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Article

Aflatoxin M1 Levels in Commercial Cows’ Milk in Mexico: Contamination and Carcinogenic Risk Assessment

by
Araceli Monter-Arciniega
1,
Nelly del Socorro Cruz-Cansino
1,*,
Araceli Castañeda-Ovando
2,
Angélica Saraí Jiménez-Osorio
3,
Salvador Ramón Tello-Solís
4,
Carlos Jiménez-Pérez
5 and
Gabriela Mariana Rodríguez-Serrano
5,*
1
Área Académica de Nutrición, Universidad Autónoma del Estado de Hidalgo, San Agustín Tlaxiaca 42160, Mexico
2
Área Académica de Química, Universidad Autónoma del Estado de Hidalgo, Mineral de la Reforma 42184, Mexico
3
Área Académica de Enfermería, Universidad Autónoma del Estado de Hidalgo, San Agustín Tlaxiaca 42160, Mexico
4
Departamento de Química, Universidad Autónoma Metropolitana, Ciudad de México 09340, Mexico
5
Departamento de Biotecnología, Universidad Autónoma Metropolitana, Ciudad de México 09340, Mexico
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2025, 15(11), 6106; https://doi.org/10.3390/app15116106
Submission received: 22 April 2025 / Revised: 24 May 2025 / Accepted: 27 May 2025 / Published: 29 May 2025
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Abstract

:
Aflatoxin M1 (AFM1), a toxic metabolite of aflatoxin B1 (AFB1), is commonly found in cow’s milk (CM) when dairy cattle consume contaminated feed. Chronic exposure to AFM1 poses serious health risks, particularly for children. This study aimed to quantify AFM1 levels in commercial CM available in Mexico and assess the associated carcinogenic risk across six age groups. A total of 23 milk samples were collected between March and July 2023 from supermarkets and convenience stores. AFM1 was measured using high-performance liquid chromatography (HPLC). Estimated daily intake (EDI), carcinogenic risk index (CRI), and Monte Carlo simulations (MCS) to model exposure were employed. AFM1 was detected in all samples, with 80% exceeding the legal limits set by Mexican and European regulations. The highest CRI values were observed in children aged 1–4 years (CRI > 5 × 103), indicating an elevated risk. There were no statistically significant differences in AFM1 concentration among different milk or thermal treatments. A cluster analysis revealed patterns related to AFM1 content, price, and processing methods. The widespread detection of AFM1 raises a public health concern, especially for vulnerable populations. This underscores the need for targeted regulatory strategies and improved control of aflatoxins in the dairy production chain.

