Objective: To establish a two reference substances for determination of multiple component method for six components (salvianic acid A, protocatechuic aldehyde, caffeic acid, rosmarinic acid, lithospermic acid and salvianolic acid B)of Danshen formula granules, and to verify the accuracy and feasibility of this method in he quality evaluation of Danshen formula granules. Methods: HPLC method was used with acetonitrile as mobile phase A, using 0.05% phosphoric acid solution as mobile phase B, and gradient elution (0-15 min, 10%A→20%A; 15-40 min, 20%A→25%A; 40-50 min, 25%A→30%A; 50-50.1 min, 30%A→10%A;50.1-60 min, 10%A), The flow rate was 1 mL·min-1, column temperature was 40 ℃, detection wavelength was 286 nm, and injection volume was 10 μL. Four different types of C18 chromatographic columns were used to measure the actual retention time of the six components of Danshen formula granules, and the average value of the retention time of each component in each chromatographic column was calculated to obtain the standard retention time of each component. Protocatechuic aldehyde (peak 2) and rosmarinic acid (peak 4) were selected as the reference substances for the determination. The chromatographic peaks of each component were accurately located by linear calibration using two reference substances,and the position of each component in unknown chromatographic column could be predicted in the same method. The methodology was validated. Taking rosmarinic acid as reference, the relative correction factors of the remaining components were calculated, and each component was quantified and compared with the results measured by the external standard method. Results: The contents of salvianic acid A, protocatechuic aldehyde, caffeic acid, rosmarinic acid, shikonic acid and salvianolic acid B in the collected 6 batches of samples were 3.82-6.54 mg·g-1, 0.44-1.14 mg·g-1, 42.75-179.49 mg·g-1, 2.20-2.63 mg·g-1, 3.11-3.49 mg·g-1, 42.41-53.74 mg·g-1, respectively. The double-standard linear correction method can accurately predict the retention time of the component to be tested on the unknown chromatographic column, and the deviation between the predicted result and the actual value was smaller than that of the relative retention time method. There was no significant difference between the content results using the relative correction factor method and the external standard method. Conclusion: The simultaneous determination of 6 phenolic acids in Danshen formula granules was feasible and accurate by two reference substances for determination of multiple components method, and the method was simple, accurate, economical, providing a new idea for the content determination of 6 phenolic acids in Danshen formula granules.
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