Kang Kyo Bin, Ryu Jayoung, Cho Youngwoong, Choi Sang-Zin, Son Miwon, Sung Sang Hyun
College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul, 08826, Republic of Korea.
Research Planning & Management Department, Dong-A ST Co., Ltd., Yongin, 17073, Republic of Korea.
Phytochem Anal. 2017 May;28(3):185-194. doi: 10.1002/pca.2659. Epub 2016 Dec 1.
DA-9801, a standardised 50% aqueous ethanolic extract of a mixture of Dioscorea japonica and D. nipponica, is a botanical drug candidate for the treatment of diabetic neuropathy, which finished its US phase II clinical trials recently. An advanced quality control method is needed for further development of DA-9801, considering its high contents of both primary and secondary metabolites.
Development of a quality assessment strategy for DA-9801, based on the combination of UHPLC-QTOF/MS, HPLC-ELSD, and H-NMR spectroscopy.
The method was developed and tested with 15 batch products of DA-9801. The steroidal saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS and were quantified with the validated HPLC-ELSD method. Primary metabolites of DA-9801 were identified and profiled using H-NMR spectrometry. The batch-to-batch equivalence of DA-9801 was tested with the H-NMR spectra using spectral binning, correlation analysis, and principal component analysis.
Six major saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS. Among them, protodioscin and dioscin were quantified by the validated HPLC-ELSD method. Twenty-six metabolites were identified in H-NMR spectra. The similarity between DA-9801 batches could be evaluated with the NMR spectra of DA-9801. The H-NMR method also revealed that two Dioscorea species contributed distinct amino acids to the contents of DA-9801.
This study validates the effectiveness of UHPLC-QTOF/MS, HPLC-ELSD, and H NMR-combined method for quality control of DA-9801 and its crude materials. Copyright © 2016 John Wiley & Sons, Ltd.
DA-9801是日本薯蓣和穿龙薯蓣混合物的标准化50%乙醇水提取物，是一种用于治疗糖尿病神经病变的植物药候选物，该药物最近完成了其在美国的II期临床试验。鉴于DA-9801中初级和次级代谢产物含量都很高，因此需要一种先进的质量控制方法来进一步开发该药物。
基于超高效液相色谱-四极杆飞行时间质谱（UHPLC-QTOF/MS）、高效液相色谱-蒸发光散射检测器（HPLC-ELSD）和氢核磁共振波谱（¹H-NMR）的组合，开发一种DA-9801的质量评估策略。
该方法用15批DA-9801产品进行了开发和测试。通过UHPLC-QTOF/MS对DA-9801的甾体皂苷进行了初步鉴定，并用经过验证的HPLC-ELSD方法进行了定量。使用¹H-NMR光谱法对DA-9801的初级代谢产物进行了鉴定和分析。使用光谱分箱、相关分析和主成分分析，通过¹H-NMR光谱对DA-9801的批次间等效性进行了测试。
通过UHPLC-QTOF/MS初步鉴定出DA-9801的六种主要皂苷。其中，原薯蓣皂苷和薯蓣皂苷通过经过验证的HPLC-ELSD方法进行了定量。在¹H-NMR光谱中鉴定出26种代谢产物。DA-9801批次之间的相似性可以通过DA-9801的NMR光谱进行评估。¹H-NMR方法还表明，两种薯蓣属植物为DA-9801的成分贡献了不同的氨基酸。
本研究验证了UHPLC-QTOF/MS、HPLC-ELSD和¹H NMR组合方法对DA-9801及其原材料质量控制的有效性。版权所有©2016约翰威立父子有限公司。
