Best Practices for OINDP Pharmaceutical Development Programs Leachables and Extractables

1. Best Practices for OINDP Pharmaceutical Development Programs Leachables and Extractables VII. Special Case Compound Classes PQRI Leachables & Extractables Working Group PQRI Training Course September 20-21, 2006 Washington, DC

2. “Special Cases” • PAHs - Polyaromatic Hydrocarbons • Also referred to as PNAs (Polynuclear Aromatics) • N-Nitrosamines • 2-Mercaptobenzothiazole

3. PAHs/PNAs as Leachables in OINDP • Historically, the primary source of PNAs is carbon black which is used as a filler in certain types of rubber (mostly sulfur cured). • There is some potential for other PNA sources (e.g. naphthalene contamination). • Some PNAs are known or suspect cancer causing agents (e.g. benzo(a)pyrene). FDA interest in MDIs traces back to the late 1980s. • Levels of PNAs in MDIs which employ “black rubber” seals are typically on the order of ng to low µg/canister. • The FDA historically requires that all elastomers in MDIs be evaluated and controlled for PNAs. • Analytical methods typically involve GC/MS. PQRI Training Course

4. Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(e)pyrene Benzo(a)pyrene Indeno(123-cd)pyrene Dibenzo(ah)anthracene Benzo(ghi)perylene PNAs Typically Analyzed and Controlled(EPA Method 610 list) PQRI Training Course

5. Structures of Some Typical PNAs naphthalene phenanthrene pyrene benzo(a)pyrene benzo(ghi)perylene

6. Trace Organic Analysis PQRI Training Course

7. PNA Analysis in Rubber – Possible Method • Slice (or grind) a measured weight of critical rubber components. • Add prepared rubber to a boiling flask with a measured volume of organic solvent (e.g. toluene). • Extract via reflux for a pre-optimized time period (likely 24 hours or greater). • Remove solvent and reduce in volume. • Analyze by GC/MS (for example). • Note that internal standards can be added at various points in the overall process. PQRI Training Course

8. PNA Analysis in a Suspension Metered Dose Inhaler Drug Product - Possible Method • Cool sample MDI canisters (one or several for a composite sample) over dry ice. • Open canister(s) and filter contents to remove suspended drug particles. • Note that filter assembly and catch flask must be cold. • Wash filter contents with organic solvent. • Evaporate sample to dryness. • Dissolve residue in a measured quantity of a suitable organic solvent (e.g. toluene). • Analyze by GC/MS (for example). • Note that internal standards can be added at various points in the overall process. PQRI Training Course

9. PQRI Training Course

10. A GC/MS System PQRI Training Course

11. Reference • The information summarized in the following slides related to PNA leachables studies is detailed in the following reference: Norwood, D.L., Prime, D., Downey, B.P., Creasey, J., Sethi, S.K., Haywood, P., Analysis of polycyclic aromatic hydrocarbons in metereds dose inhaler drug formulations by isotope dilution gas chromatography/mass spectrometry, Journal of Pharmaceutical and Biomedical Analysis, 13(3), 293-304, 1995. PQRI Training Course

12. GC/MS Analysis of Target PNAs PQRI Training Course

13. EI Spectra of Pyrene and D10-Pyrene • Note stability of the molecular ions • Note the characteristic presence of doubly-charged molecular ions PQRI Training Course

14. Benzo(e)pyrene and Benzo(a) pyrene PQRI Training Course

15. Representation Linearity1 and Linearity of Recovery2 Results for a Drug Product Assay (0.05-2.5µg/inhaler) PQRI Training Course

16. Limit of Detection/Quantitation Results for Selected Target PNAs PQRI Training Course

17. PNA Profile of an MDI Drug Product PQRI Training Course

18. PNAs as Leachables in Metered Dose Inhalers PQRI Training Course

19. N-Nitrosamines as Leachables in OINDP • Historically, the formation of “nitrosamines” in rubber involves sulfur curing agents (e.g. thiurams). • The issue of N-nitrosamines in rubber goes back to late 1970s/early 1980s with concern over their presence in baby bottle rubber nipples. FDA became involved in the issue. Official analytical methods for rubber developed and validated. • FDA interest in MDIs (and other OINDP) traces to the early 1990s. • Levels of nitrosamines in MDIs which employ “black rubber” seals are typically on the order of ng/canister. • The FDA historically requires that all elastomers in MDIs be evaluated and controlled for nitrosamines. • Analytical methods typically involve GC with “Thermal Energy Analysis” detection (GC/TEA). PQRI Training Course

