Determination Of Urea, Uric Acid And Creatinine In Human Serum Using Isotope-Dilution Liquid Chromatography

Determination of human serum urea, uric acid and creatinine is a key index in clinical detection and is providing a basis for disease diagnosis. Various methods were used in clinical laboratory for the determination of urea, uric acid and creatinine at present. However lacking of comparability of interlaboratory measurement results, it is necessary to establish accurate and reliable reference methods, develop relevant standard material for sufficing clinical testing.In this paper,13C,15N2-urea was used as an internal standard and acetonitrile as the protein precipitating agent. A triple quadrupole mass spectrometry with electrospray ion source was used for monitoring the target ions (m/z 61.1-44,64.1-46) with its multiple reaction monitoring mode (MRM). Bracketing method was used for quantification. Conditions of pre-treatment, liquid chromatography separation, mass spectrometry detection and measurement conditions for the determination of urea in serum by liquid chromatography-tandem mass spectrometry isotope dilution method were discussed.Two concentrations of serum urea was determined by using the method, the relative standard deviation (RSD) were less than 1%. The method was validated by using the NIST standard reference material 909b Level I, the bias was-0.38% within the given uncertainty. The limit of detection of this method was 0.55μg/g (3 times the signal to noise ratio), the average recovery was 100.1% and 99.9%. The method was further confirmed by participating in RELA2009 comparison of values of serum urea. A comprehensive analysis of the impact factors in measurement uncertainty was performed. The uncertainty was 1.2% and 0.84% for the two concentrations separately.This well-characterized LC-IDMS/MS method for serum urea, which demonstrates reliable, simple, good accuracy and precision, can be qualified as a candidate reference measurement procedure that can be used for providing an accuracy basing on which routine methods for urea.China National Institute of Metrology organized the Co-validation for Uric Acid and Creatinine in Serum. Isotope-dilution liquid chromatography/tandem mass spectrometry was applied to the determination. Both national metrology institute of Japan and Korea got the results which were well agreed with NIM China.