Public call for data on fatty acid composition of bovine milk

Deadline: 15 July 2026

15 January 2026
Call for data

Objective of the call

WHO is seeking global data on the fatty acid composition of bovine milk samples, with a focus on butyric acid (C4:0) and trans-fatty acids (sum of C18:1, C18:2 (including 9c,11t-CLA), and C18:3 TFAs). Data submissions from all countries are welcome. 

Background

Trans-fatty acids (TFAs) are unsaturated fatty acids with at least one double bond in the trans configuration. Dietary TFAs primarily originate from two sources: industrially produced TFAs (IP-TFAs) and ruminant TFAs (R-TFAs). WHO recommends limiting TFA intake below 1% of total energy consumption to reduce cardiovascular risk (1). IP-TFAs are mainly formed through the partial hydrogenation of vegetable oils. Partially hydrogenated oils (PHOs) can contain up to 60% TFAs. IP-TFAs are also produced during refining, deodorizing and deep-frying. Foods made with PHOs (e.g. some margarines, shortenings, baked goods, and fried foods) are the major dietary source of TFA. R-TFAs occur naturally in meat and dairy (typically 2–6%) from biohydrogenation of fatty acids by microorganisms in the rumen of ruminant animals (e.g. cows and sheep).

In 2018, WHO called for global elimination of IP-TFA and introduced the REPLACE action framework and modules to guide countries in removing IP-TFAs and replacing them with healthier fats (2,3). WHO’s recommended two best-practice policy alternatives are: 1) a mandatory limit of 2 g IP-TFA per 100 g total fat in all foods; and 2) a ban on the production or use of PHO as an ingredient in all foods. WHO has actively supported countries in strengthening TFA laboratory capacity by publishing the reference and simplified laboratory protocols (4,5) for analyzing TFAs and conducting regional and national workshops.

Some countries have requested WHO’s guidance on analytically differentiating IP-TFAs and R-TFAs. Although no universally accepted laboratory method currently exists to distinguish IP-TFAs from R-TFAs, in 2021, the European Commission’s Joint Research Centre (EC-JRC) proposed a decision-tree-based approach to estimate IP-TFA content in foods containing mixtures of PHOs, refined oils, frying oils and ruminant fats (6). This relies on measuring the amounts (g/100g fat) of butyric acid (C4:0) or C18:2 cis-9, trans-11 (9c,11t-CLA) by a standardized method (AOAC 2012.13 / ISO 16958 | IDF 231:2015) (7, 8) and applying conversion factors derived from milk and tallow fatty-acid profiles to estimate R-TFA.

WHO convened a roundtable consultation in 2024 to explore the possibility of providing global guidance on this topic. The consultation reviewed the EC-JRC approach (6) and acknowledged that, although it was an estimation, it was the only available method currently and a reasonable starting point. It was concluded that its global application would depend on whether the estimated conversion factors could be applied to food products outside the European region.   

Scope of requested data

Fatty acid composition of bovine milk and milk products. We are primarily interested in data on whole milk. However, data on anhydrous milk fat and whole milk powder are also welcomed, if available.

Whole milk samples should be collected from bulk milk (tanker milk) or from retail sources derived from bulk milk, rather than from individual dairy farms or herds, in order to avoid extreme values associated with specific feeding practices.

Analytical approach

Please use one of the four procedures (A, B, C or D described below) outlined in the WHO Reference Protocol (4) to obtain the fatty acid composition of bovine milk samples, including the levels of C4:0, and total trans-fatty acids (C18:1 TFA + C18:2 TFA (including 9c,11t-CLA) + C18:3 TFA). The section numbers (bold, italic and underlined) refer to those in the  WHO Reference Protocol.

  • Procedure A (following AOAC 2012.13 / ISO 16958 | IDF 231:2015 (7,8))

    Before transesterification, add a mixture of C11:0 FAME and C13:0 TAG as the internal standard, following Section 8.1.3 of the WHO Reference Protocol.

    Use the procedure described in Section 13.2. This procedure directly transesterifies bovine milk samples to FAME using 5% CH₃ONa–CH₃OH.

  • Procedure B (adapted from AOAC Official Method 996.06 (9))

    Before fat extraction, add an internal standard—either C13:0 triacylglycerol (TAG) or C21:0 TAG—to the test sample, as specified in Section 8.1.1 or 8.1.2 of the WHO Reference Protocol.

    Use the procedure described in Section 11.4.2 for extracting fat from bovine milk and milk products.

    After extraction, methylate the fat (together with the internal standard) to form fatty acid methyl esters (FAMEs) using the 7% BF₃–CH₃OH reagent, following Section 12.1.

  • Procedure C (following AOCS Official Method Ce 2b-11 (10))

    As with Procedure B, add C13:0 TAG or C21:0 TAG as the internal standard prior to transesterification, following Section 8.1.1 or 8.1.2 of the WHO Reference Protocol.

