目的:建立HPLC-MS法对盐沙苑子配方颗粒中化合物A、杨梅素-3-O-β-D-葡萄糖苷、化合物B、沙苑子苷B、沙苑子苷A进行定性分析,考察双标线性校正(LCTRC)法预测保留时间的可行性,并对LCTRC法和相对保留时间(RRT)法2种替代对照品定性方法进行比较。方法:采用HPLC-MS法,以乙腈为流动相A,以0.1%甲酸水溶液为流动相B,梯度洗脱(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),流速为1.0 mL·min-1,柱温为35 ℃,检测波长为266 nm。通过对照品保留时间及样品的紫外光谱、质谱数据进行样品色谱峰定位,在18根不同品牌或型号的C18色谱柱上,测定盐沙苑子配方颗粒中5个成分的实际保留时间,计算标准保留时间(SRT),以化合物A和沙苑子苷A为双标化合物,使用LCTRC法预测保留时间,并在另外3根C18色谱柱上进行方法验证。同时以中间峰化合物B为参照物,使用RRT法预测保留时间。结果:建立了LCTRC法对化合物A、杨梅素-3-O-β-D-葡萄糖苷、化合物B、沙苑子苷B、沙苑子苷A进行定性分析,5个成分在17根色谱柱上的SRT分别为14.433、19.556、34.071、37.662、39.643 min,线性关系良好(r>0.995)。与RRT法相比,LCTRC法预测保留时间的准确度更高,色谱柱适用范围更广。结论:作为一种新的替代对照品定性方法,LCTRC法能较好地辅助色谱峰定性,值得应用和推广。
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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