A novel and reliable method for tetrahydrobiopterin quantification: Benzoyl chloride derivatization coupled with...

Tetrahydrobiopterin (BH₄) is a crucial cofactor for nitric oxide synthase, acylglycerol mono-oxygenase and aromatic amino acids hydroxylases. Its significant function for redox pathways in vivo attracted much attention for long. However, because of the oxidizable and substoichiometric nature, analysis of BH₄ has never been truly achieved with adequate sensitivity and applicability. In the present work, we pioneeringly stabilized BH₄ by derivatizing the active secondary amine on five-position with benzoyl chloride (BC). Benefiting from the favorable chemical stability and excellent mass spectrometric sensitivity of the product (BH₄-BC), ultra-sensitive and reliable quantification of endogenous BH₄ in plasma was achieved using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In such methodology, BH₄-BC-d₅ was introduced as stable isotopic internal standard. And the limit of quantification (LOQ) could reach 0.02 ng mL^-1. In the end, after investigation of plasma BH₄ in healthy volunteers (n = 38), we found that the levels of BH₄ were significantly and negatively correlated to age. Comparing with all the other existed strategies, the present method was obviously superior in sensitivity, specificity and practical applicability. It could be expected that this work could largely promote the future studies in BH₄-related fields.