Development and Validation of an LC–MS/MS Method for Simultaneous Determination of Gamma-Aminobutyric Acid (GABA), Glutamic Acid, and Glutamine in Fermentation Supernatant

A selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous, derivatization-free determination of gamma-aminobutyric acid (GABA), glutamic acid, and glutamine in fermentation supernatant. This hydrophilic interaction liquid chromatography–MS/MS approach offers simplified sample preparation and simultaneous quantification of GABA with its metabolic precursors. Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH Amide column, with internal standard normalization using isotopically labeled analogs (GABA-d2 and glutamic acid-d5) improving calibration robustness. Method validation following ICH Q2(R1) guidelines confirmed linearity (R2 ≥ 0.99; ≥0.9999 for GABA and glutamic acid with internal-standard normalization) over validated ranges of 4–70 μM (GABA), 1–100 μM (glutamic acid), and 1–9 μM (glutamine), limits of detection of 0.17–0.25 μM, lower limits of quantification of 1–4 μM, intra-day precision ≤7.9% RSD and inter-day precision ≤4.9% RSD for GABA and glutamic acid (between-day 10–17% RSD for glutamine, which, quantified without an isotopically labeled internal standard, is reported as a fit-for-purpose, semiquantitative measure), between-day accuracy within ±15% bias at every level except the glutamic acid lower limit of quantification, where the +18.6% bias falls within the ±20% criterion applicable at that level, and negligible carryover. The validated method was applied to quantify GABA, glutamic acid, and glutamine in fermentation supernatant from bacterial strains cultivated under various conditions. This practical analytical tool supports process optimization and quality control in microbial fermentation for functional food production. The contribution is the de novo development and ICH Q2(R1) validation of this established technique for MRS-based fermentation supernatant, including baseline resolution of GABA from its isobaric isomer α-aminobutyric acid, and its application across lactic-acid-bacteria fermentations.