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

Abstract

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.

Graphical Abstract

Keywords:

• Tetrahydrobiopterin
• Method Development
• Pharmacokinetics
• LC-MS
• ApoE mice

Acknowledgements

The authors would like acknowledge Professor R. M. Wadsworth from University of Strathclyde for his kindness in providing the BH4 analog for the study.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

Authors would like to thank USM short-term grant [6315287].