🍎 Fatty acid composition of oils and fats (including erucic acid, trans isomers, omega-3 acids) — separation of fatty acid methyl esters (FAME) on a polar capillary column with a flame ionization detector, calculation from area percentages, with correction factors or with an internal standard, PN-EN ISO 12966-4

Food chemistry PN-EN ISO 12966-4

Overview

WHAT THE STANDARD COVERS. Scope from the PKN catalogue card of PN-EN ISO 12966-4:2026-08 (current edition), in full (our translation of the Polish card text): “This document specifies a method for the determination of fatty acid methyl esters (FAME) derived by transesterification or esterification from fats, oils and fatty acids by capillary gas chromatography (GLC). This document allows FAME from C4 to C24 to be separated, including saturated FAME, cis- and trans-monounsaturated FAME and cis- and trans-polyunsaturated FAME. This document is applicable to crude, refined, partially or fully hydrogenated fats, oils and fatty acids of animal and vegetable origin and to fats extracted from foodstuffs. This document does not apply to milk and milk products (or fat coming from milk and milk products) or to products supplemented with conjugated linoleic acid (CLA). This document does not apply to di-, tri-, polymerized, hydroxylated and oxidized fatty acids, fats and oils. A method for the determination of the FAME composition expressed as area % in liquid vegetable oils is proposed in Annex E.” We do not have the text of the 2026 edition. The technical content below is taken from the preview of the 2015 edition (ISO 12966-4:2015, first edition) from the cover to the beginning of clause 12 (test report, page 7) — this is the edition all laboratories cite in their scopes; only the “Scope” field of its card relates to the 2026 edition.

WHAT THE 2026 EDITION CHANGED — AS FAR AS CAN BE SEEN FROM THE “SCOPE” FIELDS OF BOTH PKN CARDS. (1) Range of separation: 2015 — “from C8 to C24”, 2026 — “from C4 to C24”. (2) Application: 2026 adds “fats extracted from foodstuffs”. (3) Exclusion of milk: 2015 — the method “is not suitable” for fats and oils from ruminant milk and products with added CLA, and milk and milk products (fat from them) are not within the scope; 2026 — the document “does not apply” to milk and milk products (fat from them) or products supplemented with CLA — the exclusion remains in both editions. (4) Exclusion of acids: 2026 adds hydroxylated acids. (5) New Annex E (2026) — FAME composition as area % in liquid vegetable oils. Card of 2026: 52 pages, card of 2015 (English version) — 32 pages. Whether the chromatographic conditions, formulae, column check or precision have changed is NOT GIVEN — we have not read the 2026 text.

