2.3.4. Robustness and Ruggedness of the Aflatoxin Extraction Procedure

For robustness data, Groups II and V (slurry extraction method at final dilution factor of 1247) had HorRat values of less than 2 (Table 3), indicating stable extraction efficiency precision of the improved aflatoxin test procedure under intra-laboratory repeatability conditions suggesting more satisfactory robustness compared to the other extraction conditions (see raw data at Dataset S1. Aflatoxin recovery data of supplementary files). For ruggedness results, HorRat value for Group X (final dilution factor = 2500) was greater than 1 but less than 2, while, for Group XI (final dilution factor = 1247), it was less than 1 (Table 4), indicating more stable precision under reproducibility conditions for Group XI (see raw data at Dataset S2. Precision data of supplementary files). This suggests that the improved aflatoxin test procedure has more satisfactory ruggedness (Group XI) compared to the other extraction conditions. Efficiency of extraction solvent stored at ambient temperature were 79%, 80% and 74%, respectively (see raw data at Dataset S4. Robustnessruggedness data of supplementary files), for freshly prepared, one- and three-month-old 80% acetonitrile:water (80:20, *v*/*v*). The HorRat value associated with these data was 0.22, suggesting minimal variability indicating stability of the solvent as an extraction solvent for AFB1 at ambient temperature for three months and satisfactory method ruggedness.


**Table 6.** Linearity of aflatoxin B1 standard solutions in modified acetonitrile extract solvent: phosphate buffered saline tween 20 mixture (100:650, *v*/*v*). The values are mean of duplicate analysis.

These data, taken together, suggest that the wet milling procedure (slurry method associated with final dilution factor of 1247) had satisfactory robustness as demonstrated by suitable repeatability of extraction efficiency, while stability of extraction solvent in storage at ambient temperature for three months and good within-laboratory reproducibility indicated its acceptable ruggedness [52,54,76]. Good robustness and ruggedness indicate satisfactory stability of our improved aflatoxin test procedure.
