Received on August 12, 2005
Accepted on March 24, 2006

Endocrinology and Metabolism

Comprehensive Detection of Disorders of Purine and Pyrimidine Metabolism by HPLC with Electrospray Ionization Tandem Mass Spectrometry

¹ Division of Metabolic Diseases, Department of General Pediatrics, University Children's Hospital Heidelberg, Heidelberg, Germany
² Laboratory of Metabolism, Department of General Pediatrics and Adolescent Medicine, University Children's Hospital Freiburg, Freiburg, Germany
³ University of California, San Diego, La Jolla, CA

^* To whom correspondence should be addressed. E-mail: Susen.Hartmann{at}med.uni-heidelberg.de.

Background: Clinical presentation and disease severity in disorders of purine and pyrimidine metabolism vary considerably. We present a method that allows comprehensive, sensitive, and specific diagnosis of the entire spectrum of abnormalities in purine and pyrimidine metabolism in 1 analytical run.

Methods: We used reversed-phase HPLC electrospray ionization tandem mass spectrometry to investigate 24 metabolites of purine and pyrimidine metabolism in urine samples from healthy persons and from patients with confirmed diagnoses of inherited metabolic disorders. Urine samples were filtered and diluted to a creatinine concentration of 0.5 mmol/L. Stable-isotope-labeled internal standards were used for quantification. The metabolites were analyzed by multiple-reaction monitoring in positive and negative ionization modes.

Results: Total time of analysis was 20 min. Recovery (n = 8) of a compound after addition of a known concentration was 85%-133%. The mean intraday variation (n = 10) was 12%. The interday variation (n = 7) was 17%. Age-related reference intervals were established for each compound. Analysis of patient urine samples revealed major differences in tandem mass spectrometry profiles compared with those of control samples. Twelve deficiencies were reliably detected: hypoxanthine guanine phosphoribosyl transferase, xanthine dehydrogenase, purine nucleoside phosphorylase, adenylosuccinate lyase, uridine monophosphate synthase, adenosine deaminase, adenine phosphoribosyl transferase, molybdenum cofactor, thymidine phosphorylase, dihydropyrimidine dehydrogenase, dihydropyrimidinase, and -ureidopropionase.

Conclusion: This method enables reliable detection of 13 defects in purine and pyrimidine metabolism in a single analytical run.