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-ss-aminoisobutyric acid, and N:-carbamyl-ss-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 micromol/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 micromol/L). Recoveries were 89-99% at 100-200 micromol/L and 95-106% at 1 mmol/L in liquid urines, and 93-103% at 100-200 micromol/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.
van Lenthe
H
H
Academic Medical Center, University of Amsterdam, Emma Children's Hospital, and the Department of Clinical Chemistry, PO Box 22700, 1100 DE Amsterdam, The Netherlands.
van Kuilenburg
A B
AB
Ito
T
T
Bootsma
A H
AH
van Cruchten
A
A
Wada
Y
Y
van Gennip
A H
AH
D002851:Chromatography, High Pressure Liquid; D006801:Humans; D010209:Paper; D011743:Pyrimidines / Q000378:metabolism / Q000652:urine*; D015203:Reproducibility of Results; D013048:Specimen Handling; D021241:Spectrometry, Mass, Electrospray Ionization; D013941:Thymine / Q000378:metabolism / Q000652:urine; D014498:Uracil / Q000378:metabolism / Q000652:urine
D011743:Pyrimidines; D014498:Uracil; D013941:Thymine