A microliter method for the gas chromatographic determination of long-chain non-esterified fatty acids in human serum or plasma

Non-esterified fatty acids (NEFA) from C₁₂ to C₂₄ are assayed in human serum or plasma in a four-step procedure: extraction, volume reduction, methylation and gas chromatography. NEFA are extracted with chloroform—heptane—methanol from 50–100 μl of serum or plasma buffered with phosphate. After adding ethyl acetate the volume of the extract is reduced under partial reflux to 5–7 μl. Potassium carbonate, methyl iodide and a crown ether are added to the dry concentrate and the NEFA are selectively methylated with a yield of 100% by heating in a microrefluxer for 10 min. Gas chromatography is carried out with 1 μl of the reaction mixture on a packed column by temperature-programmed operation. Thirteen individual fatty acids are determined in sera of normal adults. The coefficients of variation for 24 determinations of a pooled serum were 2.7% for the total NEFA content and 3–10% for most of the individual NEFA.