The analyses of Aflatoxin was performed using HPLC based on the following conditions: Mobile solvent :(Acetonitrile: Methanol: Water (15: 25: 60)), Flow Rate:1.2 mL/min, Column: Stainless Steel,5µm Reversed Phase C-18, Column Oven Temperature:(39 oC), Detector: (Fluorescence detector with Excitation wavelength of 365 nm, emission wavelength of 440 nm, Integrator: derivatization is achieved by hydroxylation from water of the mobile phase under UV light at 265 nm wave length when the HPLC conditions are fulfilled, directly inject prepared standard aflatoxin solutions.
The calibration curve is constructed by plotting peak area of a series of calibration standards against the concentration of the standards as shown Table 3. The concentration of the unknown samples is derived from interpolation of the curve.
Table 3
The series standard solutions of aflatoxin and peak area responses
standards ppm | Peak area | Standard ppm | Peak area |
AFB1 | AFG1 | AFB2 | AFG2 |
0.8 | 183663 | 34619 | 0.2 | 149588 | 39968 |
2 | 313752 | 120859 | 0.5 | 197234 | 82237 |
4 | 561920 | 221159 | 1 | 380472 | 169735 |
8 | 1120292 | 390339 | 2 | 744677 | 300249 |
16 | 2053828 | 763272 | 4 | 1353906 | 565174 |
20 | 2399730 | 917051 | 5 | 1578128 | 670691 |
The calibration curves for aflatoxin (B1, B2, G1 and G2) as shown in Fig. 2. The analyzed working solution gives excellent values of regression coefficient for each aflatoxin. Goodness of fit (R2) values was ˃0.996. This value gives as evidence of an acceptable fit of the data to the regression line. Higher regression coefficient shows high correlation between concentration of aflatoxin standard and their responses (peak area).
The optimized analytical methods were validated in terms of LOD, LOQ, recoveries, and linearity for aflatoxin (AFG2, AFG1, AFB1 and AFB2) in peanut sample. Extraction, chromatographic and detection conditions were optimized in order to increase sample sensitivity.
Table 4
Parameters of linear regression measured for Aflatoxins in HPLC, LOD and LOQ
AFs | Linear equations | R2 | LODn | LOQn |
B1 | 8e− 06x − 0.8623 | 0.9967 | 0.06 | 2 |
B2 | 3e-06x − 0.255 | 0.9960 | 0.148 | 0.46 |
G1 | 2e-05x − 0.4916 | 0.9979 | 0.312 | 1.34 |
G2 | 8e-06x – 01837 | 0.9975 | 0.2 | 0.5 |
As shown in Table 4, Limit of detection of the aflatoxin were, 0.2 µg/kg for aflatoxin G2, 0.312 µg/kg for aflatoxin G1, 0.148 µg/kg for aflatoxin B2 and 0.06 µg/kg for aflatoxin B1 respectively, and LOQ for aflatoxin range from 0.46 to 2 µg/kg, respectively.
The average percentage recoveries of aflatoxin ranged from 81.83–108.16% (Table 5). A reasonably high recovery of the most important method used is efficient and can be employed successfully for the reliable analysis of aflatoxin in peanut sample.
Table 5
Recovery test of aflatoxins in peanut samples
Afs | AF levels added µg/kg | Result found µg/kg | Recovery % | RSD % |
B1 | 8.00 | 8.65 | 108.16 | 0.743 |
B2 | 2.00 | 1.93 | 96.5 | 2.547 |
G1 | 8.00 | 7.31 | 91.45 | 3.507 |
G2 | 2.00 | 1.63 | 81.83 | 2.905 |
The average percentage recoveries of the methods ranged from 108.16, 96.5, 91.45 and 81.83%, for aflatoxin (B1, B2, G1 and G2), respectively. The recoveries obtained from this study were within the range between 70 and 125%, which were acceptable according to (AOAC,2005) as cited in (Kortei et al., 2021). Percent relative standards deviation (%RSD) values were obtained from the analysis of each sample four times in the same day. The %RSD ranged from 0.743 to 3.507.These values indicate that good precision of the data of analytical method was used.
As shown in Fig. 3, HPLC chromatogram of the method blank, counts versus retention time showed that, clear chromatogram and doesn’t have any specified visible chromatographic peaks. Which means that free of any aflatoxin cross contamination from extraction solvents and equipment’s, so, this describes that the quality of the employed method. The chromatogram also demonstrated that the procedure was suitable for the determination of aflatoxins in the sample.
In Fig. 4, the mixture of aflatoxin standards have clear and excellent chromatographic peaks for aflatoxin G2, G1, B2 and B1 with almost the same retention time (min) at various standard aflatoxin concentrations 2, 5, 10, 20, 40 and 50 ppb with A, B, C, D, E, and F chromatograms, respectively.
