🍎 Determination of aflatoxin M1 in milk (including low-fat and skimmed) and milk powder — the sample is passed through a column with antibodies against aflatoxin M1, the matrix is washed off with water, the toxin is eluted and determined by high-performance liquid chromatography with fluorescence detection against standards of 0.05–0.80 ng/ml; limit of detection 0.005 µg/kg in milk and 0.05 µg/kg in milk powder, limit of quantification 0.01 and 0.1 µg/kg, PN-EN ISO 14501

Food chemistry PN-EN ISO 14501

Overview

WHAT THE STANDARD COVERS. We read the text of the standard in the iTeh sample of ISO 14501:2021 | IDF 171:2021 (English text): title page, contents, ISO and IDF forewords, cautions, Clauses 1–5 and Clause 6 up to 6.10 (HPLC apparatus) — the sample ends on page 4. We do not read the rest of the apparatus, the procedure (preparation of milk and milk powder, column, purification, HPLC conditions, calibration curve, injection scheme), calculations, precision, test report or Annex A with interlaboratory results; we know their titles from the contents.

ACCORDING TO THE TEXT OF ISO 14501:2021. The third edition was prepared by ISO/TC 34 “Food products”, Subcommittee SC 5 “Milk and milk products”, with the International Dairy Federation (IDF), in collaboration with CEN/TC 302 “Milk and milk products — Methods of sampling and analysis” (Vienna Agreement); it replaces the second edition ISO 14501 | IDF 171:2007. Main change according to the foreword: the lack of detailed explanation in some clauses led to different execution of the method in laboratories — they were clarified on the basis of routine users’ practice. Contents: 8 Procedure (8.2 preparation of test samples: milk, milk powder; 8.3 immunoaffinity column preparation; 8.4 purification; 8.5 HPLC: pump setting, chromatographic performance, calibration curve, analysis of purified extracts and injection scheme), 9 Calculation and expression of results (9.1 skimmed milk, 9.2 skimmed milk powder), 10 Precision, 11 Test report, Annex A (informative) “Results of an interlaboratory trial”. Cautions: aflatoxins are carcinogenic to humans (IARC/WHO statement); the laboratory is protected from daylight and standard solutions from light (e.g. with aluminium foil); non-acid-washed glassware can cause losses of aflatoxin M1 from aqueous solutions — new glassware is soaked for several hours in dilute acid (e.g. H2SO4 2 mol/l) and rinsed to pH 6–8; wastes and glassware are decontaminated according to IARC procedures. Scope (1): determination of aflatoxin M1 in milk and milk powder; lowest level of validation 0.08 µg/kg for whole milk powder, i.e. 0.008 µg/l for reconstituted milk; limit of detection 0.05 µg/kg for milk powder and 0.005 µg/kg for liquid milk; limit of quantification 0.1 µg/kg for milk powder and 0.01 µg/kg for liquid milk; the method also applies to low-fat and skimmed milk and to such milk powders. The standard has no normative references (2). Definition (3.1): aflatoxin M1 content — concentration (µg/l) or mass fraction (µg/kg) determined by the procedure of this standard. Principle (4): the sample is passed through a column with antibodies bound to a support; the antibodies bind aflatoxin M1 in an antibody–antigen complex, other components are washed off with water, the toxin is eluted, and its amount in the eluate is determined by HPLC with fluorimetric detection. Reagents (5): immunoaffinity column with a capacity of not less than 100 ng aflatoxin M1 (2 µg/l with 50 ml of sample) and a recovery of not less than 80 % with 4 ng (80 ng/l with 50 ml) — checked regularly, at least once for every batch of columns: capacity (2.0 ml of 0.1 µg/ml stock solution to 50 ml of water, through the column, elution, HPLC) and recovery (0.8 ml of 0.005 µg/ml working solution to 50 ml; concentration not less than 0.064 µg/l; reference materials may also be used); HPLC-grade acetonitrile, 25 % and 10 % (V/V) solutions, degassed; nitrogen; calibration solution about 10 µg/ml in acetonitrile with concentration determined from the absorbance at the maximum near 350 nm (range 330–370 nm): ρ1 = A · M · 100 / (d · ε), M = 328 g/mol, ε = 1 985 m²/mol, d = 1 cm — or a certified reference material (e.g. BCR-423, 10 µg/ml in chloroform); stock solution 0.1 µg/ml in 25 % acetonitrile, well stoppered, wrapped in foil, at 1–5 °C in the dark — stable at least two months, after which the concentration is checked before use; working solutions 0.005 µg/ml in water–acetonitrile 9 : 1, prepared on the day of use, and from them e.g. 0.05; 0.10; 0.20; 0.40 and 0.80 ng/ml (other dilutions depending on the injection loop; better peak shape may be obtained when diluting with a mixture of eluent composition). Apparatus (6, up to 6.10): syringes 10 and 50 ml; vacuum system (Büchner flask, Vac-Elut system or peristaltic pump); centrifuge ≥ 2000 g; pipettes 1, 2 and 50 ml; 250 ml beakers; 100 ml volumetric flask; water baths 30 ± 2 °C, 35–37 °C and 50 ± 5 °C; Whatman No. 42 filter paper; graduated conical glass tubes 5, 10 and 20 ml; HPLC with pulse-free pump, flow about 1 ml/min, injection 20–500 µl.

