1. September 2006PQRI Training Course1 Best Practices for OINDP Pharmaceutical Development Programs Leachables and Extractables IV. Analysis of Leachables and Extractables PQRI Leachables & Extractables Working Group PQRI Training Course September 20-21, 2006 Washington, DC

2. September 2006PQRI Training Course2 Key Points to Consider ► Identification of leachables and extractables is a problem in Trace Organic Analysis. ► “Trace Organic Analysis” can be defined as the qualitative and quantitative analysis of a complex mixture of trace level organic compounds contained in a complex matrix. ► Other similar problems include:  Analysis of pollutants in environmental matrices  Organic geochemical analysis  Metabolite profiling in biological matrices

3. September 2006PQRI Training Course3 Key Points to Consider (continued) ►T►T►T►Trace Organic Analysis problems require (in general): SSSSome knowledge of the chemical nature of the analyte mixture and matrix (Supplier Information). RRRRemoval/extraction of the complex mixture of organic compounds from the matrix. SSSSeparation of the complex mixture of organic compounds into individual chemical entities. CCCCompound specific detection of the individual chemical entities within the complex mixture.

4. September 2006PQRI Training Course4 Compound Specific Detectors (from the ridiculous to the sublime) ► Single-crystal X-ray Spectrometer ► FTIR (Fourier Transform Infrared Spectrophotometer) ► NMR (Nuclear Magnetic Resonance Spectrometer) ► Mass Spectrometer  GC/MS  LC/MS

5. September 2006PQRI Training Course5

6. September 2006PQRI Training Course6 What is a Mass Spectrometer??????? ► Sample inlet system ► Ion Source ► Mass analyzer ► Detector

7. September 2006PQRI Training Course7

8. September 2006PQRI Training Course8 Sample Inlet Systems ► Direct Insertion Probes (EI/CI/FAB) ► Batch Inlet Systems (AGIS/heated septum inlets/”targets”) ► Gas Chromatographs ► Liquid Chromatographs (with or without separation in an HPLC column)

9. September 2006PQRI Training Course9 Ionization Processes ► E I -electron ionization ► C I -chemical ionization ► F I-field ionization AB -fast atom bombardment ► L SIMS -liquid secondary ion ► T SP -thermospray ► E SI -electrospray ► A PCI -atmospheric pressure CI ► M ALDI -matrix assisted laser desorption

10. September 2006PQRI Training Course10 Mass Analyzers ► Magnetic sector (high resolution) ► Quadrupole filter (“benchtop”/Triple Quad.) ► Time-of-flight (scan rate/mass range/accurate mass measurements) ► Quadrupole ion trap (MS n ) ► Ion cyclotron resonance (FTMS/ultra- high resolution/mass range/accurate mass measurements)

11. September 2006PQRI Training Course11 GC/MS – Circa Late 1980s Circa 2006

12. September 2006PQRI Training Course12 GC/MS – Electron Ionization

13. September 2006PQRI Training Course13 GC/MS Extractables Profile of an Elastomer antioxidant Coumarone-indene resin related Paraffin related

14. September 2006PQRI Training Course14 GC/MS Extractables Profile of Polypropylene Polypropylene oligomers antioxidant

15. September 2006PQRI Training Course15 Interpreting a Mass Spectrum ► Confirm molecular weight!!!!!!!!!!!  Adduct ions/multiply charged ions (LC/MS)  Alternate ionization techniques (EI/CI and APCI/ESI)  Note features of the molecular ion (obvious heteroatoms/nitrogen rule) ► Rationalize significant fragmentation processes:  Structures of fragment ions  Mechanisms for fragment ion formation

16. September 2006PQRI Training Course16 “Good” Library Search Result

17. September 2006PQRI Training Course17 “Good” Library Search Result

18. September 2006PQRI Training Course18 “Questionable” Library Search Result

19. September 2006PQRI Training Course19 “Questionable” Library Search Result

20. September 2006PQRI Training Course20 Example of Chemical Ionization (ammonia reagent gas) EI CI+ (ammonia) M +. (???) [M+NH 4 ]+

21. September 2006PQRI Training Course21 Leachables Profile – 1 Week Timepoint Expanded Section

22. September 2006PQRI Training Course22 Library Search Identification of

23. September 2006PQRI Training Course23 Leachables Profile – 1 Week Timepoint Expanded Section (2)

24. September 2006PQRI Training Course24 Library Search Identification of

25. September 2006PQRI Training Course25 Key Points to Consider ► EI is under “kinetic control”. Therefore, EI spectra are reproducible and spectral libraries can be employed (remember limitations of libraries!!!!!!!). ► CI is under “thermodynamic control”. It is useful for molecular weight confirmation but no libraries. ► GC/MS provides both a chromatographic retention time and mass spectrum (spectra) which can be compared with an authentic reference material (Good luck finding it).

