Evaluation of methylations and external/internal standard quantification of lipids using gas chromatography-mass spectrometry†
In the present work, we evaluated different widely accepted methyl derivatization procedures which are almost obligatory in gas chromatography analysis of fatty acids (FAs), both acid- and base-catalyzed ones. We determined their derivatization efficiency and rapidity and found that combined base–acid methylation, the NaOH–BF3–MeOH method, out-performed all other methods. The analyses of FAME standards and samples were performed on a conventional 30 m × 0.25 mm Wax column, with a single run of 51 min. To accelerate the analysis while maintaining resolution, a more polar 0.1 mm ID column was used to analyze 52 FAMEs. This way, we were able to obtain results within one-third of the analysis time with almost equal separation, if not better. To further evaluate the quantification methods that are performed with or without internal standards, we constructed new calibration curves by removing a known quantity from the original calibration curves, and used them to recalculate the concentration of the known removed quantity. Comparison of the calculated and actual concentration values revealed that, when performed carefully, the external standard method could achieve results that were comparable to results obtained with the internal standard method, and thus is recommended for daily analysis.