title: Simultaneous determination of thymine and its sequential catabolites dihydrothymine and β-ureidoisobutyrate in human plasma and urine using liquid chromatography-tandem mass spectrometry with pharmacokinetic application.
abstract: To study in vivo activities of dihydropyrimidine dehydrogenase, dihydropyrimidinase, and ureidoproprionase, a sensitive, accurate, selective and precise method for the determination of the endogenous pyrimidine thymine (THY) and its successive metabolites dihydrothymine (DHT) and β-ureidoisobutyric (UIB) acid in human plasma and urine has been developed and validated. Plasma or diluted urine (200 μL) was mixed with 1 mL of deuterated-THY (internal standard) in acetonitrile, then centrifuged, the supernatant evaporated, and the residue reconstituted in 150 μL 0.1% (w/w) formic acid in water. Separation was performed on a Waters Symmetry C₈ column (150 mm × 3.9 mm; 5 μm particle size), with gradient elution using a mobile phase of 0.1% (w/w) formic acid in water (phase A), and 15% (v/v) methanol in acetonitrile (phase B). The detection system was an Applied Biosystems model 3200 tandem mass spectrometer with atmospheric pressure chemical ionisation, and multiple reaction monitoring mode using the transitions: THY (m/z: 127.1-110.0), DHT (m/z: 129.1-68.9), UIB (m/z: 147.1-86.0), and deuterated-THY (m/z: 131.1-114.0). THY, DHT, and UIB eluted at 5.12 min, 5.17 min and 5.00 min, respectively. Linearity of the calibrations was established from 2 to 500 μg/L. The lower limit of quantification was 5 μg/L in plasma, and 10 μg/L in urine for THY, DHT and UIB. Ion-suppression had negligible effect. A pilot pharmacokinetic study analysed plasmas and urines from 2 healthy male subjects who each received an oral 250 mg THY dose. THY was rapidly absorbed and eliminated with an apparent biphasic log-linear profile. DHT and UIB demonstrated apparent formation-rate limited kinetics.
authors.affiliation: School of Pharmacy, The University of Queensland, QLD 4072, Australia.
authors.forename: Ming
authors.initials: M
authors.lastname: Ni
authors.forename: John
authors.initials: J
authors.lastname: Duley
authors.forename: Rani
authors.initials: R
authors.lastname: George
authors.forename: Bruce
authors.initials: B
authors.lastname: Charles
authors.forename: Catherine
authors.initials: C
authors.lastname: Shannon
authors.forename: Ross
authors.initials: R
authors.lastname: McGeary
authors.forename: Ross
authors.initials: R
authors.lastname: Norris
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013941:Thymine / Q000097:blood / Q000493:pharmacokinetics* / Q000652:urine; D014508:Urea / Q000031:analogs & derivatives* / Q000097:blood / Q000493:pharmacokinetics / Q000652:urine
chemical_list: C066926:beta-ureidoisobutyric acid; D014508:Urea; D013941:Thymine