Quantitative and confirmatory analysis of veterinary drug residues in food of animal origin by UPLC- MS/MS after QuEChERS clean-up Michelle Whelan 1,2 Martin Danaher 1, Ambrose Furey 2. Ashtown Food Research Centre 1, Cork Institute of Technology 2.
Topics Covered ProSafeBeef Food Safety Background Anti-parasitic Method Development Existing method New method Sample preparation Method Validation Milk, Liver and Muscle Method Performance Future Work
ProSafeBeef
ProSafeBeef Advancing Beef Safety and Quality through Research and Innovation: European Framework Programme 6 The ProSafeBeef consortium is multidisciplinary and comprised of 41 participants from research institutes, universities, private companies and industry organizations from 13 European countries, as well as Brazil, United States, Canada, Australia and New Zealand. ProSafeBeef is divided into 7 separate management/research areas Piller 1 - Quantitative Risk assessment for microbial and chemical contaminants
Aim of Chemical Contaminants section 1.4 of Pillar 1 Develop assays for anthelmintic residues Transfer method to project partners, QUB, AFBI, IAEA, and Microbioticos Inter-laboratory studies Survey of EU beef (n = samples) Exposure assessment Quantitative risk assessment of chemical residues in beef Dissemination
Europe Climates
Sampling Plan ProSafeBeef Important to identify the key anthelmintic drugs and their usage in different animal species Also need knowledge of the time of application Risk based approach is more likely to identify non-compliant results and will give greater consumer protection Sampling is biased and may portray a negative image of food
Food Safety
Food Safety Main concern over benzimidazoles is due to their teratogenic properties (males and females) Research has shown that OFZ causes birth defects if used in pregnant ewes Secondary metabolite is generally the most toxic Oxfendazole more toxic than fenbendazole Hydroxy-mebendazole more toxic than mebendazole
Background National Reference Laboratory for anthelmintic agents. Veterinary drug residues are tested for at AFRC as part of the National Monitoring Plan. Anthelmintics important in Ireland due to the extensive nature of farming In early 28: Four separate LC methods for anthelmintics in milk and liver (n=19 residues). No method for flukicide and levamisole Desired multi method for anthelmintics in milk, liver and muscle Developed a multi method mid 28 and demonstrated it at CRL in May 29
Anthelmintics Anthelmintics drugs are used for controlling the following worms: Nematodes (roundworms) Cestodes (tapeworms) Trematodes (Flukes) Three main classes of drugs used for the treatment are: Benzimidazoles Avermectins Flukicides Economic losses Weight loss Poor wool growth and quality Reduced milk production
Method development
List of Analytes Abamectin Mebendazole Levamisole Doramectin Hydroxy Mebendazole Coumaphos Emamectin Amino Mebendazole Coumaphos-Oxon Eprinomectin Flubendazole Rafoxanide Ivermectin Hydroxy Flubendazole Oxyclozanide Moxidectin Amino Flubendazole Niclozamide Cambendazole Fenbendazole Nitroxinil Albendazole Fenbendazole Sulphone Bithionol Albendazole Sulphone Oxfendazole Clorsulon Albendazole Sulphoxide Triclabendazole Closantel Albendazole amino sulphone 5 Hydroxy- Thiabendazole Thiabendazole Oxibendazole Morantel Haloxon Triclabendazole Sulphone Triclabendazole Sulphoxide
Existing LC-MS/MS method Quechers sample preparation was developed in USDA by Brian Kinsella LC-MS/MS method was developed using Agilent 1 and an Applied Biosystems API 3 using a Prodigy C18 column, (15 x 3, 5 µm) Two injections (pos and neg modes) 24 mins each. Two internal standards Only detects TCB and TCB-SO LOQ 5ppb Kinsella et al.