1. Introduction

Cow’s milk (CM) ranks among the most extensively consumed food products globally, attributable to its nutritional benefits and availability to all demographic groups. It serves as a principal source of high-quality protein, essential vitamins, and readily absorbable calcium [1,2]. Nevertheless, despite its nutritional importance, milk presents potential health risks as a medium for foodborne contaminants, notably aflatoxin M1 (AFM1). AFM1 is a hydroxylated metabolite of aflatoxin B1 (AFB1) produced in the liver of dairy cattle subjected to feed contaminated with Aspergillus flavus and Aspergillus parasiticus [3,4]. In addition, climatic conditions, insufficient feed storage, and inadequate quality control measures significantly contribute to the contamination of animal feed with AFB1, particularly in tropical and subtropical regions [5,6]. The AFM1 is excreted into milk and dairy products, where it can persist despite pasteurization and ultra-high temperature (UHT) treatments [7,8]. AFM1 has been classified by the International Agency for Research on Cancer (IARC) as a Group 1 human carcinogen, attributed to its genotoxic, immunosuppressive, and mutagenic properties [3]. Chronic exposure to AFM1 is associated with an elevated risk of hepatocellular carcinoma, particularly among populations with high consumption of milk and limited dietary diversity [9,10]. A significant inverse correlation has been established between the risk of carcinogenicity and body weight, particularly among children from birth to 26 months of age in Nepal who were chronically exposed to aflatoxins. This reinforces that lower body mass predisposes individuals to a higher relative exposure [11].
Evidence suggests that the toxic effects of AFM1 are more pronounced in pediatric populations than adults, attributable to their lower body weight, greater milk consumption, and the development of physiological systems with limited capacity to metabolize and excrete toxins [10,12,13]. Prolonged exposure to AFM1 during the early years of life has been associated with hepatocellular damage, immunosuppression, altered nutrient absorption, and growth retardation [14,15]. Furthermore, even low-dose exposure may impede protein synthesis and liver enzyme functionality, potentially leading to long-term developmental disorders [16,17].
The presence of AFM1 in milk and dairy products has been widely reported in several countries using various analytical approaches. In Greece, Maggira et al. (2021) compared commercial enzyme-linked immunosorbent assay kits with a high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method developed to analyze AFM1 in raw milk, underlining the importance of selecting context-specific analytical procedures [18]. Similarly, Kolarič and Šimko (2023), in Slovakia, validated an HPLC-FLD method with pretreatment in an immunoaffinity column that met the analytical performance criteria established by European standards [19]. In Hungary, routine monitoring of AFM1 in both raw and processed milk was conducted using commercial test kits, highlighting the need for continuous monitoring along the entire dairy production chain [20].
In the Latin American context, Quevedo-Garza et al. (2018) investigated AFM1 levels in liquid milk sold in Monterrey, Mexico, reporting concentrations that exceeded European regulatory limits [21]. Additionally, other studies have documented the presence of AFM1 in raw, pasteurized, and UHT milk samples throughout Mexico [22,23,24,25,26,27]. In particular, Álvarez-Díaz et al. (2022) reported concentrations of AFM1 exceeding the national regulatory threshold of 0.5 μg/L in commercially available dairy products [22,28]. These results underscore the significance of region-specific risk assessments and the necessity of continuous monitoring in various dairy markets.
While this study emphasizes the issue of AFM1 contamination in commercial milk in Mexico, it is essential to note that AFM1 contamination in dairy products has been extensively documented globally, underscoring its international significance. Researchers have reported elevated detection rates of AFM1, frequently surpassing established regulatory limits. This evidence affirms that AFM1 contamination is not restricted to particular regions but represents a pervasive issue associated with feed contamination, unsatisfactory storage conditions, and climatic influences [29,30,31,32,33]. Consequently, local evaluations like the one presented in this study are crucial for delineating exposure risks within specific national contexts. These studies provide essential data regarding prevalence; a paucity of research assesses the carcinogenic risk of AFM1 based on age-specific consumption patterns, which is vital for informing regulatory and public health strategies [34,35]. Therefore, accurate quantification necessitates using highly sensitive and reliable analytical techniques, such as HPLC-FLD, which is esteemed for its precision and reproducibility in detecting AFM1 [36,37]. In addition to quantification, the carcinogenic risk index (CRI) estimation provides a more comprehensive perspective by incorporating AFM1 concentrations alongside actual consumption patterns and body weight across diverse population groups [10,38]. This methodology aids in prioritizing regulatory actions and meaningfully assessing public health risks. Consequently, this study aimed to quantify the levels of AFM1 present in various brands of CM sold in Mexico and evaluate the cancer risks associated with its consumption among different age groups.

2. Materials and Methods

2.1. Chemicals and Reagents

The analytical standard of AFM1 at a 100 μg/mL concentration in acetonitrile (ACN) was procured from Sigma-Aldrich, located in St. Louis, MI, USA. HPLC-grade ACN and methanol were acquired from J.T. Baker, Center Valley, PA, USA. Ultrapure water was generated utilizing a Milli-Q system from Millipore, Burlington, MA, USA. All chemicals employed in this study were of analytical or chromatographic grade.

2.2. Sample Collection

Between March and July 2023, 23 samples of commercial CM (A–W) were collected in supermarkets and convenience stores in Pachuca, Hidalgo, Mexico. Selected brands of pasteurized milk (A–E), UHT (F–W), where the E, U, V samples are organic milk, which are nationally distributed and commercially available in several regions. Table S1 provides detailed characteristics of the milk, including sample ID, classification, production site, origin, certification, and price. All CM samples were transported, refrigerated, and stored at 4 °C until analysis.

2.3. Preparation of Standard Solution

A stock solution (1 µg/mL) was prepared from a standard concentration of AFM1 (100 µg/mL). The stock solution was diluted in ACN to obtain working stock solutions of a standard calibration curve of 0.01–0.5 µg/mL.

2.4. Aflatoxin M1 Extraction

The liquid–liquid AFM1 extraction (LLE) employed was adapted from previously reported procedures [39,40], with the following modifications. Five milliliters of each CM were utilized; 1 g of NaCl was incorporated to facilitate the samples’ dissolution, followed by adding 3.8 mL of ACN. The resultant mixture was subsequently vortexed utilizing a Vortex® stirrer (S1-0256, Scientific Industries, Alcobendas, Madrid, Spain) at maximum speed for 1 min and permitted to rest for 15 min. Thereafter, it underwent centrifugation (Avanti J-E, Beckman Coulter Life Sciences, Nyon, Vaud, Switzerland) at 6000 rpm for 7 min at a temperature of 4 °C. The supernatant was subsequently collected, and 5 mL of deionized water and 2 mL of chloroform were incorporated, followed by homogenization. The mixture was again subjected to centrifugation under identical conditions as previously described. The organic phase was dried under reduced pressure employing a rotary evaporator (R-300, BÜCHI® Labor Technik, Essen, North Rhine-Westphalia, Germany). Finally, the CM was reconstituted with 400 µL of the mobile phase and filtered through a 0.45 µm polytetrafluoroethylene membrane (Sigma-Aldrich®, St. Louis, MI, USA).

