An analytical procedure for the rapid and selective derivatization of free fatty acids into methyl esters directly in plasma without transmethylation of lipid-bound fatty acids was developed for their analysis by gas chromatography-mass spectrometry. The methyl esters of free fatty acids were obtained by reaction with methyl iodide in the solution of dipolar aprotic solvents and in the presence of solid bases. The mechanism of the methylation reaction with these reagents was investigated. Optimal conditions for the selective methylation of free fatty acids were established using different dipolar aprotic solvents and different solid bases. The possible transmethylation of covalently bonded fatty acids from plasma lipids has been investigated under different experimental conditions in order to be avoided. Total methylation of free fatty acids was achieved in 1 min at room temperature using methyl iodide and anhydrous potassium carbonate or sodium carbonate in dimethyl sulfoxide. Under these conditions, transmethylation of lipid-bound fatty acids was avoided. The methyl esters can be injected directly from the reaction solvents. A plasma volume of 50 μL was used without special purification. The detection limits were around 0.1 ng/μL. The proposed method avoids the drawbacks of the previous methods used for the one-step analysis of individual free fatty acids in human plasma.
Keywords: Free fatty acids analysis; Gas chromatography-mass spectrometry; Human plasma; Selective methylation.
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