Separation and quantitation of long-chain free fatty acids in human serum by high-performance liquid chromatography.
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.
Kargas
G
G
School of Pharmacy, University of Wisconsin-Madison 53706.
Rudy
T
T
Spennetta
T
T
Takayama
K
K
Querishi
N
N
Shrago
E
E
D000328:Adult; D000873:Anthracenes; D001322:Autoanalysis; D002851:Chromatography, High Pressure Liquid; D005215:Fasting / Q000097:blood; D005230:Fatty Acids, Nonesterified / Q000097:blood* / Q000302:isolation & purification; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male
D000873:Anthracenes; D005230:Fatty Acids, Nonesterified; D005456:Fluorescent Dyes; C027347:9-diazomethylanthracene