Rapid Determination of Orotic Acid in Urine by Liquid Chromatography–Electrospray Tandem Mass Spectrometry

Abstract

Orotic acid (ORA) is an important biochemical marker for uridine monophosphate synthase deficiency, an autosomal recessive disease characterized by macrocytic hypochromic megaloblastic anemia, growth retardation, orotic aciduria, and crystalluria (1). In addition, patients with urea cycle diseases excrete increased amounts of urinary ORA (2). Thus, ORA aciduria is observed in patients with ornithine carbamoylasetransferase deficiency (OCTD), an X-linked disorder, and could reveal heterozygosity after a protein load, and in citrullinemia, argininosuccinic aciduria, and argininemia (2)(3). Currently there are two widely accepted approaches to the determination of urinary ORA, stable-isotope-dilution gas chromatography–mass spectrometry (GC/MS), and ion-exchange HPLC methods with ultraviolet detection, each of which has difficulties and limitations (4)(5)(6)(7). Ito et al.(8) recently described a liquid chromatography–electrospray tandem mass spectrometry (LC-ESI-MS/MS) method for a large number of urinary metabolites, including ORA. In the present work we describe a more specific, high-throughput, sensitive stable-isotope-dilution LC-ESI-MS/MS method for the determination of urinary ORA. ORA was purchased from Sigma, and [1,3-15N2]orotic acid used as internal standard (IS) was obtained from Cambridge Isotope Laboratories. Acetonitrile and glacial acetic used for HPLC were purchased from Fisher Scientific. Syringe-driven membrane filter units were of LCR type (0.45 μm pore size) obtained from Millipore. Glass autosampler vials (2-mL capacity) and 300-μL vial inserts were purchased from Agilent. Urine samples were from pediatric patients investigated for metabolic diseases. Adult urine samples were from healthy laboratory personnel. All urine samples were stored at −20 °C until analysis. MS/MS analysis was carried out on a Micromass QuattroLC triple quadrupole mass spectrometer interfaced with a Z-spray ESI source and equipped with a Jasco HPLC pump Model PU-980 and a Jasco Model AS-950 autosampler. The ESI source was operated in the negative-ion mode with the source kept at 150 …