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Journal of ZheJiang University(Medical Science)  2011, Vol. 40 Issue (1): 7-11    DOI: 10.3785/j.issn.1008-9292.2011.01.002
Determination of quercetin metabolism in UGT1A3 cDNA-expressing cells by RP-HPLC
YAO Yan,ZHANG Xia,LIU Yao,YU Lu-shan,JIANG Hui-di,ZENG Su
Department of Pharmaceutical Analysis and Drug Metabolism,College of Pharmaceutical Sciences,Zhejiang University,Hangzhou 310058,China
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Abstract  Objective: To develop a RP-HPLC method for the determination of quercetin in UGT1A3 cDNA-transfected cells.
Methods: The lysate of cells transfected with human recombinant uridine 5-diphosphate glucuronosyltransferases UGT1A3 cDNA was co-incubated with quercetin,the reaction was terminated with acetonitrile,and luteolin was used as internal standard. The determination was performed on a C18 reversed phase column with a mobile phase of methanol-0.1% formic acid (V/V) at a flow rate of 1.0 ml/min. The gradient elution was as follows: 0-25 min(30∶70-80∶20,methanol:0.1% formic acid),>25-25.5 min(80∶20),>25.5-27 min(80∶20-30∶70),>27-30 min(30∶70). A UV-VIS detector was operated at 368 nm.
Results: The standard curve was linear over the concentration range of 5-200 μmol/L (r=0.9999). The limit of detection was 1.25 μmol/L(S/N≥3),and the limit of quantification was 5 μmol/L (S/N >10,RSD=6.99%). The method afforded recoveries of 99.1%-103.5%,and precisions for inter- and intra-assay were <2.5% and <8%,respectively. In addition,kinetic analysis indicated that the Km,Vmax and CL int (Vmax /Km) values for quercetin glucuronide were (62.95±13.16)μmol/L,(284.50±24.35)nmol·min-1·g-1 and 4.52 ml·min-1·g-1,respectively.
Conclusion: The method established is accurate and simple and suitable for the determination of quercetin in UGT1A3 cDNA-expressed cells.

Key wordsQuercetin/anal;       Chromatography, high pressure liquid;       Glucuronosyltransferase/ biosyn;       Recombinases     
Published: 25 January 2011
Cite this article:

. Determination of quercetin metabolism in UGT1A3 cDNA-expressing cells by RP-HPLC. Journal of ZheJiang University(Medical Science), 2011, 40(1): 7-11.

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方法:Bac-to-Bac系统表达的人UGT1A3重组酶细胞破碎液与槲皮素共孵育之后,以乙腈沉淀蛋白,采用木犀草素为内标,以Phenomenex Luna C18柱为分析柱,甲醇(A)-0.1%甲酸溶液(B)为流动相梯度洗脱:0~25 min(30∶70-80∶20,A∶B),>25~25.5 min(80∶20),>25.5~27 min(80∶20-30∶70),>27~30 min(30∶70);流速1.0 ml/min,紫外检测波长为368 nm。
结果:槲皮素在5~200 μmol/L内线性关系良好(r=0.9999),检测限为1.25 μmol/L(S/N≥3),定量限为5 μmol/L (S/N >10,RSD=6.99%),方法回收率达99.1%~103.5%,日内、日间RSD分别<2.5%和<8%。另测得UGT1A3催化槲皮素的Km为(62.95±13.16)μmol/L,Vmax为(284.50±24.35)nmol·min-1·g-1,清除率Vmax/Km为4.52 ml·min-1·g-1。

关键词: 槲皮素/分析,  色谱法,  高压液相,  葡糖醛酸基转移酶/生物合成,  重组酶类 
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