The retention time for all different types of concentration of aflatoxin standards were slightly vary at 7.9, 9.7, 11.5 and greater than 12 min for aflatoxin G2, G1, B2 and B1, respectively.
The concentration of each Aflatoxin was obtained by using the peak-area integration of chromatogram of peanut sample. All the samples were determined and the results indicated those roasted peanut samples are contaminated by total aflatoxin concentrations ranged from 14.05 µg/kg to 445.18 µg/kg.
For each mean aflatoxin contamination levels in roasted peanut samples ranged from 2.6 ± 0.081–246.42 ± 0.082, 0.64 ± 0.012–23.41 ± 0.016, 9.02 ± 0.008–156.10 ± 0.041,1.49 ± 0.048–19.25 ± 0.04 µg/kg for AFB1, AFB2, AFG1, AFG2, respectively (Table 6).
Table 6
HPLC result of mean ± sd µg/kg aflatoxins in roasted peanut samples.
Aflatoxins | Arada | Azezo | Tseda |
AFB1 | 246.42± 0.082 | 2.60 ± 0.081 | 4.29 ± 0.007 |
AFB2 | 23.41 ± 0.016 | 0.66 ± 0.008 | 0.64 ± 0.012 |
AFG1 | 156.10 ± 0.041 | 9.02 ± 0.008 | 10.17 ± 0.016 |
AFG2 | 19.25 ± 0.04 | 1.77 ± 0.012 | 1.49 ± 0.048 |
Total Mean aflatoxins | 445.18 ± 0.012 | 14.05 ± 0.028 | 16.59 ± 0.008 |
The result of roasted peanut sample of Arada AFB1 is the highest 246.42µg/kg) and AFG2 is the lowest 19.25µg/kg, in Azezo sampling sites, AFG1 is the highest 9.02µg/kg and AFB2 is the lowest 2.60µg/kg and for Tseda AFG1 is the highest 10.17µg/kg while AFB2 is the lowest 4.29µg/kg. In all aflatoxin types of Arada roasted peanut determined sample have highest than Azezo and Tseda as shown in Table 6. The amount of AFB1 is the highest (246.42µg/kg) in all sample site and other aflatoxins, while that of the AFG2 is the lower (19.25µg/kg). According to European Union, the maximum level of AFB1 and aflatoxin total was 2 and 4 µg/kg, respectively as cited by (Kumar et al., 2021). All the analyzed samples exceeded tolerance limit of European Union. moreover, US Food and Drug Administration (FDA) and World Health Organization (WHO) tolerance limit of aflatoxin total was 20 µg/kg (Joint, Organization, and Additives, 2017). In this case, Azezo (14.05 µg/kg) and Tseda (16.59 µg/kg) roasted peanut sample results are acceptable, whereas Arada (445.18 µg/kg) roasted peanut samples are extremely exceeded recommended maximum permissible and highly contaminated by aflatoxin. Therefore, Arada sampling area needs great attention.
The chromatogram Fig. 6, shows aflatoxin were detected in all peanut samples and counts versus retention time of peanut samples as shows that there were large chromatographic peak areas observed in Arada site than Azezo and Tseda. The acquisition times of all Aflatoxins in peanut sample were approximately between (6.7 to 10.6 min).
Comparison of AFs results of the current study with other studies
When comparing this research finding with other similar studies conducted in different countries on peanut sample, almost all peanut samples were contaminated with aflatoxins. Studies from other parts of world have shown high levels of aflatoxin contamination in peanut samples. Greater quantities of aflatoxin have been reported for groundnut samples from Kenya (250 µg/kg), Nigeria (216.1 µg/Kg), and Brazil (103.8 µg/kg) ( Nii et al., 2021) and Oliveira et al., 2009).
Many countries now regulate that aflatoxin of human health concern, by setting maximum tolerable levels for these toxins in food. In Ethiopia, the current maximum residue levels for aflatoxins set by Ethiopian Standard Agency (ESA) is 10µg/kg (Mamo et al., 2020). Peanut are crops for the bulk of the population in Ethiopia. According to Ethiopian Standard Agency, the determined total aflatoxins in roasted peanut samples values in this study ranged from 14.05-445.18 µg/kg which were above the permissible limits. The results obtained in this study were higher than the values reported in Amare et al 5-250 µg/kg and lower than Alemayehu et al 15 − 11,865 µg/kg and Mamo et al., 2020 which is 4 µg/kg.
The study done by Fuffa and Urga pointed to a surge in aflatoxin levels in cereals obtained in the Southern Peoples Nations and Nationalities, Oromia and Harari Regional States were total of 595 cereal food samples collected and determined also screened for aflatoxin contamination. The highest levels of aflatoxin B1and G1 were observed in peanut samples 738µg/kg and 201µg/kg respectively (Fuffa and Urga, 2001).
