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GALENO RESEARCH UNIT

GALENO RESEARCH UNIT. RUA LATINO COELHO, 1301 PARQUE TAQUARAL CAMPINAS, SP 13087-010. Dúvidas denucci@dglnet.com.br Arquivo Abef-2003.ppt Site www.gdenucci.com. Nitrofurans & Bound Residues. Drug Bound residue. Furazolidone AOZ Furaltadone AMOZ Nitrofurantoin AHD

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GALENO RESEARCH UNIT

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  1. GALENO RESEARCH UNIT RUA LATINO COELHO, 1301 PARQUE TAQUARAL CAMPINAS, SP 13087-010

  2. Dúvidas denucci@dglnet.com.br Arquivo Abef-2003.ppt Site www.gdenucci.com

  3. Nitrofurans & Bound Residues Drug Bound residue Furazolidone AOZ Furaltadone AMOZ Nitrofurantoin AHD Nitrofurazone SEM

  4. Structures

  5. EXTRACTION 1g of homogenised muscle Addition of Internal Standard (IS), mix and stand for 15min 5 mL of 0.2M HCl and 50µL of 100mM 2-NBA 16hrs at 37oC

  6. AOZ Detection of AOZ o-NBA

  7. NPAOZ

  8. Neutralization: 0.3M Na3PO4 and 0.2M NaOH 4 mL of Ethyl Acetate Mix for 20 min and centrifuge at 2000xg for 10min Repeat 4 mL of Ethyl Acetate, Mix for 20 min and centrifuge at 2000xg for 10min Dry the Ethyl Acetate under N2 atmosphere at 45oC Resuspension in mobile phase and LC/MS/MS analysis

  9. CHROMATOGRAPHIC PARAMETERS Column: C18 (4um, 150 mm x 4.6 mm) Flow: 1 mL/min Injection volume: 50µL Mobile Phase A: 99% water, 0.1% acetic acid Mobile Phase B: 9.9% water, 90% acetonitrile, 0.1% acetic acid Temperature: RT

  10. Channel (s) Mass Daughter Dwel NPAHD 249 134 0.2 NPAOZ 236 134 0.2 NPAMOZ 335 291 0.2 NPSEM 209 166 0.2 NPAMOZD5 340 296 0.2 NPAOZD4 240 134 0.2 Ms/Ms PARAMETERS Equipment: API4000 Ion Mode: ES+

  11. STANDARD CURVEAMOZ

  12. STANDARD CURVEAOZ

  13. STANDARD CURVEAHD

  14. STANDARD CURVESEM

  15. VALIDATION - QMC X API4000 n=8 QMC AMOZ AOZ 0.50 1.00 0.50 1.00 Mean 0.53 0.95 0.49 1.03 SD 0.08 0.13 0.06 0.10 Precision 15.58% 13.57% 12.05% 9.65% Accuracy 106.75% 95.29% 98.00% 103.43% n=8 API4000 AMOZ AOZ 0.50 1.00 0.50 1.00 Mean 0.50 0.98 0.50 0.99 SD 0.03 0.06 0.03 0.06 Precision 5.93% 5.88% 5.50% 5.95% Accuracy 100.78% 98.18% 100.65% 98.51%

  16. VALIDATION - QMC X API4000 n=8 QMC AHD SEM 0.50 1.00 0.50 1.00 Mean 0.50 1.03 0.50 1.01 SD 0.18 0.28 0.21 0.21 Precision 35.69% 26.88% 42.50% 20.35% Accuracy 99.25% 102.71% 99.75% 101.29% n=8 API4000 AHD SEM 0.50 1.00 0.50 1.00 Mean 0.50 0.99 0.50 0.98 SD 0.04 0.06 0.02 0.11 Precision 8.26% 5.82% 4.88% 11.49% Accuracy 100.35% 99.28% 100.15% 97.81%

