# 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

## TL;DR

This paper presents a reliable and eco-friendly method to measure glycerol phenylbutyrate in biological samples and identifies a new degradation product.

## Contribution

A novel degradation product of glycerol phenylbutyrate is identified under forced degradation conditions.

## Key findings

- The method is validated for use in pharmaceutical and biological samples with high recovery rates.
- Glycerol phenylbutyrate is unstable under acidic, alkaline, and oxidative conditions.
- The method shows excellent efficiency and practicality for analytical laboratories.

## Abstract

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; v/v) (≈ 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.