Validated Spectrofluorimetric and Derivative Spectrophotometric Method for the Determination of Nitisinone in Pharmaceutical Dosage Form

Background: Nitisinone is a potent inhibitor of 4-hydroxyphenylpyruvate dioxygenase, the second enzyme involved in the tyrosine catabolic pathway. It is used for treating hereditary tyrosinemia type 1. Enzyme deficiency results in the accumulation of metabolites such as maleylacetoacetate and fumarylacetoacetate. The accumulation of these harmful metabolites can damage the liver and kidneys. Inhibiting 4-hydroxyphenylpyruvate dioxygenase with nitisinone reduces these metabolites. Objectives: Until now, simple and convenient spectrofluorimetric and spectrophotometric methods for determining nitisinone in capsules as a pharmaceutical product have not been developed and validated. These methods facilitate affordable quality control in environments with limited resources. Methods: To achieve simpler and more accessible methods to determine the amount of nitisinone in its pharmaceutical dosage forms, this study utilized a spectrofluorimetric method based on the fluorescence quenching of fluorescein following the charge transfer complex formation of nitisinone with fluorescein at pH 6. Furthermore, zero, second, and third-order derivative spectrophotometric methods were also used for nitisinone determination. Results: Beer’s law was obeyed for the spectrofluorimetric and spectrophotometric methods (concentration ranges of 0.1 - 3 µg/mL and 0.1 - 5 µg/mL, respectively). Conclusions: The developed methods were employed to assess nitisinone in capsule form as a pharmaceutical dosage form and were compared with a high-performance liquid chromatography (HPLC) method. Statistical analysis indicated no significant difference between the results obtained from these methods.