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-OCH(3)](+) (ion 282) to [M-ROH+H](+) and [M-ROH-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.
Subject(s)
Podophyllotoxin/analysis , Podophyllotoxin/isolation & purification , Spin Labels , Antineoplastic Agents, Phytogenic/analysis , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Chromatography, High Pressure Liquid , Cyclic N-Oxides/chemistry , Molecular Structure , Podophyllotoxin/analogs & derivatives , Podophyllotoxin/chemical synthesis , Podophyllotoxin/chemistry , Spectrometry, Mass, Electrospray Ionization , Stereoisomerism , Tandem Mass SpectrometryABSTRACT
A rapid and stable 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, determination, and identification of eight pairs of diastereoisomers of podophyllotoxin and its esters at C-2 position. The separation was carried out on BDS Hypersil C18 column with CH3OH-CH3CN-H2O as the mobile phase in a gradient program. Interestingly, every 2alpha-H compound migrated before its corresponding 2beta-H epimer under optimum conditions. Also, the [M+NH(4)](+) of all eight pairs of compounds was observed in the HPLC-ESI/MS spectra. The characteristic elimination from the precursor protonated ions and the product ions at m/z 397, 313, 282, and 229 were the common diagnostic masses. The ion ratios of relative abundance [M-ROH+H](+) (ion 397) to [M+NH(4)](+), [A+H](+) (ion 313) to [M-ROH+H](+), and [M-ROH-ArH+H](+) (ion 229) to [M-ROH+H](+) in the ESI/MS/MS spectra of each pair of diastereoisomers of the lignans specifically exhibited a stereochemical effect. Thus, by using identical sample solutions and chromatographic conditions (including the same columns and gradient programs), the combination of DAD and MS/MS data permitted the separation and identification of the eight pairs of diastereoisomers of the podophyllotoxin and its esters in the mixture. The method could be used in rapidly identifying the purity and monitoring of the epimerization of 2-H of podophyllotoxin and its analogues from natural products, chemical reactions, and pharmaceutical metabolism.