Degradation Behavior Of Penicillin G In Citrus And Its Effect On Fruit Quality

Citrus is one of the most important fruit crops in China with the cultivate and production ranking frist in the world.However,the development of citrus industry is threatened seriously by quarantine diseases such as citrus Huanglongbing and ulcer disease which can be effectively prevented by foliar application or trunk injection of antibiotics.Antibiotic residue in animal-derived foods is generally of concern due to widely used in animal disease control or as feed additives to promote animal growth.Antibiotic residues in plant-derived foods also pose a threat to human health because antibiotic can remain on agricultural products and in the environment after being used to control plant diseases and insect pests.However,there is less attention to this issue and less research on it at present.This study mainly introduced an analytical method for rapid detection of penicillin G and its metabolites?penillic acid and penilloic acid?in citrus by dispersive solid-phase extraction and ultra performance liquid chromatography tandem mass spectrometry?DSPE/UPLC-MS/MS?,and the stability of the standard solution of the three analytes and the storage stability in citrus were studied.A field trail during in 2017-2018 was carried out in Beibei,Chongqing to study the degradation dynamics and final residue of penicillin G in citrus?Miyagawa Satsuma Mandarin and Newhall Navel Orange?.Besides,the effects of different concentrations of penicillin G on the nutritional quality of citrus fruits were studied.The main results are as follows:1.A rapid determination method of penicillin G and its metabolites?penillic acid and penilloic acid?residues in citrus by DSPE/UPLC-MS/MS was developed.Extraction of these analytes in citrus sample was performed with 80%acetonitrile and purified with C₁₈.The analytes were separated by a Acquity UPLC^?HSS T3 column utilizing gradient elution with 0.1%formic acid solution and acetonitrile,and finally detected by tandem mass spectrometry in positive ESI mode?ESI⁺?.Identification and quantitation were achieved by UPLC-MS/MS with multi-reaction monitoring?MRM?and matrix-matched standard curve method.The results showed that three analytes could achieve effective and rapid separation within 5 min.Good linearity was obtained in the analytes concentration range of 5-2 000?g/L with correlation coefficients?r²?larger than 0.9981.The LODs were 0.5-1?g/kg and LOQs were 5-10?g/kg.The recoveries of penillic acid and penilloic acid in pulp,peel and fruit were 84.8%-103.5%and 83.6%-107.4%at 5-500?g/kg with RSD were 3.6%-9.6%and 4.3%-9.6%,respectively.The recoveries of penicillin G in pulp,peel and fruit were 76.7%-101.4%with RSD was 3.5%-9.5%at 10-500?g/kg.The method is rapid,simple,sensitive and accurate,and is suitable for the determination of penicillin G and its two metabolite residues in citrus.2.The storage stability of different concentrations?1,10 and 100 mg/L?of penicillin G and its metabolite standard solutions at-50,4 and 25?were studied,and the storage stability in citrus peel,pulp and whole fruit was studied.The results show that the stability of penicillin G and its metabolite standard solution is negatively correlated with temperature.Penilloic acid has the highest stability,while penicillin G and penillic acid are inferior.The concentration has a great influence on the stability of penicillin G and high concentration is less stable.It is recommended that the mixed standard solution of penicillin G and its metabolites be stored at 25,4 and-50°C for no more than 1,3 and 120 d,respectively.The storage stability in citrus results showed that penicillin and its metabolites could be stably present in citrus for 90-120 d at-18°C,It should be measured within 90 d after the field sample is taken to accurately detect penicillin G and its metabolites.3.A field trial of 2017-2018 was carried out in Beibei,Chongqing to study the degradation of penicillin G in citrus.Field degradation behavior test showed that penicillin G is easily degraded in citrus.The metabolite is mainly penilloic acid and can be stably existed for a long time.Penicillin G has a fast degradation rate in citrus with short half-life ranging from 1.7-2.7 d.The half-life on Satsuma Mandarin?1.7-2.3 d?is slightly smaller than that on Newhall navel orange?2.0-2.7 d?.When citrus was grown to half the size of mature fruit,6 g/L and 9 g/L penicillin G potassium were sprayed 2-3times with 7 d interval on Satsuma Mandarin and Newhall navel orange.After 7-21 d,the final residual amount of penicillin G?penicillin G+penillic acid+penilloic acid?was as follows:peel?0.162-13.376 mg/kg?>whole fruit?0.047-7.653 mg/kg?>pulp?0.015-0.701 mg/kg?,most of which remained in the peel.The final residual amount of penicillin G and its metabolites in citrus is penilloic acid>penicillin G>penillic acid,and penilloic acid is the main residue.The residual amount of penicillin G and its metabolites in pulp is much lower than that of the peel.Based on the final residue test data,the residual dietary chronic risk of penicillin G in Newhall navel orange was higher than that in Satsuma Mandarin,but lower?RQc<1.265%?.RQc was far less than 100%,which was within acceptable level.4.The effects of penicillin G on the quality of citrus fruits were studied by spraying 6 g/L and 9 g/L penicillin G potassium three times with 7 d interval on Miyagawa Satsuma Mandarin and Newhall navel orange.The results showed that penicillin G has different effects on different quality indicators of citrus fruits.Specifically,penicillin G significantly increased the total soluble solids?TSS?and reducing sugar content of citrus fruits,and significantly reducing vitamin C and total flavonoid content of Satsuma Mandarin,whlie there is no significant effect on total flavonoids content of Newhall navel orange and the titratable acid?TA?,The ratio of TSS to TA and the appearance quality of the fruit such as the fruit shape index,the edible rate and the juice yield.Based on the results of this study,the following conclusions can be drawn:1.A rapid determination method of penicillin G and its metabolites?penillic acid and penilloic acid?residues in citrus by DSPE/UPLC-MS/MS was developed.The method is rapid,simple,sensitive and accurate,and is suitable for the determination of penicillin G and its two metabolite residues in citrus.2.The stable periods of penicillin G and its metabolites were 1,3 and 120 d at 25,4 and-50?,respectively,and 90-120 d in citrus.3.Penicillin G is easily degraded and metabolized in citrus with a half-life of1.7-2.7 d.The main metabolite is penilloic acid,which can be stable for a long time.Penicillin G and its metabolites are mainly resided in the peel of citrus,and the chronic risk of dietary residues is low,which is within the acceptable level.4.Penicillin G has different effects on different quality indicators of citrus fruits.