**1. Introduction**

Aflatoxins are mycotoxins found in four main chemical structures: aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2); they can occur in a wide range of crops, including the major staple cereals

(e.g., maize), edible nuts and legumes and their products. The main fungal producers of aflatoxins are *Aspergillus flavus* which produces mainly AFB1, AFB2 and *Aspergillus parasiticus*, which produces all four forms. Contamination can occur before or after harvest or both. In general, AFB1 occurs at the highest levels compared to the others, and is the most toxic and a potent carcinogen [1,2]. AFB1 is converted into its hydroxylated metabolite (AFM1) by the liver enzymes of lactating animals [3]. This toxin, like the parent compound, has been categorized by the International Agency for Research on Cancer (IARC) as a group 1 toxin, a human carcinogen [2]. Due to their carcinogenity, the aflatoxins uptake through contaminated food consumption should be as low as possible, therefore the aflatoxin legislation is intended to implement the ALARA principle (As Low As Reasonably Achievable) and no threshold limit concerning the tolerable daily intake in humans has been established [4].

Evidences of aflatoxins carry over in milk, edible animal tissues and eggs have been reported, however, among foods of animal origin, milk represents the main source of human exposure to AFM1, which is the only mycotoxin which has regulatory limits in milk [4–6]. There is evidence of AFM1 occurrence in cow milk, but also in milk produced by other ruminants, such as bu ffalo, goat, sheep and camel [5]. The occurrence of AFM1 has been reported in various locations worldwide. Overall, the incidence of AFM1 in milk samples and milk products is relatively low in European countries, whereas data from Asian countries like China, Thailand and Taiwan show AFM1 occurrence in up to 100% of samples [7]. AFM1 is heat stable and processing or storage conditions are ine ffective in reducing its concentration in milk and milk products [8–10]. Several factors may a ffect the AFM1 contamination of milk, such as environmental conditions, di fferent farming and feeding practices, as well as the quality and safety control systems put in place by food/feed business operators (FBO) [9,11]. The presence of AFM1 in milk can be therefore considered as an indicator of maize chain vulnerability to fungal contamination [12].

Nowadays, there is an increasing concern for the impact of climate changes (temperature, humidity, rainfall and carbon dioxide production) on fungal behavior and consequently on aflatoxins production [11]. The application of predictive models has already given an indication of the potential increasing contamination by aflatoxins in Europe as consequence of climate changes [13]. Furthermore, a recent study using a full chain modeling approach to predict the impacts of climate change on AFB1 production in maize and its consequences on AFM1 contamination in dairy cow's milk, showed that, in the investigated scenario (i.e., Ukrainian maize), AFM1 contamination in milk is expected to be comparable or to increase in future climate scenarios [14]. Therefore, according to EFSA definition, the presence of AFM1 in milk may be considered as an "emerging risk", being a known risk for which an increasing and unpredictable pattern of exposure risk is foreseen [15].

Approximately 60 countries have already established regulatory limits for AFM1 in milk and dairy products [16]. In the EU, the maximum permitted levels for AFM1 have been set for consumable milk (50 ng/kg) [17]. In addition, an alert threshold level of 40 ng/kg calling for action is considered in some EU member states [18]. A maximum permitted level of 500 ng/kg of AFM1 in milk has been established by the US-FDA (United States Food and Drug Administration) [19] and by the Codex Alimentarius [20]. This is also the harmonized MERCOSUR limit applied in Latin America [16,21] and in several Asian countries [16].

With the publication of the General Food Law (GFL) [22] the European Union has made a new legal framework laying down the principles, obligations and definitions that apply in the field of food safety. A general principle of the GFL is that FBOs have the primary responsibility for food and feed safety. To this purpose, FBOs must implement a food safety managemen<sup>t</sup> system, based on the hazard analysis and critical control point (HACCP) principles. Regulatory limits therefore have a strong impact on contracts and procedural guidelines in the dairy industry and, as consequence, on the number of controls needed to verify milk compliance with maximum permitted levels, which may affect production costs.

A wide range of methods for the detection of AFM1 in milk and dairy products is currently available, however, achieving key analytical performances, such as sensitivity, precision and reliability, suitable to enforce regulatory limits in the low ng/kg range, is still quite challenging [23]. Screening tests can play an important role within the safety monitoring, allowing rapid decision making and interventions, also a ffecting the final price of food products. Nowadays, screening tests based on immunochromatographic assays such as dipstick or lateral flow devices, and enzyme linked immunosorbent assays (ELISA) represent the most common formats in the market [24–26]. To support FBOs in selecting the most appropriate test in relation to the intended scope, internationally recognized guidelines for screening test performance verification have been made available for instance by the AOAC Research Institute (Performance Tested MethodsSM) and USDA-GIPSA (Performance Verified Rapid Test), whereas at European level, such guidelines are set in the Commission Decision 657/2002/EC [27] and in the Commission Regulation 2014/519/UE [28], which is specifically devoted to mycotoxin screening methods.

