Validation of an isocratic HPLC method to detect 2-fluoro-beta-alanine for the analysis of dihydropyrimidine dehydrogenase activity.
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.
Serve
Kinta M
KM
School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA.
Darnell
Jennifer L
JL
Takemoto
Jody K
JK
Davies
Neal M
NM
Black
Margaret E
ME
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
D013438:Sulfhydryl Compounds; D015091:beta-Alanine; C032348:alpha-fluoro-beta-alanine; D009764:o-Phthalaldehyde; D042943:Dihydrouracil Dehydrogenase (NADP); C007638:ethanethiol; D005472:Fluorouracil