## 3 Results and Discussion
### Validation of the Method of Extraction and Analysis  
The HS-SPME method for safrole determination in cowpea beans was validated, and the results fulfilled the guidelines by SANTE/11813/2017 [49]. Untreated bean samples (blank) and samples treated with safrole were exposed to the extraction method and analysis to assess selectivity. Then, the resulting chromatograms were compared (Figure 4). No interference was noticeable in the retention time of the compound of interest, therefore supporting the selectivity of the technique.  
The LOD of the method was determined considering three times the signal-noise (\(S/N=3\)) baseline of the safrole-free cowpea-bean samples (blank) in a GC/FID analysis. In turn, the LOQ was calculated assuming a signal at least ten times higher than the noise (\(S/N=10\)). The resulting LOD and LOQ values (0.0057 \(\upmu\)g kg\({}^{-1}\) and 0.019 \(\upmu\)g kg\({}^{-1}\) of cowpea beans, respectively) were considerably lower than the maximum concentration allowed for safrole usage in the food industry. The LOD represents the lowest concentration of an analyte in a sample that can be detected, but not necessarily quantified, under the stated conditions of the test. The LOQ is the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the testing conditions [49; 53].  
The response linearity of the method was determined by assessing cowpea-bean samples fortified at seven different concentrations of safrole (1 \(\times\) LOQ, 5 \(\times\) LOQ, 10 \(\times\) LOQ, 50 \(\times\) LOQ, 100 \(\times\) LOQ, 150 \(\times\) LOQ, and 200 \(\times\) LOQ). After fortification, the samples (5.0 g) were submitted to the extraction and analysis method in triplicate. The resulting data were plotted in an analytical curve with seven points (\(n=3\)), relating the analyte areas to the corresponding concentrations. The analytical curve for safrole extraction from cowpea beans was significant for the linear model (\(y=\text{ax}+\text{b}\)) (\(P<0.0001\)). The linearity of the method ranged from 0.019 to 3.80 \(\upmu\)g kg\({}^{-1}\) of cowpea beans, with a coefficient of determination above 0.99 (\(R^{2}=0.9990\)). The analytical curve indicates the relationship  
Figure 4: In red, chromatogram obtained from the analysis of beans fortified with safrole (t\({}_{\text{R}}=9.0\) min) and black, chromatogram of white matrix + methanol after extraction and analysis by HS-SPME-GC/FID, (\(n=3\)).  
between the theoretical analyte concentration in the sample and the chromatographic area [54].  
The precision of the method was investigated via repeatability assays. Samples of cowpea beans were fortified at three levels of concentration of safrole (1 \(\times\) LOQ, 5 \(\times\) LOQ, and 10 \(\times\) LOQ) in six replicates. The repeatability was determined by the coefficient of variation (CV) of the concentrations obtained experimentally by HS-SPME-GC/FID. These procedures were carried out on the same day, with the same equipment, and by the same analyst.  
Precision and accuracy were appraised at the levels of 0.019, 0.095, and 0.190 \(\mu\) kg\({}^{-1}\) of cowpea beans (Table 3). The average recovery ranged from 99.26 to 104.85% at these three concentrations, thus within the acceptable standards for chromatographic methods (70-120%) [49]. The HS-SPME-GC/FID method showed recovery rates close to 100% and good precision, with a CV equal to or less than 14.82%. In residue analysis, coefficients of variation of up to 20% are accepted [49].  
HS-SPME-GC/FID has advantages over other methods because it reduces or eliminates the need for organic solvents. Besides, it makes it possible to extract and concentrate the analyte simultaneously, before not requiring additional procedures for sample cleanup [42; 43; 51].