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浙江大学学报（医学版）
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 words： Quercetin/anal；       Chromatography, high pressure liquid；       Glucuronosyltransferase/ biosyn；       Recombinases     
Published: 25 January 2011
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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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https://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2011.01.002     OR     https://www.zjujournals.com/med/Y2011/V40/I1/7


RP-HPLC法测定人尿苷二磷酸葡醛酸转移酶UGT1A3转基因细胞中槲皮素

目的：建立反相高效液相色谱法测定人重组UGT1A3转基因细胞中槲皮素，并研究该酶对槲皮素的相关代谢及动力学参数测定。
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。
结论：建立的反相高效液相色谱方法准确、简便；可用于槲皮素在人UGT1A3转基因细胞中的代谢研究。

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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