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Dúvidas denucci@dglnet.br Arquivo metabolite Site gdenucci

Dúvidas denucci@dglnet.com.br Arquivo metabolite .ppt Site www.gdenucci.com. Clarithromycin and OH-Clarithromycin. Std 0.02ug/mL + IS in plasma ext. VB269. 6901VA2005rj Sm (Mn, 2x1). 3.43 5845. 100. MRM of 3 Channels ES+ 837.57 > 679.47 3.27e4 Area. %. 0.

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Dúvidas denucci@dglnet.br Arquivo metabolite Site gdenucci

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  1. Dúvidas denucci@dglnet.com.br Arquivo metabolite.ppt Site www.gdenucci.com

  2. Clarithromycin and OH-Clarithromycin Std 0.02ug/mL + IS in plasma ext VB269 6901VA2005rj Sm (Mn, 2x1) 3.43 5845 100 MRM of 3 Channels ES+ 837.57 > 679.47 3.27e4 Area % 0 6901VA2005rj Sm (Mn, 2x1) 2.53 718 100 MRM of 3 Channels ES+ 764.44 > 158.21 4.08e3 Area % 0 6901VA2005rj Sm (Mn, 2x1) 3.43 1937 100 MRM of 3 Channels ES+ 748.42 > 158.27 1.08e4 Area % 0 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

  3. Clarithromycin BE study 3000 Test formulation Klaricid 500mg 2000 Plasma Concentration (ng/mL) 1000 0 0 4 8 12 16 20 24 36 48 Time (h)

  4. Clarithromycin and OH-Clarithromycin LOQ Precision Accuracy 20 ng/mL 6.3 91.2 Intra-batch 14-OH Clarithromycin 20 ng/mL 13.0 95.3 Inter-batch 14-OH Clarithromycin LOQ Precision Accuracy 20 ng/mL 6.5 87.7 Intra-batch Clarithromycin 20 ng/mL 5.0 93.0 Inter-batch Clarithromycin

  5. Clarithromycin and OH-Clarithromycin • Internal standard (roxithromycin) • Liquid-liquid extraction (dEE-dClM) • Remove organic layer and evaporate (N2) • Ressuspend in mobile phase

  6. Statistical Analysis for BE 90% CI Parameters OH-Clarithromycin Clarithromycin 91.1 - 103.7 94.2 - 108.1 AUC % ratio (0 - last) AUC % ratio 93.9 - 107.2 91.0 - 103.4 (0 - inf) 92.3 - 116.8 C % ratio 87.5 - 108.7 max

  7. ClarithromycinII

  8. Erythomycin daughter ion mass spectrum.

  9. Clarithromycin daughter ion mass spectrum.

  10. Hydroxyclarithromycin daughter ion mass spectrum.

  11. Concentration Mean

  12. ClarithromycinII • Internal standard (erythromycin) • Liquid-liquid extraction (diethyl ether/dichloromethane) • The upper organic layer + removed solvent (N2) • Ressuspend in mobile phase

  13. Diazepam and Nordiazepam QL1 - 5 ng/mL in Plasma 12-Sep-2002 01:14:48 8502VA1019 Sm (Mn, 2x2) MRM of 3 Channels ES+ 318.21 > 182.1 1.02e5 Area 2.42 22498 100 % 0 8502VA1019 Sm (Mn, 2x2) MRM of 3 Channels ES+ 285.23 > 193.2 8.00e3 Area 3.69 1536 100 % 0 8502VA1019 Sm (Mn, 2x2) MRM of 3 Channels ES+ 271.13 > 140.03 4.79e3 Area 3.08 885 100 % 0 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

  14. Concentration Mean Diazepam Valium 300 Test formulation 200 (ng/mL) Concentration 100 0 0 4 8 12 16 20 24 48 96 144 192 240 Time (h)

  15. 40 30 20 10 0 0 4 8 12 16 20 24 Concentration MeanNordiazepam Valium Test formulation (ng/mL) Concentration 48 96 144 192 240 Time (h)

