Objective: To establish an HPLC-MS method for qualitative determination of compound A, myricetin- 3-O-β-D-glucoside, compound B, complanatoside B and complanatoside A in salted Astragali Complanati Semen dispensing granules, in order to detect the feasibility of linear calibration with two reference substances (LCTRC). And the two alternative qualitative methods of reference were compared between LCTRC and the relative retention time method (RRT). Methods: HPLC-MS method was adopted, the mobile phase consisted of acetonitrile (A)-0.2% phosphoric acid aqueous solution(B) with gradient elution(0-5 min, 10%A→15%A; 5-15 min, 15%A; 15-45 min, 15%A→25%A; 45-65 min, 25%A→45%A) at a flow rate of 1.0 mL·min-1. The column temperature was 35 ℃ and the detection wavelength was 266 nm. The retention time of the 5 components in salted Astragali Complanati Semen dispensing granules were determined on 18 C18 columns of different brands or types. The chromatographic peak was identified by retention time of reference and UV spectral and mass spectrum data of sample. The standard retention time (SRT) was calculated. Both compound A and complanatoside A were taken as the two reference substances to forecast the retention time of the other three components by LCTRC. Another 3 C18 column was used for methodology validation. Compound B was used as the reference substance to forecast the retention time of the other 4 components by RRT. Results: Compound A, myricetin-3-O-β-D-glucoside, compound B, complanatoside B and complanatoside A were qualitatively analyzed by LCTRC. The retention time of the 5 components on 17 chromatographic columns had good linear relationship (r>0.995), and the SRT of them was 14.433, 19.556, 34.071, 37.662, 39.643 min respectively. LCTRC was more accurate and suitable for more chromatographic columns than RRT. Conclusion: As a new alternative reference method, LCTRC can be used to assist the chromatographic peak identification, worthy of application and promotion.
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