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

To develop a method for determination of creatinine in human urine by isotope dilution high performance liquid chromatography/tandem mass spectrometry.

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.

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.

The determination method is successfully applied to measure the concentration of creatinine in human urine with efficiency, accuracy and high sensitivity.