20. Target N-nitrosamines • N-nitrosodimethylamine • N-nitrosodiethylamine • N-nitrosodi-n-butylamine • N-nitrosomorpholine • N-nitrosopiperidine • N-nitrosopyrrolidine

21. N-nitrosamine Formation X = NO+, N2O3, N2O4, NOZ (Z = Cl, Br, I, thiocyanate) PQRI Training Course

22. N-nitrosamine Analysis in Rubber(AOAC Method 987.05) • Place 5g cut rubber sample in 250mL flask with 100mL methylene chloride and 100mg propyl gallate, and hold for 17-18h. • Transfer solvent and rubber sample to a Soxhlet extractor. • Spike in internal standard. • Extract for 1 hour. • Add 100mL 5N NaOH and 2g Ba(OH)2 to flask and carefully distill methylene chloride (discard). Continue distilling 70mL of aqueous distillate into a sepratory funnel. • Add 300mg anhydrous Na2CO3 to funnel, followed by 50mL methylene chloride. Extract (repeat twice more). Combine extracts in sepratory funnel. • Pass through anhydrous Na2SO4 (to dry), into a Kuderna_Danish apparatus (with appropriate washes). • Concentrate to approximately 4mL. • Remove from KD and further concentrate to 1.0mL with a nitrogen stream. • Analyze by GC/TEA. PQRI Training Course

23. N-nitrosamine Analysis in Rubber(AOAC Method 987.05) Steam distillation Soxhlet extraction Extract concentration Image provided by Rubber Consultants PQRI Training Course

24. Principles of Thermal Energy Analysis Detection • N-nitrosamines elute from a GC column into a pyrolyzer, where they undergo pyrolysis and release nitrosyl radicals (NO.). The pyrolysis temperature is set low enough so that nitro-compounds will not pyrolyze. • Nitrosyl radicals are then oxidized with ozone in a reaction chamber to give electronically excited NO2*. • The NO2* decays back to ground state releasing a photon at a characteristic wavelength. • This process is known as “chemiluminescence”. • Sensitivity is further increased through use of a filter-photometer for detection. PQRI Training Course

25. A GC/TEA System – Schematic Diagram ozone pyrolysis vacuum cold trap 450oC detector GC -130oC electronics PQRI Training Course

26. A GC/TEA System Image provided by Rubber Consultants PQRI Training Course

27. A GC/TEA System Image provided by Cardinal Health PQRI Training Course

28. GC/TEA N-nitrosamines - Separation PQRI Training Course

29. GC/TEA N-nitrosamines - Sensitivity 10 ng/mL PQRI Training Course

30. Some Typical Limit of Detection/Quantitation Results for Target N-nitrosamines • AOAC Method 987.05 LOQs target acceptance criteria of NMT 10ppb (ng/g) for an individual N-nitrosamine. • Based on the LOQs for rubber, MDI methods should target LOQs around 1 ng/canister. PQRI Training Course

31. N-nitrosamines in OINDP – Points to Consider • N-nitrosamines are usually associated with sulfur-cured black rubber. • Even with the sensitivity and selectivity of the GC/TEA, other peaks are often noted in OINDP leachables profiles. • N-nitrosamines are very light sensitive, which suggests a possible procedure for identifying “non-nitrosamine” GC/TEA peaks. PQRI Training Course

32. Analysis of Mercaptobenzothiazole (MBT) Compounds from Sulfur Cured Rubber by a Liquid Chromatography – Tandem Mass Spectrometry (LC-MS-MS) Method Tianjing Deng*, Shuang Li, Xiaoya Ding and Song Klapoetke PPD 8551 Research Way Middleton, WI 53562 * Corresponding author PQRI Training Course

33. Mercaptobenzothiazole (MBT) and other benzothiazoles are common vulcanization accelerators for rubber materials that are used in pharmaceutical container/systems, such as the gaskets in the pressurized Metered-Dose Inhaler (pMDI). MBT is of particular concern since it is considered a potential carcinogen and has been shown to migrate into drug formulations. • Due to the toxicological concern and leachability of MBT and other benzothiazoles, analytical methods have been developed to study these types of compounds in the fields of food additives and contaminants (1), contact dermatitis caused by the rubbers (2), as well as pharmaceutical packaging systems (3). MBT can be analyzed by gas chromatography (4) but many other benzothiazoles are thermally-labile and readily decomposed in the GC inlet. HPLC methods are commonly used to study PQRI Training Course