Current UPLC-MS/MS method Modified sample preparation developed in USDA to detect lower levels Scaled up Clean up step Concentration step using DMSO LC-MS/MS method was developed using Acquity UPLC and Waters MicroMass Quattro Premier XE using a HSS T3 column, ( x 2.1 mm; 1.8 µm) ONE injections (covers both pos and neg modes) 12.5 mins 21 internal standards, 15 Deuterated Detects all the TCB metabolites LOQ < 2ppb
UPLC Conditions Column: HSS T3 ( x 2.1 mm; 1.8 µm) Column Temp. : 6 C MPA :.1 CH 3 COOH in H 2 + ACN (9 + 1, v/v) MPB : 5 mm Ammon. Form. in MeOH + ACN (75 + 25, v/v) Flow rate:.6 ml min -1 Injection: 5 µl Run time: 12.5 min Time A B Curve. 1.5 6 5. 5 5 6 7. 1 9 6 8.5 1 9 6 8.51 6 9.5 6 9.51 6 12.5 6
Sample Preparation for non-mrl Substances Extraction 1 g sample + 12 ml MeCN Homogenise (only for liver and muscle) Add 4g MgSO 4 and 1g NaCl Shake for 1 minute and Centrifuge for 12 minutes Clean up and Concentration Pour supernatant into centrifuge tube containing 1.5g MgSO 4 and.5g C 18 Mix for 3 seconds and Centrifuge for 1 minutes Transfer 6 mls to a test tube containing 25µl of DMSO Evaporate MeCN under nitrogen (6 C) Analysis Filter the extracts and add to auto sampler vial. Inject
Sample Preparation for MRL Substances Extraction 1 g sample + 12 ml MeCN Homogenise (only for liver and muscle) Add 4g MgSO 4 and 1g NaCl Shake for 1 minute and Centrifuge for 1 minutes Clean up and Concentration Add 1ml of the supernatant into micro-centrifuge tube containing 15mg MgSO 4 and 5mg C 18 Mix for 3 seconds and Centrifuge for 2 minutes Analysis Filter the extracts and add to auto sampler vial. Inject
Method validation
Method Validation According to 22/657/EC guidelines. Specificity was carried out using 2 blank samples. Linearity: >.98 Recovery Experiments: - 1, 1.5, 2 times the chosen level, (or).5, 1, 1.5 times MRL Within laboratory repeatability with single analyst - 1, 1.5, 2 times the chosen level, (or).5, 1, 1.5 times MRL Within laboratory reproducibility with three analysts - 1, 1.5, 2 times the chosen level, (or).5, 1, 1.5 times MRL CCα and CCβ values were calculated at 1, 1.5, 2 times the chosen level, or.5, 1, 1.5 times MRL. S/N ratio is typically > Ion Ratios very consistent and reproducible through-out runs.
Recovery at validated levels for Muscle 16. 1 1.5 2 14. 12.. 8. 6. 4. 2..
Thiabendazole 5-hydroxy-thiabendazole 34. 32. 3. 28. 26. 24. 22. 2. 18. 16. 14. 12. 1. 8. 6. 4. 2.. Within Laboratory Reproducibility - Muscle - Benz.5 1 1.5 Albendazole-sulphone Albendazole-amino-sulphone Cambendazole Fenbendazole Oxfendazole Fenbendazole-sulphone Flubendazole Amino-flubendazole Hydroxy-flubendazole Mebendazole Amino-mebendazole Hydroxy-mebendazole Oxibendazole Triclabendazole-sulphoxide Triclabendazole-sulphone Albendazole-sulphoxide CV
Moxidectin 34. 32. 3. 28. 26. 24. 22. 2. 18. 16. 14. 12. 1. 8. 6. 4. 2.. Within Laboratory Reproducibility - Muscle - Fluke - Aver.5 1 1.5 Bithionol Clorsulon Closantel Morantel Niclosamide Nitroxynil Oxyclozanide Rafoxanide Coumaphos Coumaphos-oxon Haloxon Abamectin Doramectin Emamectin Eprinomectin Ivermectin Levamisole CV
Recovery at validated levels for Milk 18. 16. 14. 1 1.5 2 12.. 8. 6. 4. 2..
