Determination of Quercetin and Kaempferol in Crotalaria ferruginea by QAMS
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摘要: 为建立适用于响铃草质量控制的槲皮素和山柰素含量的一测多评测定方法，采用高效液相色谱HPLC法，使用C18色谱柱（4.6 mm×250 mm，5 μm），乙腈：0.4%醋酸（40：60）为流动相，检测波长360 nm，柱温30℃，流速1.0 mL·min-1，检测槲皮素和山柰素含量。以槲皮素为内标物，测定其与山柰素的相对校正因子（RCF），并进行含量计算，实现一测多评；同时采用外标法（ESM）测定响铃草中2种有效成分的含量，将一测多评法（QAMS）与外标法（ESM）进行比较。结果表明，8批响铃草药材中山柰素含量的一测多评法测定结果均与外标法无显著性差异。说明该方法操作简便，快速，重复性好，结果准确可靠，可为进一步完善响铃草的质量标准提供参考。Abstract: To establish a QAMS methodology for simultaneous determination of quercetin and kaempferol contents in Crotalaria ferruginea, HPLC was applied on a C18 column (4.6 mm×250 mm, 5 μm) with acetonitrile:0.4% CH3COOH (40:60) for the mobile phase. The column temperature was kept at 30℃ and flow rate at 1.0 mL·min-1. The eluate was detected at the wavelength of 360 nm. Quercetin was used as the internal standard to establish the relative correction factor (RCF) for the quantitative analysis of multi-components on QAMS. The RCF of kaempferol was calculated. The contents of the two compounds were measured by both the external standard method and QAMS to verify the validity of the proposed QAMS methodology. The resulting RCF showed good durability on both quercetin and kaempferol determinations. There were no significant differences between the data obtained by QAMS and by the external standard method. Consequently, this new approach was considered simple, rapid and applicable for quality control on raw material and processed products involving C. ferruginea.
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Key words:
- Crotalaria ferruginea /
- quercetin /
- kaempferol /
- QAMS /
- HPLC /
- relative correction factors
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图 1 响铃草中槲皮素和山柰素高效液相色谱
注：A为混合对照品，B为供试品。1为槲皮素，2为山柰素。
Figure 1. HPLC chromatograms for quercetin and kaempferol in Crotalaria ferruginea
表 1 槲皮素和山柰素的加样回收率试验
Table 1. Recovery on quercetin and kaempferol of QAMS method
待测成分 样品量
/g已知量
/mg加入量
/mg测得量
/mg回收率
/%平均回收率
/%RSD
/%槲皮素 3.0005 0.4304 0.4000 0.8281 99.42 98.47 1.19 3.0003 0.4303 0.4000 0.8228 98.12 3.0004 0.4304 0.4000 0.8188 97.1 3.0003 0.4303 0.4000 0.8251 98.71 3.0002 0.4303 0.4000 0.8197 97.34 3.0003 0.4303 0.4000 0.8307 100.1 山柰素 3.0005 0.1667 0.2000 0.1983 98.31 97.88 1.52 3.0003 0.1666 0.2000 0.1967 97.54 3.0004 0.1667 0.2000 0.2023 100.32 3.0003 0.1666 0.2000 0.1949 96.64 3.0002 0.1665 0.2000 0.1939 96.11 3.0003 0.1666 0.2000 0.1983 98.33 
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表 2 不同仪器和色谱柱测定山柰素的相对保留时间
Table 2. RTR of kaempferol by using different instruments and columns
色谱仪器 色谱柱 相对保留时间的比值 Agilent 1260型 Agilent ZORBAX SB-C18 1.525 Phenomenex Gemini C18 1.475 Thermo ODS C18 1.534 赛默飞UltiMate300型 Agilent ZORBAX SB-C18 1.544 Phenomenex Gemini C18 1.553 Thermo ODS C18 1.561 平均值 1.532 RSD/% 2.01
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表 3 响铃草中槲皮素和山柰素的测定
Table 3. Contents of quercetin and kaempferol in C.ferruginea
[单位/(mg · g-1)] 产地 槲皮素ESM 山柰素 ESM QAMS 相对误差/% 广西 0.1431 0.0282 0.0284 0.709 广东 0.0913 0.0312 0.0314 0.641 贵州遵义 0.0392 0.0491 0.0496 1.018 福建 0.0464 0.0783 0.0786 0.383 贵州安顺 0.2712 0.2501 0.2511 0.400 云南 0.0221 0.0795 0.0774 -2.642 浙江 0.0264 0.0414 0.0421 1.691 四川 0.0347 0.0584 0.0576 -1.370 注：相对误差=(QAMS计算值-ESM实测值)/ ESM实测值× 100%
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