Khan Shazia, Ali Athar, Ahmad Shahzad, Abdin Malik Zainul
Centre for Transgenic Plant Development, Department of Biotechnology, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi, 110062, India.
Biomed Chromatogr. 2015 Oct;29(10):1594-603. doi: 10.1002/bmc.3465. Epub 2015 Mar 31.
Artemisinin (AN) and artemisinic acid (AA), valuable phyto-pharmaceutical molecules, are well known anti-malarials, but their activities against diseases like cancer, schistosomiasis, HIV, hepatitis-B and leishmaniasis are also being reported. For the simultaneous estimation of AN and AA in the callus and leaf extracts of A. annua L. plants, we embarked upon a simple, rapid, selective, reliable and fairly economical high performance thin layer chromatography (HPTLC) method. Experimental conditions such as band size, chamber saturation time, migration of solvent front and slit width were critically studied and the optimum conditions were selected. The separations were achieved using toluene-ethyl acetate, 9:1 (v/v) as mobile phase on pre-coated silica gel plates, G 60F254 . Good resolution was achieved with Rf values of 0.35 ± 0.02 and 0.26 ± 0.02 at 536 nm for AN and 626 nm for AA, respectively, in absorption-reflectance mode. The method displayed a linear relationship with r(2) value 0.992 and 0.994 for AN and AA, respectively, in the concentration range of 300-1500 ng for AN and 200-1000 ng for AA. The method was validated for specificity by obtaining in-situ UV overlay spectra and sensitivity by estimating limit of detection (30 ng for AN and 15 ng for AA) and limit of quantitation (80 ng for AN and 45 ng for AA) values. The accuracy was checked by the recovery studies conducted at three different levels with the known concentrations and the average percentage recovery was 101.99% for AN and 103.84% for AA. The precision was analyzed by interday and intraday precision and was 1.09 and 1.00% RSD for AN and 1.22 and 6.05% RSD for AA. The analysis of statistical data substantiates that this HPTLC method can be used for the simultaneous estimation of AN and AA in biological samples.
青蒿素（AN）和青蒿酸（AA）是有价值的植物药分子，是著名的抗疟药物，但它们对癌症、血吸虫病、艾滋病毒、乙型肝炎和利什曼病等疾病的活性也有报道。为了同时测定黄花蒿植株愈伤组织和叶片提取物中的AN和AA，我们采用了一种简单、快速、选择性好、可靠且经济的高效薄层色谱（HPTLC）方法。对诸如条带大小、展开缸饱和时间、溶剂前沿迁移和狭缝宽度等实验条件进行了严格研究，并选择了最佳条件。使用甲苯 - 乙酸乙酯（9:1，v/v）作为流动相，在预涂硅胶板G 60F254上进行分离。在吸收 - 反射模式下，在536 nm处AN的Rf值为0.35±0.02，在626 nm处AA的Rf值为0.26±0.02，实现了良好的分离度。该方法在AN浓度范围为300 - 1500 ng、AA浓度范围为200 - 1000 ng时，分别与r(2)值0.992和0.994呈线性关系。通过获得原位紫外叠加光谱验证了该方法的特异性，通过估计检测限（AN为30 ng，AA为15 ng）和定量限（AN为80 ng，AA为45 ng）值验证了灵敏度。通过在三个不同水平上进行已知浓度的回收率研究来检查准确性，AN的平均回收率为101.99%，AA的平均回收率为103.84%。通过日间和日内精密度分析精密度，AN的相对标准偏差（RSD）为1.09%和1.00%，AA的RSD为1.22%和6.05%。统计数据分析证实，这种HPTLC方法可用于生物样品中AN和AA的同时测定。
