Analysis of pyrimidine synthesis "de novo" intermediates in urine and dried urine filter- paper strips with HPLC-electrospray tandem mass spectrometry.
BACKGROUND
The concentrations of the pyrimidine "de novo" metabolites and their degradation products in urine are useful indicators for the diagnosis of an inborn error of the pyrimidine de novo pathway or a urea-cycle defect. Until now, no procedure was available that allowed the analysis of all of these metabolites in a single analytical run. We describe a rapid, specific method to measure these metabolites by HPLC-tandem mass spectrometry.


METHODS
Urine or urine-soaked filter-paper strips were used to measure N-carbamyl-aspartate, dihydroorotate, orotate, orotidine, uridine, and uracil. 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 de novo metabolites and their degradation products were measured within a single analytical run of 14 min with lower limits of detection of 0.4-3 micromol/L. The intra- and interassay variation for urine with added compounds was 1.2-5% for urines and 2-9% for filter-paper extracts of the urines. Recoveries of the added metabolites were 97-106% for urine samples and 97-115% for filter-paper extracts of the urines. Analysis of urine samples from patients with a urea-cycle defect or pyrimidine degradation defect showed an aberrant metabolic profile when compared with controls.


CONCLUSIONS
HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders affecting the pyrimidine de novo pathway. The use of filter-paper strips could facilitate collection, transport, and storage of urine samples.
van Kuilenburg
André B P
AB
Academic Medical Center, University of Amsterdam, Emma Children's Hospital and Departments of Clinical Chemistry, Amsterdam, The Netherlands. a.b.vankuilenburg@amc.uva.nl
van Lenthe
Henk
H
Löffler
Monika
M
van Gennip
Albert H
AH
D056807:Argininosuccinic Aciduria; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000652:urine; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D020163:Ornithine Carbamoyltransferase Deficiency Disease; D010209:Paper; D011743:Pyrimidines / Q000378:metabolism / Q000652:urine*; D012016:Reference Values; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
D011743:Pyrimidines; D042943:Dihydrouracil Dehydrogenase (NADP)