Bryn Shurmer, M.Sc. Andrew Harris, M.Sc. Perry Martos, Ph.D., CIH

1. The Qualitative, Quantitative and Confirmatory Determination of Controlled (and Prohibited) Drugs and Pesticides in Food Using LC-MS/MS Bryn Shurmer, M.Sc. Andrew Harris, M.Sc. Perry Martos, Ph.D., CIH Trace Organics and Pesticide Section Laboratory Services Division University of Guelph Guelph, Ontario, Canada

2. Overview:Tandem MS and Food Safety • Laboratory Services at the University of Guelph • -Agonists in Beef • Malachite Green in Fish • Require sub ppb detection limits using LC-MS/MS. For small molecule analyses this ensures data are irrefutable – a consistent theme with international approach to calling “positives” for legal concerns. • Conclusions

4. Laboratory Services • Number of sections • Microbiology, Food Chemistry, Microscopy, Dairy Testing…. • Trace Organics and Pesticides • Regulatory – ISO 17025 • Food - pesticide and drug residues • PMRA (SCC Accredited - GLP) • OECD guidelines • Unique analyses for on-campus researchers

5. General Parameters • Drug Residues (more than 15 different drug classes) • Food (muscle, liver, fish tissue, etc.) • Thyroid, diaphragm • Pesticide Residues (over 260 compounds) • Food (fruits, vegetables, muscle) • New Pesticides and Drugs - almost all analyses require LC-MS/MS

6. LC-MS/MS Equipment Sciex 4000 Sciex 4000 QTrap Waters – Quattro Ultima

7. SCIEX 4000 QTrap

8. Waters Quattro Ultima

9. Analysis of Clenbuterol With LC-MS/MS

10. Clenbuterol • Formula: C12 H18 Cl2 N2O • Monoisotopic mass 276.0796

11. Consequences • Growth promoter in livestock • Used in humans as a performance enhancing drug • Test for clenbuterol in retina and confirm in liver (pharmakodynamic studies proved retina was best target organ to test for residues – about 1 g sample size) • Farmer’s product is held and if tested positive, then destroyed, but…there are very serious human health concerns to contend with…

12. Health Consequences

15. Analysis

16. Instrument Conditions Positive Electrospray Ionization on Waters Quattro Ultima TIC of 3 MRM Transitions 277>203 277>168 277>132

17. Blank retina BA Feb 25 2004 11 Sm (SG, 2x5) 5: MRM of 3 Channels ES+ TIC 100 5.55e3 11.85 10.47 % 0 Time 10.25 10.50 10.75 11.00 11.25 11.50 11.75 12.00 12.25 12.50 12.75 13.00 Clenbuterol

18. 2- Fortified blank retina (10ng/g) BA Feb 25 2004 12 Sm (SG, 2x5) 5: MRM of 3 Channels ES+ 11.72 TIC 100 1.27e4 Clenbuterol % 12.74 0 Time 10.25 10.50 10.75 11.00 11.25 11.50 11.75 12.00 12.25 12.50 12.75 13.00

19. Test retina sample X BA Feb 25 2004 17 Sm (SG, 2x5) 5: MRM of 3 Channels ES+ 11.73 TIC 100 4.80e4 Clenbuterol % 0 Time 10.25 10.50 10.75 11.00 11.25 11.50 11.75 12.00 12.25 12.50 12.75 13.00

20. Clenbuterol Calibration by MRM (2 to 20 ng/g)

21. Analysis of Malachite Green With LC-MS/MS

22. Scope of the Problem http://www.cnn.com/2004/TECH/science/09/27/environment.salmon.reut/ • Used as anti-microbial, anti-fungal, and anti-parasitic treatment for fish

23. Scope of the Problem http://www.cnn.com/2004/TECH/science/09/27/environment.salmon.reut/ • “SANTIAGO, Chile (Reuters) -- A cargo of farmed Chilean salmon tainted with a potentially harmful toxin and rejected for import in Europe has returned to Chile and could be re-exported to a country with less stringent laws, consumer and environmental groups warned.”

24. This is the latest in a series of tainted shipments that has Chile's $1.15 billion-a-year salmon industry on the defensive. Fish and fish products are one of Chile's top exports. • Health authorities in Holland rejected a container bound for France, which was packed with 20 tons of frozen salmon in August after detecting the fungicide malachite green, which is used to control molds that kill the eggs of farmed fish. • The use of malachite green, which can cause cancer, is prohibited in many parts of the world. Chile banned it in 2002.

