title: LC-MS/MS method for simultaneous analysis of uracil, 5,6-dihydrouracil, 5-fluorouracil and 5-fluoro-5,6-dihydrouracil in human plasma for therapeutic drug monitoring and toxicity prediction in cancer patients.
abstract: The chemotherapeutic drug 5-fluorouracil (5-FU) is widely used for treating solid tumors. Response to 5-FU treatment is variable with 10-30% of patients experiencing serious toxicity partly explained by reduced activity of dihydropyrimidine dehydrogenase (DPD). DPD converts endogenous uracil (U) into 5,6-dihydrouracil (UH(2) ), and analogously, 5-FU into 5-fluoro-5,6-dihydrouracil (5-FUH(2) ). Combined quantification of U and UH(2) with 5-FU and 5-FUH(2) may provide a pre-therapeutic assessment of DPD activity and further guide drug dosing during therapy. Here, we report the development of a liquid chromatography-tandem mass spectrometry assay for simultaneous quantification of U, UH(2) , 5-FU and 5-FUH(2) in human plasma. Samples were prepared by liquid-liquid extraction with 10:1 ethyl acetate-2-propanol (v/v). The evaporated samples were reconstituted in 0.1% formic acid and 10 μL aliquots were injected into the HPLC system. Analyte separation was achieved on an Atlantis dC(18) column with a mobile phase consisting of 1.0 mm ammonium acetate, 0.5 mm formic acid and 3.3% methanol. Positively ionized analytes were detected by multiple reaction monitoring. The analytical response was linear in the range 0.01-10 μm for U, 0.1-10 μm for UH(2) , 0.1-75 μm for 5-FU and 0.75-75 μm for 5-FUH(2) , covering the expected concentration ranges in plasma. The method was validated following the FDA guidelines and applied to clinical samples obtained from ten 5-FU-treated colorectal cancer patients. The present method merges the analysis of 5-FU pharmacokinetics and DPD activity into a single assay representing a valuable tool to improve the efficacy and safety of 5-FU-based chemotherapy.
authors.affiliation: Institute of Clinical Chemistry, Inselspital, Bern University Hospital, and University of Bern, Switzerland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Büchel
authors.forename: Peter
authors.initials: P
authors.lastname: Rhyn
authors.forename: Stefan
authors.initials: S
authors.lastname: Schürch
authors.forename: Claudia
authors.initials: C
authors.lastname: Bühr
authors.forename: Ursula
authors.initials: U
authors.lastname: Amstutz
authors.forename: Carlo R
authors.initials: CR
authors.lastname: Largiadèr
mesh_terms: D000368:Aged; D000964:Antimetabolites, Antineoplastic / Q000008:administration & dosage / Q000009:adverse effects / Q000097:blood; D002853:Chromatography, Liquid / Q000379:methods*; D015179:Colorectal Neoplasms / Q000097:blood* / Q000188:drug therapy; D016903:Drug Monitoring / Q000379:methods*; D004355:Drug Stability; D005260:Female; D005472:Fluorouracil / Q000008:administration & dosage / Q000009:adverse effects / Q000031:analogs & derivatives / Q000097:blood* / Q000493:pharmacokinetics; D006801:Humans; D016014:Linear Models; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014498:Uracil / Q000031:analogs & derivatives / Q000097:blood* / Q000493:pharmacokinetics
chemical_list: D000964:Antimetabolites, Antineoplastic; D014498:Uracil; C019389:5-fluorodihydrouracil; D005472:Fluorouracil