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 microliter of serum or plasma buffered with phosphate. After adding ethyl acetate the volume of the extract is reduced under partial reflux to 5--7 microliter. 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 microliter 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.