Specific methylation of plasma nonesterified fatty acids in a one-step reaction.

Analysis of plasma nonesterified fatty acids (NEFA) by gas-liquid chromatography requires procedures that are both lengthy and cumbersome. A 45-min direct methylation procedure was carried out at 24-29 degrees C on 150 microliter of plasma added with an internal standard in 5.0 ml of methanol-acetyl chloride 50:1 (v/v). To stop the reaction, 3 ml of 6.0% K2CO3 was added. After addition of 150 microliter of hexane, shaking and centrifugation, an aliquot of the upper phase was injected into the gas chromatograph. The specificity of the methylation reaction for NEFA without hydrolysis of other classes of plasma lipids was substantiated with appropriate standards. This one-step specific methylation procedure is superior to currently used methods.