Comparative evaluation of native and fluorinated amino acid probes in stereoselective plasma profiling: A...

Stereoselective plasma profiling of amino acid enantiomers has been proposed as a useful approach for investigating organ-specific transport processes. However, probe-dependent metabolic and/or distributional perturbations may affect the interpretation of stereoselective plasma concentration profiles. Therefore, we compared the plasma concentration profiles of a non-native trifluoromethylated alanine analog (DL-Ala-F₃) with those of two deuterium-labeled native amino acids, alanine (DL-Ala-d₃) and serine (DL-Ser-d₃). The three probes were intravenously co-administered to rats (20 mg/kg each, n = 3). Enantiomer-specific quantification was performed using a validated crown ether-based chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which approach demonstrated adequate separation (resolution, Rs ≥ 2.52), linearity (r² > 0.995). DL-Ala-d₃ and DL-Ser-d₃ exhibited pronounced enantiomeric imbalance at 5 min post-dose, with D/L ratios of 10.1 and 7.0, respectively, reflecting substantially lower plasma concentrations and more rapid disappearance of the L-enantiomers. In contrast, DL-Ala-F₃ showed nearly identical D- and L-enantiomer concentrations at 5 min post-dose (D/L = 1.0), indicating preservation of the initial enantiomeric balance. Thereafter, stereoselective elimination became evident, with D-Ala-F₃ disappearing faster than L-Ala-F₃ but more slowly than the D-enantiomers of the deuterium-labeled probes. Although the mechanisms underlying these differences remain unclear, the plasma concentration profiles of non-native DL-Ala-F₃ appeared less susceptible to probe-dependent metabolic and/or distributional perturbations than those of the deuterium-labeled native probes. This proof-of-concept study highlights the importance of probe selection in minimizing potential bias when investigating enantiomer-dependent organ-specific transport processes.