PaO体外活力测定需要Fd作还原剂.通过丙酮沉淀法、DEAE柱层析等制备菠菜Fd,并将其应用于HPLC-荧光法研究PaO催化的Pheidea降解反应.结果表明:经DEAE柱层析纯化得到的是氧化型Fd,而未经DEAE柱层析得到的是还原型Fd,还原型Fd可以参与Phei...PaO体外活力测定需要Fd作还原剂.通过丙酮沉淀法、DEAE柱层析等制备菠菜Fd,并将其应用于HPLC-荧光法研究PaO催化的Pheidea降解反应.结果表明:经DEAE柱层析纯化得到的是氧化型Fd,而未经DEAE柱层析得到的是还原型Fd,还原型Fd可以参与Pheidea降解反应,色谱峰面积可以表示PaO的活力;FCC的HPLC洗脱时间是5.1min,遮光条件下FCC的半衰期是34.66min,PaO在20℃催化Pheidea降解的活力较高.小麦幼苗离体暗诱导衰老过程中PaO活力变化幅度较大,暗处理5d其活力增加24.47倍, PaO Chl降解途径在麦类作物叶片衰老过程中普遍存在.展开更多
A simple and sensitive high performance liquid chromatography with fluorescence detection （HPLC-FD） has been developed for simultaneous quantification of doxorubicin （DOX） and its dipeptide conjugate prodrug （PDO...A simple and sensitive high performance liquid chromatography with fluorescence detection （HPLC-FD） has been developed for simultaneous quantification of doxorubicin （DOX） and its dipeptide conjugate prodrug （PDOX） in mice plasma. The chromatographic separation was carried out on an Amethyst C18-H column with gradient mobile phase of 0.1% formic acid and 0.1% formic acid in acetonitrile at a flow rate of 1.0 mL/min. The excitation and emission wavelengths were set at 490 and 550 nm, respectively. The method was comprehensively validated. The limits of detection were low up to 5.0 ng/mL for DOX and 25.0 ng/mL for PDOX. And the limits of quantification were low up to 12.5 ng/mL for DOX and 50 ng/mL for PDOX, which were lower than those for most of the current methods. The calibration curves showed good linearity （R2 〉 0.999） over the concentration ranges. The extraction recoveries ranged from 84.0% to 88.2% for DOX and from 85.4% to 89.2% for PDOX. Satisfactory intra-day and inter-day precisions were achieved with RSDs less than 9.1%. The results show that the developed HPLC-FD method is accurate, reliable and will be helpful for preclinical pharmacokinetic study of DOX and PDOX.展开更多
文摘PaO体外活力测定需要Fd作还原剂.通过丙酮沉淀法、DEAE柱层析等制备菠菜Fd,并将其应用于HPLC-荧光法研究PaO催化的Pheidea降解反应.结果表明:经DEAE柱层析纯化得到的是氧化型Fd,而未经DEAE柱层析得到的是还原型Fd,还原型Fd可以参与Pheidea降解反应,色谱峰面积可以表示PaO的活力;FCC的HPLC洗脱时间是5.1min,遮光条件下FCC的半衰期是34.66min,PaO在20℃催化Pheidea降解的活力较高.小麦幼苗离体暗诱导衰老过程中PaO活力变化幅度较大,暗处理5d其活力增加24.47倍, PaO Chl降解途径在麦类作物叶片衰老过程中普遍存在.
基金supported by the National Natural Science Foundation of China (Grant nos. 21375101, 81573384 and 91417301)Natural Science Foundation of Hubei Province, China (No. 2014CFA077)Innovation Seed Fund and Translational Medical Research Fund of Wuhan University School of Medicine, China
文摘A simple and sensitive high performance liquid chromatography with fluorescence detection （HPLC-FD） has been developed for simultaneous quantification of doxorubicin （DOX） and its dipeptide conjugate prodrug （PDOX） in mice plasma. The chromatographic separation was carried out on an Amethyst C18-H column with gradient mobile phase of 0.1% formic acid and 0.1% formic acid in acetonitrile at a flow rate of 1.0 mL/min. The excitation and emission wavelengths were set at 490 and 550 nm, respectively. The method was comprehensively validated. The limits of detection were low up to 5.0 ng/mL for DOX and 25.0 ng/mL for PDOX. And the limits of quantification were low up to 12.5 ng/mL for DOX and 50 ng/mL for PDOX, which were lower than those for most of the current methods. The calibration curves showed good linearity （R2 〉 0.999） over the concentration ranges. The extraction recoveries ranged from 84.0% to 88.2% for DOX and from 85.4% to 89.2% for PDOX. Satisfactory intra-day and inter-day precisions were achieved with RSDs less than 9.1%. The results show that the developed HPLC-FD method is accurate, reliable and will be helpful for preclinical pharmacokinetic study of DOX and PDOX.
