Development of an analytical method for determining linoleic and stearic acid in sheep serum samples

Abstract

Sample preparation is an important step in the extraction of analytes from complex matrices such as biological samples. The lipids present in these samples can be found associated with lipoproteins, which makes it difficult to determine the profile of fatty acids in living organisms. Traditional analysis techniques such as the method of Folch et al. (1957), although they are still widely used, have several disadvantages such as the large amount of organic solvents used, large number of stages, degradation of compounds with unsaturation and high toxicity. In this sense, miniaturized techniques are alternatives capable of overcoming the

impossibilities of traditional methods. Among them, there is the low density dispersive liquid- liquid microextraction (LD-DLLME), which uses low density solvent, less toxic to the

environment and in small quantities. Thus, the objective of the work was to develop, validate and apply an analytical methodology for the extraction and determination of linoleic acid (AL) and stearic acid (AE) in sheep blood serum samples. The developed method used LD-DLLME with an esterification and quantification step by gas chromatography with flame ionization detector (GC/FID). The extraction was optimized through the planning of mixtures with the Scheffé polynomial model. The generalized inverse of Moore-Penrose was applied to Scheffé's complete cubic model (MCC), to determine the three dij coefficients concomitantly enabling the chemical interpretation of the model. The optimization evaluated the variables of the LD-DLLME dispersion medium volume (0.017% MgCl2 saline), extractor solvent volume (toluene) and dispersant solvent volume (methanol) and the results indicated that the best extraction condition was 1400 μL of the dispersion medium, 400 μL of extractor solvent and 1200 μL of dispersant solvent. The proposed method was validated for extraction of linoleic acid (AL) and for stearic acid (AE) based on the guidelines of the EURACHEM/2014 guide. The evaluation of the matrix effect (EM) for the developed method was classified as high and thus, all quantitative evaluations were performed using the standard addition method. The performance of the method demonstrated selectivity, sensitivity and precision adequate to be applied to real samples, showing an average recovery of 98.54% and 103.83% for the LA and LA respectively. LD-DLLME proved to be superior to the method of Folch et al. (1957), hitherto used in the determination of fatty acids in sheep blood serum samples. Through the analysis in real samples it can be inferred that the developed method is efficient in the monitoring of these substances in the body of ruminants.