Sequential determination of triglycerides and free fatty acids in biological fluids by use of a continuous pretreatment module coupled to a gas chromatograph.

A continuous system coupled to a gas-liquid chromatograph was used for the sequential determination of triglycerides and free fatty acids in serum and urine. The module provides compositional information and hence more detailed information on lipid metabolism changes in patients suffering metabolic disorders. Lipids in biological samples are manually extracted in methanol-n-hexane and introduced into the flow system; free fatty acids are then separated by retention on an ion-exchange resin and triglycerides (not retained) are transesterified with acetyl chloride in methanol. The resulting methyl esters are continuously injected into the gas chromatograph and determined by using a flame ionization detector. In a second step, retained free fatty acids are eluted and derivatized (also with acetyl chloride in methanol) and subsequently determined similarly as the triglycerides. The proposed method was applied to the determination of triglycerides in a lipid control serum; free fatty acids were determined in a human pool serum by the proposed method and compared with the volumetric method used in clinical practice. The results obtained in both instances showed good agreement between the results provided for triglycerides and free fatty acids. The proposed method was also applied to urine samples; a parallel recovery study was also made in order to assess the performance of the method.