Bioanalysis of tetrahydrobiopterin with liquid chromatographic-mass spectrometric and its application for pharmacokinetics in apolipoprotein E knockout mice

Decreased level of endogenous tetrahydrobiopterin (BH4) has been associated with endothelial dysfunction, which is involved in vascular diseases. Despite its chemical instability due to auto-oxidation, exogenous supplementation of BH4 can reverse this dysfunction. Thus, a sensitive and simple liquid chromatographic-mass spectrometric (LC-MS) method was developed and validated for the measurement of BH4 in plasma after its oral or intravenous administration in apolipoprotein E (ApoE) knockout mice. Human plasma samples were prepared from protein precipitation, and chromatographic separation was performed on a C18 column with methanol-water mobile phases and a run time of 7 minutes with ion detection at m/z 242. The limit of quantitation was 10 ng/mL and inaccuracy and imprecision were <= 15%. In the presence of antioxidant dithioerythritol, recovery of BH4 was 80% and stable for 24 h post sample collection. Following oral or intravenous administration of BH4 in ApoE mice, the maximum concentrations detected were 102 and 483 ng/mL, respectively. Area under the curve values for oral BH4 were three time lower than those of intravenous BH4. The half-life of BH4 was 1.6 h, and it demonstrated low oral bioavailability (15%). Results of this study provide the basis for further studies of future clinical applications of BH4.