One-step rapid extractive methylation of plasma nonesterified fatty acids for gas chromatographic analysis

The present report describes a one-step method for the derivatization and extraction of nonesterified fatty acids in plasma with subsequent analysis by conventional capillary gas-liquid chromatography or gas-liquid chromatography-mass spectrometry. The procedure requires 200 microliters of citrated plasma, dilution with 200 microliters of methanol containing a suitable internal standard, and rapid methylation (10 min) with ethereal diazomethane. An aliquot (60%) of the ether layer is subsequently removed, taken to dryness with nitrogen gas, and the residue is dissolved in a small volume of hexane (usually 50 microliters) for chromatographic analysis (taking 1 microliter for on-column injection). Samples are ready for analysis within 15 min after initial preparation of the plasma. The method has been found to be simple and rapid, providing clean fatty acid profiles. Although the method has been tested with 200 microliters of rat and human plasma, it can easily be adapted to a 40 microliters plasma sample if the esterified plasma extract is suspended in a smaller volume of hexane and/or a larger aliquot of the extract were to be injected into the gas chromatograph through use of a splitless injector.