HPLC-DAD, HPLC-ESI/MS~n Study On Diastereoisomers Of Nature Anticancer Agents Podophyllotoxin Lignans

Podophyllotoxin is a well-known natural product due to its long history of use in folk medicine and the biological activity of its many derivatives. In particular, its glucopyranodide derivatives such as etoposide (VP-16), teniposide (VM-26), and etopofos have been widely used in the chemotherapy of cancers. Research on structure-activity relationships (SARs) indicated that the biological activity and toxicity of podophyllotoxin and its derivatives are stereochemically specific. In this dissertation, novel HPLC-DAD and HPLC-ESI/MSⁿ methods were developed and successfully applied in rapid separation, determination and identification of the diastereoisomers of podophyllotoxin and its analogues at 2 and 4 position. The method could be used in rapidly identifying the purity and monitoring of the epimerization of 2-H, 4-H of podophyllotoxin and its analogues from natural products, chemical reactions, and pharmaceutical metabolism.I 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. Interestingly, every 2α-H compound migrated before its corresponding 2β-H epimer under optimum conditions. In the HPLC-ESI/MS spectra, each pair of diastereoisomers of the derivatives in the mixture was directly confirmed and identified by [M+NH₄]⁺ ions and ion ratios of relative abundance of [M-ROH+H]⁺ (ion 397) to [M+NH₄]⁺. 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 characteristic elimination from the precursor protonated ions and the product ions at m/z 313, 282, and 229 were the common diagnostic masses. The ion ratios of relative abundance [M-ROH+H]⁺ (ion 397) to [M+NH₄]⁺, [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. The method could be rapidly identified the each pair of diastereoisomer of the lignans.On the basis of the above study, 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. The results support the research of the above study, revealing that provided a basis for identifying spin-labeled diastereoisomers of podophyllotoxin and its analogues at C-2 position in the mixture. Meanwhile, using HPLC-DAD, HPLC-ESI/MS, ESI/MS/MS, seven pairs of epipodophyllotoxin and its esters were separated and identified. The characteristics of the chromatography and mass spectrometry were studied and given a reasonable explaination. Moreover, six pairs of natural podophyllotoxin were separated and identified by HPLC-DAD and CD.II Spin-labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, configurations of these spin-labeled derivatives cannot be determined by Nuclear Magnetic Resonance (NMR) methods. HPLC-ESI/MS and ESI/MS methods were developed and validated for the separation, identification of five pairs of diastereoisomers of spin-labeled derivatives of podophyllotoxin at C-4 position. It was found that the migration behaviors of the compounds with different types were regular, that is, every 4β-H compound migrated before its corresponding 4α-H epimer under optimum conditions. In the HPLC-ESI/MS spectra, each pair of diastereoisomers of the lignans in the mixture was directly confirmed and identified by [M+H]⁺ ions and EIC. Off-line ESI-MS were performed to study the relationship between the structural characteristics and ESI/MS fragmentation pathways of the diastereoisomers at C-4 of the lignans. The ion ratios of relative abundance [M-ROH+Na]⁺ to [M+Na]⁺ in the ESI/MS spectra of each pair of diastereoisomers of the lignans specifically exhibited a stereochemical effect. Undoubtedly, for the Spin-labeled nitroxide derivatives of podophyllotoxin, this is a novel separation and identification method.III Podophyllum emodi Wall var. Chinesis. which is existed in the western China. Plant containing podophyllotoxin analogues have been used as folk remedies in traditional medicinal of many diverse cultures. Compounds extracted from P. Wall var. Chinesis. in three extraction methods were analyed by HPLC-DAD, HPLC-ESI/MS. Eighteen components were unambiguously identified or tentatively characterized based on their retention times, UV spectra and mass spectra in comparison with the data from the reference standards. The results may be useful for the quality control of P. Wall var. Chinesis.and its related preparations.