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

Abstract

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 microM in plasma, less than 0.82 microM in DBS and from 0.2 to 1.4 mmol/mol of creatinine in urine. A patient with citrullinemia showed ORA levels of 133 microM in plasma and 39 microM 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.

Reference intervals for orotic acid in urine, plasma and dried blood spot using hydrophilic interaction liquid chromatography–tandem mass spectrometry

“…Baker (Deventer, The Netherlands). OA standard was purchased from Sigma-Aldrich (Steinheim, Germany), and [1,[3][4][5][6][7][8][9][10][11][12][13][14][15] N 2 ]-OA, used as internal standard (IS), was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Filter paper for DBS preparation was of grade 903 and was purchased from Whatman GmbH (Dassel, Germany).…”

“…Urine, plasma, and DBS samples were analyzed by HILIC-MS/MS performed according to the method previously described [8]. Briefly, chromatography was performed by a column packed with sub-2 m particles (Zorbax RX-SIL, 1.8 m, 50 mm × 2.1 mm id, Agilent Technologies, USA), and OA was eluted by an isocratic mobile phase constituted of ACN/water (90/10) containing 4 mM of ammonium acetate.…”

“…Using this method, OA levels in plasma and DBS from a patient affected by citrullinemia were at least fifty times higher than normal controls [8]. These rare data make it difficult to define safety limits of OA in blood and its usefulness as a biomarker of disease; moreover, reference intervals in blood of healthy humans are completely lacking.…”

“…Analysis of urinary OA provides an integrated picture of OA production over time, rather than the snapshot that is provided by analysis of a single plasma sample [7]. Although the OA in urine represents worldwide the reference test for differential diagnosis and for the monitoring of disorders above mentioned, patients affected by urea cycle defects also show elevated OA levels in plasma or blood [3,4,8], and only few affected patients with these disorders have been carefully investigated analyzing OA in blood or plasma. Increased concentration of OA in plasma and blood has been found in ASL [4], and OTC defects [9].…”

Pre-analytic evaluation of volumetric absorptive microsampling and integration in a mass spectrometry-based metabolomics workflow

“…Nowadays, the use of DBS sampling has become a common practice in several clinical applications, for the diagnosis of an inborn error of metabolism [2][3][4] and for therapeutic drug monitoring and clinical drug development [5,6], including toxicology [7].…”