Validation of lipolysis product extraction from aqueous/biological samples, separation and quantification by thin-layer chromatography with flame ionization detection analysis using O-cholesteryl ethylene glycol as a new internal standard.
A general and easily accessible method for the extraction followed by the simultaneous separation and quantitative determination of triacylglycerols, diacylglycerols, monoacylglycerols and free fatty acids has been improved and optimized based on existing protocols using liquid-phase extraction and thin-layer chromatography coupled to flame ionization detection (TLC/FID Iatroscan). After lipid extraction in the presence of a suitable new synthetic internal standard, namely CholE1, a single elution step using n-heptane/diethyl ether/formic acid (55:45:1, v/v/v) was applied. This method was validated in line with international bioanalytical method validation guidelines using two different matrix systems: purified water and human gastro-intestinal fluid. Overall, the assay was found to have high levels of precision with coefficients of variation ranging from 1.48% to 11.0% and accuracy ranging from -13.3% to +5.79% RE. The confidence limits of the lipid mean recovery rates varied between 89.9% and 104%. This method is therefore highly suitable for quantifying the lipolysis products generated in vitro during the hydrolysis of various fats and oils by digestive lipases, as well as those collected from the gastro-intestinal tract in the course of human clinical studies on lipid digestion.
Cavalier
Jean-François
JF
CNRS-Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse, UPR 9025, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. jfcavalier@ifr88.cnrs-mrs.fr
Lafont
Dominique
D
Boullanger
Paul
P
Houisse
David
D
Giallo
Jacqueline
J
Ballester
Jean-Michel
JM
Carrière
Frédéric
F
D053000:Analytic Sample Preparation Methods / Q000592:standards; D002784:Cholesterol / Q000031:analogs & derivatives*; D002855:Chromatography, Thin Layer; D005026:Ethylene Glycols; D005410:Flame Ionization; D005766:Gastrointestinal Contents / Q000737:chemistry; D006801:Humans; D008055:Lipids / Q000032:analysis* / Q000592:standards; D008066:Lipolysis; D012015:Reference Standards; D014280:Triglycerides / Q000378:metabolism
D005026:Ethylene Glycols; D008055:Lipids; C543684:O-cholesteryl ethylene glycol; D014280:Triglycerides; D002784:Cholesterol