Aflatoxin B1 (AFB1) which is known the determine concentration is acceptable in Azezo site (2.60 µg/kg) and comparable with Tseda site (4.29 µg/kg), but Arada site (246.42 µg/kg) was extremely high as compared to the Ethiopian Standard Agency (4 µg/kg) as reported by Mamo et al., 2020). In South Korea, the levels of peanuts aflatoxin B1, was detected in the range of 0.2–28.0 µg/kg (Chun et al., 2007) which was comparable with Azezo and Tseda. However, the aflatoxinB1 concentration in Arada sampling site was exceeding.
This variation of total aflatoxin contamination observed in this study may be by the use of low improper processing that allows cross contamination, storage and preservation practices. A research done by Mutegi et al., (2013), storage and packaging materials for roasted peanuts contribute to the higher level of aflatoxins due to poor ventilation resulting in the humidity increase that favors fungal growth. The other reason may be due to once the crop becomes infected under field conditions, the fungal growth continues with increasing at post-harvest and storage conditions (Wagacha et al., 2008). Omer et al, (2001), reported the correlation between humid local storage conditions and high levels of aflatoxins concentrations in peanut products.
Risk assessment of Total Aflatoxins via consumption of peanuts
Due to genotoxic carcinogenic potential, there was no established safe level for exposure of aflatoxins (EFSA, 2007). Therefore, the Estimated Daily Intakes (EDI) of the total aflatoxins in the peanut samples was 0.531, 0.299 and 0.229µg/Kgbw/day for children, adolescents, and adults respectively. For the Margin of Exposure (MOE), values of 753.3, 1337.8 and 1746.7 were recorded respectively (Table 7). The average potency of the aflatoxin was 0.00825 ng Aflatoxins/kgbw/day and produced a population risk of 4.4 × 10− 3, 2.5 × 10− 3 and 1.9 × 10− 3 respectively (Table 6) which was estimated by the European Food Safety Authority (EFSA) based on carcinogenicity data in rats exposed to aflatoxins.
In Table 7, MOE was used to consider possible safety concerns arising from the presence of roasted peanut in different in human ages. According to European Food Safety Authority (EFSA, 2007), when MOE is < 10,000 indicates occurrence of a larger human risk. Even in the consumer only scenario, the MOE of aflatoxins in this study was above this safe lower limit.
In MOE approach possible safety concerns that arise from the existence of genotoxic and carcinogenic substances in food are used by risk assessors at the EFSA. When the calculated MOE is ≤ 10,000, the EFSA estimates the existing carcinogenic risk as low and suggests that these substances be treated with low priority (Barlow et al., 2006).
Table 7
Evaluation of risk for total aflatoxin via consumption of peanut samples
Sample | Age | Ave.AF total(µg/kg) | Body weight(kg) | EDI(µg/kg.bw/day) | MoE | Ave.potency(ng/kg bw/day) | Cancer risk |
peanut | Childrens (5–11 years) | 158.60 | 26 | 0.531 | 753.3 | 0.00825 | 0.0044 |
Adolescents (12–18 years) | 158.60 | 46 | 0.299 | 1337.8 | 0.00825 | 0.0025 |
Adults (19–60 years) | 158.60 | 60 | 0.229 | 1746.7 | 0.00825 | 0.0019 |
Daily average consumption of the peanut is 0.087kg/day
Risk Management Advice
We suggest that Ethiopian’s government needs to continue efforts to reduce dietary exposure of aflatoxins. On the basis of the risk assessment results, the Ethiopian government should control dietary exposure to aflatoxins. Based on EU when the higher levels of total aflatoxins were changed from 15 to 4 ppb, aflatoxins exposure risk reduced and increase the MOE of aflatoxins beyond the safe lower limit. Hence, aflatoxins exposure risk will not create a health problem.
The most effective way of controlling aflatoxin is to monitor food and its raw ingredients. In addition, contamination reduced in susceptible commodities to an acceptable level by strengthening border control, carrying out strict periodic monitoring, and imposing fines on noncompliant products, and accumulation, while preventing significant health and economic losses. The results of this assessment may be useful to the Ethiopian Standard Agency (ESA) to improve supervision of product improvement by peanut product manufacturers, ensure border inspections are carried out, and realize effective imported food control. When production activities improved and appropriate storage conditions sustained, aflatoxins in peanut foodstuffs can be managed. The government organization give direction and controlling mechanism in food companies in testing aflatoxins. As a result, by creating the awareness/education of producers and consumers the health risk potential of aflatoxin may be reduced.