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

Nico  G. Dumandan,; Adonis  A. Yanos,; Andrei  D. Dela Mar,; Annie Cita  T. Kagaoan,; Al Jerome  A. Magsino,

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

Nico G. Dumandan | Adonis A. Yanos | Andrei D. Dela Mar | Annie Cita T. Kagaoan | Al Jerome A. Magsino

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 -1 -1 mL and 0.07–1.35μg mL , respectively. Acquisition in selected-ion monitoring (SIM) mode improved signal-to-noise relative to full-scan, enabling reliable quantification of lowabundance 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.

References:

Alinafiah, S. M., Azlan, A., Ismail, A., & Ab Rashid, N. K. M. (2021). Method development and validation for omega-3 fatty acids (DHA and EPA) in fish using GC-FID. Molecules, 26(21), 6592.
Cantwell, H. (Ed.). (2025). Eurachem guide: The fitness for purpose of analytical methods – A laboratory guide to method validation and related topics (3rd ed.). Eurachem.
Castro-Gómez, P., Fontecha, J., & Rodríguez-Alcalá, L. M. (2014). A high-performance direct transmethylation method for total fatty acids assessment in biological and foodstuff samples. Talanta, 128, 518–523.
Dissanayake, T. T., Crawford-Clark, S., Bowen, B. J., Henderson, L. C., Barrow, C. J., & Adcock, J. L. (2025). Comparison of methods for the preparation of fatty acid methyl esters for the analysis of omega-3 rich oils by gas chromatography. Journal of the American Oil Chemists’ Society, 102(9), 1351–1361.
Dumandan, N. (2026). Data set for analytical validation of a GC–MS method employing an in situ acid-catalyzed transmethylation for fatty acid quantification in aquaculture biological matrices [Data set]. Figshare.
Fagerquist, C. K., Neese, R. A., & Hellerstein, M. K. (1999). Molecular ion fragmentation and its effects on mass isotopomer abundances of fatty acid methyl esters ionized by electron impact. Journal of the American Society for Mass Spectrometry, 10(5), 430–439.
González, A. G., & Herrador, M. Á. (2007). A practical guide to analytical method validation, including measurement uncertainty and accuracy profiles. TrAC Trends in Analytical Chemistry, 26(3), 227–238.
Gerhardtova, I., Jankech, T., Majerova, P., Piestansky, J., Olesova, D., Kovac, A., & Jampilek, J. (2024). Recent analytical methodologies in lipid analysis. International Journal of Molecular Sciences, 25(4), 2249.
Ichihara, K., & Fukubayashi, Y. (2010). Preparation of fatty acid methyl esters for gas–liquid chromatography. Journal of Lipid Research, 51(3), 635–640.
Indarti, E., Majid, M. I. A., Hashim, R., & Chong, A. (2005). Direct FAME synthesis for rapid total lipid analysis of fish oil and cod liver oil. Journal of Food Composition and Analysis, 18(2–), 161–170.
Iverson, S. J., Lang, S. L. C., & Cooper, M. H. (2001). Comparison of the Bligh and Dyer and Folch methods for total lipid determination in marine tissues. Lipids, 36(11), 1283–1287.
Kerwin, J. L., Wiens, A. M., & Ericsson, L. H. (1996). Identification of fatty acids by electrospray mass spectrometry and tandem mass spectrometry. Journal of Mass Spectrometry, 31(2), 184–192.
Korban, A., 膶abala, R., Egorov, V., Bosáková, Z., & Charapitsa, S. (2022). Evaluation of variation in relative response factors of GC–MS with internal standard methods and application to alcoholic product quality control. Talanta, 246, 123518.
Perez-Palacios, T., Solomando, J. C., Ruiz-Carrascal, J., & Antequera, T. (2022). Improvements in the methodology for fatty acids analysis in meat products: One-stage transmethylation and fast-GC method. Food Chemistry, 371, 130995.
Vetter, W., & Thurnhofer, S. (2007). Analysis of fatty acids by mass spectrometry in the selected ion monitoring mode. Lipid Technology, 19(8), 184–186.
Yin, M., & Wang, X. (2024). Impact of temperature fluctuations on fatty acid composition and nutritional quality of tilapia fillet (Oreochromis niloticus). Journal of Food Measurement and Characterization, 18, 1679–1689.