## 3 Results and Discussion
### 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 \(\upmu\)g/L, with an R\({}^{2}\) value of 0.9982, and a retention time of 9.16 \(\pm\) 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 \(\upmu\)g/L and the LOQ value of 0.008 \(\upmu\)g/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 \(\upmu\)g/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  
Figure 1: Geographical distribution of AFM1 concentrations (\(\upmu\)g/L) in commercial CM samples collected in Mexico. Data is presented as mean \(\pm\) standard deviation.  
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.