HPLC-一测多评法结合色差原理分析不同生长年限北苍术药材的质量

Quality Analysis of Atractylodes chinensis with Different Growth Years by HPLC-QAMS Combined with Color Difference Principle

目的:建立同时测定北苍术中白术内酯Ⅱ、β-桉叶醇、苍术素、苍术酮含量的方法,并结合色差原理评价不同生长年限北苍术药材的质量。方法:采用高效液相色谱(HPLC)法进行测定,色谱柱为Agilent Eclipse XDB-C18,流动相为乙腈-0.2%磷酸水溶液(梯度洗脱),流速为1.0 mL/min,检测波长分别为208 nm(白术内酯Ⅱ、β-桉叶醇)、340 nm(苍术素)和220 nm(苍术酮),进样量为15μL。以苍术素为内参物,采用一测多评法建立白术内酯Ⅱ、β-桉叶醇、苍术酮的相对校正因子,并计算不同生长年限北苍术药材中各成分的含量;同时,采用外标法测定上述成分含量,并与一测多评法结果进行比较。基于色差原理对不同生长年限北苍术药材粉末进行颜色测定,采用Pearson相关分析法对苍术药材中上述4种成分含量与其颜色进行相关性分析。结果:白术内酯Ⅱ、β-桉叶醇、苍术素、苍术酮的分离度均大于1.5,检测质量浓度线性范围分别为1.01~10.10、3.30~33.00、4.40~44.00、5.34~53.40μg/mL,精密度、重复性、稳定性试验的RSD均小于2%,平均加样回收率为101.34%~104.67%(RSD<1.5%,n=6);以苍术素为内参物,白术内酯Ⅱ、β-桉叶醇、苍术酮的平均校正因子分别为3.8967、5.9282、9.7279,RSD分别为0.35%、2.89%、0.36%(n=6)。采用一测多评法与外标法测得的24批北苍术药材中3种成分(除苍术素外)含量的相对偏差在0.03%~1.45%之间,表明2种方法测定结果一致,且各成分含量随药材生长年限的增加而升高。北苍术药材中白术内酯Ⅱ、β-桉叶醇、苍术素、苍术酮与其颜色亮度(L*)、色差值(E*ab)呈显著负相关(P<0.01),与红绿色度(a*)、黄蓝色度(b*)呈显著正相关(P<0.01)。结论:HPLC-一测多评法可用于北苍术药材中白术内酯Ⅱ、β-桉叶醇、苍术素、苍术酮的含量测定;药材生长年限越长,各成分含量越高;药材颜色与各成分含量显著相关,且颜色偏暗黄棕色的北苍术药材的有效成分含量较高。

OBJECTIVE:To simultaneously determine the contents of atractylenolideⅡ,β-eudesmol,atractyloxin and atractylone in Atractylodes chinensis,and to evaluate the quality of A.chinensis with different growth years combined with color difference principle.METHODS:HPLC method was adopted.The determination performed on Agilent Eclipse XDB-C18 column with mobile phase consisted of acetonitrile-0.2%phosphoric acid(gradient elution)at the flow rate of 1.0 mL/min;the detection wavelengths were set as 208 nm(atractylenolideⅡ,β-eudesmol),340 nm(atractyloxin)and 220 nm(atractylone);the sample size was 15μL.Using atractyloxin as reference,QAMS was adopted to establish relative correction factors(RCFs)of atractylenolideⅡ,β-eudesmol and atractylone;the content of each component in A.chinensis with different growth years were calculated.The contents of above 4 components were determined by external standard method and then compared with the results of QAMS.The color difference values of A.chinensis powder were measured based on color difference principle.The correlation analysis of above 4 components content with color was carried out by Pearson correlation analysis.RESULTS:The separation degree of atractylenolideⅡ,β-eudesmol,atractyloxin and atractylone in A.chinensis was higher than 1.5.The linear range were 1.01-10.10,3.30-33.00,4.40-44.00,5.34-53.40μg/mL,respectively.RSDs of precision,reproducibility and stability tests were all lower than 2%,while the average recovery rates were 101.34%-104.67%(RSD<1.5%,n=6).Using atractyloxin as reference,RCFs of atractylenolideⅡ,β-eudesmol and atractylone were 3.8967,5.9282,9.7279,with RSD of 0.35%,2.89%,0.36%(n=6),respectively.Relative deviation of 3 components(except for atractyloxin)in 24 batches of A.chinensis ranged 0.03%-1.45%between QAMS and external standard method,which indicated that the results of two methods were consistent,and the content of each component increased with the increase of growth years.AtractylenolideⅡ,β-eudesmol,atractyloxin and atractylone in A.chinensis had significant negative correlation with its color shade(L*),total color difference(E*ab)(P<0.01),and significant positive correlation with color red-green direction(a*),color yellow-blue direction(b*)(P<0.01).CONCLUSIONS:The established HPLC-QAMS method can be used for the determination of atractylenolideⅡ,β-eudesmol,atractyloxin and atractylone in A.chinensis.The longer the growth period is,the higher each component content is.The color of A.chinensis is closely related to the content of each component,and the content of effective components is higher in A.chinensis with dark yellowish brown color.