HPLC-DAD and HPLC-ESI-MS separation, determination and identification of the spin-labeled diastereoisomers of podophyllotoxin

Abstract

Spin-labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, the 2-H configurations of these spin-labeled derivatives cannot be determined by nuclear magnetic resonance (NMR) methods. In the present paper, a high-performance liquid chromatography-diode array detection (HPLC-DAD) and a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI/MS/MS) method were developed and validated for the separation, identification of four pairs of diastereoisomers of spin-labeled derivatives of podophyllotoxin at C-2 position. In the HPLC-ESI/MS spectra, each pair of diastereoisomers of the spin-labeled derivatives in the mixture was directly confirmed and identified by [M+H]+ ions and ion ratios of relative abundance of [M-ROH+H]+ (ion 397) to [M+H]+. When the [M-ROH+ H]+ ions (at m/z 397) were selected as the precursor ions to perform the MS/MS product ion scan. The product ions at m/z 313, 282, and 229 were the common diagnostic ions. The ion ratios of relative abundance of the [M-ROH+H]+ (ion 397) to [M+H]+, [A+H]+ (ion 313) to [M-ROH+H]+, [A+H-OCH3]+ (ion 282) to [M-ROH+H]+ and [MROH-ArH+H]+ (ion 229) to [M-ROH+H]+ of each pair of diastereoisomers of the derivatives specifically exhibited a stereochemical effect. Thus, by using identical chromatographic conditions, the combination of DAD and MS/MS data permitted the separation and identification of the four pairs of diastereoisomers of spin-labeled derivatives of podophyllotoxin at C-2 in the mixture. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.