ACCORDING TO THE 2015 EDITION (clauses 1–11). The standard was prepared by ISO/TC 34/SC 11 “Animal and vegetable fats and oils”; the first edition replaced ISO 5508:1990 and ISO 15304:2002 (technically revised). The ISO 12966 series: part 1 — guidelines on modern gas chromatography of FAME, part 2 — preparation of methyl esters, part 3 — preparation of methyl esters using trimethylsulfonium hydroxide (TMSH), part 4 — determination by capillary gas chromatography. Normative references: ISO 661 (preparation of test sample), ISO 3696 (water), ISO 6353 (reagents), ISO 12966-2 and ISO 12966-3. Principle: FAME are separated on a highly polar stationary phase according to chain length, degree of unsaturation and the geometry and position of the double bonds. Reagents: reference mixtures of pure FAME and/or oils of known composition for identification; fats and oils with certified composition, e.g. reference material BCR 162; reference FAME, in particular cis and trans isomers of octadecenoic (oleic), octadecadienoic (linoleic) and octadecatrienoic (α-linolenic) acids — trans isomers of linoleic and α-linolenic acids can be prepared in the laboratory with p-toluenesulfonic acid. For a result in grams per 100 g a FAME internal standard is needed (external calibration with mixtures of acids is also possible); examples according to the type of fat: C11:0, C17:0, C19:0, C21:0, C23:0; an additional analysis of the sample without the standard is recommended to check the natural content of the acid used as standard, which is taken into account in the calculation; the purity of the standard is confirmed by TLC, HPLC, GC or another suitable technique. Example solutions: C21:0 FAME (methyl heneicosanoate, purity > 99 %) 5,0 mg/ml in iso-octane or MTBE; C21:0 triacylglycerol (triheneicosanoin, purity > 99 %) 5,0 mg/ml in chloroform or 2 mg/ml in toluene (dissolves after slight warming, crystallizes in the refrigerator); the TAG standard is added before the preparation of esters, the FAME standard before or after, and different chain lengths of the two allow the derivatization efficiency to be assessed; a TAG standard hard to dissolve in the cold requires hot methylation according to ISO 12966-2:2011 (4.3–4.5); solvents other than iso-octane are removed after adding the TAG standard. Solvents: iso-octane, MTBE, chloroform, n-hexane, n-heptane, toluene. Apparatus: gas chromatograph with flame ionization detector, split or splitless injector (on-column and PTV also possible) and data acquisition system; fused silica capillary column 100 m × 0,25 mm with 100 % cyanopropyl silicone phase (e.g. SP-2560 or CP-Sil 88), film thickness 0,20 µm — such a column separates most trans and cis C18:1 isomers; when this separation is not needed a 50 m or 60 m column is sufficient, other phases (e.g. BPX70, DB-23, SP-2380) are possible with a possible change in elution order, short 10–15 m columns — for fast GC with limited information; 10 µl microsyringe; carrier gas hydrogen (recommended) or helium of at least 99,999 5 % purity, dried, with oxygen removed below 0,1 mg/kg — nitrogen as carrier gas is not acceptable; flame gases (hydrogen, air) and make-up gas (nitrogen or helium). Sampling is not part of the method (ISO 5555 recommended); the test sample is prepared according to ISO 661 and the methyl esters according to ISO 12966-2 or ISO 12966-3; the internal standard is added so that after the fat is added its mass fraction is between 0,05 and 0,10 mg standard per mg fat, and its solvent is evaporated before methylation; FAME are dissolved in n-heptane, n-hexane or iso-octane, approx. 15–20 mg/ml for split injection. Procedure: the first sample of a batch is always a blank of the solvent alone — no peaks may appear in it. Conditions proven for FAME from C4 to C24 on a 100 m column (others are possible): injector and detector 250 °C; oven from 120 °C to 240 °C at 4 °C/min and 7 min at 240 °C; hydrogen at 220 kPa column head pressure, linear velocity 30–40 cm/s, flow approx. 1,0 ml/min; split ratio 1:100; injection 1 µl (15–20 µg FAME). Column check with a FAME mixture covering the acids under investigation; if necessary the sample size, concentration or oven temperature is changed — the latter preferably by 1 °C; on all cyanopropyl silicone columns the column temperature strongly affects the elution order of, among others, 13t- and 14t-C18:1, 16t-C18:1, 14c-C18:1, 9c,12c,15t-C18:3, 11c-C20:1 and 9c,12c,15c-C18:3. Identification (10.1): by retention times against FAME standards and reference hydrogenated oils; unknown peaks are not included in the sum of areas unless confirmed to be fatty acids — they may also be summed separately; the standard notes minor co-elution of cis and trans isomers, particularly in the C18:1 region (cis-9 oleic acid). Calculations: (10.2.1) area fraction xi = Ai/ΣA × 100 — for most fats it corresponds to the fraction of triacylglycerols in grams per 100 g; the FAME-to-TAG conversion factors according to AOCS Ce 1h-05 lie between 0,9114 (C8:0) and 0,9965 (C24:1) and the standard regards them as negligible; result in grams per 100 g with one decimal place. (10.2.2) When acids with fewer than 16 carbon atoms are present (lauric fats with C10, C12, C14), the areas are corrected with factors Fi determined for each instrument from a material of certified composition: Fi = (mi × ΣA)/(Ai × Σm), wi = Fi·Ai/Σ(Fi·Ai); at the client’s request the factor may not be applied, but the report must state whether it was applied; the theoretical FID correction factors (Annex A) are not the same factors, and large differences between them require the whole system to be checked. (10.2.3) With an internal standard (e.g. when not all acids are quantified, when C4 and C6 acids are present alongside C16 and C18, or when the absolute content of an acid is needed): wi = (mIS × Fi × Ai)/(m × FIS × AIS), with the mass of the standard corrected for purity (usually 0,99); result with one decimal place. Precision (11): repeatability r and reproducibility R in Tables D.1–D.3 from an interlaboratory test (the values may not apply to other concentration ranges and matrices). The table of contents also lists clause 12 (test report) and informative Annexes A (theoretical FID correction factors), B (examples of chromatograms), C (comparison of FAME composition on two columns), D (interlaboratory trial).

