Defects in pyrimidine degradation identified by HPLC-electrospray tandem mass spectrometry of urine specimens or urine-soaked filter paper strips

Background: Urinary concentrations of thymine, uracil, and their degradation products are useful indicators of deficiencies of enzymes of the pyrimidine degradation pathway. We describe a rapid, specific method to measure these concentrations to detect inborn errors of pyrimidine metabolism. Methods: We used urine or urine-soaked filter-paper strips as samples and measured thymine, uracil and their degradation products dihydrothymine, dihydrouracil, N-carbamyl-β-aminoisobutyric acid and N-carbamyl-β-alanine. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards. Results: All pyrimidine degradation products could be measured in one analytical run of 15 min. Detection limits were 0.4-4 μmol/L. The intraassay imprecision (CV) of urine samples with added compounds was 1.3-12% for liquid urines and 1.0-10% for filter-paper extracts of the urines. The interassay imprecision (CV) was 3-11% (100-200 μmol/L). Recoveries were 89-99% at 100-200 μmol/L and 95-106% at 1 mmol/L in liquid urines and 93-103% at 100-200 μmol/L and 100-106% at 1 mmol/L in filter-paper samples. Correct identifications of deficiencies of the pyrimidine-degrading enzymes were readily made with urine samples from patients with known defects. Conclusions: HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders of the pyrimidine degradation pathway and filter-paper samples allow easy collection, transport and storage of urine samples. (C) 2000 American Association for Clinical Chemistry.