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Technical Briefs

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

¹ Emma Children's Hospital and Department of Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

^* To whom correspondence should be addressed. E-mail: a.b.vanKuilenburg{at}amc.uva.nl.

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.