32. 3. 28. 26. Within Laboratory Reproducibility - Milk - Benz.5 1 1.5 24. 22. 2. 18. 16. CV 14. 12. 1. 8. 6. 4. 2..
3. 28. 26. Within Laboratory Reproducibility - Milk - Fluke and Aver 1 1.5 2 CV 24. 22. 2. 18. 16. 14. 12. 1. 8. 6. 4. 2..
Recovery at validated levels for Liver 16. 14. 1 1.5 2 12.. 8. 6. 4. 2..
CV 3. 28. 26. 24. 22. 2. 18. 16. 14. 12. 1. 8. 6. 4. 2.. Within Laboratory Reproducibility - Liver - Benz 1 1.5 2
CV 42. 4. 38. 36. 34. 32. 3. 28. 26. 24. 22. 2. 18. 16. 14. 12. 1. 8. 6. 4. 2.. Within Laboratory Reproducibility - Liver - Fluke - Aver 1 1.5 2
CCα and CCβ for Milk milk LOR/MRL CC CCβ µg/kg µg/kg Albendazole 17.4 116.61 Albendazole-sulphoxide 122.75 145.5 Albendazole-sulphone 14.77 115.75 Albendazole-amino-sulphone 14.52 119.96 Cambendazole 2.7 1.2 Fenbendazole 1 1.79 12.16 Oxfendazole 1 12.5 13.78 Fenbendazole-sulphone 1 1.84 11.81 Flubendazole 2 1.53 2.61 Amino-flubendazole 2.82 1.41 Hydroxy-flubendazole 2.8 1.36 Mebendazole 2.7 1.19 Amino-mebendazole 2.92 1.56 Hydroxy-mebendazole 2.69 1.17 Oxibendazole 2 1.95 3.32 Triclabendazole 2.71 1.2 Triclabendazole-sulphoxide 2.9 1.53 Triclabendazole-sulphone 2 1.33 2.26 Thiabendazole 2 1.65 2.81 5-hydroxy-thiabendazole 2 1.49 2.54
CCα and CCβ for Milk Milk LOR/MRL CC CCβ µg/kg Levamisole 2 1.26 2.14 Bithionol 4 1.59 2.71 Clorsulon 4.87 1.48 Closantel 2 1.7 1.82 Morantel 5 57.89 71.15 Niclosamide 2.74 1.27 Nitroxynil 2.66 1.12 Oxyclozanide 1 14.36 2.1 Rafoxanide 2.8 1.36 Coumaphos 2.82 1.4 Coumaphos-oxon 2.87 1.48 Haloxon 2.89 1.52 Abamectin 2.77 1.31 Doramectin 2 1.13 1.92 Emamectin 2.74 1.26 Eprinomectin 2 22.3 24.61 Ivermectin 2.74 1.25 Moxidectin 4 45.4 51.88
CCα and CCβ for Bovine Liver Liver LOR/MRL CC CCβ µg/kg µg/kg Albendazole 192.3 1173.85 Albendazole-sulphoxide 1385.8 3844.29 Albendazole-sulphone 1396.67 243.42 Albendazole-amino-sulphone 151.59 1169.45 Cambendazole 2.32.55 Fenbendazole 5 552.81 62.57 Oxfendazole 5 537.2 589.62 Fenbendazole-sulphone 5 588.83 674.82 Flubendazole 2.87 1.49 Amino-flubendazole 2.87 1.49 Hydroxy-flubendazole 2.25.43 Mebendazole 2.21.36 Amino-mebendazole 2.87 1.49 Hydroxy-mebendazole 2.26.44 Oxibendazole 2.85 1.45 Triclabendazole 25 287.99 335.75 Triclabendazole-sulphoxide 25 438.55 1116.29 Triclabendazole-sulphone 25 1225.1 1825.22 Thiabendazole 2.39.66 5-hydroxy-thiabendazole 2.67 1.13
