Orotic acid quantification in dried blood spots and biological fluids by hydrophilic interaction liquid chromatography tandem mass spectrometry

Orotic acid (ORA) is an intermediate metabolite in the pathway of pyrimidine nucleotides; its urinary excretion is useful to diagnose the hereditary orotic aciduria and some hyperammonemic inherited defects of urea cycle enzymes and amino acid transporters. ORA analysis is based on stable isotope dilution by GC-MS or LC-MS/MS methods. We developed a fast assay that measures the ORA in dried blood spots (DBS), plasma and urine using hydrophilic interaction LC-MS/MS. Within- and between-day analytical imprecision (CV%) of three quality control levels, in plasma, DBS and urine, ranged from 0.8 to 14.1%, while the inaccuracy ranged from 13.5 to 9.4%. In healthy children (n = 20), ORA concentrations were less than 0.69 mu M in plasma, less than 0.82 mu M in DBS and from 0.2 to 1.4 mmol/mol of creatinine in urine. A patient with citrullinemia showed ORA levels of 133 mu M in plasma and 39 mu M in DBS. A patient with hyperammonemia-hyperornithinemia-homocitrullinemia (HHH) syndrome presented a urinary ORA level of 9.1 mmol/mol of creatinine. The method is potentially able to discriminate affected patients from reference subjects; the clinical validation should be expanded on a higher number of patients.