WHO reference protocol for measuring fatty acids in foods, with emphasis on monitoring trans-fatty acids originating from partial hydrogenation of edible oils

Overview

This revised document, entitled “WHO laboratory protocol – Reference protocol for measuring fatty acids in foods, with emphasis on monitoring trans-fatty acids originating from partial hydrogenation of edible oils” comprehensively includes validated procedures for fat extraction, gas chromatography (GC) peak identification, and determination of FAME flame ionization detector response. It also includes validated alternatives for internal standards, methylating agents, solvents for preparing FAMEs, GC parameters for determination of FA composition, and FAME reference standards. Furthermore, this protocol includes two sample preparation procedures in which the FA are methylated directly in the food sample before extraction, significantly reducing the time and cost of analysis. Throughout this document “methylation” is defined as the generic process of converting FA to FAME regardless of their free or bound status. The described procedures allow the determination of the TFA content as grams (g) per 100 g of food, grams per 100 g total FAs and grams per 100 g total fat. For measuring TFA in grams of FA per 100 g of food, an internal standard should be added to the test food sample portion before fat extraction or combined methylation/extraction. For the differences among the original WHO reference protocol, the WHO simplified protocol and the updated WHO reference protocol, please refer to Table 1 of this document. 

The updated WHO reference protocol retains the following sections outlined in the 2020 WHO reference protocol and 2022 WHO simplified protocol: clinical relevance, dietary trans fats, sampling plan, sample collection and storage, sample preparation for analysis, safety precautions and laboratory equipment. This updated WHO reference protocol replaces the original WHO global reference protocol. This updated edition of the WHO reference protocol is a compendium of validated procedures for conducting FAME analysis, allowing laboratories to choose the most suitable procedure for their specific analytical needs. While this protocol is also inclusive of all the procedures outlined in the WHO simplified protocol (except for the fat extraction procedure using solvents in section 6.3.2), the simplified protocol will remain as a standalone protocol as it is a useful summary of the fit-for-purpose procedures, and is recommended for laboratories operating on a limited budget and which are aiming to obtain the minimal data required for government surveillance and monitoring activities of fats and oils externally added during food preparation and processing.