34. MBT MBTS   In this study, a method using liquid chromatography with tandem mass spectrometer (LC-MS-MS) was developed to analyze MBT in the sulfur cured rubber. The method is capable of detecting ng level of MBT in the rubber extracts. This study demonstrates the feasibility of using detector with high selectivity, such as LC-MS-MS method, for extractable/leachable with special toxicological concern that requires greater sensitivity and specificity. PQRI Training Course

35. RReferences: 1Barnes, K.A., Castle L., Damant, A. P., Read, W. A., and Speck, D. R., Food Additives and Contaminants, Vol. 20, No. 2, 196-205 (2003). 2Hansson, C., Bergendorff, O., Ezzelarab, M., and Sterner, O., Contact Dermatitis, 36, 195-200, (1997). 3Gaind, V. S., and Jedrzejczak, K., Journal of Analytical Toxicology, Vol. 17, 34-37, (1993). 4Niessen, W. M. A., McCarney, C. C., Moult, P. E.G., Tjaden, U. R., and Van der Greef, J., Journal of Chromatography, 647, 107-119, (1993). 5Mathieu, C., Herbreteau, B., Lafosse, M., Morin, Ph., Renaud, M., Cardinet, C., and Dreux, M., J. High Resol. Chromatogr., 23, (9), 565-566, (2000). 6 PQRI Training Course

36. Method Conditions: HPLC Parameters Mobile Phase: Water:Methanol:Formic acid 20:80:0.05 (v/v/v) Flow Rate: 0.2 mL/min Column: Waters Symmetry C18, 3.5 m, 2 x 100 mm Column Temperature: 40°C Autosampler Temperature: Ambient Injector Volume: 5 L PQRI Training Course

37. “Bench-top” LC/MS Systems Time-of-flight Linear ion trap Triple Quadrupole PQRI Training Course

38. Triple Quadrupole Mass Spectrometer PQRI Training Course

39. MS-MS Spectrum of MBT Mercaptobenzothiazole (MBT) PQRI Training Course

40. MS-MS Spectrum of MBTS Dibenzothiazyl Disulfide (MBTS) PQRI Training Course

41. Mass Spectrometer: PE Sciex API 2000/API365 Triple Quadruple Mass Spectrometer PQRI Training Course

42. MRM Chromatogram of Extraction Blank: MBT (blue trace) and MBTS (red trace) Selectivity/Specificity PQRI Training Course

43. MRM Chromatograms of MBT (blue) and MBTS (red) in the 500 ng/mL standard solution. PQRI Training Course

44. MRM Chromatograms of MBT (blue) and MBTS (red) in the 30 min TBME Extract. PQRI Training Course

45. Extraction Method • Hansson et al. studied the extraction of MBT/MBTS using different solvents. They found out that Methyl tert-Butyl Ether (MTBE) is a good solvent for MBT/MBTS due to its: • Powerful extraction medium • Low toxicity • Inertness to MBT/MBTS • High volatility • In this study, the rubber was cut into 3 x 3 mm squares. One gram of the rubber was extracted with 10 mL MTBE for 30 minutes by sonication. After extraction, the extract was diluted to different volume using Methanol: Water 50: 50 diluent to give varying MBT concentrations and filtered using glass fiber syringe filters for LC-MS study. PQRI Training Course

46. The Extraction Study of MBT by MTBE from Sulfur-Cured Rubber PQRI Training Course

47. Linearity Plot of MBT (50 – 1000 ng/mL) PQRI Training Course

48. Repeatability Calculated MBT Concentration (PPM) in Three Replicates of Extract. PQRI Training Course

49. LOQ/LOD The DL of MBT was calculated using S/N ratio = 3. DL = 6 ng/mL in the solution or 12 pg on column. MRM Chromatogram of MBT Standard (50 ng/mL) PQRI Training Course

50. Accuracy - Filter Study A Filter study was conducted to verify that the syringe filter used in the sample preparation did not reduce the recovery of MBT and MBTS. Three 500 ng/mL standards were analyzed before and after the filtration and the area responses of MBT and MBTS were compared. The percent differences between the filter and non-filtered samples are less than 2.5% indicating that filtration does not affect the method accuracy. PQRI Training Course