title: Validation of an isocratic HPLC method to detect 2-fluoro-beta-alanine for the analysis of dihydropyrimidine dehydrogenase activity.
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.
authors.affiliation: School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA.
authors.forename: Kinta M
authors.initials: KM
authors.lastname: Serve
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Darnell
authors.forename: Jody K
authors.initials: JK
authors.lastname: Takemoto
authors.forename: Neal M
authors.initials: NM
authors.lastname: Davies
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Black
mesh_terms: D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000378:metabolism*; D004355:Drug Stability; D005472:Fluorouracil; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D009764:o-Phthalaldehyde / Q000737:chemistry
chemical_list: D013438:Sulfhydryl Compounds; D015091:beta-Alanine; C032348:alpha-fluoro-beta-alanine; D009764:o-Phthalaldehyde; D042943:Dihydrouracil Dehydrogenase (NADP); C007638:ethanethiol; D005472:Fluorouracil