Analytical Validation of a GC–MS Method Employing an In Situ Acid-Catalyzed Transmethylation for Fatty Acid Quantification in Aquaculture Biological Matrices

Abstract

Accurate fatty acid quantification in aquaculture feeds and biological tissues is essential for nutritional evaluation and lipid metabolism studies. In routine practice, GC–MS-based fatty acid analysis often relies on multi-step workflows involving lipid extraction followed by derivatization, which may increase analytical complexity and handling-related variability. To address these methodological challenges, a validated analytical workflow is reported for quantitative fatty acid profiling using in situ hydrochloric acid–catalyzed transmethylation coupled with gas chromatography–mass spectrometry (GC–MS). An established acid-catalyzed transmethylation reaction was employed directly within the sample matrix, eliminating any prior lipid-extraction step. Validation performed under ISO/Eurachem guidelines confirmed excellent linearity (R2 > 0.99 for all analytes), high precision (intra-day RSD 1.72–8.69%, inter-day RSD 1.77–10.15%), and acceptable recovery (81.5–118.3%). Sensitivity was high, with LOD and LOQ values of 0.02–0.41μg mL^-1 and 0.07–1.35μg mL^-1, respectively. Acquisition in selected-ion monitoring (SIM) mode improved signal-to-noise relative to full-scan, enabling reliable quantification of low- abundance polyunsaturated fatty acids. The workflow performed consistently across feed material, fish oils, and lyophilized tissue samples, with stable retention behavior and maintained ion-ratio fidelity. Collectively, this work provides the first ISO/Eurachem-validated demonstration that in situ HCl-catalyzed transmethylation supports extraction-free and analytically equivalent FAME quantification in complex matrices, establishing an eco-efficient and high-throughput platform for routine application in lipid chemistry and related biochemical investigations.