## 3 Results and Discussion
### Comparison between the ELISA and UPLC/FLD Method  
The comparison among different approaches for a specific analytical determination is essential when a laboratory aims at implementing a new procedure. Point-to-point comparisons, mainly based on the available validation parameters and the application to commercial/real samples, are extremely useful for evaluating the pros/cons of each approach, as well as relating to the specific need, ability, equipment, and expertise of a laboratory [13; 31; 32]. However, to date, there is no available comparison regarding screening and confirmatory approaches in mycotoxin determination. Concerning the performances of analytical methods for the research of drug residues and environmental contaminants, European legislation (Decision No. 2002/657/EC) provides indications about both screening and confirmatory methods. Technically, confirmatory methods are generally more sophisticated and expensive with respect to screening. They can be used as a first analytical approach; however, where possible, it is extremely cheaper and faster to use screening methods. Indeed, screening methods allow for greater productivity when the analysis of a large amount of samples in a brief time is required. In this study, the determination of the T-2 and HT-2 toxins by using the ELISA method required less than 60 min for a single analysis, while UPLC/FLD required a longer time (about 240 min). Thus, it is possible to suggest that the scheme illustrated in Supplementary Figure S1 as an effective approach for a laboratory. After having arrived in a laboratory, the sample should be \"screened\" by using ELISA, and only in case of suspect positivity it should be analyzed by a confirmatory method. The screening validation, in fact, aims to eliminate \"false negative\" responses through the determination of specific CC\(\beta\), which is the smallest substance content that can be detected, identified, and/or quantified in a sample with the probability of a \(\beta\)-error.The \(\beta\) error, that should be \(<5\%\), is \"the probability that the sample is truly non-compliant even though a compliant measurement has been obtained\" [20].It should be noted that no false positive or false negative results were observed in this study. The \(\beta\)-error calculated during this study was \(<\)5%. It resulted in compliance with the European requirements, since the maximum percentage of \(\beta\)-error permitted by Decision No. 2002/657/EC is 5%. The samples were considered suspected positive if their concentrations were \(\geq\)MRL (maximum residue limit, as defined by a related regulation) subtracted from two times the repeatability SD of the method; in this case: 250 \(-\) 24.32 = 225.68 ug kg\({}^{-1}\). This approach was developed by considering the main focus of this study, which was the comparison between two analytical methods. Thus, the coefficient of 1.64, usually adopted for evaluating CC\(\alpha\) and CC\(\beta\), was rounded up to 2 in order to increase the number of samples useful for comparison. Regarding \"suspected positive\" samples, the UPLC/FLD approach, after screening analysis, was characterized by an absolute reliability thanks to its high selectivity, high accuracy, and low measurement uncertainty. From Table 4, it is possible to appraise a point-to-point comparison between two approaches developed in this study. As stated above, this type of comparison is considered very useful for laboratories involved in food control activities, since it shows the most significant pros and cons of each approach, thus simplifying the choice of a lab manager. Some characteristics, such as method selectivity and sensitivity, are obviously better for instrumental approaches, especially regarding fluorometric detection coupled to solid phase immunoaffinity extraction, which guarantees the absence of interfering signals and very low limits of quantification.