*3.3. Effect of Cooking Time on Residues of Amphenicols and Metabolites in Livestock and Poultry Meat*

The residue levels of amphenicols and metabolites in the meat blocks of livestock and poultry cooked by boiling for varying time periods are presented in Table 5. It can be seen from the table that the residue concentrations of CAP, TAP, FF and FFA in pork, beef, lamb and chicken gradually decreased with the prolonged boiling time. Within 25 min, the depletion rates of the four drugs were 38.55–75.75% in pork, 47.60–100% in beef, 20.18–100% in lamb and 39.31–50.19% in chicken. This is consistent with the results

reported by Shakila et al. [15] and Filazi et al. [10], which showed that boiling reduced CAP residues in shrimps and FF and FFA residues in eggs, and the loss was strongly correlated with heating time. In addition, Table 5 also shows that the elimination rates of the four drugs in different livestock and poultry meat matrices were different during the boiling process. CAP, TAP and FF were removed faster in beef and lamb and relatively slower in pork and chicken, while FFA was removed faster in beef, pork and chicken and was removed the slowest in lamb.

**Table 5.** Effect of boiling time on residues of amphenicols and metabolites in livestock and poultry meat.


Different lowercase letters in the same column indicate significant differences (*p* < 0.05) in the concentration of the same compound in the same species of livestock and poultry meat between different boiling times. **\*** indicates that the drug residue concentrations do not meet the requirements of non-detectable (CAP) or lower than MRLs (TAP, FF (sum of FF and FFA)), as stipulated by the Chinese standard GB 31650-2019 and (EU) No 37/2010 after boiling.

The effects of different deep-frying times on the residue levels of the four amphenicols and metabolites in livestock and poultry meat are shown in Table 6. The residue concentrations of CAP and TAP in pork, beef, lamb and chicken showed a decreasing or first increasing and then decreasing trend with frying time, while FF and FFA showed a decreasing trend in all four livestock and poultry meats. These findings indicate that the effect of heat treatment on amphenicols and metabolites is matrix-dependent. In addition, the reason for the elevated CAP and TAP residue concentrations in some livestock and poultry meat at the beginning of the deep-frying process may be related to the rapid water loss and evaporation from the meat at the initial stage with less drug loss [16]. Residue concentrations of CAP, TAP, FF and FFA in pork decreased by 6.70–41.29% within 5 min of frying, while those in beef, lamb and chicken decreased by 43.07–61.14%, 5.45–57.16% and 15.20–40.27%, respectively. One notable result worth emphasizing was that all four drugs were removed at the fastest rate in beef during deep-frying, as in the case of boiling. The reason may be that beef has a high water content, and heating results in the most

severe loss of quality (Figure 2) due to the disruption of its water-retaining protein spatial structure, the tightening of myogenic fibers and reduced water-binding capacity [17,18]. It is known that the decrease in the water binding capacity of meat increases drug degradation [19]. Therefore, it can be speculated that the highly reduced contents of amphenicols and metabolites in beef may be at least partially attributed to the significant decrease in the water-binding capacity of the meat caused by heating. In addition, the thermal treatment itself can affect the drug's chemical structure and its solubility in tissues [9].

**Table 6.** Effect of deep-frying time on residues of amphenicols and metabolites in livestock and poultry meat.


Different lowercase letters in the same column indicate significant differences (*p* < 0.05) in the concentration of the same compound in the same species of livestock and poultry meat between different deep-frying times. **\*** indicates that the drug residue concentrations do not meet the requirements of non-detectable (CAP) or lower than MRLs (TAP, FF (sum of FF and FFA)), as stipulated by the Chinese standard GB 31650-2019 and (EU) No 37/2010 after deep-frying.

It is evident from the above that boiling and deep-frying can effectively reduce the concentrations of four amphenicols and metabolites in livestock and poultry meat and that drug residue levels continue to decrease with the prolongation of cooking time. The loss of amphenicols and metabolites during cooking questions their stability when heated. Shakila et al. [15] have reported that CAP is an unstable drug destroyed during cooking and boiling. Tian [20] detected seven degradation products and metabolites of CAP in cooked mussels containing CAP, and structures were proposed for six. Similarly, Franje et al. [11] demonstrated that FF residues in chicken meat degraded to produce TAP in water at 100 ◦C by identifying the degradation structures of amphenicols after processing. The loss of amphenicols and metabolites observed in the present study after boiling and deepfrying suggests that they might have been destroyed or degraded to other substances. Moreover, it is also possible that the drug migrated from the livestock and poultry meat tissue into the surrounding liquid or meat juices during cooking, resulting in decreased residual concentration.

There was a difference in the effect of microwaving on the concentration of residues of amphenicols and metabolites in livestock and poultry meat over time compared with boiling and deep-frying (Table 7). From 0 to 1.25 min, CAP, TAP, FF and FFA showed an increasing or first decreasing and then increasing trend in pork, beef, lamb and chicken. At 1.25 min, the concentrations of all four drugs increased in the livestock and poultry meat matrices compared with the control groups. The increase rate was 8.43–43.84% for pork, 23.16–33.27% for beef, 6.50–80.29% for lamb and 26.66–135.92% for chicken. This result is inconsistent with the previous proposal by Nashwa et al. [21] that microwave heating was the most effective method for reducing drug residues in meat. In this experiment, microwaves did not cause a reduction in the levels of residues of amphenicols and metabolites. The reason for this may be that microwave processing caused a large amount of rapid water evaporation from livestock and poultry meat and a serious loss of quality (Figure 2c), which significantly reduced the water content of the meat while the drug abatement was at a low level. Overall, the livestock and poultry meat matrices are equivalent to being concentrated, and, therefore, the concentration of drug residues in the samples was elevated [22].

**Table 7.** Effect of microwave time on residues of amphenicols and metabolites in livestock and poultry meat.


Different lowercase letters in the same column indicate significant differences (*p* < 0.05) in the concentration of the same compound in the same species of livestock and poultry meat between different microwave times. **\*** indicates that the drug residue concentrations do not meet the requirements of non-detectable (CAP) or lower than MRLs (TAP, FF (sum of FF and FFA)), as stipulated by the Chinese standard GB 31650-2019 and (EU) No 37/2010 after microwaving.
