Journal of Chromatography B 2010-07-01

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

Kinta M Serve, Jennifer L Darnell, Jody K Takemoto, Neal M Davies, Margaret E Black

Index: J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 878(21) , 1889-92, (2010)

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Abstract

The efficacy of the chemotherapeutic drug 5'-fluorouracil is reduced by catabolism to 2'-fluoro-beta-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 beta-mercaptoethanol, and the resulting products assayed. Calibration curves were linear over a range of 10-200 microg/ml and the method was successfully applied to the examination of DPD activity in cultured cells.Copyright 2010 Elsevier B.V. All rights reserved.