Comprehensive study of Tetrahydrobiopterin autoxidation: Bridging electrochemical and thermodynamic approaches with tandem mass spectrometry

The 5,6,7,8-tetrahydrobiopterin (H 4 B) cofactor is essential for several enzymatic functions, including phenylalanine-4-hydroxylase (PAH, EC 1.14.16.1). However, H 4 B is highly unstable and spontaneously oxidizes in aqueous solution at room temperature, which complicates its measurement in clinical and research settings and has led to its exclusion from many routine analytical protocols. This study addresses the need for a quantitative description of H 4 B autoxidation and for practical strategies to mitigate analytical artefacts in solution and in urine. A kinetic study using flow injection analysis–tandem mass spectrometry (FIA–MS/MS) was carried out to monitor H 4 B signal decay under different media and antioxidant compositions, and the data were interpreted within a pseudo-first-order kinetic framework. Autoxidation was fastest in aqueous solvent and slower in urine, with rate constants further modulated by the presence of ascorbic acid (H 2 A) and glutathione (GSH). Equimolar H 2 A and GSH, and excess GSH, significantly slowed H 4 B decay but did not fully suppress oxidation, indicating partial stabilization rather than complete protection. Cyclic voltammetry and thermodynamic analysis provided complementary information on redox potentials, intermediates and thermodynamic affinity, linking changes in reaction rate to the underlying redox equilibria. The combined kinetic, electrochemical and thermodynamic results show that matrix composition and antioxidant content critically influence H 4 B stability in solution and in urine. While antioxidants such as H 2 A and GSH attenuate spontaneous oxidation, careful control of sample handling, timing and storage remains essential. The mechanistic insight obtained her e supports the rational design of stabilization and preparation protocols for future LC–MS/MS assays targeting H 4 B in bioanalytical and diagnostic applications. • H4B autoxidation kinetics were mapped in solution and urine. • FIA-MS/MS, voltammetry and modelling give a unified redox picture. • Antioxidants GSH and ascorbate slow H4B decay without full stabilisation. • Urine matrix partially stabilises H4B and modulates antioxidant effects. • Practical guidelines are proposed for handling H4B in bioanalytical assays.