4.2.5. Determination of Limits of Detection (LOD) and Quantification (LOQ)

To counteract matrix effect of chicken feed, we used blank measurement approach to determine LOD and LOQ [81,82]. Briefly a blank specimen was quantified for AFB1 in five measurements and mean OD value and standard deviation obtained. From this, an OD value of mean OD minus 2 standard deviations and mean OD minus 5 standard deviations were calculated to get two OD values which were extrapolated from the standard curve to get LOD and LOQ respectively. Details are given below. Two acetonitrile extracts of the blank specimen prepared by conventional dry milling procedure were pooled, then diluted 100-fold in PBST to attain baseline level of analyte signal and used for LOD and LOQ determination at two assay conditions. Six 100 µL extract aliquots were diluted 100-fold in PBST (Group XII), while the other five 100 µL aliquots were first diluted 5-fold in acetonitrile:water (80:20, *v*/*v*) and then 10-fold in PBS-T (for Group XIII). All the aliquots were further diluted 10-fold in PBST to attain final dilution factor of 1000 for Group XII and 500 for Group XIII for dry milling procedure, before analysis by ELISA. For wet milling procedure, five 25 g fresh feed aliquots were separately water slurried and blended, two 1.3 g analytical portions from each water slurry, weighed and diluted 66.5-fold in acetonitrile:water (80:20, *v*/*v*), contents agitated and extract harvested (Section 4.1.4). The extract was further diluted 66.5-fold in PBS-T to attain baseline level of analyte signal, then diluted 7.5-fold in PBS-T and assayed by ELISA to give a final dilution factor of 1247 (Group XIV) for wet milling procedure. The limit values were calculated from mean absorbance of the 0-standard, B<sup>0</sup> [63] minus 2-fold (for LOD) and 5-fold (for LOQ) the StDev of absorbance [81] of replicate wells of the blank samples analyzed by ELISA. The means of the concentrations corresponding to the %B/B<sup>0</sup> value of B<sup>0</sup> minus 2-fold, and 5-fold the standard deviation generated from the calibration curve was taken as the lowest detectable (LOD) and quantifiable (LOQ) concentration of AFB1.