2.5. Chromatographic Analysis of Aflatoxin M1

For the separation, identification, and detection of AFM1, an HPLC-FLD (LC-20AD) was utilized. This system comprised a solvent supply unit, an automatic sampler (model: SIL-20AC XR, Shimadzu Corporation, Kyoto, Japan), a fluorescence detector (RF-20A XS), a column heater (CTO-20A), a degassing unit (DGU-20A 5R), and a communication module (model: CBM-20A, Shimadzu Corporation, Kyoto, Japan). The separation was conducted utilizing a reverse phase C18 column (dimensions: internal diameter 4.6 mm, length 250 mm, particle size 5 µm, pore size 120 Å; ODS HypersilTM®, Thermo Fisher Scientific, Powai, Mumbai, India, maintained at 30 °C, and a mobile phase consisting of water and ACN (60:40, v/v), operated under isocratic conditions at a flow rate of 0.4 mL/min and an injection volume of 5 µL. the total run time was 15 min injection. AFM1 was detected at an excitation wavelength of 365 nm and an emission wavelength of 435 nm. The chromatographic data were processed using LabSolutions® software, version 5.87.1 (Shimadzu Corporation, Kyoto, Japan).

2.6. Reliability and Analytical Effectiveness of the Method

The analytical procedure for quantifying AFM1 in milk was validated in accordance with the guidelines established by the International Council for Harmonization. This validation considered the following parameters: linearity, limit of detection (LOD), limit of quantification (LOQ), selectivity, precision, and recovery [41]. Linearity was evaluated by constructing a calibration curve from standard solutions of AFM1 in acetonitrile, which spanned a concentration range of 0.01 to 0.5 μg/L. Duplicate calibration points were prepared, and the determination coefficient (R2) to assess the linear response was used. The LOD and LOQ were computed based on the standard deviation of the intercept point (Sy) and the slope (b) of the calibration curve, utilizing the following equations:
LOQ = 3.3 × Sy/b
LOQ = 10 × Sy/b
Selectivity was evaluated by analyzing blank CM samples subjected to the identical liquid–liquid extraction (LLE) procedure outlined in Section 2.4 to ensure the absence of retention time interference peaks associated with AFM1. Accuracy was ascertained through the calculation of the relative standard deviation from three repeated injections of CM. Recovery was assessed by supplementing a previously analyzed CM sample with a quantifiable baseline concentration of AFM1 and a predetermined amount of standard solution (1.0 μg/L). The spiked CM underwent triplicate processing employing uniform extraction and quantification methods. To compute the recovery of the introduced standard, the native concentration was deducted from the total measured value. Subsequently, the recovery rate was established utilizing the following equations:
Theoretical total concentration = Cnative + Cadded
Recovered standard = Cmeasured − Cnative
Recovery (%) = (Cmeasured − Cnative/Cadded) × 100
where Cnative = initial concentration of AFM1 in the unfortified CM; Cadded = known concentration of the standard spiked; and Cmeasured = final concentration determined after spiking.

2.7. Risk Assessment for Exposure to Aflatoxin M1

2.7.1. Estimated Daily Intake

The estimated daily intake (EDI) of AFM1 was calculated to determine the amount of AFM1 ingested per unit of body weight each day, using the following equation:
EDI = C × IR/BW
where C = mean concentration of AFM1 in milk (µg/L); IR = daily milk intake (L/day); and BW = average body weight of the exposed individual (kg).
Age groups were defined according to national food risk assessment guidelines and WHO population classifications: 1–2 years, 3–4 years, 5–9 years, 10–19 years, 20–59 years, and >60 years. Body weight (BW) and intake rate (IR) for each group were derived from the Encuesta Nacional de Salud y Nutrición (ENSANUT) (2022), Norma Oficial Mexicana-031-SSA2-1999, and World Health Organization growth standards [42,43,44]. Population-weighted averages were calculated from national anthropometric data, considering sex-specific differences (Table 1). Milk intake rates were based on national averages reported for liquid milk consumption by age group, with a value of 0.5 L/day for young children, adjusted proportionally for older groups as reported in ENSANUT, 2022 [42]. These values were utilized to stratify exposure levels by age and sex, allowing for a more accurate assessment of the dietary risk associated with the intake of AFM1 through CM. The concentration of AFM1 was determined through chromatographic analysis of commercial CM samples.

