Carbon-13-isotopomics and metabolomics of fatty acids from triacylglycerols: overcoming the limitations of GC-C-IRMS for short- and medium-acyl chains.

Carbon-13 isotopomics of triacylglycerol (TAG) fatty acids or free fatty acids in biological matrices holds considerable potential in food authentication, forensic investigations, metabolic studies, and medical research. However, challenges arise in the isotopic analysis of short- and medium-chain (C4 to C10) fatty acid methyl esters (SMCFAMEs) through gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The high volatility of these esters results in losses during their preparation, leading to isotopic fractionation. Moreover, the methoxy group added to acyl chains requires the correction of δ¹³C values, thereby increasing the uncertainty of the final results. Analyzing free fatty acids (FFAs) addresses both issues encountered with SMCFAMEs. To achieve this objective, we have developed a new protocol enabling the isotopomics of individual fatty acids (FAs) by GC-C-IRMS. The same experiment also provides the FA profile, i.e., the relative percentage of each FA in the TAG hydrolysate or its concentration in the studied matrix. The method exhibited high precision, as evidenced by the repeatability and within-lab reproducibility of results when tested on TAGs from both animal and vegetal origins. Compared to the analysis of FAMEs by GC-C-IRMS, the current procedure also brings several improvements in alignment with the principles of green analytical chemistry and green sample preparation. Thus, we present a two-in-one method for ¹³C-isotopomic and metabolomic biomarker quantitation within quasi-universal TAG compounds, encompassing the short- and medium-acyl chains.