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 (200μL) was mixed with 1mL of deuterated-THY (internal standard) in acetonitrile, then centrifuged, the supernatant evaporated, and the residue reconstituted in 150μL 0.1% (w/w) formic acid in water. Separation was performed on a Waters Symmetry® C8 column (150mm×3.9mm; 5μm 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 5.12min, 5.17min and 5.00min, respectively. Linearity of the calibrations was established from 2 to 500μg/L. The lower limit of quantification was 5μg/L in plasma, and 10μg/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 250mg THY dose. THY was rapidly absorbed and eliminated with an apparent biphasic log-linear profile. DHT and UIB demonstrated apparent formation-rate limited kinetics.