2.7.2. Monte Carlo Simulation for Estimating Carcinogenic Risk Index

To account for the variability and uncertainty associated with exposure to AFM1 through milk consumption, a Monte Carlo simulation (MCS) was performed to estimate the CRI across different age groups of the Mexican population. The simulation was based on the following equation, derived from the European Food Safety Authority (EFSA) (2020) and Kuiper-Goodman (1990) [10,38]:
CRI = EDI × EF × ED × CSF/AT
where EDI = estimated daily intake of AFM1 (µg/kg bw/day); EF = exposure frequency (days/year, assumed to be 365); ED represents exposure duration in years, defined by age group; CSF, or cancer slope factor for AFM1, is 0.04 (µg/kg bw/day)−1, as proposed by Kuiper-Goodman (1990) [38] and endorsed by the EFSA (2020) [10]; and AT = averaging time for carcinogens (25,550 days = 70 years × 365 days).
The AFM1 concentration was modeled using a triangular distribution based on experimental data: minimum: 0.02 µg/L; maximum: 7.62 µg/L; and mean: 1.26 µg/L. The triangular distribution of AFM1 was used for 10,000 iterations in each age group for simulation details. For each iteration, a random value of the AFM1 concentration was generated and used to calculate the EDI and CRI. The simulation produced descriptive statistics for CRI: mean CRI, 95 percentile (P95) CRI, and maximum CRI.

2.8. Cluster Analysis

A hierarchical clustering analysis was performed to investigate patterns among commercial CM samples based on four variables: AFM1 concentration (μg/L), retail price (MXN/L–Mexican peso per liter), type (regular or organic), and heat treatment (pasteurized or UHT). Before the clustering process, the dataset underwent standardization utilizing Z-scores to mitigate the effects of scale differences among the variables. The clustering procedure employed the minimum variance method of Ward, with Euclidean distance serving as the similarity metric. This multivariate technique facilitated the identification of natural groupings predicated upon similarities in pollution levels, pricing, and production characteristics. All analyses were conducted utilizing Minitab® 17 Statistical Software State College, PA, USA.

2.9. Statistical Analysis

All experimental procedures for the extraction, identification, and quantification of AFM1 were performed in triplicate. The data were expressed as means ± standard deviations. Variations in AFM1 concentrations among the CM were evaluated using one-way analysis of variance, followed by Tukey’s test to discern statistically significant differences between individual samples. A significance threshold of p < 0.05 was established as statistically significant. All statistical analyses were executed using SPSS software, version 25.0 (IBM® Corp, Chicago, IL, USA).

3. Results and Discussion

3.1. Sample Origin and Overview of AFM1 Contaminatión

The geographical sources of the 23 CM samples underscore the extensive distribution and integration of the dairy market across Mexico. The CM available in Hidalgo is sourced from nine distinct states: Aguascalientes (one sample), Durango (six), Guanajuato (one), Hidalgo (two), Jalisco (one), Mexico City (five), Nuevo León (two), Puebla (one), and the State of Mexico (four) (Table S1), situated in the central and northern regions, thereby indicating substantial interregional supply chain interconnectivity. The average concentration of AFM1 detected for each CM sample of different state allowed a more precise visualization and identification of regional differences in contamination levels of this mycotoxin (Figure 1). This scenario carries significant implications for food safety management, as contamination issues, particularly concerning AFM1, cannot be solely attributed to the state where the product is sold; instead, they may also reflect production and storage practices in other regions of origin. Regulatory monitoring programs should therefore concentrate on local retail establishments and encompass the entirety of the dairy supply chain, from primary production on the farm to post-harvest handling and industrial processing, particularly in producing regions including Durango and the State of Mexico. In Section 3.3 details the identification and quantification of AFM1 in the collected CM samples.

