Sensitive method for the quantification of urinary pyrimidine metabolites in healthy adults by gas chromatography–tandem mass spectrometry

Abstract

Enzyme deficiencies in pyrimidine metabolism are associated with a risk for severe toxicity against the antineoplastic agent 5-fluorouracil. To assess whether urinary levels of pyrimidines and their metabolites can be used for predicting patients’ individual phenotype, a new gas chromatographic–tandem mass spectrometric method was developed which allows the simultaneous determination of uracil and thymine and their metabolites dihydrouracil, dihydrothymine, β-ureidopropionic acid, β-ureidoisobutyric acid, and the amino acids β-alanine and β-aminoisobutyric acid in human urine. Small aliquots (2–20 μl) of the urine samples were evaporated and derivatized to the tert.-butyldimethylsilyl derivatives before quantification, using the respective stable isotope-labelled analogues as internal standards. Analytical variation was acceptable with an intra-day imprecision (RSD) below 10%, for β-ureidoisobutyric acid below 15%. The method was used for investigating the stability of urine samples and the influence of urine collection at different times.