STATUS (as of 03.10.2026). In the PKN search (query “14501”, 03.10.2026) PN-EN ISO 14501:2021-10 in the Polish and English versions (“Wprowadza: EN ISO 14501:2021 [IDT], ISO 14501:2021 [IDT]”) carries no withdrawal note; PN-EN ISO 14501:2009 (Polish version) — “Wycofana i zastąpiona przez PN-EN ISO 14501:2021-10 – wersja angielska”, and earlier editions (2007, 2002) — withdrawn and replaced by later ones; other results (PN-EN 14501 on blinds and shutters, PN-B-14501 on mortars) concern other fields. Card of the Polish version: publication date 12.01.2023, 16 pages, KT 35 Mleka i Przetworów Mlecznych, ICS 67.100.10, “Zastępuje PN-EN ISO 14501:2009 – wersja polska”, no withdrawal note. In the iTeh catalogue (read 03.10.2026) ISO 14501:2021 has the status “Published”, stage 90.93 (International Standard confirmed).

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, data up to 17.09.2026, read 03.10.2026). The number 14501 appears in 5 records at 4 laboratories: AB 400, AB 451, AB 465 (2), AB 515. In the current PCA documents (read 03.10.2026; AB 451 issue no. 26 of 24.11.2025, AB 465 issue no. 31 of 23.12.2025, AB 400 issue no. 37 of 07.08.2026, AB 515 issue no. 30 of 12.08.2026) the number appears in 5 lines at the same 4 laboratories; in each document the number of page markers equals the number of PDF pages, without repetitions; a search of all PCA documents downloaded from the BIP on 03.10.2026 found no laboratories outside the database copy. Form “PN-EN ISO 14501:2021-10” (AB 451, AB 515), “PN-EN ISO 14501” without year (AB 400) and “PN-EN ISO 14501” after “Procedury badawcze” (AB 465 — food; for feed additionally with PN-EN 15792). None of these items is suspended. The “Laboratories” tab (query “PN-EN ISO 14501”) shows all 4 laboratories — checked by a tab query on the labcoda.pl production server on 03.10.2026.

WHAT THE LABORATORIES TEST (items in PCA). Object: milk, condensed milk, milk powder, including for infants and young children (AB 515); milk-based food products, including for young children and infants, milk (AB 451); food (AB 400); food and feed (AB 465). Characteristic: aflatoxin M1 content (AB 400, AB 451, AB 515), mycotoxin content (AB 465). Ranges: (0.004–0.32) µg/l or µg/kg (AB 515), (0.017–0.24) µg/kg (AB 451); AB 400 and AB 465 give no range. Technique: HPLC with fluorescence detection; for feed at AB 465 — HPLC with spectrophotometric and/or fluorescence detection.

