2.3.2. Limits of Detection (LOD) and Quantification (LOQ) Values

LOD and LOQ values associated with different sample preparation procedures are shown in Table 5 and raw data in Supplementary Materials (Dataset S3. LOD and LOQ data). The wet milling (slurry) procedure exhibited the strongest and most stable signals with the highest signal-to-noise ratio. These conditions yielded the lowest LOD (7.5 ppb) and the only value below the EU legal limit for AFB1 residues in animal feed of 10 ppb [23]. LOD and LOQ values associated with the slurry procedure were 7.5 and 16.0 ppb, respectively.

According to EU requirement, the maximum allowable AFB1 content in complete feed is 50 ppb (adult cattle, sheep and goats), 20 ppb (adult pigs and poultry) and 10 ppb (young animals) [23]. The LOD of our method is 7.5 ppb, well below the lowest regulatory limit. Other quantification methods can be incorporated with an obvious advantage of lowering LOD if so desired. Alternatively, levels below LOD can be estimated by extrapolation using a company program [68], substituting the values with the LOD divided by the square root of two [69] or replacing by half the LOD value if the below LOD results are less than 60% of the data [70,71], the latter being an approach used by several workers [72–75].

**Table 5.** Limits of detection (LOD) and quantification (LOQ) values associated with dry and wet (water slurring) sample homogenization procedures for chicken feed at different extraction conditions (dilution factors). The LOD and LOQ values were derived from signal and noise of baseline native aflatoxin content expressed as absorbance.


R is n = number of replicates; OD, optical density; B0, average OD of the 0 standard; Mean, average of B<sup>0</sup> OD values; StDev, standard deviation of OD values of blank feed material.