  17. VALIDATION API 4000 - 0.3 AND 0.5 g/kg n=8 AMOZ AOZ 0.30 0.50 0.30 0.50 Mean 0.38 0.50 0.35 0.50 SD 0.07 0.05 0.06 0.06 Precision 19.24% 9.21% 17.05% 11.61% Accuracy 126.42% 100.00% 117.46% 99.58% n=8 AHD SEM 0.30 0.50 0.30 0.50 Mean 0.26 0.50 0.30 0.50 SD 0.05 0.04 0.08 0.02 Precision 20.43% 8.26% 28.05% 4.88% Accuracy 88.25% 100.35% 99.21% 100.15%

  18. 0.5g/kg - QUATTRO MICRO AMOZD5 AMOZ

  19. 0.5g/kg - API4000 AREA 825000 AMOZD5 AREA 84200 AMOZ

  20. 0.5g/kg - QUATTRO MICRO AOZD4 AOZ

  21. 0.5g/kg - API4000 AREA 366000 AOZD4 AREA 74900 AOZ

  22. 0.5g/kg - QUATTRO MICRO AHD SEM

  23. 0.5g/kg - API4000 AREA 11100 AHD AREA 26100 SEM

  24. AMOZ - SUSPECT AREA 33800 0.3 g/kg AREA 14200 <0.3 g/kg

  25. AOZ - SUSPECT AREA 17500 0.3 g/kg AREA 13400 <0.3 g/kg

  26. MAPA SAMPLES ANALYSED 27-05-2003 TOTAL = 3629 SAMPLES POSITIVES = 0.3% SUSPECTS = 1.1% NEGATIVES 98.6%

  27. Projetos Nitrofurano Outras drogas

  28. Nitrofuranos Validação da Metodologia para outras Matrizes Avaliação da real toxicidade causada pelos nitrofuranos

  29. Outras Drogas Estabelecer metodologia para detecção de outras drogas (cloranfenicol, nitroimidazólicos, dapsona)

  30. Avaliação da Toxicidade - Testes in vitro - Testes in vivo

  31. Testes in-vitro • Testes de Genotoxicidade: • Teste de Ames • Teste de Micronúcleo(eritrócitos de rato) • Teste em Linfoma de rato (timidina-quinase)

  32. Ames Test (Salmonella typhimurium) • Bacterial reverse mutation assays are widely used to evaluate the mutagenic potential of chemicals, formulations, or extracts. The most common assay involves the use of amino acid-requiring strains of Salmonella typhimurium and is commonly referred to as the "Ames Test". • In the absence of an external amino acid source (histidine for Salmonella), the cells cannot grow to form colonies unless a reversion of the mutation allows the synthesis of the amino acid to resume. •  Spontaneous reversions occur with each of the strains. However, some compounds induce a mutagenic response and thereby increase the number of revertant colonies substantially over the spontaneous reversion level.

  33. The bacterial reverse mutation assay involves the measurement of revertant colony frequencies that are obtained when Salmonella strains are treated in the presence and absence of a test article. Positive and negative controls are included for each strain tested. Metabolic activation using rat liver S9 is included to mimic the in vivo activity of the liver enzymes in transforming some pro-mutagens to mutagens

  34. Effect of CRIS031 in Ames test using S. typhimurium strain (TA 98)

  35. Effect of CRIS031 in Ames test using S. typhimurium strain (TA 100)

  36. Effect of CRIS031 in Ames test using S. typhimurium strain (TA 1535)

  37. Effect of CRIS031 in Ames test using S. typhimurium strain (TA 1537)

  38. Conclusion “The compound CRIS031 showed no mutagenic effects on Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 neither in the presence nor in the absence of a metabolically active microsomal S9 fraction from rat liver using standard Ames test.”

  39. Avaliação do potencial tóxico em Humanos • Verificar a biodisponibilidade dos nitrofuranos • - Verificar a biodisponibilidade de seus metabólitos

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