超高效液相色谱指纹图谱法和一测多评法评价不同产地西红花质量

Quality Evaluation of Croci Stigma from Different Producing Areas Based on HPLC Fingerprints and QAMS

目的 建立西红花药材的超高效液相色谱(UPLC)指纹图谱及测定药材中苦番红花素、西红花苷-Ⅰ、西红花苷-Ⅱ、西红花苷-Ⅲ、西红花苷-Ⅳ5种活性成分含量的一测多评(QAMS)法,评价不同产地西红花药材的质量。方法 采用UPLC法建立西红花药材的指纹图谱,色谱柱为Waters Acquity UPLC HSS T3柱(100 mm×2.1 mm,1.8μm),流动相为乙腈-水(梯度洗脱),流速为0.3 mL/min,检测波长为254 nm(0~8 min,苦番红花素),440 nm(8~25 min,西红花苷-Ⅰ、西红花苷-Ⅱ、西红花苷-Ⅲ、西红花苷-Ⅳ),柱温为25℃,进样量为4μL。利用相似度评价、聚类分析、正交偏最小二乘法-判别分析(OPLS-DA)分析药材样品的质量差异性。以西红花苷-Ⅰ为内参物,建立西红花苷-Ⅰ与苦番红花素、西红花苷-Ⅱ、西红花苷-Ⅲ、西红花苷-Ⅳ的相对校正因子(f_(s/i)),测定各成分的含量,并与外标法测定结果比较。结果 建立了30批西红花药材的UPLC指纹图谱,确定了11个共有峰,指认峰1为苦番红花素,峰4为西红花苷-Ⅰ,峰6为西红花苷-Ⅱ,峰10为西红花苷-Ⅲ,峰7为西红花苷-Ⅳ;30批西红花药材指纹图谱的相似度为0.983~0.999。30批西红花药材可聚为4类,并呈现一定的产地规律性;OPLS-DA共确定5种西红花差异性成分,变量重要性投影(VIP)值从大到小依次为峰8>峰9>峰11>峰10(西红花苷-Ⅲ)>峰2。5种成分的fs/i的RD均低于2.0%,且不同色谱柱、色谱仪、流速和柱温对fs/i影响较小(RSD均<2.0%)。与外标法结果的相对平均偏差均小于2.0%,无显著差异(P> 0.05)。结论 所建立的方法准确、便捷,可用于不同产地西红花药材的质量评价。

Objective To establish an ultra-high performance liquid chromatography(UPLC)fingerprints of Croci Stigma and a quantitative analysis of multi-components by single-marker(QAMS)method for the content determination of five active ingredients(picrocrocin,crocinⅠ,crocinⅡ,crocinⅢ,and crocinⅣ)in Croci Stigma,and to evaluate the quality of Croci Stigma from different producing areas.Methods UPLC method was used to establish the fingerprint of Croci Stigma,and the chromatographic column was Waters Acquire UPLC HSS T3 column(100 mm×2.1 mm,1.8μm),the mobile phase was acetonitrile-water(gradient elution),the flow rate was 0.3 mL/min,the detection wavelengths were 254 nm(0-8 min,picrocrocin)and 440 nm(8-25 min,crocinⅠ,crocinⅡ,crocinⅢ,and crocinⅣ),the column temperature was 25℃,and the injection volume was 4μL.The quality differences of Croci Stigma were evaluated by similarity evaluation,cluster analysis(CA),and orthogonal partial least squares-discriminant analysis(OPLS-DA).The relative correction factors(fs/i)of picrocrocin,crocinⅡ,crocinⅢand crocinⅣwere established with crocinⅠas internal reference,and then the content of each component was determined and compared with the results of the external standard method(ESM).Results UPLC fingerprints of thirty batches of Croci Stigma were established,eleven common peaks were determined,and five components were identified,namely picrocrocin(peak 1),crocin-Ⅰ(peak 4),crocinⅡ(peak 6),crocin-Ⅲ(peak 10),and crocin-Ⅳ(peak 7).The similarity of fingerprints of 30 batches of Croci Stigma was in the range of 0.983-0.999,and 30 batches of Croci Stigma could be clustered into four categories and showed a certain geographical pattern.Five different components of Croci Stigma were identified by the OPLS-DA,with a variable importance of projection(VIP)value ranking as peak 8>peak 9>peak 11>peak 10(crocinⅢ)>peak 2.The RDs of the fs/i of five components were lower than 2.0%,and the influence of different chromatographic columns,chromatographs,flow rates,and column temperatures on fs/i was relatively less(RSD<2.0%).The relative average deviation between the content of the five components in 30 batches of Croci Stigma determined by the QAMS and the ESM was lower than 2.0%,with no significant difference(P>0.05).Conclusion The established method is accurate and convenient,which can be used for the quality evaluation of Croci Stigma from different producing areas.