Establishment of HPLC fingerprint and determination method of three index components of classical herbal formula substance benchmarks of Xiebai Powder

Yan-Shuai WANG

ABSTRACT

Objective:

To establish the HPLC fingerprint and the determination method of three index components of the classical herbal formula substance benchmarks of Xiebai Powder.

Methods:

Fingerprint chromatographic conditions were as following detection wavelength 254 nm/325 nm, column temperature 35 ℃; flow rate 0.8 mL/min; injection volume 25 μL; mobile phase consisting of 0.1% aqueous formic acid (A) and acetonitrile (B); binary gradient elution 0-20 min, 5%-10% B; 20-33 min, 10%-15% B; 33-50 min, 15%-20% B; 50-95 min, 20%-58% B. Ten batches of substance benchmarks of Xiebai Powder fingerprints were collected and evaluated by the Chinese Pharmacopoeia Committee "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software. Chromatographic conditions of content determination detection wavelength 237 nm, column temperature 30 ℃; flow rate 1.0 mL/min; injection volume 5 μL; mobile phase consisting of 0.1% aqueous phosphoric acid (A) and acetonitrile (B) for binary gradient elution 0-10 min, 5%-20% B; 10-18 min, 20%-60% B; 18-26 min, 60%-100% B; 26-38 min, 100% B; 38-41 min, 100%-5% B; 41-45 min, 5% B.

Results:

Based on the matching results, 55 common peaks were determined at a wavelength of 254 nm, and 57 common peaks were determined at a wavelength of 325 nm. Three substances, mulberroside A (S), liquiritin and ammonium glycyrrhizinate, were identified in the common peaks. After methodological research, its precision, stability and reproducibility were good. Ten batches of substance benchmarks of Xiebai Powder fingerprints were evaluated with reference fingerprints, and their similarities were greater than 0.9. The average recovery rates of mulberroside A (S), liquiritin and ammonium glycyrrhizinate were 97.82%, 97.40% and 105.81%, respectively. The RSD (n = 6) was 4.41%, 2.51% and 1.19%, respectively, which met the require of 2015 edition of the Chinese Pharmacopoeia. The three components had good linearity in the range of 25.25-2525 ng, 25-2 500 ng and 8.5-850 ng, respectively. The method had good precision, stability and repeatability. The contents of 10 batches of substance benchmarks of Xiebai was determined. The content of mulberry A was 11.6-35.5 mg/g, the content of liquiritin was 0.1-1.6 mg/g, and the content of glycyrrhizic acid was 0.3-2.5 mg/g. The range of the contents of these ingredients was large, which indicated that the quality of mulberry husks and licorice herbs from different places was quite different.

Conclusion:

The establishment of the HPLC fingerprint and the determination method of three index components of the classical herbal formula substance benchmarks of Xiebai Powder provided some bases for the study of the quality standard of substance benchmarks of Xiebai Powder.