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Dúvidas denucci@dglnet.br Arquivo Analytical Methodologies Site gdenucci

Dúvidas denucci@dglnet.com.br Arquivo Analytical Methodologies .ppt Site www.gdenucci.com. Bioequivalence. Two formulations of the same drug product produce equivalente bioavailability profiles. BIOAVAILABILITY CAN BE DETERMINED BY MEASUREMENTS OF.

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Dúvidas denucci@dglnet.br Arquivo Analytical Methodologies Site gdenucci

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

  2. Bioequivalence Two formulations of the same drug product produce equivalente bioavailability profiles

  3. BIOAVAILABILITY CAN BE DETERMINED BY MEASUREMENTS OF • Concentration of the active drug or metabolite(s) in biological fluid as a function of time. • Urinary excretion of the active drug or metabolite(s) as a function of time. • Any appropriate acute pharmacological effect.

  4. Pharmacokinetic parameters 8 6 4 2 0 Cmax Concentration tmax 0 3 6 9 12 Hours

  5. Pharmacokinetic parameters 8 6 4 2 0 Concentration AUC 0-12h 0 3 6 9 12 Hours

  6. Metodologias para análise de drogas • Ensaios biológicos. • Testes colorimétricos. • Radioimunoensaio. • Espectrofotometria (uv visível, i.v, fluorescência). • Espectrometria de massa. • Ressonância magnética nuclear. • Cromatografia de camada delgada. • Cromatografia gasosa. • Cromatografia líquida de alta pressão • LC-MS-MS

  7. Metodologia para dosagem em líquidos biológicos • H.P.L.C. • H.P.L.C. acoplado a MS (LC-MS-MS).

  8. Topics - The Mass spectrometry • The choice of the Internal Standard • The ionization methods • Sample preparation • The ion suppression experiment • Practical Examples • Conclusion

  9. What is the Mass Spectrometry? • Measure the mass (mass-to-charge ratio) • Analytical technique for: • identify unknown compounds • elucidate structure and chemical properties • quantitation of chemical substances • Good for detection of very small concentration (typically 10-12 - 10-15 moles)

  10. How does the mass spectrometer work?

  11. Triple Quadrupole Mass Spectrometer Set up

  12. Mass Spectrometry - Ionization Methods • Electron Impact (EI) • Chemical Ionization (CI) • Atmospheric Pressure Ionization (API) • ElectroSpray Ionization (ESI) • Atmospheric Pressure Chemical Ionization (APcI) • Matrix Assisted Laser Desorption Ionization (MALDI) • Fast Atom Bombardment (FAB)

  13. Why use API-MS? • Cover a high range of molecular weight • Compatible with a broad range of compounds • Several types of inlets can be used (flow injection, HPLC, CE) • Rugged and easier to use

  14. Atmospheric Pressure Ionization Types • Electrospray (ESI): uses chemical fields to generate charged droplets. It can be positive (ES+) or negative (ES-). • Pneumatically Assisted Electrospray: same as above but use a pneumatic nebulization • APcI: where the solvent acts as the CI reagent gas to ionize the sample • APPI (Atmospheric Pressure PhotoIonization): Krypton Lamp producing UV light ionizes gas phase analytes or dopants with subsequent gas-phase reaction

  15. Internal Standard (I.S.) • Similar structure (Analogues) • the analyte and the I.S. should have the same functional groups • attention in the extraction phase • ionization conditions may be different • Deuterated isotopes • the deuterated analyte • not always commercially available

  16. Sample Extraction • Liquid/Liquid • two phases (aqueous/organic solvent) • faster • cheaper • Protein precipitation • Very simple • Fast • “dirty” samples • SPE (Solid Phase Extraction) • cartridges • Easier for automatization • More expensive

  17. Typical procedure for Liquid/Liquid Extraction • Dispense the sample into appropriate clean glass tubes; • Dispense the I.S. solution and vortex-mix the samples for approximately 10 s.; • Add the organic solvent mixture (e.g. Diethyl-Ether/Hexane 80:20) and vortex-mix the samples for 40 s.; • Remove the organic layer, transfer it into anaother glass tube and evaporate it into a Nitrogen stream; • Reconstitute the samples in a fixed volume of mobile phase, vortex-mix for 15 s. and transfer it into the HPLC vial

  18. Typical procedure for Solid Phase Extraction (SPE) • Add the sample into appropriate tubes; • Dispense the corresponding I.S. and vortex-mix samples; • Apply the samples to individual SPE cartridges (previously pre-conditioned with methanol and water). Using a light positive pressure the samples are slowly drawn through the cartridges; • Wash the cartridges (usually using water); • Apply positive pressure to ensure to dry the cartridges; • The extract is then eluted form the cartridges using an appropriate eluent and collected into glass test tubes; • Transfer the eluted solution into the HPLC vials;

  19. Liquid/Liquid Fluoxetine Fluconazole Captopril Omeprazole Paroxetine Diazepam Bromazepam Midazolam Nimodipine Amlodipine SPE Cephalexin Cefaclor Amoxicilin Didanosine Acyclovir Losartan Enalapril Lisinopril Some examples of use in bioequivalence studies • Protein precipitation • Methyl-DOPA

  20. Ion Suppression • MS signal suppression • Caused by any interference (e.g. salt, proteins, etc.) • Non-specific extraction • The effects of the sample preparation in the ionization process should be evaluated

  21. Ion suppression experimentExperimental set-up

  22. Ion suppression experiment • Infusion pump: an standard concentration of an analyte/I.S. mix (e.g. 50 ug/mL at 50 uL.min) • HPLC pump: normal mobile phase at the flow rate specified for the assay • Extract a blank sample and inject it into the system • See what happen with the MS signal

