HPLC-electrospray tandem mass spectrometry for rapid determination of dihydropyrimidine dehydrogenase activity.
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.
van Kuilenburg
André B P
AB
Emma Children's Hospital and Department of Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. a.b.vanKuilenburg@amc.uva.nl
van Lenthe
Henk
H
Zoetekouw
Lida
L
Kulik
Willem
W
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
D042943:Dihydrouracil Dehydrogenase (NADP)