کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
9749076 1493800 2005 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Development and validation of a microsomal online cytochrome P450 bioreactor coupled to solid-phase extraction and reversed-phase liquid chromatography
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Development and validation of a microsomal online cytochrome P450 bioreactor coupled to solid-phase extraction and reversed-phase liquid chromatography
چکیده انگلیسی
The development and validation of an online cytochrome P450 (CYP)-based bioreactor coupled to automated solid-phase extraction (SPE) and gradient HPLC separation is described. The analytical method was checked on intra- and inter-day repeatability of the ethoxyresorufin-O-demethylation (EROD) reaction with CYP 1A1/1A2 containing β-NF induced rat liver microsomes as an enzyme source. These experiments showed that CYP activity was linearly decreased with 16% over an 11 h period. Inter-day measurements had a CV of 9.1%. Furthermore, Km and Vmax values of the EROD reaction, measured with the bioreactor, were 2.72 ± 0.46 μM and 7.9 ± 0.5 nmol/min/mg protein, respectively. These were in good correspondence with Km and Vmax values, measured with standard batch assay, which amounted 0.66 ± 0.08 μM and 6.4 ± 0.2 nmol/min/mg protein respectively. In conclusion the newly developed analytical method can be used effectively and at a microliter scale for online generation, extraction and separation of metabolites.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography A - Volume 1075, Issues 1–2, 20 May 2005, Pages 205-212
نویسندگان
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