  16. Diazepam and Nordiazepam LOQ Precision Accuracy 5 ng/mL 14.9 96.1 Intra-batch Diazepam 5 ng/mL 2.2 95.0 Inter-batch Diazepam LOQ Precision Accuracy 5 ng/mL 15.0 84.6 Intra-batch Nordiazeazepam 5 ng/mL 7.3 93.0 Inter-batch Nordiazeazepam

  17. Diazepam and Nordiazepam • Liquid-liquidextraction(hexane/dichloromethane) • Freeze –70 oC • Remove organic layer + evaporate (N2) • Ressuspend in mobile phase

  18. Statistical Analysis for BE 90% CI Parameters Nordiazepam Diazepam 89.00 - 106.65 88.70 - 108.91 AUC % ratio (0 - last) AUC % ratio 102.55 - 169.37 88.81 - 108.43 (0 - inf) 88.28 - 107.52 C % ratio 50.27 - 72.42 max

  19. OH-Simvastatin Std 0.10ng/mL OH-Simvastatin + IS in plasma extr. VB269 3901CC5005 Sm (Mn, 2x2) MRM of 2 Channels ES- 435.42 > 115.27 2.15e4 Area 3.22 2572 100 % 0 3901CC5005 Sm (Mn, 2x2) MRM of 2 Channels ES- 421.34 > 319.34 1.50e5 Area 3.20 16977 100 % 0 3901CC5005 Sm (Mn, 2x2) 3.20 19535 100 MRM of 2 Channels ES- TIC 1.71e5 Area % Time 0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

  20. OH-Simvastatin LOQ Precision Accuracy 0.1 ng/mL 7.6 92.5 Intra-batch OH- Simvastatin 0.1 ng/mL 3.9 96.4 Inter-batch OH-Simvastatin

  21. OH-Simvastatin • Internal standard (OH-Lovastatin) • Liquid-liquid extraction (dEE/n-hexane) • Vortex-mixing • Remove the organic phase & evaporate (N2). • Ressuspend in mobile phase

  22. Simvastatin 2.93 MRM of 2 Channels ES+ 436.2 > 198.8 3.13 1.94 1.76 2.36 0.80 2.08 1.54 3.87 0.49 0.03 0.15 0.31 0.88 0.72 2.30 2.69 3.75 1.86 2.8 3.0 3.2 3.4 3.6 3.8 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.70 MRM of 1 Channels ES+ 405.3 > 199.1 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 Time, min

  23. Simvastatin • Internal standard (Lovastatin) • Liquid-liquid extraction (diethyl ether/hexane) • Freeze -70ºC (5 min) • Transfer organic phase + evaporate (N2) • Ressuspende in mobile phase

  24. Simvastatin LOQ Precision Accuracy 0.1 ng/mL 10.7 110.9 Intra-batch Simvastatin 0.1 ng/mL 4.6 105.1 Inter-batch Simvastatin

  25. Name: Simvastatin Active metabolite: OH-simvastatin Method: Chemical hydrolysis Simvastatin and OH-Simvastatin

  26. OH-Simvastatin • Hydrolysis from the Simvastatin • Unstable compound, easily convert to Simvastatin (lactone stable form) • Characterized by IR and MS

  27. CID Scan mass spectrum of OH-Lovastin

  28. CID Scan mass spectrum of OH-Simvastatin

  29. 0.1 ng/mL 4.51 441.20 > 325.20 100 % 0 3.85 427.10 > 325.20 100 % 0 1.48 435.40 > 114.60 100 % 0 1.46 421.30 > 319.20 100 % 0 1.46 100 TIC % Time 0 1.00 2.00 3.00 4.00 5.00 6.00 8.00 Simultaneous determination of Simvastatin and OH-Simvastatin

  30. Simultaneous determination of Simvastatin and OH-Simvastatin Total run time: 8.0 min. LOQ: 0.093 ng/mL Precision: 7.0 % Accuracy: 7.1 %