3.2. Reliability of the Method

The LLE analytical method, which has been adapted from previously published procedures, offers a dependable alternative for extracting AFM1 from milk, achieving a recovery rate exceeding 80% [39,40]. This method is particularly advantageous when access to immunoaffinity columns is restricted or when high CM sample yields are required. The analytical method underwent evaluation to ascertain its reliability and suitability for detecting trace levels in dairy matrices. The calibration curve illustrated linearity within the concentration range of 0.01 to 0.5 μg/L, with an R2 value of 0.9982, and a retention time of 9.16 ± 0.06 min (Figures S1 and S2). Triple analyses of various CM samples, which were processed under uniform extraction conditions, substantiated the method’s selectivity. No interference peaks were detected during the retention time of AFM1. The calculated LOD value of 0.002 μg/L and the LOQ value of 0.008 μg/L were adequate for identifying levels beneath the regulatory maximum limits established by European Union and Mexican standards [28,45]. Furthermore, the method’s selectivity was confirmed through chromatographic analysis of blank and fortified CM samples that were processed using the identical LLE protocol.
Figure S3 shows representative chromatograms: a CM at a known concentration of AFM1 labeled as blank (a), a CM sample enriched with 1.0 μg/L of AFM1 (b), and a commercial CM with the highest concentration of AFM1 identified in this study (c). All chromatograms were obtained under identical HPLC-FLD conditions and exhibited consistent retention times with no interfering peaks observed, thereby affirming the method’s specificity and matrix applicability. The recovery of the enriched CM was 81.6%, which falls within the acceptable range for mycotoxin analysis in complex food matrices, typically between 70% and 120%, as stipulated by international criteria [46]. The performance parameters obtained in this study are comparable to those cited in previous work utilizing solvent-based extraction methods and immunoaffinity column cleaning procedures, thereby confirming the validity of the analytical approach despite employing a more accessible LLE protocol [19,39]. Although the immunoaffinity column is commonly regarded as the reference standard due to its high specificity, it frequently incurs higher costs and single-use limitations [47]. Similarly, solid phase extraction provides good reproducibility and the potential for automation but necessitates meticulous method development to minimize matrix effects [48,49]. QuEChERS-based protocols have also been employed to detect multiple mycotoxins; however, they may lack the selectivity necessary to detect AFM1 at trace levels [50,51]. In this context, the results indicate that the combination of liquid–liquid extraction represents a valid and practical alternative for determining a single analyte, particularly in resource-limited environments. Moreover, the absence of interference peaks corroborated the method’s selectivity, and the intraday reproducibility (relative standard deviation < 5%) underpinned the accuracy in quantifying AFM1.

3.3. Identification and Quantification of Aflatoxin M1

All 23 commercial CM samples analyzed contained detectable AFM1 levels, with an average concentration of 1.64 µg/L. Notably, 73.91% of the CM exceeded the maximum permissible limits established by Mexican regulations (0.5 µg/L) and European regulations (0.05 µg/L) [28,45]. Sample B exhibited the highest concentration at 7.62 µg/L, significantly surpassing the concentrations found in the other CM samples. This particular CM originated from a nationally distributed milk brand processed in Durango, Mexico, a region characterized by a semi-arid climate, with low rainfall (less than 500 mm) and extreme temperatures [52]. In conjunction with suboptimal agricultural and storage practices, these climatic conditions may facilitate the proliferation of aflatoxin-producing fungi such as Aspergillus spp. in crops utilized as dairy cattle feed, including maize and sorghum [53,54]. Furthermore, the application of silage in dairy operations presents additional risks if storage conditions are not meticulously controlled, thereby heightening the likelihood of aflatoxin formation [55]. Inadequate storage infrastructure in regions supplying feed ingredients may also contribute to heightened AFM1 levels [56], as evidenced by the findings in sample B.
Mycotoxin contamination may occur at various stages of animal feed production, encompassing pre-harvest, drying, and storage processes [57]. Insufficient agricultural practices, inadequate management, and adverse environmental conditions contribute to the formation of mycotoxins, which can disseminate throughout fungal colonies, contaminating spores, hyphae, and the surrounding substrate [58,59]. Consuming these contaminated foods remains the primary route of exposure to mycotoxins in dairy cattle, particularly in semi-arid regions [59,60,61].
The remaining 26.8% of CM, which included A, I, J, L, M, and O, remained compliant with legal limits, demonstrating an average AFM1 concentration of 0.18 µg/L (Figure 2). The present study’s findings align with prior research conducted across various regions. Maggira et al. (2021) identified fluctuations in AFM1 levels in raw milk obtained from distinct locations within Greece, indicating that geographical factors and management practices may significantly affect contamination levels [18]. Similarly, Kolarič and Šimko (2023) substantiated the efficacy of analytical methods for detecting AFM1 in dairy products in Slovakia, emphasizing the necessity of reliable techniques for monitoring this mycotoxin [19]. Furthermore, the research conducted by Buzás et al. (2023) in Hungary highlighted the urgent need for continuous monitoring to assess exposure to AFM1 among the general population [20]. In Mexico, there are limited studies evaluating the incidence of AFM1 in milk intended for human consumption. The most recent report documented a concentration of 0.03 µg/L in CM, which remains well within national legal thresholds [22,28]. The presence of AFM1 in milk primarily reflects the contamination of dairy cattle feed with AFB1, which is commonly found in cereals, oilseeds, flours, and by-products utilized in feed formulation. Seasonal factors may also impact the concentration of AFM1 in milk [62,63]. Elevated levels have been reported during the autumn–winter period compared to the spring–summer period [64]. This trend correlates with a decreased availability of fresh forage and an increased reliance on dry stored feed, which is more susceptible to fungal contamination under moisture conditions and inadequate storage [64]. Given that the CM samples in this study were collected during the winter, the heightened AFM1 concentrations observed in 80% of the CM may be partially attributed to this seasonal vulnerability.