CCα and CCβ for Bovine Liver Liver LOR/MRL CC CCβ µg/kg Levamisole 2.3.52 Bithionol 4.43.74 Clorsulon 4.51.87 Closantel 2.45.77 Morantel 5 57.89 71.15 Niclosamide 2.59 1. Nitroxynil 2.38.65 Oxyclozanide 1 14.36 2.1 Rafoxanide 2.43.73 Coumaphos 2.63 1.7 Coumaphos-oxon 2 1.79 3.4 Haloxon 2 3.31 5.64 Abamectin 2 1.6 2.73 Doramectin 2 1.51 2.57 Emamectin 2 1.7 1.83 Eprinomectin 2 22.3 24.61 Ivermectin 2 1.9 1.86 Moxidectin 4 45.4 51.88
CCα and CCβ for Muscle Muscle LOR/MRL CC CCβ µg/kg µg/kg Albendazole 5.26.44 Albendazole-sulphoxide 5.54.91 Albendazole-sulphone 5.41.7 Albendazole-amino-sulphone 5.25.42 Cambendazole 5.24.4 Fenbendazole 5.26.44 Oxfendazole 5.34.58 Fenbendazole-sulphone 5 1.12 1.89 Flubendazole 5.51.86 Amino-flubendazole 5.4.67 Hydroxy-flubendazole 5.29.48 Mebendazole 5.26.44 Amino-mebendazole 5.58.98 Hydroxy-mebendazole 5.35.59 Oxibendazole 5.24.41 Triclabendazole 1.7 1.17 Triclabendazole-sulphoxide 1 2.38 4.2 Triclabendazole-sulphone 1 3.27 5.51 Thiabendazole 5.28.48 5-hydroxy-thiabendazole 5.5.85
CCα and CCβ for Muscle Muscle LOR/MRL CC CCβ µg/kg Levamisole 5.15.25 Bithionol 25 5.1 8.9 Clorsulon 25 1.21 17.23 Closantel 25 8.75 14.77 Morantel 25 4.66 7.87 Niclosamide 5.65 1.1 Nitroxynil 25 7.1 11.84 Oxyclozanide 1 1.99 3.37 Rafoxanide 5 1.72 2.9 Coumaphos 5.61 1.4 Coumaphos-oxon 5.33.56 Haloxon 5.84 1.41 Abamectin 5 1.9 1.85 Doramectin 5 1.5 1.77 Emamectin 5.92 1.55 Eprinomectin 5.69 1.17 Ivermectin 5 1.92 3.22 Moxidectin 5 2.57 4.31
Year 1 Survey Results
Survey Results Year 1 - PSB EU Beef very safe 97.78 2.22 5 beef steaks 97.78 of samples residue free No risk to the consumer 1.22 -> Clos.2 -> Iver.2 -> Abz.2 -> Moxi.2 -> Eprino.2 -> Leva Iver 1 sample non-compliant contained ivermectin (no-mrl)
Method performance
LC Versus UPLC-MS/MS for Milk Time HPLC Analysis (36 samples) UPLC-MS/MS (36 samples) No. analytes 19 38 No. runs 4 1 Sample Prep. (days) 6 1 Run time (min) 68.1 (33 + 35.1) 12.5 No. injections 144 64 Analysis time (h) 81.8 13.3 Processing time (days) 3 1 Totals (days) 12.5 2.5
Application to incurred milk Sample No. Analyte Zu score 1 Blank - 2 Sum ABZ -.698 3 Sum FBZ -.927 4 Sum TCB +.73 5 Levamisloe -.19 6 Levamisle Sum TCB +.57 +.198
Application to incurred muscle Provisional Results Sample No. Analyte Zu score 1 Blank - 2 Sum ABZ Sum TCB 3 Sum ABZ Sum MBZ 4 Levamisole Sum FBZ Sum TCB 5 Levamisole Sum FBZ 6 Sum FBZ Sum MBZ -1.1 +.49 -.23 -.95 -.14 -.869 +.129 -.1 -.346 -.4 -.254
Overlay of 38 Analytes 2ppb
