Optimized HPLC-DAD in simultaneous determination of 4 flavonoids in Apocynum venetum / 第二军医大学学报

ABSTRACT

Objective: To establish an optimized HPLC method for simultaneous determination of rutin, hyperoside, isoquercitrin, and quercetin in Apocynum venetum obtained from different locations. Methods: The separation was performed on a Shiseido C18 (3.0 μm, 30mmX100mm) column by gradient elution with acetonitrile methanol = 10 1 as the mobile phase A and 0.1% formic asid as the mobile phase B (0-4 min, 5% → 16%A; 4-15 min, 16% → 23%A; 15-20 min, 23% → 35%A; 20-22 min, 35% → 55%A) at a flow rate of 0.6 ml • min-1. The detection wave length of DAD was set at 360 nm, and the column temperature was 30°C. The injection volume was 5 μl. Results: Rutin, hyperoside, isoquercitrin, and quercetin were separated at base line within 22 min with good linearity (r = 0.9999), with the linearity range being 0.308 4 ~ 30.84 μg • ml-1 (r = 0.9999), 0.877 6-87.76 μg • ml-1 (r=0.9999), 0.9020 ~ 90.20 μg • ml-1(r = 0.9999),and 0.2498 ~ 24.98 μg • ml-1 (r = 0.9999), respectively. The result of intra-day and inter-day precisions, limits of detection and quantitation were all within the normal ranges. The recovery rates (n = 3) were 102.0%, 96.40%, and 103.8% for rutin, 98.50%, 101.0%, and 102.9% for hyperoside, 98.40%, 100.4%, and 101.4% for isoquercitrin, and 104.4%, 103.1%, and 103.5% for quercetin. The contents of the above 4 flavonoids were determined in Apocynum venetum from 9 different locations. Conclusion: The method developed in this study is rapid, simple, accurate, reliable, and with good repeatability; the method provides evidence for the quality control of Apocynum venetum.