*2.4. Optimization of the FPIA*

The influence of methanol concentration and reaction time on the assay's performance was studied to evaluate the applicability of the method. OTA is typically extracted from cereals with methanol. The dyes that were applied in this experiment were methanol-sensitive substances; various physicochemical statuses can importantly affect the cross-linkage between the antigens and antibodies [19,20]. As the methanol concentration rose from 0% to 40%, the half maximal inhibitory concentration (IC50) of the experiment slightly decreased (Figure 5a). The reaction time was studied at a concentration of 40% methanol. The best level of sensitivity was attained at 0–10 min; the IC50 values increased with the increase of the assay time (Figure 5b). Therefore, 40% methanol and 20 min reaction time were regarded as the optimum assay conditions and were applied in the subsequent experiments.

**Figure 5.** Normalized correction curves of the optimum FPIA implemented with OTA reference solutions with different methanol content (**a**) and reaction time (**b**) (*n* = 3).

Based on the optimum conditions, we established the OTA calibration curve within the concentration range of 0.03–0.78 ng/mL. When the correlation coefficient was equivalent to 0.996, the limit of detection (LOD) and IC50 were 0.02 and 0.09 ng/mL, respectively (Figure 6). Due to the dilution ratio of rice being 20:1, the linear detection range of this method for rice in the actual detection was 0.60–15.60 ng/mL.

**Figure 6.** The standard curve of OTA.

#### *2.5. Evaluation of FPIA*

The recovery, intra-assay, and inter-assay tests were carried out to assess the performance of the FPIA method. The recovery tests in rice samples spiked with OTA standards of 0.5, 5, and 50 μg/kg concentration (Table 1) yielded an average recovery range of 70–110% with a relative standard deviation of equal to or less than 20%. The recovery and reproducibility values of this method proved that it fits the mycotoxin screening and testing methods' validation and performance criteria standard set by the EU for the control of mycotoxin in food products [4].


**Table 1.** Relative standard deviations and average recoveries of OTA from spiked rice samples obtained by FPIA.