  23. 100 MRM of 2 Channels ES+ % 247 > 204.2 -15 100 237 > 194.2 % -15 Time 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Ion suppresion experiment

  24. 2.32 100 MRM of 2 Channels ES+ % 247 > 204.2 -15 2.37 100 237 > 194.2 % -15 Time 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Ion suppresion experiment

  25. Bioequivalence studies: Examples

  26. Determination of Amoxicilin using Cefadroxil as internal standard • Analytical column: Genesis C18, 4µm (150 mm x 4.6 mm i.d.) • Mobile phase: 50% Acetonitrile/50% Water & 10mM formic acid • Ionization mode: Positive electrospray (ES+) • Ions were monitored by MRM (Multiple Reaction Monitoring) • Sample Extraction: Solid/Liquid (Oasis SPE cartridges)

  27. Amoxicilin M.W. 365.40 g/mol Cefadroxil M.W. 363.38 g/mol Chemical Structures

  28. m/z 364 m/z 366 m/z 349 m/z 208 Fragmentation routes for Amoxicilin and Cefadroxil

  29. Daughters of 366 348.9 100 % 113.7 366.2 208.2 159.9 304.7 330.6 234.4 276.7 0 366.3 100 349.0 % 104.4 388.0 0 Da/e 100 150 200 250 300 350 400 Mass Spectra - Amoxicilin

  30. 208.0 100 Daughters of 364 % 157.9 347.1 0 364.0 100 % 0 M/z 100 200 300 400 500 600 Mass Spectra - Cefadroxil

  31. Amoxicillin and Cefadroxil chromatograms Amoxicilin 0.05 mg/mL 2.63 366.00 > 349.20 100 % 0 Cefadroxil 0.05 mg/mL 2.63 100 364.00 > 207.80 % Time (min) 0 1.00 2.00 3.00 4.00 5.00

  32. 3.49 Response 0 mg/mL 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Amoxicillin Calibration Curve

  33. Total run time: 4.0 min. • LOQ: 50 ng/mL • Precision: 15.4 % • Accuracy: - 6.5 %

  34. The determination of RSD 921 using RSD 921-D3as I.S. • Analytical column: Genesis C18, 4µm (150mm x 4.6mm i.d.) • Mobile phase: 50% Acetonitrile + 10mM Formate • Ionization mode: Positive electrospray (ES+) • Ions were monitored by MRM (Multiple Reaction Monitoring) • Sample Extraction: Liquid/Liquid (Diethyl ether/Hexane 80:20)

  35. RSD 921 M.W. 356.54 g/mol RSD 921-D3 M.W. 359.52 g/mol Chemical Structures

  36. m/z 357 m/z 360 m/z 286 m/z 289 Fragmentation routes for RSD921 and RSD921-D3

  37. RSD 921 and RSD 921-D3 Chromatograms RSD 921 360.20 > 289.10 1.84 100 % 0 RSD 921- D3 357.20 > 286.00 1.85 100 % 0 Time 0.50 1.00 1.50 2.00 2.50

  38. 4.97 Response ng/mL 0 5.0 10.0 15.0 20.0 Calibration Curve - RSD

  39. 286.3 Daughters of 357 100 % 0 357.4 100 % m/z 0 60 100 140 180 220 260 300 340 380 420 Mass Spectra - RSD

  40. 289.3 100 360.4 % 82.3 0 114.9 100 Daughters of 360 289.2 % 147.0 80.6 209.1 0 Da/e 50 100 150 200 250 300 350 400 450 Mass Spectra - RSD-D3

  41. Total run time: 3.0 min. • LOQ: 0.1 ng/mL • Precision: 4.9 % • Accuracy: 2.16 %

  42. Determination of Enalaprilat using Enalaprilat-phenyl-D5 as internal standard • Analytical column: Genesis C18, 4µm (150 mm x 4.6 mm i.d.) • Mobile phase: 50% acetonitrile in 10 mM acetic acid + 20 mM ammonium acetate aqueous solution • Ionization mode: Positive electrospray (ES+) • Ions were monitored by MRM (Multiple Reaction Monitoring) • Sample Extraction: Solid/Liquid (Oasis SPE cartridges)

  43. Enalaprilat M.W. 348.39 g/mol Enalaprilat-phenyl-D5 M.W. 353.42 g/mol Chemical Structures

  44. m/z 354 m/z 349 m/z 211 m/z 206 Fragmentation routes for Enalaprilat and Enalaprilat-phenyl-D5

  45. Daughters of 349 206 100 % 160 102 303 116 349 0 Scan ES+ 349 100 % 350 371 64 91 81 206 185 429 303 415 214 0 Da/e 50 100 150 200 250 300 350 400 450 500 Enalaprilat Mass Spectra

  46. 12.9 Response ng/mL 0 50.0 100.0 150.0 200.0 Calibration Curve

  47. Total run time: 4.0 min. • LOQ: 0.5 ng/mL • Precision: 6.9 % • Accuracy: 2.75 %

  48. Determination of Diclofenac using ES + • Analytical column: Genesis C8, 4µm (150 mm x 4.6 mm i.d.) • Mobile phase: 50% acetonitrile / 50% water & 10 mM acetic acid • Ionization mode: Positive electrospray (ES+) • Ions were monitored by MRM (Multiple Reaction Monitoring) • Sample Extraction: Liquid/Liquid (diethyl ether/dicloromethane 70:30)

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