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- WHO reference protocol for measuring fatty acids in foods, with emphasis on monitoring trans-fatty acids originating from partial hydrogenation of edible oilsPublication . World Health Organization Nutrition and Food Safety (NFS)Executive summary: In December 2020, the World Health Organization (WHO) first developed and published a laboratory protocol for measuring trans-fatty acids (TFAs) in foods2 (hereinafter referred to as the “WHO reference protocol”). Although the WHO reference protocol was successfully implemented in several laboratories worldwide, some laboratories, especially those operating with a limited budget, had challenges implementing it. These laboratories requested that the procedures be simplified, and suitable alternative methods and chemicals be included in the protocol so that it is easier to use and globally applicable. In response to the requests, WHO organized an expert consultation meeting in June 2022, based on which the Simplified Protocol for Measuring TFA content (hereinafter referred to as the “WHO simplified protocol”) was developed.3 This fit-for purpose protocol can be implemented by all laboratories including those with limited budgets, and provides most of the data that are required for governments’ surveillance and monitoring activities to check the trend of TFA content of fats and oils added during food preparation and processing. This will ensure that the fats and oils used in preparation comply with regulations for TFA elimination. Regarding the key differences between the original WHO reference protocol and the WHO simplified protocol, please refer to Table 1 in the report of the WHO expert consultation held in June 2022. When developing the WHO simplified protocol, new data and information were obtained on the procedures for measuring fatty acids (FA). It was realized that the WHO reference protocol needs to be revised to provide up-to-date, comprehensive and validated options for conducting full fatty acid methyl ester (FAME) analysis. A 2-day, virtual WHO Expert Consultation was held on 19 and 22 September 2023 to update the WHO reference protocol.5 In doing so, all the critical steps of the WHO reference protocol and the WHO simplified protocol were reviewed. During this expert consultation, validated alternative procedures were recommended for increasing the versatility of the protocol and expanding its ability to analyse all types of food products that could contain TFAs. 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.