    Use the procedure described in Section 13.1. This procedure directly converts (transesterifies) bovine milk samples to FAMEs using 0.5 M NaOH–CH₃OH followed by 14% BF₃–CH₃OH.

  • Procedure D (no addition of the internal standard)

    This procedure does not require the addition of the internal standard.

For converting the milk samples to FAMEs, use either Procedure B (i.e. Section 11.4.2 for fat extraction and Section 12.1 for methylation) or Procedure C (i.e. Section 13.1 for direct transesterification). 

(Please note that this procedure determines the fatty acid composition as a weight percentage of total fatty acids, whereas Procedures A, B and C allow the determination of the fatty acid composition as g per 100 g of the test sample as well as a weight percentage of total fatty acids.)

GC Analysis

The resulting FAME from any of the three procedures should be analyzed by gas–liquid chromatography (GC) following Section 14.2.2 of the WHO Reference Protocol (4). GC analysis is then followed by calculation of the fatty acid composition, including C4:0 and total TFA (sum of C18:1, C18:2 (including 9c,11t-CLA), and C18:3 trans isomers), relative to the added IS (except for Procedure D). 

Calculation

For convenience, please use dedicated spreadsheets for calculating fatty acid composition.

These can be downloaded here:

Download spreadsheet

There are four spreadsheets 1–4 in the Excel.

  • Use spreadsheet 1, if Procedure A was used.
  • Use spreadsheet 2, if 13:0 TAG was used as the internal standard in either Procedure B or C.
  • Use spreadsheet 3, if 21:0 TAG was used as the internal standard in either Procedure B or C.
  • Use spreadsheet 4, if an internal standard was not used as per Procedure D.

Reporting Data

Please use the spreadsheets for reporting data. The data to be reported include a detailed description of the food sample (name, date collected, place collected, brand name, sample ID, and date analyzed). All fatty acids present at ≥0.02 g per 100 g of total fatty acids should be reported.

Include a GC chromatogram for each analyzed sample with all identified peaks clearly labelled. Labelling should follow the format shown in Figures 1–18 of the WHO Reference Protocol (4).
Ensure that labels include the complete GC profile as illustrated in Figures 1-18 (4)—from solvent front to C22:6 n-3—along with enlarged segmented views covering:

  • Solvent to C18:0
  • C18:0 to C18:3 n-3 (9c,12c,15c-C18:3)
  • C18:3 n-3 to C22:6 n-3 (4c,7c,10c,13c,16c,19c-C22:6)

Data Submission

Please complete the spreadsheets and send them together with GC chromatograms to Dr Rain Yamamoto ([email protected]).

If you have any questions, please contact [email protected].

References

  1. World Health Organization. Saturated fatty acids and trans-fatty acids intake for adults and children. WHO guidelines. Geneva, Switzerland, 2023
  2. WHO plan to eliminate industrially produced trans-fatty acids from global food supply [news release]. World Health Organization; 14 May 2018
  3. World Health Organization. REPLACE trans fat: an action package to eliminate industrially produced trans-fatty acids. Geneva, Switzerland, 2021
  4. WHO reference protocol for measuring fatty acids in foods, with emphasis on monitoring trans-fatty acids originating from partial hydrogenation of edible oils. Geneva, Switzerland, 2025
  5. World Health Organization. Simplified protocol for measuring trans-fatty acid content as a percentage of total fatty acids in food products. Geneva, Switzerland, 2023
  6. Ulberth, F. and Wenzl, T. Analytical approach for checking the compliance of fats and oils against the regulated limit for industrial trans fatty acids (Commission Regulation (EU) 2019/649), EUR 30767 EN, Publications Office of the European Union, Luxembourg, 2021, ISBN 978-92-76-40304-3, doi:10.2760/102265, JRC125335
  7. AOAC official method 2012.13: Determination of labeled fatty acids content in milk products, infant formula, and adult/pediatric nutritional formula by Capillary gas chromatography.  In: Official Methods of Analysis, of AOAC INTERNATIONAL (22nd edition). Gaithersburg, Maryland: AOAC International; 2016 
  8. International Standard ISO 16958| IDF 231:2015(E), first edition 2015-11: Milk, milk products, infant formula and adult nutritionals – determination of fatty acids composition – capillary gas chromatographic method. Geneva: International Organization for Standardization; 2015. Brussels, International Dairy Federation
  9. AOAC official method 996.06: Fat (total, saturated, and unsaturated) in foods – hydrolytic extraction gas chromatography method, first action 1996, revised 2001. In: Official methods of analysis, 18th edition. Gaithersburg, Maryland: AOAC INTERNATIONAL; 2005 
  10. AOCS official method Ce 2b-11: Direct methylation of lipids in foods by alkali hydrolysis. In: Official methods and recommended practices of the AOCS, seventh edition. Champaign, Illinois: American Oil Chemists’ Society; 2017