Clinical Chemistry
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Clinical Chemistry 53: 528-530, 2007. First published February 1, 2007; 10.1373/clinchem.2006.074096
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(Clinical Chemistry. 2007;53:528-530.)
© 2007 American Association for Clinical Chemistry, Inc.

Technical Briefs

HPLC-Electrospray Tandem Mass Spectrometry for Rapid Determination of Dihydropyrimidine Dehydrogenase Activity

André B.P. van Kuilenburga, Henk van Lenthe, Lida Zoetekouw and Willem Kulik

Emma Children’s Hospital and Department of Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;

aaddress correspondence to this author at: Academic Medical Center, Laboratory Genetic Metabolic Diseases, F0-224, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; fax: 31-206962596, e-mail a.b.vanKuilenburg{at}amc.uva.nl)


Abstract

Background: Patients with a partial dihydropyrimidine dehydrogenase (DPD) deficiency have an increased risk of developing severe 5-fluorouracil–associated toxicity. We developed a rapid and specific method to measure the DPD activity in peripheral blood mononuclear cells using HPLC tandem-mass spectrometry (HPLC-MS/MS).

Methods: The activity of DPD was measured with thymine as the substrate, followed by reversed-phase HPLC combined with electrospray ionization MS/MS and detection of the product dihydrothymine with multiple-reaction monitoring. Stable-isotope labeled dihydrothymine was used as the internal standard.

Results: Dihydrothymine was measured within an analytical run of 10 min, with a lower limit of quantification of 54 µg/L (0.4 µmol/L). The intraassay and interassay variations of the DPD activity assay were both <7%. A linear correlation (R2 = 0.980; P <0.001) was observed between the HPLC-MS/MS data and those obtained with a reference method using radiolabeled thymine. There were no systematic differences between the 2 methods, and both methods yielded similar results.

Conclusion: The analysis of the DPD activity with HPLC-MS/MS is rapid, accurate, and sufficiently sensitive to be used as a screening method for patients with a DPD deficiency.






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Copyright © 2007 by the American Association for Clinical Chemistry.