Introduction
Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1), a mycotoxin primarily produced by the fungi Aspergillus flavus and Aspergillus parasiticus. These fungi frequently contaminate raw materials used in animal feed production, such as corn, wheat, sorghum, peanuts, and other cereals and oilseeds, especially under conditions of water stress, high temperatures, and poor storage (Movassaghghazani & Shabansalmani, 2024; IARC, 2002).
When an animal ingests feed contaminated with AFB1, it is absorbed in the gastrointestinal tract and transported to the liver, where it undergoes metabolic hydroxylation mediated mainly by cytochrome P450 enzymes (CYP1A2 and CYP3A4), resulting in the formation of AFM1. This metabolite is excreted in the milk of dairy animals, particularly cows, and remains stable during the processing and storage of dairy products, including pasteurization and sterilization (Battacone et al., 2009; Anfossi et al., 2013; Universidad de Jaén, 2017).
It is estimated that between 1 % and 6 % of the ingested AFB1 is converted into AFM1 and excreted in milk, with the carry-over transfer rate varying according to the AFB1 dose, animal species, individual milk production, and the animal’s physiological state (Gimeno, 2004; Battacone et al., 2009).
AFM1 is classified by the International Agency for Research on Cancer (IARC) in Group 2B (possibly carcinogenic to humans), while AFB1 is classified in Group 1 (carcinogenic to humans). Although AFM1 is less carcinogenic than AFB1, it exhibits a high level of genotoxic activity, capable of damaging the genetic material (DNA) of cells, causing mutations, chromosomal breaks, and alterations in cellular replication (Shibahara et al., 1995; IARC, 2002).
Given that milk and dairy products are widely consumed foods, especially by vulnerable populations such as children, pregnant women, and the elderly, the presence of AFM1 poses a significant risk to public health. For this reason, the regulation and analytical control of this mycotoxin are essential in the food chain.
Origin and contamination factors
Fungal production and predisposing conditions
Fungal contamination of raw materials occurs both in the field and during storage. The factors that favor the growth of Aspergillus spp. and the synthesis of aflatoxins include:
- Temperature: Between 25 and 35 °C is optimal for fungal growth.
- Relative humidity: Above 70-85 %, or a grain moisture content exceeding 13-14 %.
- Water stress: Droughts followed by rainfall favor crop infection in the field.
- Physical damage: Damage from insects, rodents, or mechanical impacts facilitates the entry of the fungus into the grain.
- Climate change: Rising average temperatures and drought episodes increase the prevalence of aflatoxins in temperate zones, including Europe (Battacone et al., 2009; Chhaya et al., 2023).
AFM1 stability in dairy products
A relevant characteristic of AFM1 is its remarkable thermal stability. Standard technological processes applied to milk, such as pasteurization (72 °C/15 s), UHT (135-140 °C/2-5 s), and sterilization, reduce the concentration of AFM1 by only 10 to 20 %, which is insufficient for complete elimination. Furthermore, AFM1 is slightly more soluble in the aqueous phase than in fat, meaning that skimming is not an effective reduction method. In cheesemaking, up to 40-50 % of AFM1 can become concentrated in the curd, posing an additional risk in solid dairy products (Battacone et al., 2009; Becker-Algeri et al., 2016).
Regulatory framework
Due to the risk that AFM1 poses to human health, health authorities have established maximum contamination limits in milk and its derivatives:
| Organization | Product | Maximum Limit (µg/kg) |
| European Union (Reg. (CE) 1881/2006) | Raw milk, heat-treated milk, and milk for the manufacture of dairy products | 0.050 |
| European Union | Infant formulae and follow-on formulae/foods intended for infants and young children | 0.025 |
| Codex Alimentarius (FAO/ WHO) | Milk | 0.500 |
| United States (FDA) | Milk | 0.500 |
| China | Milk and dairy products | 0.500 |
Table 1. Maximum limits of AFM1 in milk according to different regulatory bodies.
The European Union applies one of the strictest limits worldwide (0.050 µg/kg). EFSA carries out periodic assessments of dietary exposure to AFM1 in the population, especially in vulnerable groups, and may revise these limits based on new scientific evidence (EFSA, 2020).
Milk as an analytical matrix
Matrix complexity
In analytical chemistry, milk is recognized as a complex matrix because it contains a wide variety of components that can interfere with the detection of the analyte of interest. Its main components are:
- Fat: Must be removed before analysis, generally via centrifugation and filtration, as it interferes with extraction and can clog chromatographic systems.
- Proteins: Especially casein and whey proteins, which can co-precipitate with the analyte or generate interfering signals. They are removed through precipitation (using heat, acetonitrile, or acids) or by filtration using size-exclusion membranes.
- Lactose: Can interfere with highly sensitive techniques such as HPLC-MS/MS due to matrix effects (suppression or enhancement of analyte ionization).
- Mineral salts and vitamins: Present in lower concentrations, their interference is generally negligible in mycotoxin analysis (Universidad de Murcia, n.d.).
Advantages over other matrices
Despite its complexity, milk offers certain analytical advantages compared to solid matrices such as cereals or animal feed. Being a homogeneous liquid matrix, analyte transfer during extraction is more efficient and reproducible. Furthermore, sample representativeness is higher, as it is easier to obtain representative aliquots from the batch. Moreover, extract clean-up procedures are generally less laborious than those required for solid samples.
