HPLC-electrospray tandem mass spectrometry for rapid determination of dihydropyrimidine dehydrogenase activity

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. © 2007 American Association for Clinical Chemistry.