HPLC法测定人尿中右美沙芬及去甲右美沙芬的含量及CYP2D6表型区分研究

Determination of Dextromethorphan and Dextrophan in Human Urine by HPLC and Study on the Discrimination of Phenotype of Cytochrome CYP2D6

目的：建立以高效液相色谱法测定人尿中右美沙芬及去甲右美沙芬含量的方法，并行CYP2D6表型区分。方法：尿样经酶水解，酸、碱提取纯化，直接进样。其中色谱柱为Waters Nova—Pak Phenyl，流动相为乙腈-水（60：40），流速为1．0mL·min^-1，激发波长为280nm，发射波长为310nm，柱温为25℃。结果：右美沙芬、去甲右美沙芬尿药浓度分别在0．065～4．148（r=0．9985）、0．353～22．592（r=0．9997）μg·mL^-1范围内线性关系良好，最低检测限分别为0．016、0．018μg·mL^-1，平均加样回收率各为90．5％、93．5％，日内RSD各≤3．26％、≤3．47％，日间RSD各≤3．65％、≤4．05％。8位受试者均为CYP2D6快代谢型。结论：本方法简便可靠，适用于人尿中右美沙芬及去甲右关沙芬含量测定和CYP2D6多态性的表型研究。

OBJECTIVE： To establish an HPLC method for the determination of dextromethorphan and dextrophan in human urine and to discriminate phenotype of CYP2D6. METHODS： The urine samples were hydrolyzed by enzyme, distilled and purified by acid and alkali before direct introduction. HPLC system was adopted using Waters Nova- Pak Phenyl column with mobile phase consisted of acetonitrile - water （60 ： 40） at a flow rate of 1.0 mL · min^-1. The fluorescence detection was performed with excitation wavelength at 280 nm and emission wavelength at 310 nm. The column temperature was 25℃ . RESULTS： The linear ranges of dextromethorphan and dextrophan were 0. 065～4.148μg·mL^-1（ r = 0. 998 5） and 0.3S3～22. 592 μg·mL^-1（ r = 0.999 7）, respectively. The lower detection limits were 0.016μg·mL^-1 and 0.018μg·mL^-1, respectively. The average recoveries were 90.5% and 93.s%, resepctively; the intra- day RSD were no more than 3.26% and 3.47% and inter- day RSD were no more than 3.65% and 4.05%, respectively. All the 8 subjects were extensive metabolizers （EM） . CONCLUSION： The method is simple and reliable, and suitable for the determination of both dextromethorphan and dextrophan in human urine and as well as the study of the phenotype of CYP2D6 polymorphisms.