Development and Application of an UHPLC-MS/MS Method for Comparative Pharmacokinetic Study of Eight Major Bioactive Components from Yin Chen Hao Tang in Normal and Acute Liver Injured Rats

Abstract

Yin Chen Hao Tang (YCHT) is one of the most famous hepatoprotective herbal formulas in China, but its pharmacokinetic investigation in model rats has been rarely conducted. In this study, the hepatic injury model was caused by intraperitoneal injections of carbon tetrachloride (CCl₄), and YCHT was orally administered to the model and normal rats. An ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established to analyze the plasma pharmacokinetics of eight major bioactive ingredients from YCHT in both the normal and liver injured rats. The calibration curves presented good linearity (r > 0.9981) in the concentration range. The relative standard deviation (RSD%) of inter- and intraday precision was within 9.55%, and the accuracy (RE%) ranged from -10.72% to 2.46%. The extraction recovery, matrix effect, and stability were demonstrated to be within acceptable ranges. The lower limit of detection (LLOD) and lower limit of quantitation (LLOQ) were around 0.1 ng/mL and 0.5 ng/mL, respectively, which were much lower than those in other related researches. Results reveal that there are significant differences in the pharmacokinetics of scoparone, geniposide, rhein, aloe-emodin, physcion, and chrysophanol in hepatic injured rats as compared to those in control except for scopoletin and emodin. Our experimental results provide a meaningful reference for the clinical dosage of YCHT in treating liver disorders, and the improvement of LLOD and LLOQ can also broaden the range of our method's application, which is very suitable for quantitating these eight compounds with low levels.

Figure 1

Representative DMRM chromatograms for scoparone, scopoletin, geniposide, aloe-emodin, rhein, emodin, chrysophanol, physcion, and their internal standards glibenclamide and wogonin in rat plasma samples. Panel A: a blank plasma sample; panel B: a blank plasma sample spiked with bioactive compounds and ISs at the LLOQ; panel C: a rat plasma sample collected at 30 min after drug administration.

Figure 2

Biochemical and physiological parameters in the experimental rats. (a) Biochemical parameters of normal and model rats. (b) Representative photomicrographs of histopathological studies of livers stained with hematoxylin and eosin (100 ×).

Figure 3

Mean concentration-time profiles of (a) scoparone, (b) scopoletin, (c) geniposide, (d) aloe-emodin, (e) rhein, (f) emodin, (g) chrysophanol, and (h) physcion based integrated concentration in control and model rat plasma after oral administration of YCHT (n = 6).