Combined Application of UHPLC-QTOF/MS, HPLC-ELSD and 1 H-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: Chromatography, High Pressure Liquid - methods; Dioscorea - chemistry; Diosgenin - analogs & derivatives; Diosgenin - analysis; Magnetic Resonance Spectroscopy - methods; Mass Spectrometry - methods; Plant Preparations - analysis; Plant Preparations - standards; Quality Control; Reproducibility of Results; Saponins - analysis; Spectrometry, Mass, Electrospray Ionization - instrumentation; Spectrometry, Mass, Electrospray Ionization - methods
• ISSN: 0958-0344; 1099-1565
• DOI: 10.1002/pca.2659

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.