Simultaneous Separation and Identification of the Diastereoisomers of Epipodophyllotoxin and its Esters by Liquid Chromatography-Diode Array Detection and -Electrospray Ionization Mass Spectrometry

Epipodophyllotoxin and its derivatives are important derivatives of podophyllotoxin which are used in cancer chemotherapy. The conditions for the separation and identification of a mixture of seven diastereoisomer pairs at the C-2 position of epipodophyllotoxin and its esters were optimized using both high-performance liquid chromatography-diode array detection (HPLC-DAD) and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI/MS/MS). The separation was carried out on a BDS Hypersil C18 column with methanol-acetonitrilewater as the mobile phase in a gradient elution program. In the HPLC-ESI/MS spectra, the [M+Na]+ ions of all seven pairs were observed. The ion ratios of the relatively abundant ions of [M-ROH+H]+ (ion 397 or 383) to [M+Na]+, [A+H]+ (ion 313) to [M-ROH+H]+, and [A+H-OCH3]+ (ion 282) to [M-ROH+H]+ of each diastereoisomer pair of the lignans specifically exhibited a stereochemical effect. Thus, using identical chromatographic conditions, the combination of DAD and MS permits the separation and identification of seven diastereoisomers of epipodophyllotoxin at the C-2 position. The results of this work may serve as an effective tool for the separation and detection of other epipodophyllotoxin derivatives and their analogues.