Combined Application of UHPLC‐QTOF/MS, HPLC‐ELSD and 1H–NMR Spectroscopy for Quality Assessment of DA‐9801, A Standardised Dioscorea Extract

• Published in: Phytochemical analysis 2017-05, Vol.28 (3), p.185-194
• Main Authors: Kang, Kyo Bin, Ryu, Jayoung, Cho, Youngwoong, Choi, Sang‐Zin, Son, Miwon, Sung, Sang Hyun
• Format: Article
• Language: English
• Subjects: 1H–NMR spectroscopy; Amino acids; Correlation analysis; DA‐9801; HPLC‐ELSD; Liquid chromatography; Metabolites; Principal components analysis; quality assessment; Quality control; Secondary metabolites; Spectrometry; Spectroscopy; UHPLC‐QTOF/MS
• ISSN: 0958-0344; 1099-1565
• DOI: 10.1002/pca.2659

Introduction 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. Objective Development of a quality assessment strategy for DA‐9801, based on the combination of UHPLC‐QTOF/MS, HPLC‐ELSD, and 1H–NMR spectroscopy. Methods 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 1H–NMR spectrometry. The batch‐to‐batch equivalence of DA‐9801 was tested with the 1H–NMR spectra using spectral binning, correlation analysis, and principal component analysis. Results 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 1H–NMR spectra. The similarity between DA‐9801 batches could be evaluated with the NMR spectra of DA‐9801. The 1H–NMR method also revealed that two Dioscorea species contributed distinct amino acids to the contents of DA‐9801. Conclusion This study validates the effectiveness of UHPLC‐QTOF/MS, HPLC‐ELSD, and 1H NMR‐combined method for quality control of DA‐9801 and its crude materials. Copyright © 2016 John Wiley & Sons, Ltd. 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. In this paper, a quality assessment strategy for DA‐9801 was proposed and validated based on the combination of UHPLC‐QTOF/MS, HPLC‐ELSD, and 1H–NMR spectroscopy, to evaluate the contents of primary and major steroidal saponins.