Title Page
Contents
ABSTRACT 10
1. Introduction 14
2. Materials and Methods 17
2.1. Animals 17
2.2. Test substance and Content 20
2.3. Administration of test substance 21
2.4. Sample collection and autopsy 22
2.5. Chemicals and reagents 23
2.6. Standard solutions preparation 24
2.7. Sample preparation 26
2.7.1. Muscle, Liver, and Kidney 26
2.7.2. Fat 27
2.8. LC-MS/MS instrumentation and conditions 30
2.9. Method validation 32
2.9.1. Specificity and Linearty 32
2.9.2. Accuracy and Precision 34
2.9.3. Recovery 34
2.9.4. Limits of Detection and quantification 34
2.9.5. Stabilities 35
3. Results and Discussion 36
3.1. Animals 36
3.2. Contents 36
3.3. Administration of test substance 38
3.4. Sample preparation 41
3.5. LC-MS/MS instrumentation and conditions 44
3.6. Validation and result of residue analysis 46
3.6.1. Specificity and Linearity 46
3.6.2. Accuracy and Precision 53
3.6.3. Recovery 58
3.6.4. Limits of Detection and quantification 61
3.6.5. Stabilities 64
3.6.6. The result of residue analysis 68
3.7. Setting of withdrawal period 76
4. Conclusions 80
References 81
Abstract (in Korean) 85
Table 1. Experimental design for residue study 19
Table 2. Working solution preparation for analysis of Piperazine 25
Table 3. Preparation of muscle, liver, kidney, and fat samples spiked with Piperazine for validation of residue analysis method 25
Table 4. LC-MS/MS condition for the analysis of Piperazine 31
Table 5. Preparation of muscle, liver, kidney, and fat samples spiked with Piperazine for validation of residue analysis method 33
Table 6. Contents of Piperazine product A, B 37
Table 7. Contents of piperazine product A in Feed and Contents of piperazine product B in Water 37
Table 8. Body weight and dose of Piperazine in the Fx and Wx treatment groups during acclimation 39
Table 9. Body weight of Fx and Lx treatment groups 39
Table 10. Intra-day accuracy and precision of DNS-Cl-Piperazine spiked into chicken muscle 54
Table 11. Inter-day accuracy and precision of DNS-Cl-Piperazine spiked into chicken muscle 54
Table 12. Intra-day accuracy and precision of DNS-Cl-Piperazine spiked into chicken liver 55
Table 13. Inter-day accuracy and precision of DNS-Cl-Piperazine spiked into chicken liver 55
Table 14. Intra-day accuracy and precision of DNS-Cl-Piperazine spiked into chicken kidney 56
Table 15. Inter-day accuracy and precision of DNS-Cl-Piperazine spiked into chicken kidney 56
Table 16. Intra-day accuracy and precision of DNS-Cl-Piperazine spiked into chicken fat 57
Table 17. Inter-day accuracy and precision of DNS-Cl-Piperazine spiked into chicken fat 57
Table 18. Recovery and precision of DNS-Cl-Piperazine spiked into chicken-muscle 59
Table 19. Recovery and precision of DNS-Cl-Piperazine spiked into chicken-liver 59
Table 20. Recovery and precision of DNS-Cl-Piperazine spiked into chicken-kidney 60
Table 21. Recovery and precision of DNS-Cl-Piperazine spiked into chicken-fat 60
Table 22. Freezer storage stability of DNS-Cl-Piperazine spiked into chicken tissues 65
Table 23. After-preparation storage stability of DNS-Cl-Piperazine spiked into chicken tissues 66
Table 24. Stock solution stability of Piperazine (10 ng/mL) 67
Table 25. Validation of analytical method for determination of DNS-Cl-Piperazine in Chickens 71
Table 26. Piperazine residue depletion in the tissues of broiler chickens after a single oral administration (Fx) 72
Table 27. Piperazine residue depletion in the tissues of broiler chickens after a single oral administration (2x) 73
Table 28. Piperazine residue depletion in the tissues of broiler chickens after a single oral administration (Control) 74
Table 29. Piperazine residue depletion in the tissues of broiler chickens after a single oral administration 75
Figure 1. Chemical structures of Piperazine (PPZ) 16
Figure 2. Sample preparation of Piperazine 29
Figure 3. Body weight changes of Fx and Wx treatment groups 40
Figure 4. Comparison of recovery rates for formic acid and acetonitrile ratios in chicken 43
Figure 5. The recovery of muscle, liver, kidney, and fat in chicken using the finally determined preparation method 43
Figure 6. Chromatogram of DNS-Cl-Piperazine in blank (top) and spiked-chicken muscle at 100 μg/kg (bottom) 47
Figure 7. Chromatogram of DNS-Cl-Piperazine in blank (top) and spiked-chicken liver at 100 μg/kg (bottom) 48
Figure 8. Chromatogram of DNS-Cl-Piperazine in blank (top) and spiked-chicken kidney at 100 μg/kg (bottom) 49
Figure 9. Chromatogram of DNS-Cl-Piperazine in blank (top) and spiked-chicken fat at 100 μg/kg (bottom) 50
Figure 10. Calibration curve for DNS-Cl-Piperazine in chicken muscle 51
Figure 11. Calibration curve for DNS-Cl-Piperazine in chicken liver 51
Figure 12. Calibration curve for DNS-Cl-Piperazine in chicken kidney 52
Figure 13. Calibration curve for DNS-Cl-Piperazine in chicken fat 52
Figure 14. Chromatogram of DNS-Cl-Piperazine spiked-chicken muscle, liver, kidney, and fat for LOD 62
Figure 15. Chromatogram of DNS-Cl-Piperazine spiked-chicken muscle, liver, kidney, and fat for LOQ 63
Figure 16. Plot of withdrawal time calculation for muscle, liver, kidney, and fat of broiler chickens treated with Piperazine (Fx) 78
Figure 17. Plot of withdrawal time calculation for muscle, liver, kidney, and fat of broiler chickens treated with Piperazine (Wx) 79