STATUS (cards as of 02.10.2026). The card of PN-EN ISO 12966-4:2026-08 (English version) has no “Withdrawn” header — this edition is CURRENT: published 31-08-2026, 52 pages, price group U, Food, Agriculture and Forestry Sector, KT 92 for Oilseeds, Vegetable and Animal Fats and their By-products, ICS 67.200.10, “Introduces: EN ISO 12966-4:2026 [IDT], ISO 12966-4:2026 [IDT]”, “Replaces: PN-EN ISO 12966-4:2015-07 - Polish version, PN-EN ISO 12966-4:2015-07 - English version”. There is no Polish version of the 2026 edition in the catalogue: the PKN search for “PN-EN ISO 12966-4” (02.10.2026) shows three items — 2026-08 (English) and 2015-07 (Polish and English). Cards of PN-EN ISO 12966-4:2015-07 in both versions: “Withdrawn and replaced by PN-EN ISO 12966-4:2026-08 - English version”, withdrawal date 31-08-2026 (English version — published 10-07-2015, 32 pages; Polish — 03-10-2022, 28 pages); the 2015 edition replaced PN-EN ISO 5508:1996 and PN-EN ISO 15304:2003 (with corrigendum AC:2005). What the change from 2015 to 2026 means for the result: according to the “Scope” fields — separation range from C4 instead of from C8, fats extracted from food directly in scope, Annex E with area percentages for vegetable oils; the exclusion of milk and CLA products remains; the rest we do not know. We have not checked the status of ISO 12966-4:2026 on the ISO website. Legislation: Commission Regulation (EU) 2023/915 on maximum levels for certain contaminants in food (consolidated text of 19.08.2026 — the latest according to the CELLAR database; point 2.1 has the same wording as in the original act, OJ L 119 of 5.5.2023) gives for erucic acid, including erucic acid bound in fat, maximum levels in g/kg: vegetable oils and fats — 20,0; camelina oil, mustard oil and borage oil — 50,0; mustard (condiment) — 35,0; it does not indicate a method of determination (the number 12966 does not appear anywhere in the consolidated text).

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, data up to 17.09.2026, read 02.10.2026). The number 12966-4 appears in 17 records of 9 laboratories: AB 343, AB 515, AB 552, AB 562 (3 each), AB 444, AB 775, AB 819, AB 1179, AB 1679 (1 each). Current scopes on the PCA website (read 02.10.2026, scope issues from 24.11.2025 to 18.09.2026): the number stands in 13 lines — 3 each at AB 515 and AB 562, 1 at each of the other seven (at AB 343 and AB 552 one entry of reference documents covers several objects, which the copy splits into separate records). Edition: everywhere 2015-07, at AB 444 without date (“PN-EN ISO 12966-4”) — so all 9 laboratories cite today the edition withdrawn on 31.08.2026. None of these items is suspended (at AB 1179 the footnotes on the suspension of part of the scope from 24.04.2026 to 21.10.2026 concern lines in bold italics — the lines with 12966-4 are not set that way). The “Laboratories” tab searches for entries BEGINNING WITH “PN-EN ISO 12966-4” and shows all 9 laboratories — checked with the tab query on the production server labcoda.pl on 02.10.2026: 9 (under “EN ISO 12966-4” and “ISO 12966-4” — 0).

