Position-specific isotope fragmentation differential quantification (PIF-DQ) of phenylalanine positional isotopomers: A rapid UPLC-MS/MS approach for urine analysis in the IAAO method.

Authors

Ippei Suzuki, Mai Matsumoto, Keiko Komikado, Ryosei Sakai, Yoichi Hatamoto

Published in

Journal of chromatography. A. Volume 1785. Pages 467311. Jul 30, 2026. Epub Jul 30, 2026.

Abstract

The indicator amino acid oxidation (IAAO) method is a minimally invasive approach for estimating amino acid and protein requirements using a ¹³C-labeled indicator amino acid. In IAAO studies using l-[1-¹³C]phenylalanine ([1-¹³C]Phe), accurate determination of tracer-derived enrichment relative to unlabeled phenylalanine is important for calculating the tracer-to-tracee ratio (TTR). A key analytical challenge is distinguishing the administered [1-¹³C]Phe tracer from naturally occurring M + 1 phenylalanine isotopologues. In this study, we developed and validated a novel analytical method, termed Position-specific Isotope Fragmentation Differential Quantification (PIF-DQ), using ultra-high performance liquid chromatography-tandem mass spectrometry for the simultaneous quantification of l-phenylalanine (Phe) and its isotopologues in human urine. By optimizing collision energy, we established specific multiple reaction monitoring transitions to differentially quantify the species: m/z 166.1 > 120.2 for Phe; m/z 167.1 > 78.1, selective for the phenyl-group labeled Natural-(M + 1)-Phe; and m/z 167.1 > 77.1 for Total-(M + 1)-Phe (the sum of natural and tracer isotopologues). The method demonstrated good linearity (R² > 0.995), sensitivity (LLOQ ≤ 0.0004 µg/mL), accuracy (recoveries of 94.0-111.0 %), and precision (RSD < 13.1 %) over a 4.5-minute run time. Application to urine samples from an IAAO study successfully distinguished the tracer-derived elevation in the Total-(M + 1)-Phe/Phe ratio from the stable natural abundance ratio, demonstrating the robustness of the method against physiological variations like urinary concentration.

PMID:
42541922
Bibliographic data and abstract were imported from PubMed on 02 Aug 2026.

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