Objective: Establishment of a HPLC method for six chemical components in Saposhnikovia divaricata (prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisamminoside, sec-O-glucosylhamaudol, psoralen, and imperatorin), comparison of the advantages and disadvantages of the dual-standard linear calibration method and the relative retention time method, and investigation of the feasibility of the linear calibration using two reference substances for qualitative analysis of multiple components in Saposhnikovia divaricata.
Methods: A HPLC method was employed, using 21 different brands and models of C18 columns. The mobile phase consisted of acetonitrile (A) and 0.02% phosphoric acid aqueous solution (B), with gradient elution (0-20 min, 18%A; 20-45 min, 18%A-50%A; 45-60 min, 50%A). The column temperature was set at 30 ℃, the flow rate was 1 mL·min^-1, and the detection wavelength was 254 nm.
Results: The results predicted by the linear calibration using two reference substances were accurate and superior to those of the relative retention time method, and the six components exhibited good linear relationships within their respective ranges.
Conclusion: The established linear calibration using two reference substances accurately predicts the retention times of the target components. This method is stable and reliable, providing a reference for the establishment of digital standards in traditional Chinese medicine.