WHAT THE LABORATORIES DETERMINE (items in PCA). Erucic acid — AB 343 (vegetable oils and fats (0,1–7,0) % by weight, infant formulae (0,1–1,5) %, mustard (10–50) %), AB 552 (oils (0,1–6,0) % of fat, foods for special nutritional purposes (0,1–3,0) %, mustard (10,0–55,0) % of fat, each also with “erucic acid content in the product (from calculation)”), AB 562 (oils and fats (0,2–10,0) %, infant formulae (0,2–2,0) % together with linoleic acid (0,2–50,0) %), AB 775 (rapeseed (0,1–5,0) %). Full fatty acid composition with a list of acids — AB 515 (from C6:0 to C24:1, range (0,08–100) %, from calculation among others the sums of saturated, mono- and polyunsaturated acids, trans isomers, omega-3, -6, -9, CLA, essential fatty acids and conversions per portion or kcal), AB 819 (from C4:0 to C22:6, (0,1–75,0) %, with sums “from calculation”), AB 1179 (from C4:0 to C22:6, (0,06–100) % and g/100 g, with sums “from calculation”); without a list — AB 444 (“fatty acid composition” and “butyric acid content”, food), AB 1679 (vegetable oil as food and liquid biofuel, (0,1–63,0) % (m/m)), AB 562 (proportion of omega-3 acids, (0,2–50,0) %). Technique in all items: gas chromatography with flame ionization detection (GC-FID).

NEXT TO THIS STANDARD (the same PCA items). PN-EN ISO 12966-2:2017-05 (preparation of methyl esters) — all 9 laboratories, including with exclusions or named points: AB 562 — excluding points 5.2, 5.3 and 5.5; AB 1679 — excluding points 5.4 and 5.5; AB 819 — point 5.3; AB 1179 — point 5.2; AB 444 without date. PN-EN ISO 12966-1:2015-01 (guidelines), with or without corrigendum AC:2015-06 — 7 laboratories (all except AB 343 and AB 444). PN-ISO 1444:2000 (free fat in meat) — AB 343. In-house procedures — AB 1179 (IB-17 issue 4 of 10.03.2021, IB-05 issue 6 of 12.05.2021). Status (cards as of 02.10.2026): PN-EN ISO 12966-2:2017-05 (Polish version, published 14-11-2022), PN-EN ISO 12966-1:2015-01 (Polish version, published 06-07-2021, with additional element AC:2015-06) and PN-ISO 1444:2000 — cards without a “Withdrawn” header. Preparation of methyl esters according to PN-EN ISO 12966-2, the PN-EN ISO 12966-1 guidelines and free fat in meat according to PN-ISO 1444 have separate entries in the Knowledge Base.

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. In the choice of sample: both versions of the scope exclude milk and milk products (milk fat) and products supplemented with CLA, and the 2015 edition also fats from ruminant milk — yet the scopes citing this standard include: “Milk and milk products” (AB 562, omega-3 acids), “Butyric acid content” in food (AB 444), “CLA content” among the quantities from calculation (AB 515) and infant formulae (AB 343, AB 515, AB 562); AB 819 excludes milk, its products and fats of milk origin in the object. A chromatogram of such a sample can be calculated, but the result is not within the scope of the standard. With short-chain acids: the 2015 scope speaks of separation from C8 to C24, the conditions in 9.2 of the same edition — of FAME from C4 to C24, and the 2026 scope — from C4; C4:0 and C6:0 appear in the scopes of AB 515, AB 819 and AB 1179 with the 2015 edition. In the measurement: co-elution of cis and trans isomers in the C18:1 region and the effect of column temperature on the elution order of trans isomers of C18:1, C18:3 and C20:1 — a change of the oven temperature by a few degrees can swap peak order and name assignment; a 50–60 m column does not separate most trans and cis C18:1 isomers, and a phase other than cyanopropyl silicone may change the elution order; nitrogen as carrier gas is not acceptable; the first run of a batch — a blank, without peaks. In the calculation: the area percentage (10.2.1) equals “grams per 100 g” only for most fats — with acids below C16 correction factors determined for the own instrument are needed, and not applying them must be stated in the report; unknown peaks are not included in the sum unless confirmed to be fatty acids; with an internal standard its natural content in the sample is checked by a separate analysis and taken into account. Quantities “from calculation” (sums of saturated, trans, omega-3, content in the product) are calculations from the results of this method: the proportion in the product additionally requires the fat content, determined by another method (at AB 343, PN-ISO 1444 — a standard for meat — stands next to it); the maximum levels of erucic acid in Regulation (EU) 2023/915 are given in g/kg. The edition and points of PN-EN ISO 12966-2 chosen by the laboratory (methylation) are part of the result — see the entry for that standard.

