Ahmad Ajaz, Amir Mohd, Alshadidi Abdulrahman A, Hussain Muhammad Delwar, Haq Anzarul, Kazi Mohsin
Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia.
Department of Natural Product & Alternative Medicines, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, Dammam 1982, Saudi Arabia.
Saudi Pharm J. 2020 Apr;28(4):487-494. doi: 10.1016/j.jsps.2020.02.011. Epub 2020 Mar 4.
The current study was executed for method development, validation and to estimate the concentration of protopine in methanolic extract of by high-performance thin-layer chromatography (HPTLC). Isolation of bioactive compounds was carried out using the mobile phase, toluene:ethyl acetate:diethyl amine (8:2.5:0.5 ), and detected at wavelength 290 nm. This method was validated for precision, specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), etc. The calibration range was found to be 500-5000 ng/spot for the bioactive compound. Protopine was separated with an R value of 0.22 ± 0.03. The method was validated for linearity (r2 ≥ 0.996 ± 0.082), accuracy 98.75-102.12%), and RSD of precision (0.49-2.07) with a calibration curve range of 500.00-5000.00 ng/spot. The LOD and LOQ were found to be 83.92 ng/spot and 254.30 ng/spot., respectively. The Central Composite design expert was applied for the validation of robustness. Three independent variables such as the volume of toluene in solvent system, chamber saturation time and wavelength were investigated. The results indicated that a slight change in these variables had no significant effect on the peak response. This developed HPTLC method is simple, precise, robust, specific, rapid, and cost effective. It could be used for quality control study and quantification of protopine in the plant extract and different herbal formulations containing the plant species.
本研究旨在通过高效薄层色谱法（HPTLC）进行方法开发、验证并测定[提取物名称]甲醇提取物中原托品碱的浓度。使用甲苯：乙酸乙酯：二乙胺（8:2.5:0.5）的流动相进行生物活性化合物的分离，并在290 nm波长处进行检测。该方法针对精密度、特异性、线性、检测限（LOD）、定量限（LOQ）等进行了验证。发现生物活性化合物的校准范围为500 - 5000 ng/斑点。原托品碱的Rf值为0.22±0.03。该方法在500.00 - 5000.00 ng/斑点的校准曲线范围内，线性（r2≥0.996±0.082）、准确度（98.75 - 102.12%）和精密度的相对标准偏差（RSD，0.49 - 2.07）均得到验证。采用中心复合设计专家系统对稳健性进行验证。研究了溶剂系统中甲苯体积、展开缸饱和时间和波长这三个自变量。结果表明，这些变量的轻微变化对峰响应没有显著影响。所开发的HPTLC方法简单、精确、稳健、特异、快速且成本效益高。它可用于植物提取物以及含有该植物物种的不同草药制剂中原托品碱的质量控制研究和定量分析。
