Comparison of Different Extraction Conditions Associated with Wet Milling Procedure

The 25 g aliquots were water-slurried and paired 1.3 g slurry analytical samples weighed out as described above (Section 4.1.4). To each of the 1.3 g slurry analytical sample, 130 mL acetonitrile water (80:20, *v/v*) was added for Group III (dilution factor = 100), 65 mL acetonitrile:water (80:20, *v/v*) for Group IV (dilution factor = 50) and 86.5 mL acetonitrile:water (80:20) was added Group V (dilution factor = 66.5), and the mixture was agitated as described in Section 4.1.4. The extracts were diluted 10-fold for Groups III (total dilution factor = 2500) and IV (final dilution factor = 1250) and 7.5-fold for Group V (final dilution factor = 1247) in PBST. The extracts were analyzed for AFB1 by ELISA (Section 4.1.5) and the results of paired test portions for each slurry sample averaged. Mean background level was calculated, subtracted from each of the AFB1 levels of "20 and 100 ppb" replicates and these results expressed as percent recovery. For each group (Groups I–V), mean percent recovery, StDev and RSD of the replicates were calculated. Outlier values in the percent recovery data were identified on Excel 2013 using the following formula:

$$1\text{st quartile-1.5 IQR} \le \text{x} \ge \text{3rd quartile} + 1.5\text{IQR} \tag{5}$$

where IQR is the interquartile range.

Outlier identification criterion also included recommended mean recovery range of 75–125% by European Commission and variance predicted by the modified Horwitz equation [50–53,63]. Where more than one out of five replicates were identified as outliers, the data were discarded and spiking and recovery procedure repeated [52,53].