Overlay of Internal Standards
ABZ and metabolites 2 ppb Pos Ctl 2 5.54 4416 6: MRM of 19 Channels ES+ 266.7 > 191.3 (ABZ) 9.71e5 6: MRM of 19 Channels ES+ 5.54 266.7 > 234 (ABZ) 35989 8.9e5 5: MRM of 16 Channels ES+ 298.1 > 266.2 (ABZ-SO2) 1.33e6 3.61 23364 5: MRM of 16 Channels ES+ 3.61 298.1 > 159.8 (ABZ-SO2) 29136 4.83e5 5: MRM of 16 Channels ES+ 3.31 282.24 > 24.1 (ABZ-SO) 19838 2.94e5 5: MRM of 16 Channels ES+ 3.31 282.24 > 159.6 (ABZ-SO) 8797 1.36e5 Time
Closantel & Rafoxanide 2ppb Pos Ctl 2 6.83 2415 9: MRM of 22 Channels ES- 66.85 > 315.1 (Closantel) 7.24e4 9: MRM of 22 Channels ES- 6.83 66.84 > 126.9 (Closantel) 4438 1.38e5 9: MRM of 22 Channels ES- 7.9 623.79 > 344.83 (Rafox) 35 8.4e4 9: MRM of 22 Channels ES- 7.9 623.79 > 126.87 (Rafox) 3673 9.52e4 Time
Clorsulon 4 ppb, Nitroxynil 2 ppb Pos Ctl 2 3.33 664 4: MRM of 5 Channels ES- 379.8 > 343.95 (Clorsulon) 1.2e4 4: MRM of 5 Channels ES- 3.33 377.7 > 341.95 (Clorsulon) 128 1.6e4 4: MRM of 5 Channels ES- 3.22 288.9 > 161.95 (Nitrox) 6684 9.86e4 4: MRM of 5 Channels ES- 3.22 288.9 > 126.86 (Nitrox) 6885 9.95e4 Time
Avermectins 2ppb Pos Ctl 2 7.86 561 1: MRM of 13 Channels ES+ 916.6 > 593.35 (Dora) 1.84e4 1: MRM of 13 Channels ES+ 7.86 916.6 > 331.3 (Dora) 816 2.72e4 1: MRM of 13 Channels ES+ 7.57 915.15 > 298.15 (Eprino) 387 2.2e4 1: MRM of 13 Channels ES+ 7.57 915.15 > 144.6 (Eprino) 493 2.23e4 1: MRM of 13 Channels ES+ 7.31 886.54 > 158.1 (Ema) 78444 3.26e6 1: MRM of 13 Channels ES+ 7.31 886.54 > 126.5 (Ema) 463 1.98e5 Time
Avermectins 2 ppb Pos Ctl 2 7.83 497 1: MRM of 13 Channels ES+ 64.25 > 528.4 (Moxi) 1.77e5 1: MRM of 13 Channels ES+ 7.83 64.25 > 498.3 (Moxi) 3129 1.15e5 1: MRM of 13 Channels ES+ 8.16 892.25 > 569.45 (Iver) 1658 4.67e4 1: MRM of 13 Channels ES+ 8.16 892.25 > 37.35 (Iver) 156 4.14e4 1: MRM of 13 Channels ES+ 7.67 89.5 > 567.1 (Aba) 137 4.31e4 1: MRM of 13 Channels ES+ 7.68 89.5 > 35.15 (Aba) 146 5.47e4 Time
Future Work Year 2 samples ProSafeBeef Risk Assessment from analysis of beef samples (RIKILT) Improve precision for residues with CV >23 Compare QuEChERS against reference methods using incurred samples Stability study in solvent and Matrix
Acknowledgements Mr. Brian Kinsella (AFRC) Dr. Helen Cantwell (AFRC) Dr. Steve Lehotay (USDA, Wyndmoor) This research was supported by EU Framework VI programme on Food Quality and Safety, ProSafeBeef project FOOD-CT-26-36241