WHAT WE DO NOT GIVE. We do not have the text of the 2026 edition (other than the “Scope” field of the card) — we do not give its chromatographic conditions, formulae, column check, precision or the content of Annex E. From the 2015 edition we do not have clause 12 (test report — except item a) or Annexes A–D, including the r and R values. We do not give the preparation of esters (PN-EN ISO 12966-2, 12966-3) or the sampling rules (ISO 5555).

Method principle

The fat is converted into fatty acid methyl esters (according to ISO 12966-2 or 12966-3), and the esters are separated by capillary gas chromatography on a highly polar cyanopropyl silicone phase — according to chain length, number of double bonds and their position and geometry (cis/trans) — and detected with a flame ionization detector. Peaks are identified by retention times against standards, and the composition is calculated from peak area percentages, with correction factors determined for the instrument (acids below C16) or with an internal standard (e.g. C21:0) when the absolute content in grams per 100 g is needed.

Applications

• Erucic acid in vegetable oils and fats, mustard, rapeseed and infant formulae — AB 343, AB 552, AB 562, AB 775
• Full fatty acid profile of food (C4:0–C24:1), with sums of saturated, trans, omega-3 and omega-6 acids “from calculation” — AB 515, AB 819, AB 1179
• Fatty acid composition of vegetable oil as food and liquid biofuel — AB 1679

Key parameters

Standard

Standard number

PN-EN ISO 12966-4:2026-08 (wersja angielska; EN ISO 12966-4:2026, ISO 12966-4:2026 IDT)

Title (PL)

Oleje i tłuszcze roślinne oraz zwierzęce — Chromatografia gazowa estrów metylowych kwasów tłuszczowych — Część 4: Oznaczanie techniką kapilarnej chromatografii gazowej

Title (EN)

Animal and vegetable fats and oils — Gas chromatography of fatty acid methyl esters — Part 4: Determination by capillary gas chromatography

Step-by-step procedure

1. Sample and methyl esters

   Test sample according to ISO 661; methyl esters according to ISO 12966-2 or 12966-3; internal standard (when needed) — 0,05–0,10 mg per mg fat, its solvent evaporated before methylation. PN-EN ISO 12966-4:2026-08 current (card as of 02.10.2026); the laboratories cite the 2015-07 edition.

2. FAME solution

   FAME in n-heptane, n-hexane or iso-octane, approx. 15–20 mg/ml for split injection.

3. Blank and column check

   First in the batch — a blank of the solvent alone, without peaks; check of separation with a FAME mixture covering the acids under investigation, if necessary oven temperature corrected by 1 °C.

4. Chromatography

   E.g. injector and detector 250 °C; oven 120 °C → 240 °C (4 °C/min), 7 min at 240 °C; hydrogen 220 kPa, approx. 1,0 ml/min; split 1:100; 1 µl (2015 edition, example conditions).

5. Identification

   Retention times against FAME standards and reference hydrogenated oils; unknown peaks outside the sum unless confirmed to be fatty acids.

6. Calculation

   Area fraction xi = Ai/ΣA × 100; with acids below C16 — correction factors Fi determined for the instrument (whether applied or not — in the report); internal standard — wi = (mIS·Fi·Ai)/(m·FIS·AIS); result in g/100 g with one decimal place.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Hydrogen as carrier gas speeds up the analysis but is hazardous — the standard requires a hydrogen generator and safety devices; toxic solvents — work in a fume hood
• Chloroform — classified by the standard as a carcinogenic solvent of category 3; after n-hexane and n-heptane — warning against prolonged exposure through inhalation and swallowing
• TECHNICAL NOTE: the method does not cover milk and milk products or products supplemented with CLA (both editions); the laboratories cite the 2015-07 edition, withdrawn 31-08-2026

Frequently asked questions