Simple analytical method to determine urinary isotopic enrichment of phenylalanine by GC/EI-MS coupled with pentafluorobenzyl derivatization.

Abstract

The indicator amino acid oxidation (IAAO) technique estimates the physiological requirements for amino acids and proteins in living organisms, including humans. It involves monitoring urinary amino acids and exhaled CO₂ after ingesting 1-¹³C-labeled (carboxy-labeled) amino acids. The most common IAAO indicator amino acid is 1-¹³C-labeled phenylalanine ([1-¹³C]Phe). Its urinary concentration in test subjects ranges from below the detection limit to several μM. A simple analytical method for distinguishing trace amounts of [1-¹³C]Phe in urine from high levels of naturally occurring Phe is crucial for making IAAO tests easier. This study presents a simple and reliable approach for the simultaneous quantification of [1-¹³C]Phe and Phe in human urine using conventional GC-EI-MS. In this method, urinary phenylalanine is reacted with pentafluorobenzyl bromide in a single-phase solvent system of acetone-borate buffer without dehydration or desalting to form disubstituted pentafluorobenzyl (PFB) derivatives, which are then analyzed by GC-EI-MS (SIM). The Phe and [1-¹³C]Phe PFB derivative peaks eluted at the same retention time on the gas chromatogram but could be differentiated on the basis of fragment ions (m/z 434, 435) derived from the loss of the phenyl group ([M - 91]⁺). Correcting the interference of the m+1 isotope peak of Phe in the [M - 91] fragment (m/z 435) of [1-¹³C]Phe using the m/z 434 peak intensity and natural isotope ratio, both Phe and [1-¹³C]Phe could be quantified in the concentration range found in urine. The method was successfully applied to examine the temporal enrichment of [1-¹³C]Phe in urine samples obtained from IAAO subjects following the ingestion of a test meal containing [1-¹³C]Phe.