  31. Same extraction procedures for both assay Different MS conditions Simvastatin ionizes in positive electrospray OH-Simvastatin ionizes in negative electrospray needed to make a switch between negative and positive into the same analytical run Advantages of separate the analytical runs improves sensitivity (singnal-to-noise ratio) shorter total run time Determination of Simvastatin and OH-Simvastatin in different assays

  32. Extraction procedure • Internal standard (lovastatin/OH-lovastatin) • Liquid-liquid extraction (diethyl ether/hexane) • Freeze –70 oC for 10 min • Remove organic layer + evaporate (N2); • Ressupende in mobile phase

  33. Simvastatin assay API 2000 (Applied Biosystems) Positive mode LOQ: 0.106 ng/mL Accuracy: 106.5 % Precision: 7.7 % Total run time: 3.6 min. OH-Simvastatin assay Quattro Ultima (Micromass) Negative mode LOQ: 0.090 ng/mL Accuracy: 92.5 % Precision: 7.6 % Total run time: 3.1 min. Determination of Simvastatin and OH-Simvastatin in different assays

  34. Risperidone Standard 0.1 ng/mL in Plasma 03-Oct-2002 20:10:31 4099VA1005 Sm (Mn, 2x2) MRM of 3 Channels ES+ 411.59 > 191.435 6.97e3 Area 3.28 685 100 % -15 4099VA1005 Sm (Mn, 2x2) 2.89 184325 MRM of 3 Channels ES+ 376.3 > 165.23 7.78e5 Area 100 % -15 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

  35. Risperidone LOQ Precision Accuracy 0.1 ng/mL 13.9 90.4 Intra-batch Risperidone 0.1 ng/mL 8.8 92.6 Inter-batch Risperidone

  36. Statistical Analysis for BE 90% CI Parameters Risperidone OH-Risperidone AUC % ratio 94.26 % - 105.94 % 78.60 % - 98.28 % (0 - last) AUC % ratio 79.30 % - 98.95 % 92.54 % - 105.08 % (0 - inf) C % ratio 86.37 % - 99.09 % 71.55 % - 92.91 % max

  37. Risperidone • Internal standard (Haloperidol) • Liquid-liquid extraction (dEE/dClM) • Freeze–70 oC • Remove organic layer & evaporate (N2) • Ressuspend in mobile phase

  38. Norms - ANVISA It must be used chemical substances of reference and/or biological standards officialized by the Brazilian Farmacopeia or other authorized codes for the current law. Studies will be admitted using that its certification is proven, in the chemical substance absence of reference and/or farmacopeicos biological standards;

  39. Norms - ANVISA For the studies of bioavailability and relative bioequivalence internal standard must be used, always that chromatographic methods will be used. The impossibility of its use must be justified; for effect of this guide, analytical race is considered the complete set of standards, samples and quality control.

  40. How do get them? • Commercialy available • Chemical synthesis • Biological synthesis • Recovery from biological samples

  41. Chemical synthesis • Expensive • Technically difficult

  42. Biological synthesis • Enzymes (bacteria, microsomes, hepatocytes, etc). • Requires very good

  43. Recovery from biological samples • Previous administration to volunteer (recovery from urine)

  44. Not available Measurable (expressed as equivalents of parent)

  45. Not available Not measurable

  46. Regulatory issues • Certificate of analysis – is MS and MR enough? • Purity issues • Expiration date - stability tests?

  47. Other difficulties Same issues may applied to internal standard for metabolite measurement

  48. Method Development Aspects • Chromatography (cross talk) • Ionization characteristics (LC-MS-MS) • Extraction efficiency

  49. Simultaneous extraction of loratadine and descarboethoxyloratadine (DCL)  Step 1 • 1 mL plasma + 100 uL sodium hydroxide (1 M) + 3 mL toluene • vortex + centrifuged • aqueous phase in frozen - 30ºC • organic phase decanted

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