反相高效液相色谱法测定人尿中右美沙芬及去甲右美沙芬的含量

目的建立反相高效液相色谱法测定人尿中右美沙芬及其代谢产物去甲右美沙芬浓度的方法。方法以非那西丁为内标,尿样经水解,碱化后用正己烷-正丁醇(9:1)萃取,采用DiamonsilTMC18柱(250mm×4.6mm,5μm)分析。色谱条件:流动相为乙腈(A)-1%三乙胺(磷酸调节pH=2.2,B),梯度洗脱,0～15min,20%～35%A,流速为1.0ml/min,检测波长为280nm,柱温40℃。结果右美沙芬在0.05～2.0μg/ml范围内线性良好(r=0.9999,n=5),检测限为0.04μg/ml;去甲右美沙芬尿样浓度在0.5～20.0μg/ml范围内线性良好(r=0.9999,n=5),检测限为0.4μg/ml。两者日内、日间精密度RSD均<10%,低、中、高浓度的提取回收率在94%～108%之间。结论此方法简便准确、重复性好,适用于CYP2D6表型分析以及右美沙芬与其代谢产物的人体药代动力学研究。

RP-HPLC in determination of dextromethorphan and dextrophan in human urine:phenotype analysis of CYP2D6

Objective To establish a RP-HPLC method for determination of the concentrations of dextromethorphan and its metabolites dextrorphan in human urine.Methods Phenacetine was used as internal standard, and the urine sample was hydrolyzed by enzyme,alkalified and extracted with hexane-butanol(9:1).The separation was carried out on Diamonsil~(TM) C_(18) column (250 mm×4.6 mm,5μm) with mobile phase of acetonitrile-1% triethylamine buffer solution (pH adjusted to 2.2 with H_3PO_4).Gradient elution was done for 0-15 min(20%-35% A).The flow rate was 1.0 mt/min.The detection wavelength was set at 280 nm and the column temperature was 40℃.Results The linear ranges of dextromethorphan and dextrorphan were 0.05-2.0 μg/ml (r=0.9999,n=5) and 0.5-20.0μg/ml (r=0.9999,n=5),respectively, and their lowest detecting concentrations were 0.04 μg/ml and 0.4 μg/ml, respectively.The intra-day and inter-day precision were both less than 10%.The low,middle and high extraction recoveries were between 94%-108%.Conclusion Our method is accurate and sensitive,and is suitable for the CYP2D6 phenotype analysis and pharmacokinetic studies of dextromethorphan and its metabolites in human.