The aim of this work was to evaluate and compare the analytical performances of two commercial immunoassays (strip test immunoassay and ELISA) widely applied for the detection of AFM1 in milk. For this purpose, the Commission Regulation 2014/519/UE [28] was taken into consideration as guidance document. Analytical performances, such as precision profile, cut-o ff value, false positive and false negative rates were evaluated for each assay by single laboratory validation, whereas a verification of the results from the validation study was performed based on long-term intra-laboratory quality control (QC) data. Correlation of the results obtained with the rapid immunoassays and the AOAC O fficial Method 2000.08 was evaluated for the analysis of naturally contaminated cow milk samples. Finally, the extension of the scope of the strip test method to goa<sup>t</sup> and sheep milk was evaluated by applying the experimental design foreseen in the EU regulation [28].

#### **2. Results and Discussion**

The experimental design to evaluate analytical performances of strip test immunoassay and ELISA comprised the following steps, which were carried out in parallel for the two assays: i) single laboratory validation study to evaluate precision, cut-o ff value, false suspect and false negative rates (milk samples fortified by AFM1 were used at this stage); ii) verification of cut-o ff and precision values by long-term intra-laboratory QC study (a QC cow milk sample spiked at 50 ng/kg was used at this stage); iii) evaluation of results correlation between rapid immunoassays and AOAC O fficial Method 2000.08 (a set of naturally contaminated cow milk samples was used for this purpose). Data obtained for each step are described and discussed in the following.

## *2.1. Validation Results*

Validation experiments were performed according to the experimental design described in Section 4.6. The screening target concentration (STC) value was 50 ng/kg. Other tested mass fractions values were: blank (AFM1 ≤ 0.5 ng/kg), 25 ng/kg (50% of the STC), 75 ng/kg (150% STC). The same sample set was analyzed by the ELISA and the strip test. Results obtained from the 24 measurements performed for each validation level were taken as basis for the calculation of validation parameters: precision, cut-o ff value, false positive and false negative rate. The overall results of the statistical assessment are shown in Table 1.


**Table 1.** Analytical performances of the strip test immunoassay and enzyme linked immunosorbent assay (ELISA), as resulted by validation experiments.

1 RSDr relative standard deviation of the repeatability; 2 RSDip relative standard deviation for intermediate precision.

First, precision data were calculated for all tested concentrations. Specifically, RSDip (intermediate precision) values of 32% (strip test) and 5% (ELISA) were obtained for samples contaminated at 25 ng/kg, values of 17% (strip test) and 10% (ELISA) at 50 ng/kg, 19% (strip test) and 15% (ELISA) at 75 ng/kg. Repeatability values (RSDr) were lower than 26% in all cases. Comparable values were obtained for the two tests at STC and above STC, whereas at 50% STC (25 ng/kg) lower intermediate precision values where obtained for ELISA. This could be partially explained by the fact that ELISA was working at a level five times higher than its limit of detection (LOD, 5 ng/kg, see Section 4.4), whereas the strip test was working at its LOD (25 ng/kg see Section 4.3).

With respect to the blank samples, a high relative standard deviation of the strip test response was observed. This could be mainly explained by the fact that for the strip test assay analytical signal values below a certain fixed limit, which is set by the manufacturer, are reported as "zero concentration", whether that is true or not. This led to a high number of "zero concentration" values in blank samples generating a high standard deviation. However, in the following, it will be shown that, notwithstanding this high value, an acceptable low rate of false suspect results for the blank samples was obtained anyway, due to the good separation of test responses for blank and contaminated samples.

Overall, the obtained precision values indicated an acceptable robustness of the two test methods, also taking into consideration the very low target levels of AFM1 considered for validation.

Once intermediate precision data were available, it was possible to calculate the cut-off values. According to European legislation [28], this value is defined as the response (AFM1 mass fraction) obtained with the screening method, "above which the sample is classified as suspect", with a false negative rate of 5%. The calculated cut-off values were 37.7 ng/kg for the strip test and 47.5 ng/kg for the ELISA test, respectively. In both cases, the assay sensitivity was considered satisfactory for assessing milk contamination at levels encompassing the EU maximum limits.

Based on the cut-off values, the rate of false suspect results was estimated for samples containing AFM1 below the STC. Specifically, for samples contaminated at 50% STC (25 ng/kg) the false suspect rate was 3% for the strip test and < 0.1% for the ELISA test. Finally, the false negative rate for samples contaminated at levels above the STC (75 ng/kg in the present case) resulted to be 0.4% for strip test and 0.1% for ELISA. In both cases, the acceptability criterion of maximum 5% false negative rate was met.

The overall results indicated satisfactory kits reliability in discriminating samples contaminated at different AFM1 levels set in a very narrow working range (from ≤ 0.5 to 75 ng/kg), encompassing EU regulatory limits.

In addition, the method fitness for purpose of evaluating milk contamination at the alert threshold of 40 ng/kg was evaluated by analyzing 20 contaminated samples from two different farms. The obtained average responses were 40.0 ng/kg for the strip test and 40.2 for the ELISA, with relative standard deviation (RSDip) of 9.8% and 5.8%, respectively. The resulting cut-off levels were 33.2 ng/kg and 36.1 ng/kg. No false suspect samples resulted for blanks with respect to these cut-off values. These data demonstrated the fitness for purpose of the two tested kits in evaluating compliance of milk samples with respect to the alert threshold of 40 ng/kg.