Sample preparation
The standard pretreatment of milk for AFM1 analysis includes skimming by centrifugation (3,000–5,000 rpm, 10 min), followed by dilution or extraction of the analyte with organic solvents (methanol, acetonitrile) or via immunoaffinity columns (IAC). IAC columns exhibit high selectivity and are the reference method recommended by European regulations for extract purification before chromatographic determination (Anfossi et al., 2013; Regulation (EC) 401/2006).
Analytical Techniques for the Detection of AFM1
There are several validated analytical techniques for the detection and quantification of AFM1 in milk. The most widely used at the commercial and laboratory levels are lateral flow, ELISA, and HPLC-MS/MS. In addition to these, there are other emerging techniques such as biosensors, fluorescence spectroscopy, and PCR applied to the detection of the producing fungus.
Lateral Flow Immunochromatography (Lateral Flow Assay, LFA)
Lateral flow analysis is based on the immunochromatographic assay (ICA) technique and is characterized by being rapid, low-cost, and highly useful for field analysis. Sample pretreatment is minimal, and the test does not require specialized technical training, which facilitates its use in resource-limited settings (Anfossi et al., 2013).
It can be used qualitatively (presence/absence of the analyte) or quantitatively (analyte concentration) by using portable readers that digitize the signal from the test strips. The operating principle is based on a competitive reaction: the free analyte in the sample competes with the analyte conjugated to gold nanoparticles (or other markers) for antibodies immobilized on the membrane.
- Advantages: Speed (results in within 10 min), low cost, portability, ease of use.
- Limitations: Lower sensitivity than ELISA and HPLC-MS/MS, potential false positives due to cross-reactivity with other aflatoxins, and lower quantitative accuracy.
ELISA (Enzyme-Linked Immunosorbent Assay)
The ELISA technique is an enzyme immunoassay that quantifies the analyte based on specific antigen-antibody binding, generating a colorimetric signal measured via microplate spectrophotometry. There are two main formats for AFM1: direct ELISA (the analyte binds to the antibody immobilized on the plate) and competitive ELISA (the analyte in the sample competes with an enzyme-conjugated analyte).
There are validated commercial kits available specifically for the detection of AFM1 in milk, with a limit of detection of approximately 2 ppt and a limit of quantification of approximately 5 ppt. The analysis time ranges between 60 and 90 minutes. It requires a microplate spectrophotometer and some technical training for result interpretation.
- Advantages: High sensitivity, parallel processing of multiple samples, standardized kits.
- Limitations: Higher cost than lateral flow, potential matrix effects, and requirement for specific equipment.
HPLC-MS/MS (High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry)
HPLC-MS/MS is the reference technique for the detection and quantification of AFM1 in milk, and the method recommended by European regulatory bodies for the confirmation of positive results. It combines chromatographic separation capability (which allows AFM1 to be distinguished from other mycotoxins and matrix compounds) with tandem mass spectrometry detection, which provides high specificity by selecting characteristic precursor-to-product ion transitions for AFM1.
Its limit of detection can be lower than 0.01 µg/kg (< 10 ppt), well below the European maximum limit (ML) of 0.050 µg/kg. An additional advantage is the ability to detect multiple mycotoxins simultaneously in a single analysis, which increases the efficiency of analytical control.
- Advantages: Maximum sensitivity and specificity, multi-analyte capability, official confirmatory method.
- Limitations: High cost (equipment and maintenance), requirement for highly qualified personnel, long analysis time (> 90 min), matrix effects that require calibration with an isotopic internal standard.
| Analytical Technique | Cost | Difficulty | Time (min) | Detection Limits (ppt) | Multi-analyte |
| Lateral Flow | € | + | ≥ 10 | 6 | No |
| ELISA | €€ | ++ | > 60-90 | 2 | No |
| HPLC-MS/MS | €€€ | +++ | > 90 | < 1 | Yes |
Table 2. Comparison of analytical techniques for the detection of AFM1 in milk. Adapted from (Universidad de Jaén, 2017).
AFM1 analysis as a mycotoxin mitigation strategy
The analysis of AFM1 in milk or milk products allows the indirect estimation of the AFB1 ingested by the animal through contaminated raw materials or feed. Given that between 1 % and 6 % of the total AFB1 is metabolized into AFM1 (Gimeno, 2004; Battacone et al., 2009), it is possible to infer the contamination levels of the feed based on the milk analysis results.
This back-calculation strategy is especially useful when prior analyses of raw materials are not available, and it helps justify the need for corrective interventions in animal nutrition management.
Conclusions
Aflatoxin M1 represents a highly relevant contaminant in the dairy chain. Although less carcinogenic than AFB1, its high genotoxicity, stability against technological treatments, and widespread population exposure justify its rigorous regulation and analytical control.
For this reason, the analysis of the AFM1 biomarker in milk is considered a fundamental tool to guarantee food safety for consumers. In this scenario, understanding the analytical techniques available is essential for selecting the most appropriate method according to the analytical objective in order to select the most appropriate option depending on the context and the purpose of the analysis.
Given the high toxicity of aflatoxin B1 (AFB1) as a food contaminant, the implementation of mitigation strategies aimed at reducing its systemic bioavailability is essential, thereby mitigating the toxic effects of both the precursor molecule and its hydroxylated metabolite, aflatoxin M1 (AFM1).