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. Lower limit: according to the standard the limit of detection in liquid milk is 0.005 µg/kg and the limit of quantification 0.01 µg/kg (1) — the range of AB 515 starts at 0.004 µg/l or µg/kg, below both values; the lowest validation level of the standard is 0.008 µg/l for reconstituted milk. Matrix: the scope of the standard is milk and milk powder (1); AB 451 applies it to milk-based products, and AB 465 to food and feed with the general characteristic “mycotoxin content” — for feed together with PN-EN 15792:2012, a standard on zearalenone in feed (PKN card: publication 26.03.2012, KT 40 Pasz, no withdrawal note, read 03.10.2026), so the two standards divide different toxins there and are not alternatives. Glassware: non-acid-washed glassware adsorbs aflatoxin M1 from aqueous solutions (caution 3). Column: each batch of columns must show capacity ≥ 100 ng and recovery ≥ 80 % (5.1) — which is why the standard requires each batch of columns to be checked separately, before analysing samples. Standards: the concentration of the calibration solution is determined spectrophotometrically, and a stock solution older than two months is checked before use (5.4.1, 5.4.2). Unit: the result in µg/l (liquid milk) and in µg/kg (milk powder) are different quantities (3.1) — AB 515 gives the range “µg/l or µg/kg” with one pair of numbers.

WHAT WE DO NOT GIVE. Preparation of milk and milk powder samples, HPLC conditions, calculation formulae, precision data and interlaboratory results are not quoted — they lie outside the sample. We do not give maximum levels of aflatoxin M1 from regulations.

Method principle

A sample of milk (or reconstituted milk powder) is passed through a column with antibodies against aflatoxin M1; the toxin binds in an antigen–antibody complex, other components are washed off with water. Aflatoxin M1 is eluted and determined by high-performance liquid chromatography with fluorescence detection against standard solutions (e.g. 0.05–0.80 ng/ml), whose concentration is determined spectrophotometrically at about 350 nm.

Applications

• Aflatoxin M1 in milk (whole, low-fat, skimmed) and milk powder; LOQ 0.01 µg/kg and 0.1 µg/kg (1)
• Milk, condensed and powdered milk, also for infants and young children, milk-based products, food and feed — items in the accreditation scopes of 4 laboratories in PCA

Key parameters

Standard

Standard number

PN-EN ISO 14501:2021-10

Title (PL)

Mleko i mleko w proszku — Oznaczanie zawartości aflatoksyny M1 — Oczyszczanie za pomocą chromatografii powinowactwa immunologicznego i oznaczanie za pomocą wysokosprawnej chromatografii cieczowej

Title (EN)

Milk and milk powder — Determination of aflatoxin M1 content — Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography

Step-by-step procedure

1. Preparing laboratory and glassware

   Protection from daylight, acid-washed glassware (cautions 2, 3). PN-EN ISO 14501:2021-10 with no withdrawal note in PKN (03.10.2026).

2. Column check

   Capacity (2.0 ml of 0.1 µg/ml solution in 50 ml) and recovery (0.8 ml of 0.005 µg/ml solution in 50 ml, ≥ 0.064 µg/l) for each batch (5.1).

3. Standards

   Concentration of the calibration solution from absorbance at about 350 nm; working 0.05–0.80 ng/ml on the day of use (5.4).

4. Purification

   Sample through the column, washing with water, elution of aflatoxin M1 (4; details in Clause 8, outside the sample).

5. HPLC and result

   Fluorescence detection, calibration curve; result in µg/l or µg/kg (3.1, 4; calculations in Clause 9, outside the sample).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Aflatoxins are carcinogenic to humans — work with standard solutions in a designated area, decontamination of wastes and glassware according to IARC procedures (cautions 1, 4)
• Acetonitrile — flammable and toxic; work under a fume hood

Frequently asked questions