Determination of five chemical markers in DF formula, the herbal composition of Ephedra intermedia, Rheum palmatum, and Lithospermum erythrorhizon, Using High-performance Liquid Chromatography-ultraviolet Detection
Birang Jeong1, Jong Seong Roh2, Michung Yoon3, Yoosik Yoon4, Soon Shik Shin2, Hyun Joo Cho5, Yong Soo Kwon1, Heejung Yang1
1 Laboratory of Natural Products Chemistry, College of Pharmacy, Kangwon National University, Chuncheon 24341, Republic of Korea
2 Department of Formula Sciences and Research Center of Korean Medicine for Diabetes and Obesity, College of Korean Medicine, Dong-Eui University, Busan 47227, Republic of Korea
3 Department of Biomedical Engineering, Mok-Won University, Daejeon 35349, Republic of Korea
4 Department of Microbiology, College of Medicine, Chung-Ang University, Seoul, Republic of Korea
5 Department of Research and Development, Hamsoa Pharmaceutical Co., Ltd., Seoul, Republic of Korea
Laboratory of Natural Products Chemistry, College of Pharmacy, Kangwon National University, Chuncheon 24341
Republic of Korea
Source of Support: None, Conflict of Interest: None
Background:DF formula is a herbal preparation comprised three medicinal herbs, namely, Ephedra intermedia, Rheum palmatum, and Lithospermum erythrorhizon, which is being used for the treatment of obesity and liver fibrosis in Korean local clinics. Objective: Since the abovementioned three herbs exist with different proportions in DF formula and their chemical markers have different physiochemical properties; it is quite challenging to develop an analytical methodology for the determination of these chemical markers. Materials and Methods: For the analysis of the three herbs, five chemicals, (+)-pseudoephedrine (1) and (−)-ephedrine (2) for E. intermedia, aloe-emodin (3), and chrysophanol (4) for R. palmatum, and shikonin (5) for L. erythrorhizon, were selected for method validation of DF formula, and the analytical conditions were optimized and validated using high-performance liquid chromatography coupled with an ultraviolet detector (HPLC-UV). Results: The specificities for the five compounds 1–5 were determined by their UV absorption spectra (1–4: 215 nm and 5: 520 nm). Their calibration curves showed good linear regressions with high correlation coefficient values (R2 > 0.9997). The limits of detection of these five markers were in the range 0.4–2.1 ng/mL, with the exception of 5 (12.7 ng/mL). The intraday variability for all the chemical markers was less than a Relative standard deviation (RSD) of 3%, except for 5 (RSD = 12.6%). In the case of interday analysis, 1 (1.0%), 2 (3.1%), and 4 (3.7%) showed much lower variabilities (RSD < 5%) than 3 (7.6%) and 5 (8.2%). Moreover, the five chemical markers showed good recoveries with good accuracies in the range of 90%–110%. Conclusions: The developed HPLC-UV method for the determination of the five chemical markers of the components of DF formula was validated.
Abbreviations used: EP: (−)-ephedrine; PSEP: (+)-pseudoephedrine; HPLC: High-performance liquid chromatography; UV: Ultraviolet; LOD: Limit of detection; LOQ: Limit of quantification; RSD: Relative standard deviation.