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Separation and quantitation of long-chain free fatty acids in human serum by high-performance liquid chromatography.

Journal of chromatography (1990-04-06)
G Kargas, T Rudy, T Spennetta, K Takayama, N Querishi, E Shrago
ABSTRACT

A rapid, simple and highly sensitive reversed-phase high-performance liquid chromatographic method is described for the separation and quantitation of fatty acids in human serum using a very reactive fluorescent labeling reagent, 9-anthryldiazomethane. Quantitative esterification proceeds at room temperature without heat or catalysis. Baseline separation of nineteen select fatty acids from a standard mixture was achieved on two C18-bonded silica columns connected in tandem using stepwise gradient elution of an acetonitrile-methanol-water mobile phase. The eluent was monitored by a fluorescence detector (maximum excitation wavelength, 365 nm; maximum emission wavelength, 412 nm). The procedure was applied to the analysis of both saturated and unsaturated long-chain free fatty acids (C8 to C22) extracted from human serum. Sera from fasting and non-fasting subjects were analyzed to show the applicability of this assay to biological samples. Detection limit and recovery of free fatty acids in serum were less than 10 pmol/microliter and greater than 92%, respectively.

MATERIALS
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Brand
Product Description

Sigma-Aldrich
cis-4,7,10,13,16,19-Docosahexaenoic acid, ≥98%