基于图像分析的五倍子没食子酸薄层色谱定量研究

Thin layer chromatography quantitative analysis on gallic acid in Galla Chinensis based on image analysis technology

目的以五倍子中没食子酸为研究对象,探讨Image J软件用于薄层色谱定量的可行性。方法采用硅胶GF254薄层板,以三氯甲烷-甲酸乙酯-甲酸(5∶5∶1)为展开剂,紫外光灯(254 nm)下拍照;采用聚酰胺薄膜,以75%乙醇-冰醋酸(50∶1)为展开剂,1%三氯化铁乙醇溶液为显色剂,日光下拍照。将图像导入Image J软件进行分析,以外标两点法计算五倍子中没食子酸含量。采用HPLC法,以甲醇-0.1%磷酸溶液(15∶85)为流动相,在273 nm波长下测定五倍子中没食子酸含量。结果硅胶GF254薄层板没食子酸定量限为0.4016μg,线性范围2.855~9.515μg(r^(2)=0.9960),回收率为105.12%(RSD=3.48%);聚酰胺薄膜没食子酸定量限为0.3634μg,线性范围1.427~4.758μg(r^(2)=0.9915),回收率为103.75%(RSD=4.60%);HPLC法没食子酸的定量限为4.42 ng,线性范围0.122~0.977μg(r^(2)=0.9999),回收率为98.30%(RSD=1.40%),3种方法的精密度、重复性、稳定性RSD值均<5%。使用Image J测得的没食子酸含量与HPLC测定结果比较,最大相对误差9.30%,最小相对误差1.62%。结论Image J软件可用于薄层色谱定量分析,可作为中药质量控制方法的补充。

Objectives To discuss the feasibility of Image J in quantitative analysis on thin layer chromatography(TLC)using gallic acid in Galla Chinensis as research object.Methods Silica gel GF254 thin-layer plate was used with chloroform-ethyl formate-formic acid(5:5:1)as the developing solvent and the images were taken under ultraviolet light(254 nm).Polyamide film was used with 75%ethanol-glacial acetic acid(50:1)as the developing solvent and 1%ferric chloride ethanol solution as the chromogenic reagent,and the images were taken under sunlight.Images obtained from the above conditions were imported into Image J to analyze and calculate the content of gallic acid in Galla Chinensis by using external standard two-point method.High-performance liquid chromatography(HPLC)was used with a mobile phase of methanol-0.1%phosphoric acid solution(15:85)at a wavelength of 273 nm to determine the gallic acid content in Galla Chinensis.Results The quantitation limit of gallic acid on silica gel GF254 thin-layer plate was 0.4016μg,the linear range was 2.855-9.515μg(r^(2)=0.9960),and the average recovery was 105.12%(RSD=3.48%);the quantitation limit of gallic acid on polyamide film was 0.3634μg,the linear range was 1.427-4.758μg(r^(2)=0.9915),and the average recovery was 103.75%(RSD=4.60%).The HPLC method had a quantitative limit of 4.42 ng,a linear range of 0.122-0.977μg(r^(2)=0.9999),and a recovery rate of 98.30%(RSD=1.40%).The accuracy,repeatability and stability of RSD were all<5%.The gallic acid content measured using Image J showed a maximum relative error of 9.30%and a minimum of 1.62%compared to the HPLC results.Conclusions Image J is feasible for quantitative analysis of TLC and can be used as a complementary method for quality control of Chinese materia medica.