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 C(18) column with methanol-acetonitrilewater as the mobile phase in a gradient elution program. In the HPLC-ESI/MSspectra, the [M+Na](+) ions of all seven pairs were observed. The ion ratios of the relatively abundant ions of [M-ROH+H](broken vertical bar) (ion 397 or 383) to [M+Na](broken vertical bar) , [A+ H](broken vertical bar) ( ion 313) to [M-ROH+H](+), and [A+H-OCH(3)](+) ( 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.