LC–MS/MS Methods for Direct Measurement of Sepiapterin and Tetrahydrobiopterin in Human Plasma and Clinical Applications

Aim: Tetrahydrobiopterin (BH₄), a natural cofactor of aromatic amino acid hydroxylases, and sepiapterin, a natural precursor of BH₄, are endogenously present in human plasma. This is the first report on methods for direct quantification of sepiapterin and BH₄ in human plasma by LC-MS/MS for pharmacokinetic assessment. Materials & methods: The analytes in plasma were harvested from blood that were treated with 10% ascorbic acid (AA) to a final concentration of 1% AA. Results & conclusion: The quantification methods were validated for calibration ranges of 0.75-500 ng/ml and 0.5-500 ng/ml for sepiapterin and BH₄, respectively. Quantification of analytes was challenging due to their susceptibility to redox reactions. The validated methods were utilized successfully to support clinical development of sepiapterin.