HPLC Determination of Underivatized Fatty Acids Saponified at 37°C Analysis of Fatty Acids in Oils and Tissues

Abstract

The present study was undertaken to establish a simple and appropriate method for fatty acids determination where samples are not submitted to high temperatures during the preparative and quantification processes. We describe an alternative method in which two techniques developed separately were combined: lipids saponification at low temperature and underivatized fatty acids quantification by HPLC (high performance liquid chromatography). Total lipids fraction was extracted from oils and rat tissues using the Folch method. Lipids were saponified at 37°C for 2 hours and extracted with hexane.

Fatty acids were separated in a reverse-phase column (C₈-Shimadzu) and identified by a refractive index detector (RID-6A-Shimadzu). Fatty acid composition of oils (soybean oil, coconut fat and cocoa butter) and rat tissues (spleen and thymus) was determined. The analysis of fatty acids using this method was reproducible and the retention time, fatty acid recovery, minimal detection limit were compared to gas chromatography. The advantage of this method is that during handling and detection, fatty acids are not exposed to high temperatures, decreasing the possibility of its oxidation and racemization. Therefore, this method is recommended when fatty acids recovery is required.

Key Words:

• HPLC
• GC
• saponification at 37°C
• underivatized fatty acids
• oils
• tissues