Standards Development

Scope

This document specifies a method for the extraction and quantification of free fatty acids (FFAs) in milk and dairy products

Purpose

Free fatty acids (FFA) in dairy products have experienced a resurgence of interest in recent years due to their associated health benefits, flavour potential and antimicrobial activity, as a direct cause of hydrolytic rancidity, and also because of the importance of accurate determination for quality, research and legislative purposes.

They have also been shown to contribute to functionality in dairy products. Gas chromatographic analysis remains the technique of choice for FFA analysis; however, little advancements in method development have occurred over the last twenty years and current methods are generally limited in their application to specific dairy sample types.

The current standard ISO 14156| IDF 172:2001 which details fat extraction of milk and milk products does not include the determination of FFAs for many dairy samples, such as milk, condensed milk, dried milk products, cream or fermented milk. For samples such as cheese, a six-hour reflux is described for fat extraction. The limitations of this standard are noticeable and evident by the fact that most publications reference other methods for determination of FFAs in dairy. Thus, there is considerable scope to harmonise existing fat extraction methods, to incorporate a strategy for Total fatty acid (TFA) and free fatty acid (FFA) determination. We have made suggestions that will suffice for most dairy products, where an additional step may be required for some specific dairy products. In summary, this new method incorporates aspects of three existing ISO/IDF methods (ISO 14156|IDF 172, Milk and milk products – Extraction methods for lipids and liposoluble compounds; ISO 15884|IDF 182, Milk fat – Preparation of fatty acid methyl esters & ISO 15885 |IDF 184, Milk fat - Determination of the fatty acid composition by gas-liquid chromatography).ISO 14156|IDF 172 details the extraction of lipids in dairy products. However, this procedure does not cover FFA in the following important dairy products, such as milk, dried milk products, cream and fermented milk. From our research we believe that solvent extraction followed by solid phase extraction will suffice in most cases, however some additional procedures are required for certain sample types (milk, ice-cream and processed cheese). We propose that by separating the FFA from TFA using solid phase extraction, the resultant can be esterified separately post analysis by GC-FID. Also, the IDF 172 procedure describes a 6-hour reflux for the fat extraction from cheese. Also, the acidic esterification described in IDF 182, details the transferring of the fat extract and reagents (highly flammable) into a glass ampoule, flame sealing and then heating for 3 hours at 110 degrees. This is impractical and imposes significant risks that are unnecessary. ISO 15884|IDF 182 describes the preparations of FAMEs from glycerides using base catalysed esterification (only suitable for glyceride analysis). When dealing with samples that have high levels of lipolysis, there is an alternative procedure using an acidic catalyst (H2SO4) to methylate both glycerides and FFAs together, but this is not suitable for individual FFA determination. ISO 15885|IDF 184 is the GC analysis of FAMEs, this method is widely used but due to the volatility of methyl esters methyl butyrate can co-elute with the solvent peak resulting in quantification issues that raise the limits of quantification and limits of detection for this important FFA. Also we have noted as well as other studies that artefact peaks from the methylation agent can periodically interfere with quantification of other short chain methyl esters