HPLC analysis of Ecdysteroids in plant extracts using superheated deuterium oxide with multiple on-line spectroscopic analysis (UV, IR, 1H NMR, and MS)

HPLC, using superheated D2O as the mobile phase, combined with on-line characterization via a combination of diode array UV, H-1 NMR, FT-IR spectroscopy, and mass spectrometry has been used for the analysis of a standard of 20-hydroxyecdysone- and ecdysteroid-containing plant extracts. This combination of spectrometers enabled the on-flow collection of UV, H-1 NMR, IR, and mass spectra not only for pure 20-hydroxyecdysone (100-400 mug on column) but also the major ecdysteroids present in crude extracts of Silene otites, Silene nutans, and Silene frivaldiskyana. The ecdysteroids unequivocally identified in these extracts included 20-hydroxy-ecdysone, polypodine B, and integristerone A.