title: 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 microg/L (0.4 micromol/L). The intraassay and interassay variations of the DPD activity assay were both <7%. A linear correlation (R(2) = 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Emma Children's Hospital and Department of Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. a.b.vanKuilenburg@amc.uva.nl
authors.forename: André B P
authors.initials: AB
authors.lastname: van Kuilenburg
authors.forename: Henk
authors.initials: H
authors.lastname: van Lenthe
authors.forename: Lida
authors.initials: L
authors.lastname: Zoetekouw
authors.forename: Willem
authors.initials: W
authors.lastname: Kulik
mesh_terms: D002851:Chromatography, High Pressure Liquid; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000097:blood*; D006801:Humans; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D042943:Dihydrouracil Dehydrogenase (NADP)