同位素稀释高效液相色谱串联质谱测定尿中肌酐

Analysis of creatinine in human urine by isotope dilution high performance liquid chromatography/tandem mass spectrometry

目的建立同位素稀释高效液相色谱串联质谱测定尿中肌酐的分析方法。方法尿液样品直接用甲醇-水溶液(7∶3,V/V)稀释800倍,经0.45μm滤膜过滤净化,使用Atlantis Hilic Silica(2.1 mm×150 mm,3μm)色谱柱分离样品,甲醇-水溶液(7∶3,V/V)为流动相进行等度洗脱,电喷雾离子源正离子模式(ESI+)和多反应监测模式(MRM)采集信号,肌酐同位素内标进行定量,利用尿肌酐标准参考物质评价方法准确性,并对测定方法进行不确定度评价。结果肌酐标准曲线在0.31~5.00μg/mL范围内线性关系良好,相关系数为0.999 9;尿液中肌酐的定性检出限(LOD)为0.003 mg/mL,定量检出限(LOQ)为0.011 mg/mL;实际尿液样品在两个浓度水平的加标回收率分别为93.9%~104.0%和90.6%~93.8%,相对标准偏差为4.4%~7.6%和4.6%~7.8%(n=6);尿肌酐标准参考物质定量结果在参考限值范围内;测定结果的不确定度主要源于标准曲线、回收率和仪器允差。结论本方法可用于测定人尿液中肌酐浓度,其测定值准确可靠,灵敏度高,稳定性好。

Objective To develop a method for determination of creatinine in human urine by isotope dilution high performance liquid chromatography/tandem mass spectrometry. Methods The urine samples were directly diluted 800 fold by mixed solution of methanol and water（ 7 ∶ 3,V/V）,and filtered with 0. 45 μm filter. The chromatographic separation was carried on an Atlantis Hilic Silica column（ 2. 1 mm × 150 mm,3 μm）. Methanol and water mixture solution（ 7 ∶ 3,V/V） were used as the mobile phases in an isocratic gradient. Mass detection was then conducted by electrospray ionization in positive ion（ ESI＋） mode and multiple reaction monitoring（ MRM） mode.The concentration of creatinine was quantified by stable isotope labelled d3-creatinine internal standard. Standard reference material of urine creatinine was used to evaluate the accuracy of method,and then the uncertainty of this detection method was also evaluated.Results The calibration curve of creatinine showed a good linear relationship in the range of 0. 31 μg/mL to 5. 00 μg/mL,and the related coefficient was 0. 999 9. The limit of detection（ LOD） and limit of quantitation（ LOQ） were 0. 003 mg/mL and 0. 011 mg/mL respectively,in real urine. Recoveries rates at two levels were 93. 9%-104. 0% and 90. 6%-93. 8%,with the relative standard deviation of 4. 4%-7. 6% and 4. 6%-7. 8%（ n = 6）,respectively. The creatinine concentration of standard reference material was in accordance with the reference value. The measurement uncertainty was mainly due to standard curves,recoveries rates and permissible error of instrument. Conclusion The determination method is successfully applied to measure the concentration of creatinine in human urine with efficiency,accuracy and high sensitivity.