3.4. Carcinogenic Risk Assessment of Aflatoxin M1 Exposure

To enhance the assessment of carcinogenic risk associated with chronic exposure to AFM1 through CM, an MCS involving 10,000 iterations was conducted. The simulation utilized a triangular distribution characterized by the minimum (0.02 μg/L), mean (1.26 μg/L), and maximum (7.62 μg/L) concentrations of AFM1 observed in the CM. The findings indicated that all age demographics exhibited significant values for the CRI, with the highest mean CRI noted in children aged 1–2 years (7.92 × 103) and 3–4 years (5.07 × 103). The P95 values surpassed the internationally recognized threshold of 1 × 104 across all age groups, suggesting that a considerable segment of the population may encounter an elevated cancer risk throughout their lifetime due to chronic exposure to AFM1 via CM consumption. The comprehensive results, encompassing mean, P95, and maximum CRI values for each demographic group, are illustrated in Table 2.
Incorporating probabilistic modeling via MCS enhanced the authenticity of the risk analysis by considering inherent variability and uncertainty in both contaminant exposure and population attributes. This methodology is consistent with contemporary guidelines from the EFSA (2020) and other global organizations endorsing distribution-based risk assessment models for evaluating carcinogenic mycotoxins [10]. The finding that children under five presented CRI values far exceeding the reference threshold (1 × 104) is particularly concerning. Additionally, even adult and elderly groups showed elevated CRI levels, though to a lesser extent, indicating that this issue affects the population more broadly.
This analysis employed a CSF of 0.04 µg/kg bw/day, as proposed by Kuiper-Goodman (1990) and subsequently reaffirmed by the EFSA (2020) [10,38]. Given the structural and toxicological similarity of AFM1 to AFB1, this parameter was essential for estimating lifetime cancer risk [3]. The CRI values derived from this study are congruent with earlier research that has evaluated the cancer risks associated with AFM1 [65,66,67,68]. A probabilistic analysis in Hungary estimated chronic exposure to AFM1 in toddlers using a two-dimensional MCS [69]. The results indicated exposure levels of up to 3.79 × 103 µg/kg bw/day at the 97.5 percentile, accompanied by a hazard index >1, which signifies a non-negligible health risk for this age demographic. Similarly, a study conducted in Monterrey, Mexico, identified AFM1 in 100% of breast CM, with average concentrations measuring 1.70 × 102 µg/L. The estimated daily intake for infants varied from 1.09 × 103 to 2.02 × 102 µg/kg bw/day, which exceeded tolerable intake levels and underscored a considerable carcinogenic risk, especially for children aged 0 to 24 months [70]. The aforementioned external findings substantiate the data acquired in this study, particularly concerning CRI values among pediatric populations. Moreover, these findings underscore the pervasive nature of exposure to AFM1 and its significant implications for public health. The continual exceedance of established risk thresholds across diverse populations and nations further accentuates the necessity for enhanced surveillance, effective risk communication, and robust preventive measures to safeguard vulnerable consumers, especially infants and young children [10,71,72].

3.5. Classification of Milk Samples Through Dendrogram Analysis of Contaminants and Product Attributes

The hierarchical clustering analysis elucidated distinct groups among CM samples based on similarities in AFM1 concentration, retail price, thermal processing, and organic labeling. As illustrated in Figure 3, the CM samples are systematically organized along the X-axis. At the same time, the vertical Y-axis denotes the Euclidean distance to the square, thereby reflecting the degree of similarity among them. The groups formed at shorter distances signify CM with more comparable profiles. The B sample exhibited the highest level of AFM1, and appeared to be isolated or to establish a separate branch, indicating a distinct contamination or production profile. This pattern may represent specific regional practices, supply chain conditions, or feed quality associated with that CM. To investigate contamination patterns in commercial CM, a hierarchical cluster analysis was performed using four variables: AFM1 concentration (μg/L), price per liter (MXN), milk classification (regular or organic), and thermal treatment (pasteurized or UHT).
The generated dendrogram (Figure 3) identified three primary clusters illustrating variations in AFM1 levels and product characteristics. The first cluster included milks with low AFM1 levels (<0.5 μg/L), featuring several UHT-treated and/or organic CM samples, such as U and V. The second cluster consisted of CM with moderate AFM1 values (0.5–2.0 μg/L). In contrast, the third group contained the most contaminated CM, including sample B (7.62 μg/L), an outlier due to its concentration significantly exceeding international safety limits. The groupings above indicate that heat treatment and classification significantly influence pollution profiles. Prior research has suggested that UHT treatment may yield lower levels of AFM1 [73,74].
Nevertheless, AFM1 is widely acknowledged for its heat resistance [72,75], implying that such variations are more likely to be related to CM sample processing rather than to the degradation of the toxin. This highlights the necessity for standardized analytical procedures when comparing CM subjected to diverse heat treatments. Furthermore, the analysis has demonstrated that elevated prices do not inherently correlate with superior microbiological quality concerning AFM1 contamination. This observation aligns with prior reports indicating that brand reputation and perceived value frequently influence pricing instead of objective quality assessment [76,77]. Interestingly, organic CM tended to group within the low-risk category; nevertheless, regular CM in the same group indicate that organic labeling alone fails to serve as a sufficient predictor of reduced contamination [78,79,80]. The levels of AFM1 are predominantly influenced by factors such as feed quality, storage conditions, and climate [81,82]. However, the observed trend aligns with the notion that organic dairy systems may implement more stringent feed sourcing practices. A more extensive sampling process is required to validate this association.

