Thin layer chromatography quantitative analysis on gallic acid in Galla Chinensis based on image analysis technology
周洪亮 1黄亚威 1田明鑫 2张登山 1韩月 3张琪
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作者信息
1. 南京中医药大学附属医院药学部，南京 210029
2. 南京中医药大学第一临床医学院，南京 210046
3. 南京中医药大学附属医院妇科，南京 210029
折叠
摘要
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0.401 6 μg，线性范围2.855~9.515 μg（r 2=0.996 0），回收率为105.12%（RSD=3.48%）；聚酰胺薄膜没食子酸定量限为0.363 4 μg，线性范围1.427~4.758 μg（r 2=0.991 5），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0.122~0.977 μg（r 2=0.999 9），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 0.401 6 μg, the linear range was 2.855 - 9.515 μg (r2 = 0.996 0), and the average recovery was 105.12% (RSD=3.48%) the quantitation limit of gallic acid on polyamide film was 0.363 4 μg, the linear range was 1.427 - 4.758 μg (r2 = 0.991 5), 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 0.122-0.977 μg (r2 = 0.999 9), 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.
Abstract
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 0.401 6 μg, the linear range was 2.855 - 9.515 μg (r2 = 0.996 0), and the average recovery was 105.12% (RSD=3.48%) the quantitation limit of gallic acid on polyamide film was 0.363 4 μg, the linear range was 1.427 - 4.758 μg (r2 = 0.991 5), 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 0.122-0.977 μg (r2 = 0.999 9), 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.
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