High-performance liquid chromatographic determination of orotic acid as its methyl derivative in human urine

Described is a method for the determination of orotic acid as its methyl ester in human urine. The method involves the use of solid-phase extraction to isolate pyrimidines from urine and derivatization with methanol and sulfuric acid, followed by isocratic high-performance liquid chromatography on a reversed-phase C₁₈ column with UV absorbance detection. The assay is shown to be sufficiently sensitive for use in clinical investigations where elevated orotic acid excretion is suspected.