4. Conclusions

This study investigated the levels of AFM1 in various brands of CM available in Mexico and assessed the carcinogenic risk index across different age groups. AFM1 was detected in all CM samples analyzed, with 80% exceeding the maximum limits established by Mexican and European regulations, which raises significant public health concerns. Notably, the products under analysis are distributed nationwide and accessible to consumers across all socio-economic strata, thereby emphasizing a widespread risk of exposure throughout the country.
Cancer risk estimates derived from EDI and IRC have identified a significantly elevated susceptibility among children aged 1 to 4 during this crucial developmental phase. Monte Carlo simulations have corroborated these findings by encompassing various exposure scenarios and confirming that peak risk levels disproportionately impact younger demographics.
The findings underscore the urgent necessity to augment national surveillance, particularly regarding dairy products consumed by children. Regulatory authorities should prioritize the implementation of AFM1 limits, enhance on-farm feed management and storage protocols, and establish thorough risk-based surveillance strategies that extend beyond average exposure assessments. Future research should expand the geographic and seasonal scope of sampling and examine biomarker-based exposure evaluations, particularly within vulnerable populations.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app15116106/s1, Table S1. Characteristics of the collected samples; Figure S1. The calibration curve for AFM1 was obtained through HPLC-FLD within the range of 0.01 to 0.5 μg/L; Figure S2. HPLC-FLD chromatograms of the AFM1 standard solutions calibration curve at ascending concentrations. µg/L: micrograms per liter; min: minutes; mV: millivolt; Figure S3. Chromatograms demonstrating AFM1 in milk samples processed through liquid–liquid extraction for method selectivity, analyzed by HPLC-FLD: (a) initial concentration of AFM1 in the unfortified sample; (b) standard concentration enriched with 1.0 μg/L of AFM1; (c) sample B.

Author Contributions

Conceptualization, A.M.-A., G.M.R.-S. and S.R.T.-S.; methodology, A.S.J.-O. and C.J.-P.; software, A.M.-A. and C.J.-P.; validation, G.M.R.-S. and C.J.-P.; formal analysis, G.M.R.-S.; investigation, A.M.-A.; resources, A.C.-O.; data curation, A.C.-O.; writing—original draft preparation, A.M.-A.; writing—review and editing, S.R.T.-S.; visualization, A.S.J.-O., C.J.-P. and S.R.T.-S.; supervision, G.M.R.-S. and N.d.S.C.-C.; project administration, G.M.R.-S. and N.d.S.C.-C.; funding acquisition, G.M.R.-S. and N.d.S.C.-C. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors express their sincere gratitude to the Departamento de Biotecnologia of the Universidad Autónoma Metropolitana Unidad Iztapalapa for granting permission to utilize its facilities and equipment for the measurements conducted in this research.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AFM1Aflatoxin M1
AFB1Aflatoxin B1
CMCow’s milk
HPLC-FLDHigh-performance liquid chromatography with fluorescence detection
EDIEstimated daily intake
CRICarcinogenic risk index
MCSMonte Carlo simulations
UHTUltra-high temperature
IARCInternational Agency for Research on Cancer
ACNAcetonitrile
LLELiquid–liquid extraction
LODLimit of detection
LOQLimit of quantification
IRDaily milk intake
BWThe average body weight of the exposed individual
ENSANUTEncuesta Nacional de Salud y Nutrición
EFSAEuropean Food Safety Authority

