HPLC波长切换法同时测定清热解毒颗粒中8个成分的含量

目的: 建立高效液相色谱法同时测定清热解毒颗粒中绿原酸、栀子苷、木犀草苷、连翘苷、黄芩苷、木犀草素、黄芩素、汉黄芩素共8个成分的含量。方法: 采用Phenomenex Luna 5μ-C₁₈色谱柱(4.6 mm×250 mm,5 μm),以甲醇(A)-0.1%磷酸水溶液(B)为流动相,梯度洗脱,流速1 mL·min^-1,检测波长326 nm(0~26 min,测定绿原酸)、238 nm(26~35 min,测定栀子苷)、350 nm(35~44 min,测定木犀草苷)、230 nm(44~48 min,测定连翘苷)、276 nm(48~53 min,测定黄芩苷)、350nm(53~60 min,测定木犀草素)、276 nm(60~85 min,测定黄芩素、汉黄芩素)。结果: 各待测组分分离度良好;绿原酸、栀子苷、木犀草苷、连翘苷、黄芩苷、木犀草素、黄芩素、汉黄芩素8个成分的进样量分别在0.041 3~0.413 μg(r=0.999 6),0.034 0~0.340 μg(r=0.999 9),0.037 1~0.371 μg(r=0.999 5),0.026 4~0.264 μg(r=0.999 3),0.196 0~1.960 μg(r=0.999 8),0.010 8~0.108 μg(r=0.999 2),0.021 2~0.212 μg(r=0.999 2),0.005 7~0.057 μg(r=0.999 0)与峰面积呈良好的线性关系;加样回收率(n=6)分别为98.4%(RSD=1.6%)、99.2%(RSD=1.4%)、98.5%(RSD=1.7%)、100.7%(RSD=2.0%)、99.0%(RSD=0.9%)、98.8%(RSD=1.9%)、98.1%(RSD=1.8%)、99.9%(RSD=1.3%)。经HPLC测定,3批样品中8个成分的含量分别为绿原酸0.326~0.775 mg·g^-1,栀子苷0.167~0.581 mg·g^-1,木犀草苷0.093~0.216 mg·g^-1,连翘苷0.107~0.304 mg·g^-1,黄芩苷1.574~3.810 mg·g^-1,木犀草素0.092~0.114 mg·g^-1,黄芩素0.175~0.625 mg·g^-1,汉黄芩素0.042~0.188 mg·g^-1。结论: 经方法学验证,本方法简单、快速、灵敏、准确,可用于清热解毒颗粒中绿原酸、栀子苷、木犀草苷、连翘苷、黄芩苷、木犀草素、黄芩素、汉黄芩素8个成分的含量测定。

Objective: To develop an HPLC method for simultaneous determination of chlorogenic acid,geniposide,cynaroside,phillyrin,baicalin,luteolin,baicalein and wogonin in Qingrejiedu granules. Methods: The chromatographic separation was carried out on a Phenomenex Luna 5μ-C₁₈ column(4.6 mm×250 mm,5 μm)with a mobile phase consisting of methanol(A)-0.1% phosphoric acid solution(B)in a gradient mode at a flow rate of 1 mL·min^-1.The UV detection wavelength was set at 326 nm for chlorogenic acid in the first 26 min,238 nm for geniposide during 26-35 min,350 nm for cynaroside during 35-44 min,230 nm for phillyrin during 44-48 min,276 nm for baicalin during 48-53 min,350 nm for luteolin during 53-60 min,276 nm for baicalein and wogonin between 60 and 85 min. Results: Excellent chromatographic separation was achieved and the ranges for linear correlation of chlorogenic acid,geniposide,cynaroside,phillyrin,baicalin,luteolin,baicalein and wogonin were 0.041 3-0.413 μg(r=0.999 6),0.034 0-0.340 μg(r=0.999 9),0.037 1-0.371 μg(r=0.999 5),0.026 4-0.264 μg(r=0.999 3),0.196 0-1.960 μg(r=0.999 8), 0.010 8-0.108 μg(r=0.999 2),0.021 2-0.212 μg( r=0.999 2)and 0.005 7-0.057 μg(r=0.999 0),respectively.The average recoveries(n=6)of the eight components were 98.4%(RSD=1.6%),99.2%(RSD=1.4%),98.5%(RSD=1.7%),100.7%(RSD=2.0%),99.0%(RSD=0.9%),98.8%(RSD=1.9%),98.1%(RSD=1.8%)and 99.9%(RSD=1.3%).The eight ingredient contents in 3 batches of samples were:0.326-0.775 mg·g^-1(chlorogenic acid),0.167-0.581 mg·g^-1(geniposide),0.093-0.216 mg·g^-1(cynaroside),0.107-0.304 mg·g^-1(phillyrin),1.574-3.810 mg·g^-1(baicalin),0.092-0.114 mg·g^-1(luteolin),0.175-0.625 mg·g^-1(baicalein) and 0.042-0.188 mg·g^-1(wogonin)by HPLC detection. Conclusion: The established method is simple,rapid,sensitive and accurate,which can be applied to determine chlorogenic acid,geniposide,cynaroside,phillyrin,baicalin,luteolin,baicalein and wogonin in Qingrejiedu granules.