Measurement of urine Orotic acid and Orotidine by UPLC – MS/MS

1. Measurement of urine Orotic acid and Orotidine by UPLC – MS/MS Elisabeth Schwarz1, Nicola Longo 1,2,3,4 , Marzia Pasquali1,2,3,4 1ARUP Institute for Clinical and Experimental Pathology®, Salt Lake City, UT, 2Departments of Pathology and 3Pediatrics ; 4ARUP Laboratories, Inc, University of Utah Health Sciences Center,Salt Lake City, UT. Abstract # 76 Introduction: Sensitivity Recovery Orotic acid and orotidine are intermediates in the synthesis of pyrimidine nucleotides. They can accumulate in disorders of the synthesis of pyrimidines, hereditary orotic aciduria, and in urea cycle defects. Their determination in urine is an essential tool to aid in the diagnosis of these inherited conditions. Here we report a new method to measure urine orotic acid and orotidine using stable isotope dilution with ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Imprecision: • Table 1. Chromatographic gradient used for the separation of orotic acid and orotidine using a Waters UPLC-MS/MS system. • MS/MS ANALYSIS: the data were acquired using Selective Reaction Monitoring mode. • The transitions monitored were the following: • Orotic acid: 155 > 111, d2-Orotic acid: 157 > 113 • Orotidine: 287 > 111 • Data were analyzed using QuanLynx V4.1 SP4 software Linearity: Accuracy: Orotic acid Orotidine Method: • Sample Preparation: • Urine was de-proteinized using centrifugal filter and diluted (1:20) with 10% acetone. • 100 µL of diluted samples were transferred into a clean 96-well plate and 10 µL of 10µM d2- Orotic acid were added. • 5 µl were injected into the UPLC-MS/MS for analysis. Orotic acid and Orotidine were eluted using a gradient (Table 1) of water acidified with 0.1% formic acid and acetonitrile. • Injection to injection time was 5 min. • The chromatographic column used was a Waters HSS T3, 1.7µm, 2.1 x 100 mm column Results: Deming regression analysis: Orotic acid: Y = 1.016 X - 2.8 Orotidine: Y = 1.005 X - 0.6 Y = 0.974 X – 0.02 Y = 1.016 X – 0.2 The sensitivity of orotic acid and orotidine was 0.05µM. The method was linear up to 100µM and 200µM for orotidine and orotic acid respectively. We have evaluated samples from patients with urea cycle defects to validate the method. Their urinary excretion of orotidine and orotic acid was significantly higher than normal control (Figure 1). Conclusions: • UPLC-MS/MS allows rapid analysis of orotic acid and orotidine • The sample preparation for this analysis is simple and inexpensive. • There are no interferences observed with this method Figure 1: Chromatographic separation of Orotic acid and orotidine. Orotic acid and orotidine urine excretion in normal control. Orotic acid and orotidine urine excretion in patient with urea cycle defect. NORMAL Orotidine Orotic Acid Orotic acid Orotidine UREA CYCLE DEFECT Orotic Acid Orotidine d2 -Orotic acid