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Applsci 15 06106 g001
Figure 1. Geographical distribution of AFM1 concentrations (μg/L) in commercial CM samples collected in Mexico. Data is presented as mean ± standard deviation.
Figure 1. Geographical distribution of AFM1 concentrations (μg/L) in commercial CM samples collected in Mexico. Data is presented as mean ± standard deviation.
Applsci 15 06106 g001
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Figure 2. Quantification and identification of AFM1 by liquid chromatography in commercial milk. AFM1 μg/L: micrograms per liter of aflatoxin M1; A–W: cow’s milk; # indicates a statistically higher value compared to samples analyzed; * indicates a statistically lowest value compared to the samples. The red horizontal line indicates the maximum limit set by the Mexican standard NOM-243-SSA1-2010 (0.5 μg/L).
Figure 2. Quantification and identification of AFM1 by liquid chromatography in commercial milk. AFM1 μg/L: micrograms per liter of aflatoxin M1; A–W: cow’s milk; # indicates a statistically higher value compared to samples analyzed; * indicates a statistically lowest value compared to the samples. The red horizontal line indicates the maximum limit set by the Mexican standard NOM-243-SSA1-2010 (0.5 μg/L).
Applsci 15 06106 g002
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Figure 3. Dendrogram of commercial milk samples based on hierarchical cluster analysis. The X-axis represents individual milk samples, while the Y-axis indicates group distance, reflecting the similarity between samples based on AFM1 concentration, price per liter, milk classification (regular or organic), and heat treatment method (pasteurized or UHT).
Figure 3. Dendrogram of commercial milk samples based on hierarchical cluster analysis. The X-axis represents individual milk samples, while the Y-axis indicates group distance, reflecting the similarity between samples based on AFM1 concentration, price per liter, milk classification (regular or organic), and heat treatment method (pasteurized or UHT).
Applsci 15 06106 g003
Table 1. Characteristics of age groups and estimated daily intake of aflatoxin M1.
Table 1. Characteristics of age groups and estimated daily intake of aflatoxin M1.
Age GroupAFM1IRBWEDI
(Year)(µg/L)(L/day)(kg)
1–21.260.511.10.056
3–41.260.4514.50.039
5–91.260.422.50.022
10–191.260.35440.01
20–591.260.25680.005
>601.260.2650.004
AFM1: mean concentration of AFM1 in milk (μg/L); IR: daily milk intake (L/day); BW: body weight; EDI: estimated daily intake.
Table 2. Carcinogenic risk index.
Table 2. Carcinogenic risk index.
Age GroupMeanP95Max
(year)
1–27.92 × 1031.57 × 1023.17 × 102
3–45.07 × 1031.02 × 1022.06 × 102
5–93.56 × 1037.16 × 1031.45 × 102
10–192.12 × 1034.29 × 1038.68 × 10³
20–591.47 × 1032.97 × 1036.01 × 103
>601.72 × 1033.46 × 1036.93 × 103
CRI: carcinogenic risk index; P95: percentile 95; Max: maximum value.
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Monter-Arciniega, A.; Cruz-Cansino, N.d.S.; Castañeda-Ovando, A.; Jiménez-Osorio, A.S.; Tello-Solís, S.R.; Jiménez-Pérez, C.; Rodríguez-Serrano, G.M. Aflatoxin M1 Levels in Commercial Cows’ Milk in Mexico: Contamination and Carcinogenic Risk Assessment. Appl. Sci. 2025, 15, 6106. https://doi.org/10.3390/app15116106

AMA Style

Monter-Arciniega A, Cruz-Cansino NdS, Castañeda-Ovando A, Jiménez-Osorio AS, Tello-Solís SR, Jiménez-Pérez C, Rodríguez-Serrano GM. Aflatoxin M1 Levels in Commercial Cows’ Milk in Mexico: Contamination and Carcinogenic Risk Assessment. Applied Sciences. 2025; 15(11):6106. https://doi.org/10.3390/app15116106

Chicago/Turabian Style

Monter-Arciniega, Araceli, Nelly del Socorro Cruz-Cansino, Araceli Castañeda-Ovando, Angélica Saraí Jiménez-Osorio, Salvador Ramón Tello-Solís, Carlos Jiménez-Pérez, and Gabriela Mariana Rodríguez-Serrano. 2025. "Aflatoxin M1 Levels in Commercial Cows’ Milk in Mexico: Contamination and Carcinogenic Risk Assessment" Applied Sciences 15, no. 11: 6106. https://doi.org/10.3390/app15116106

APA Style

Monter-Arciniega, A., Cruz-Cansino, N. d. S., Castañeda-Ovando, A., Jiménez-Osorio, A. S., Tello-Solís, S. R., Jiménez-Pérez, C., & Rodríguez-Serrano, G. M. (2025). Aflatoxin M1 Levels in Commercial Cows’ Milk in Mexico: Contamination and Carcinogenic Risk Assessment. Applied Sciences, 15(11), 6106. https://doi.org/10.3390/app15116106

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