Validation of an isocratic HPLC method to detect 2-fluoro-β-alanine for the analysis of dihydropyrimidine dehydrogenase activity

The efficacy of the chemotherapeutic drug 5′-fluorouracil is reduced by catabolism to 2′-fluoro-β-alanine (FBAL), a three-step reaction in which dihydropyrimidine dehydrogenase (DPD) catalyzes the rate-limiting step. To study in vitro DPD activity, we developed and validated an isocratic, reverse-phase HPLC method to detect and quantify FBAL without using multiple columns or radiolabeled substrates. Pre-column derivatization of FBAL was performed using o-phthalaldehyde in the presence of two sulfur donors, ethanthiol or β-mercaptoethanol, and the resulting products assayed. Calibration curves were linear over a range of 10–200 μg/ml and the method was successfully applied to the examination of DPD activity in cultured cells.