Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland.
Department of Chemistry, University of Lodz, Tamka 12, 91-403 Lodz, Poland.
Talanta. 2019 Jan 15;192:263-269. doi: 10.1016/j.talanta.2018.09.036. Epub 2018 Sep 15.
A fast and precise qNMR method was developed for quantification of major bioactive constituents in the bark of horse chestnut and dry extracts prepared thereof. The method was optimised using 600 MHz spectrometer, and the final acquisition parameters (90°-pulse, acquisition time - 3.0 s, relaxation delay - 27 s, number of transients - 16) allowed for performing of quantitative experiments in under 15 min. The contents of three analytes were determined using specific H resonances at δ7.45 ppm for esculin, δ5.00 ppm for fraxin, and δ5.94 ppm for (-)-epicatechin. The validation showed good precision (RSD < 1.5%) and accuracy (95-103%), and adequate sensitivity (LODs in the range of 3.3-5.9 µg) of the measurements. The determined levels in commercial samples of Hippocastani cortex were in the range of 25.89-38.94 mg/g dry weight (dw) of the bark for esculin, 12.58-17.13 mg/g dw for fraxin and 10.42-13.96 mg/g dw for (-)-epicatechin, and in the dry extracts prepared thereof 97.02-143.51 mg/g, 45.78-58.92 mg/g and 28.07-46.29 mg/g, respectively. The obtained results were cross-validated by a HPLC-PDA method with the use of a fused-core column, and no statistical differences were found between the results obtained by both methodologies, but with the advantage of higher precision of the qNMR assay. The relevant variability in quantitative composition of the commercial samples emphasise the need to introduce quality control studies in production of preparations containing horse chestnut bark and the developed method was proved suitable for this purpose.
建立了一种快速、精确的 qNMR 方法，用于定量测定欧洲七叶树树皮及其干提取物中的主要生物活性成分。该方法使用 600MHz 光谱仪进行优化，最终的采集参数（90°脉冲，采集时间 3.0s，弛豫延迟 27s，瞬变量 16）允许在 15min 内完成定量实验。使用特定的 H 共振峰 δ7.45ppm 用于测定秦皮甲素，δ5.00ppm 用于测定七叶素，δ5.94ppm 用于测定 (-)-表儿茶素，对三种分析物的含量进行了测定。验证结果表明，该方法具有良好的精密度（RSD < 1.5%）和准确性（95-103%），以及足够的灵敏度（LOD 范围为 3.3-5.9μg）。在商业样本中，欧洲七叶树树皮的秦皮甲素含量为 25.89-38.94mg/g 干重（dw），七叶素含量为 12.58-17.13mg/g dw，(-)-表儿茶素含量为 10.42-13.96mg/g dw，在制备的干提取物中，秦皮甲素含量为 97.02-143.51mg/g，七叶素含量为 45.78-58.92mg/g，(-)-表儿茶素含量为 28.07-46.29mg/g。使用熔融核芯柱的 HPLC-PDA 方法对所得结果进行了交叉验证，两种方法所得结果无统计学差异，但 qNMR 测定法具有更高的精密度优势。商业样本中定量成分的差异强调了在生产含有欧洲七叶树树皮的制剂时需要进行质量控制研究，所开发的方法适用于该目的。
