HPLC‐DAD and HPLC‐ESI‐MS separation,determination and identification of the spin‐labeled diastereoisomers of podophyllotoxin |
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Authors: | Lei Zhao Zhen‐Ling Liu Peng‐Cheng Fan Zhi‐Wei Zhang Xiong Liu Yun‐Jing Zhan Xuan Tian |
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Affiliation: | 1. State Key Laboratory of Applied Organic Chemistry, Lanzhou Universitry, Lanzhou, China. Fax: +86‐931‐891‐2582;2. Department of Pharmacy, Gansu College of Traditional Chinese Medicine, Lanzhou, China;3. Department of Pharmacy, General Hospital of Lanzhou, Lanzhou, China |
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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 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. |
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Keywords: | Diastereoisomers separation High‐performance liquid chromatography– electrospray tandem mass spectrometry Nitroxide Podophyllotoxin Relative abundance |
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