Orotic acid (OA) is an anabolic intermediate in pyrimidine biosynthesis that originates from carbamyl phosphate. It is present only in trace amount in normal urine, but its excretion increases during pregnancy, drug treatment or because of metabolic block in the urea cycle. Several techniques have been used to measure OA. The widelyused colorimetric method is simple and quite rapid, but suffers from interfering compounds that, if not removed (Harris and Oberholzer 1980) reduce the accuracy of the assay. Because clinicians more and more often require both urinary orotic and organic acid determination, we developed a new stable isotope dilution assay for OA using the same processed urine as for organic acid analysis.Urine samples from patients and controls corresponding to 0.5 mg of creatinine were mixed with 50nmol [15Nz]orotic acid. Organic acids were isolated by solvent extraction according to DiDonato et al (1986) except that after acidification and saltsaturation with NaC1 samples were extracted once with an equal volume of tetrahydrofuran (THF) and twice, respectively with ethyl acetate and diethyl ether. Derivatization was performed with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). A silylated sample aliquot was injected into a Perkin Elmer model 8420 gas chromatograph equipped with a 30 m SPB-20, 0.25 mm ID fused capillary column and interfaced with a Perkin Elmer Ion Trap Detector. The electron impact ionization of the tri-TMS derivative of OA gave two ions with high relative intensity that were
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