Measurement of urea in human serum by isotope dilution mass spectrometry: a reference procedure

Abstract

Background: A reference measurement procedure is needed to demonstrate the traceability of results of urea measurements in human serum. We developed a measurement procedure using the principle of isotope dilution gas chromatography/mass spectrometry.

Methods: [(13)C,(15)N(2)]Urea as internal standard was added to a serum sample and equilibrated with endogenous nonlabeled urea. For the preparation of calibrators, the same amount of labeled urea was mixed with known amounts of nonlabeled urea. The serum samples were treated with ethanol to remove proteins by precipitation. The labeled and nonlabeled urea of the samples was converted into a trimethylsilyl derivative of 2-hydroxypyrimidine. The gas chromatography/mass spectrometry system was adjusted to monitor m/z 153 and 168 for the nonlabeled urea derivative and m/z 156 and 171 for the isotopically labeled analogs. The results of the determination were calculated from peak ratios by a hyperbolic calculation function based on the theory of isotope dilution analysis.

Results: The procedure was applied to control samples and patient samples and evaluated with respect to its trueness and precision. The standard uncertainty of the results was 0.47-1.72%.

Conclusions: This reference measurement procedure allows values to be assigned to controls and calibrators that are traceable to the primary urea reference material of NIST and, therefore, to the Système International unit "mole" with a low degree of uncertainty. This procedure provides a tool for the highly accurate determination of urea in control materials as well as in patient sera.