Simultaneous determination of six main components in Bushen Huoxue prescription by HPLC-CAD

The Bushen Huoxue prescription is a traditional Chinese medicine formula treating diabetic retinopathy, which was developed by our research group. Catalpol, puerarin, salvianolic acid B, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Rd are six of main effective components, which could be partly representative of this prescription. The corona charged aerosol detector (corona CAD) is one kind of universal detectors equipped with the high performance liquid chromatography (HPLC). The CAD has many advantages for the analysis of complex mixtures, but too few applications in traditional Chinese medicine compounds.

The aims of this study are to establish a method for the determination of six components in Bushen Huoxue prescription, and to increase the use of the CAD in traditional Chinese medicine compounds.

HPLC-CAD analysis was performed on an Inertsil ODS-SP (4.6 mm × 250 mm, 5 μm) with a mobile phase consisting of 0.5 % formic acid solution(A)-acetonitrile(B) at a flow rate of 1 mL/min (0-7 min, 1 % B; 7-12 min, 1 %-12 % B; 12-22 min, 12 %-19 % B; 22-40 min, 19 %-28 % B; 40-43 min, 28 %-33 % B; 43-50 min, 33 % B; 50-65 min, 33 %-42 % B). The column temperature maintained at 30 ℃, the injection volume was 20 μL, the atomization temperature mode was LOW, the filtration constant (filter) was 3.6 and data collection rate was 10 Hz. The methodology was examined and the linearity of regression of different functions was compared. Sixteen batches of samples were prepared and their contents were determined.

The six compounds showed a better linearity (R² > 0.9990) in their concentration ranges when using the linear function. The average recoveries were 99.18 %-101.30 %. Although the RSD value of puerarin and ginsenoside Rg1 was slightly out of 3 % during the average recovery investigation, all the other methodological investigations of the six components were within 3 %. The precision, stability and repeatability of the method were good. In sixteen batches of Bushen Huoxue prescription samples, the contents of six components were 0.3138 %-0.6042 % for catalpol, 0.8095 %-1.2917 % for puerarin, 0.7416 %-1.1189 % for salvianolic acid B, 0.0231 %-0.0418 % for ginsenoside Rg1, 0.0702 %-0.1724 % for ginsenoside Rb1, 0.0384 %-0.1196 % for ginsenoside Rd.

In this experiment, a method for the determination of six components in Bushen Huoxue prescription based on HPLC-CAD was established with high accuracy, good repeatability and simple operation, and it can provide references for the improvement of quality standard of the Bushen Huoxue prescription. It is reasonable and accessible for the CAD application in the determination of traditional Chinese medicine compound prescriptions.