear regression displayed good linear relationships in the ranges of 0.25–100 ng/mL for paeonol and eugenol and 0.001–5 ng/mL for piperine. The interday and intraday precision of the quality control samples exhibited relative standard deviations (RSD) <13.56%. The accuracy values ranged from −14.92% to 14.00%. The present method was successfully applied in pharmacokinetics studies following dermal administration of Huoxuezhitong rubber patch in rats. Pip's T(max) (488.00±150.73) min was greater than that of Pae (186.67±48.44) min and Eug (240.00±138.56) min, and the rank order of t(1/2) was Pae > Pip > Eug. The rank order of AUC(0-720) and C(max) was both Eug > Pae > Pip. MRT(0-∞) of Pip was higher than that of Pae and Eug. Eugenol showed a faster elimination and a shorter half-life. Paeonol showed a stronger drug reservoir function after removing the drug source.