25. Earlier this year Holland rejected a different 180-tonne shipment of salmon for the same reason and environmental group Ecoceanos says part of that shipment was later sold in Estonia. • "The food has to be destroyed, because it is contaminated and cannot be consumed," said Juan Carlos Cardenas, a veterinarian and director of Ecoceanos. • Chilean government health officials in southern Chile, where the container now sits, confirmed Holland's findings and outlawed the sale of the salmon in Chile. However, they said it was up to the company to decide whether to sell it elsewhere or have it destroyed.

26. "We have control of the container and the company must decide if it re-exports it to a country without these limitations," said health ministry official Yuri Carvajal in a statement on Friday. • Officials at a salmon producers association contacted by Reuters were not immediately available for comment. • The government has not identified the companies that produced the fish. • Chile exported $820 million worth of salmon and trout to world markets in the first half of 2004, mainly to the United States and Japan. • Chilean salmon farmers said in January their fish were among the safest to eat and contained low levels of potentially harmful chemicals -- such as polychlorinated biphenyls, or PCBs, dioxins and others -- compared to their North American counterparts.

27. Malachite Green(MG) • Formula: C23 H25 N2 • Monoisotopic weight 329.2018

28. Leucomalachite Green (LMG) • Formula: C23 H26 N2 • Monoisotopic mass 330.2096 amu

29. Analysis

30. Analysis • Target Analyte Identification • Analyte extraction and concentration • Retention time (tR) • Hits with MRM, at least three transitions, TIC of the transitions • A full spectrum would be more definitive, but is not possible for sub ppb concentrations using conventional LC-MS/MS • Sciex 4000 Q can yield full spectral detail at sub ppb concentrations (data to follow)

31. Leucomalachite Green: 331>316 331>239 331>223 331>194 Malachite Green: 329>313 329>284 329>241 329>208 Instrument Conditions Positive Electrospray Ionization on Sciex API4000QTrap TIC of 4 MRM transitions for each compound

32. Traditional Approaches to Analyze Malachite Green • 1995 – GC-MS • 1995 - LC-Post-column reaction with Pb(IV)O (5ppb) • 1997 – LC-Post-column electrochemical oxidation (25ppb) • 1997 - HPLC using electrochemistry with UV-VIS diode array and fluorescence detection • 1998 - HPLC-visible and ESP-LC-MS • 1998 - Isotope dilution LC-APCI/MS • 2003 - Ion-trap LC-MS“…very low level (<1µg/kg) quantitation of drug residues using this instrument will require additional investigation, and may be more readily achieved using an alternate platform, such as a triple quadrupole instrument” • 2004 - LC-MS/MS

33. Process – Optimization, Chromatography • Infusion, 15 µL/min of 10 to 100 ppb • MS scan • Product Ion Scan • EPI • MS/MS/MS • Chromatography

34. Optimization (Infusion)

35. MG – 15 µL/min of 100 ppb (+ve ESI, MG+) (scan)

36. LMG – 15 µL/min of 100 ppb (+ve ESI, [LMG+H]+) (scan)

37. LMG - Products of [M+H]+ 331

38. LMG – EPI (331>(scan))

39. LMG - MS/MS/MS (331>239>scan)

40. Chromatography

41. Standard (10 µL of 50 ppb mix) LMG MG

42. Precision:0.5 ppb and 25 ppb standards, by TIC of several MRM transitions

43. MG Calibration by MRM (0.4 to 25 ppb)

44. MG Calibration (0.4 to 25 ppb) (EPI)

45. LMG Calibration by MRM (0.4 to 25 ppb)

46. LMG Calibration (0.4 to 25 ppb) (EPI)

47. MRM of Standard (10 µL of 2.0 ppb) LMG MG LMG MRM of Spike (10 µL of 2.6 ppb in-vial) MG

48. MRM of Matrix Blank MRM of Negative Sample

49. MRM of Incurred Positive Sample MRM of Standard (10 µL of 2.0 ppb)

50. For Malachite GreenQuestionable Positive (by MRM, ion ratios):(there is zero tolerance for malachite green)“…freezing in fish tissue can oxidize LMG back to parent compound…” Questionable Positive (EPI) (~400 ppt)