Objective: Dihydropyrimidine dehydrogenase (DPD) catalyzes the degradation of thymine, uracil, and the chemotherapeutic drug 5-Fluorouracil. In general reverse-phase high pressure liquid chromatography is the standard method for separating 5-[2-(14)C]Fluorouracil and 5-[2-(14)C]Fluoro-5,6-dihydrouracil. However, the use of 100% aqueous solution (as HPLC mobile phase) may collapse the C-18 bonded phase and result in a retention time shift. The aim of this study is to develop a rapid, reproducible, sensitive method for screening partial DPD deficiency in healthy volunteers.
Design and methods: The activity of DPD was measured using 5-[2- (14)C]Fluorouracil (5-[2-(14)C]FUra) followed by separation of substrate and product 5-[2-(14)C]FUraH(2) with a 15 x 4.6 mm I.D., 5 microm particle size (d(p)) porous graphitic carbon (PGC) column (Hypercarb(R)) and HPLC with online detection of the radioactivity. This was standardized using the protein concentration of the cytosol (NanoOrange(R) Protein Quantitation).
Results: Complete baseline separation of 5-[2-(14)C]Fluorouracil (5-[2-(14)C]FUra) and 5-[2-(14)C]Fluoro-5,6-dihydrouracil (5-[2-(14)C]FUraH(2)) was achieved using a porous graphitic carbon (PGC) column. The detection limit for 5-[2-(14)C]FUraH(2) was 0.4 pmol.
Conclusions: By using linear gradient separation (0.1% Trifluoroacetic acid [TFA] in water to 100% Methanol) protocols in concert with PGC columns (Hypercarb(R)), we have demonstrated that a PGC column has a distinct advantage over C-18 reverse phase columns in terms of column stability (pH 1-14). This method provides an improvement on the specific assay for DPD enzyme activity. It is rapid, reproducible and sensitive and can be used for routine screening for healthy and cancer patients for partial and profound DPD deficiency before treatment with 5- FUra.