#### *2.2. Verification of Method Performances through Quality Control Data*

Verification of method performances was carried out through long-term intra-laboratory QC measurements over a period of 12 months (see Section 4.6). The results of the validation and the verification study were compared in terms of precision, recovery rates and cut-off values, as shown in Table 2. The cut-off values calculated by QC data matched very well with those obtained by validation data. Moreover, the data from the validation study as well as from the QC exercise revealed comparable values for the precision and the recovery rate, thus demonstrating sufficient ruggedness of both methods over the time and different production lots.


**Table 2.** Verification of strip test and ELISA method performances though quality control (QC) data.

1 RSDip relative standard deviation for intermediate precision.

#### *2.3. Analysis of Naturally Contaminated Samples*

The trueness of data generated by the two screening methods was evaluated by comparing them with results obtained by the reference AOAC Official Method 2000.08 on a set of raw cow milk samples naturally contaminated in the range n.d. (≤0.5 ng/kg) – 50 ng/kg AFM1.

Results are depicted in Figure 1. The two test kits performed in a similar way, and in both cases a satisfactory correlation was observed, with results provided by the reference method (*r* = 0.923 and slope = 0.84 for strip test vs HPLC and *r* = 0.924 and slope = 1.05 for ELISA vs HPLC). Irrespective of a slight overestimation of the AFM1 content in some of the blank samples (HPLC result ≤ 0.5 ng/kg), both immunoassays returned values lower than 14 ng/kg, confirming the absence of false suspect results.

**Figure 1.** Correlation between results (AFM1 mass fraction, ng/kg) obtained by strip test or ELISA and the HPLC analysis performed according to the AOAC Official Method 2000.08.

#### *2.4. Extension of Scope of the Method to Other Commodities*

Finally, the extension of the scope of the strip test method to goa<sup>t</sup> and sheep milk was evaluated by applying the experimental design foreseen in the EU regulation. The regulation foresees that "as long as the new commodity belongs to a commodity group ("milk" in the present case) for which an initial validation has already been performed, a minimum of 10 homogeneous negative control and 10 homogeneous positive control (at STC) samples shall be analyzed under intermediate precision conditions. The positive control samples shall all be above the cut-off value as calculated in validation experiments.

For these purposes, first specific calibration curves (bar codes) were generated for strip test analysis of raw goa<sup>t</sup> and raw sheep milk. Then 10 blank (negative) samples and 10 samples contaminated by AFM1 at 50 ng/kg were analyzed for each milk type. An additional sample set containing AFM1 at 25 ng/kg was also included. Results are reported in Figure 2. In both cases negative samples were correctly classified as below the cut-off. No false suspect was reported. In addition, samples contaminated at 50% STC (25 ng/kg) were all correctly classified as below the cut-off (Table 1) and no false suspect was reported. All samples contaminated at 50 ng/kg (STC) were correctly classified above the cut-off.

**Figure 2.** Results of strip test analysis of blank sheep and goa<sup>t</sup> milk samples and samples contaminated with 25 and 50 ng/kg AFM1.

The obtained data showed the applicability of the strip test immunoassay to goa<sup>t</sup> and sheep milk provided that a specific calibration curve was used.

#### *2.5. Fitness for Purpose of the Validated Immunoassays*

Validation experiments returned, for both immunoassays, fit for purpose analytical performances such as cut-o ff values (37.7 ng/kg and 47.5 ng/kg for strip test and ELISA respectively), false suspect rate for blanks (<0.1% for both assays) and false negative rate (<0.4% (for both assays). Both assays showed an intermediate precision at STC (50 ng/kg) <17% either in validation and QC measurements. However, besides analytical performances, when choosing a method for rapid mycotoxin screening, the concept of fitness for purpose also includes some practical parameters. Factors such as the time needed for analysis, the skills or level of education of the user of the method and the place where the analysis needs to be carried out are generally taken into consideration by the end users. A more comprehensive comparison of performances of mycotoxin screening tests can be found in Lattanzio et al. [29]. In the present case, the total analytical time for strip test assay was about 10 min and the use of the incubator, as well as the portable reader, made it suitable for on farm use. The ELISA involved more steps, a basic laboratory equipment and more time (approx 80 min). On the other hand, ELISA tests allow to handle up to 48 samples simultaneously (including calibrants and QC samples), while the strip test foresees only one sample per analysis/strip. ELISA can be therefore more e fficient when a large number of (sub)samples need to be analyzed in a short period of time. On the other hand, when applied in routine by experienced technicians, strip testing can be stacked to process multiple samples in a relatively short period of time, by processing 10 to 15 samples 1 min apart. Finally, concerning method transferability to unskilled personnel, the strip test appears easier to be applied by low experienced technicians, not only because the analytical protocol is less laborious, but also because the automatic calibration via QR code uploading. In principle both platforms are potentially suitable for multiplexing [30–32].
