LC-MS/MS and LC-PDA Methods for Robust Determination of Glycerol Phenylbutyrate in Biological Fluids and High-Resolution Mass Spectrometric Identification of Forced Degradation Product and Its Whiteness

In 2013, the FDA approved glycerol phenylbutyrate to treat urea cycle disorders in people who could not go through 2 months of protein restriction and/or amino acid supplementation. The paper suggested a simple, quick, and eco-friendly liquid chromatographic method to analyze glycerol phenylbutyrate in Ravicti, pharmaceutical formulation, bulk, human urine, and plasma. Also, a novel degradation product was characterized by applying severe degradation conditions, according to the ICH Q1A(R2) guideline. The liquid chromatography conditions were 0.5 mL/min flow rate and 1 mM ammonium acetate buffer:acetonitrile (25:75; <i>v</i>/<i>v</i>) (≈ pH 5.30). The system backpressure was 67 bar. A core-shell particle column (Ascentis Express F5 2.7 μm, 100 × 4.6 mm i.d.) from Supelco was used for separation. The method was fully validated according to the ICH Q2(R1) guideline. The method linearities for bulk and pharmaceutical analysis were 1.40-55.84 ng/mL for LC-PDA and 2.79-111.68 μg/mL for LC-MS/MS. Indeed, for the plasma sample, the lowest recovery was LC-PDA and LC-MS/MS achieving 94.27 and 98.20%, respectively. Moreover, in forced degradation experiments, the active substance was unstable in acid, alkali, and oxide conditions, and an elimination reaction forms the novel degradation product. Lastly, the method was evaluated to have excellent whiteness, efficiency, and practicality, making it suitable for application in all analytical method development laboratories.