用HPLC-MS/MS法测定人尿液中依普利酮的浓度

Determination of eplerenone concentration in human urine by HPLC-MS/MS method

目的:建立HPLC-MS/MS法测定人尿液中依普利酮的浓度。方法:用蛋白质沉淀法处理尿液,采用Waters Symmetry C18色谱柱(50 mm×4.6 mm,5μm),流动相为甲醇(A)-乙腈(B)-1 mmol/L乙酸铵水溶液(C),梯度洗脱(0.01min:15%A,85%C;0.8min:100%A;2.1min 50%A,50%B;2.11~3.5min:15%A,85%C);流速:1.0ml/min,柱温:40℃,采用电喷雾离子源,正离子扫描模式检测。考察该方法的专属性、标准曲线、定量下限、精密度、回收率、基质效应和稳定性。结果:依普利酮尿液浓度在5~2000ng/ml范围内线性关系良好(r>0.996 8),定量下限为5ng/ml。批内精密度RSD<4.31%,批间精密度RSD<6.69%,提取回收率在94.41%~98.47%,基质效应在83.97%~99.22%(n=6)。结论:该法准确、灵敏,重复性好,可用于依普利酮在人尿液中浓度的测定。

Objective：To establish a HPLC-MS/MS method for concentration determination of eplerenone in human urine.Methods：The urine was treated by protein precipitation.Waters Symmetry C18 column（50 mm×4.6 mm,5μm）was employed with the mobile phase consisting of methanol（A）-acetonitrile（B）-1 mmol/L ammonium acetate solution（C）,gradient elution（0.01 min,15% phase A,85% phase C;0.8 min,100% phase A;2.1 min,50% phase A,50% phase B;2.11-3.5 min,15%phase A,85% phase C）at a flow rate of 1.0 ml/min.The column temperature was 40 ℃.Mass spectrometry was performed using electro spray ionization（ESI）,combined with positive ion scanning switch.A comprehensive investigation was made on the specificity,standard curve,lower limit of quantitation,precision,recovery rate,matrix effect and stability of the substance.Results：A good linearity（r=0.996 8）of eplerenone was obtained in the concentrations range of 5-2000 ng/ml,with the lower limit of quantitation of 5 ng/ml.The rates of intra-and inter-batch precision were less than 4.31% and 6.69%,respectively.The extraction recovery rate ranged from 94.41%to 98.47%,and matrix effect was within the range of 83.97%-99.22%（n=6）.Conclusion：The method was accurate,sensitive,reproducible,and could be used for the determination of eplerenone in human urine.