Quantitative and confirmatory analysis of veterinary drug residues in food of animal origin by UPLC- MS/MS after QuEChERS clean-up

Transcription

1 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.

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

3 ProSafeBeef

4 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

5 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

6 Europe Climates

7 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

8 Food Safety

9 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

10 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

11 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

12 Method development

13 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

14 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.

15 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

16 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 ( , v/v) Flow rate:.6 ml min -1 Injection: 5 µl Run time: 12.5 min Time A B Curve

17 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

18 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

19 Method validation

20 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.

21 Recovery at validated levels for Muscle

22 Thiabendazole 5-hydroxy-thiabendazole Within Laboratory Reproducibility - Muscle - Benz 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

23 Moxidectin Within Laboratory Reproducibility - Muscle - Fluke - Aver Bithionol Clorsulon Closantel Morantel Niclosamide Nitroxynil Oxyclozanide Rafoxanide Coumaphos Coumaphos-oxon Haloxon Abamectin Doramectin Emamectin Eprinomectin Ivermectin Levamisole CV

24 Recovery at validated levels for Milk

25 Within Laboratory Reproducibility - Milk - Benz CV

26 Within Laboratory Reproducibility - Milk - Fluke and Aver CV

27 Recovery at validated levels for Liver

28 CV Within Laboratory Reproducibility - Liver - Benz

29 CV Within Laboratory Reproducibility - Liver - Fluke - Aver

30 CCα and CCβ for Milk milk LOR/MRL CC CCβ µg/kg µg/kg Albendazole Albendazole-sulphoxide Albendazole-sulphone Albendazole-amino-sulphone Cambendazole Fenbendazole Oxfendazole Fenbendazole-sulphone Flubendazole Amino-flubendazole Hydroxy-flubendazole Mebendazole Amino-mebendazole Hydroxy-mebendazole Oxibendazole Triclabendazole Triclabendazole-sulphoxide Triclabendazole-sulphone Thiabendazole hydroxy-thiabendazole

31 CCα and CCβ for Milk Milk LOR/MRL CC CCβ µg/kg Levamisole Bithionol Clorsulon Closantel Morantel Niclosamide Nitroxynil Oxyclozanide Rafoxanide Coumaphos Coumaphos-oxon Haloxon Abamectin Doramectin Emamectin Eprinomectin Ivermectin Moxidectin

32 CCα and CCβ for Bovine Liver Liver LOR/MRL CC CCβ µg/kg µg/kg Albendazole Albendazole-sulphoxide Albendazole-sulphone Albendazole-amino-sulphone Cambendazole Fenbendazole Oxfendazole Fenbendazole-sulphone Flubendazole Amino-flubendazole Hydroxy-flubendazole Mebendazole Amino-mebendazole Hydroxy-mebendazole Oxibendazole Triclabendazole Triclabendazole-sulphoxide Triclabendazole-sulphone Thiabendazole hydroxy-thiabendazole

33 CCα and CCβ for Bovine Liver Liver LOR/MRL CC CCβ µg/kg Levamisole Bithionol Clorsulon Closantel Morantel Niclosamide Nitroxynil Oxyclozanide Rafoxanide Coumaphos Coumaphos-oxon Haloxon Abamectin Doramectin Emamectin Eprinomectin Ivermectin Moxidectin

34 CCα and CCβ for Muscle Muscle LOR/MRL CC CCβ µg/kg µg/kg Albendazole Albendazole-sulphoxide Albendazole-sulphone Albendazole-amino-sulphone Cambendazole Fenbendazole Oxfendazole Fenbendazole-sulphone Flubendazole Amino-flubendazole Hydroxy-flubendazole Mebendazole Amino-mebendazole Hydroxy-mebendazole Oxibendazole Triclabendazole Triclabendazole-sulphoxide Triclabendazole-sulphone Thiabendazole hydroxy-thiabendazole

35 CCα and CCβ for Muscle Muscle LOR/MRL CC CCβ µg/kg Levamisole Bithionol Clorsulon Closantel Morantel Niclosamide Nitroxynil Oxyclozanide Rafoxanide Coumaphos Coumaphos-oxon Haloxon Abamectin Doramectin Emamectin Eprinomectin Ivermectin Moxidectin

36 Year 1 Survey Results

37 Survey Results Year 1 - PSB EU Beef very safe beef steaks of samples residue free No risk to the consumer > Clos.2 -> Iver.2 -> Abz.2 -> Moxi.2 -> Eprino.2 -> Leva Iver 1 sample non-compliant contained ivermectin (no-mrl)

38 Method performance

39 LC Versus UPLC-MS/MS for Milk Time HPLC Analysis (36 samples) UPLC-MS/MS (36 samples) No. analytes No. runs 4 1 Sample Prep. (days) 6 1 Run time (min) 68.1 ( ) 12.5 No. injections Analysis time (h) Processing time (days) 3 1 Totals (days)

40 Application to incurred milk Sample No. Analyte Zu score 1 Blank - 2 Sum ABZ Sum FBZ Sum TCB Levamisloe Levamisle Sum TCB

41 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

42 Overlay of 38 Analytes 2ppb

43 Overlay of Internal Standards

44 ABZ and metabolites 2 ppb Pos Ctl : MRM of 19 Channels ES > (ABZ) 9.71e5 6: MRM of 19 Channels ES > 234 (ABZ) e5 5: MRM of 16 Channels ES > (ABZ-SO2) 1.33e : MRM of 16 Channels ES > (ABZ-SO2) e5 5: MRM of 16 Channels ES > 24.1 (ABZ-SO) e5 5: MRM of 16 Channels ES > (ABZ-SO) e5 Time

45 Closantel & Rafoxanide 2ppb Pos Ctl : MRM of 22 Channels ES > (Closantel) 7.24e4 9: MRM of 22 Channels ES > (Closantel) e5 9: MRM of 22 Channels ES > (Rafox) e4 9: MRM of 22 Channels ES > (Rafox) e4 Time

46 Clorsulon 4 ppb, Nitroxynil 2 ppb Pos Ctl : MRM of 5 Channels ES > (Clorsulon) 1.2e4 4: MRM of 5 Channels ES > (Clorsulon) e4 4: MRM of 5 Channels ES > (Nitrox) e4 4: MRM of 5 Channels ES > (Nitrox) e4 Time

47 Avermectins 2ppb Pos Ctl : MRM of 13 Channels ES > (Dora) 1.84e4 1: MRM of 13 Channels ES > (Dora) e4 1: MRM of 13 Channels ES > (Eprino) e4 1: MRM of 13 Channels ES > (Eprino) e4 1: MRM of 13 Channels ES > (Ema) e6 1: MRM of 13 Channels ES > (Ema) e5 Time

48 Avermectins 2 ppb Pos Ctl : MRM of 13 Channels ES > (Moxi) 1.77e5 1: MRM of 13 Channels ES > (Moxi) e5 1: MRM of 13 Channels ES > (Iver) e4 1: MRM of 13 Channels ES > (Iver) e4 1: MRM of 13 Channels ES > (Aba) e4 1: MRM of 13 Channels ES > (Aba) e4 Time

49 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

50 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