26. September 2006PQRI Training Course26 LC/MS Interfaces/Ionization Processes ► Moving belt/wire (transport device) ► Thermospray (unique ionization process) ► Continuous flow FAB (transport device) ► Particle-beam (transport device) ► Electrospray (unique ionization process) ► APCI (transport device/ionization process)

27. September 2006PQRI Training Course27 A “Modern” LC/MS System

28. September 2006PQRI Training Course28 “Bench-top” LC/MS Systems

29. September 2006PQRI Training Course29 Schematic of an Orthogonal API Source

30. September 2006PQRI Training Course30

31. September 2006PQRI Training Course31 Features of Electrospray ► Often reflects solution chemistry (multiply charged ions observed) ► Operates at atmospheric pressure ► Uses a strong electric field ► Flow-rate (optimum performance at uL/min) ► Soft-ionization process ► Extremely rugged

32. September 2006PQRI Training Course32 Features of APCI ► Gas phase ionization process ► Operates at atmospheric pressure ► Flow-rate (optimum performance at mL/min) ► Uses a corona discharge ► Soft-ionization process ► Extremely rugged

33. September 2006PQRI Training Course33 APCI LC/MS Extractables Profile(s) UV @280 nm TIC (APCI+)

34. September 2006PQRI Training Course34 Mass Chromatograms (Diphenylamine Acetone Condensate) UV m/z 170 (diphenylamine) m/z 212 (isopropyldiphenylamine) m/z 210 (dimethyldihydroacridine)

35. September 2006PQRI Training Course35 APCI+ Mass Spectrum of Isopropyldiphenylamine [M+H]+ [M+H+AcCN]+

36. September 2006PQRI Training Course36 Irganox 1010 m/z 1199.5 UV TIC

37. September 2006PQRI Training Course37 APCI+ Mass Spectrum Irganox 1010 [M+Na]+ [M+K]+

38. September 2006PQRI Training Course38 APCI- Mass Spectrum Irganox 1010 (no induced fragmentation) [M-H]-

39. September 2006PQRI Training Course39 APCI- Mass Spectrum Irganox 1010 (induced fragmentation in the ion source) [M-H]-

40. September 2006PQRI Training Course40 Structure and Fragmentation of Irganox 1010 in Negative Ion APCI - - m/z 957 m/z 205

41. September 2006PQRI Training Course41 APCI- Mass Spectrum of Stearic Acid [M-H]-

42. September 2006PQRI Training Course42 Triple Quadrupole Mass Spectrometer

43. September 2006PQRI Training Course43 MS/MS Spectrum of Irganox 1076 (ESI+; products of m/z 531) [M+H]+ [M+H-56]+ [M+H-2X56]+

44. September 2006PQRI Training Course44 Structure and Fragmentation of Irganox 1076 in Positive Ion APCI (MS/MS) +H + m/z 475 m/z 419

45. September 2006PQRI Training Course45 MS/MS Spectrum of Tetramethylthiuram Disulfide (ESI+; products of m/z 241) [M+H]+

46. September 2006PQRI Training Course46 Structure and Fragmentation of Tetramethylthiuram Disulfide in Positive Ion APCI (MS/MS) +H + m/z 241 m/z 196 m/z 120 m/z 88m/z 44 + + + +

47. September 2006PQRI Training Course47

48. September 2006PQRI Training Course48 ESI+ TOF Mass Spectrum of TMTMS [M+H]+ [M+Na]+ [2M+Na]+

49. September 2006PQRI Training Course49 Expanded ESI+ TOF Mass Spectrum of TMTMS [M+Na]+

50. September 2006PQRI Training Course50 Elemental Composition Report for TMTMS from ESI+ TOF-MS Single Mass Analysis - displaying only valid results Tolerance = 10.0 PPM / DBE: min = -0.5, max = 20.0 Monoisotopic Mass, Odd and Even Electron Ions 43463 formula(e) evaluated with 3 results within limits (all results (up to 1000) for each mass) Minimum: -0.5 Maximum: 2.0 10.0 20.0 Mass Calc. Mass mDa PPM DBE Formula 231.0052 231.0052 0.0 0.2 8.0 12C4 1H2 14N9 23Na 32S 231.0051 0.1 0.6 9.5 12C11 1H7 14N2 32S2 231.0051 0.1 0.6 9.5 12C11 1H7 14N2 32S2 231.0060 -0.8 -3.6 1.5 12C6 1H12 14N2 23Na 32S3 231.0060 -0.8 -3.6 1.5 12C6 1H12 14N2 23Na 32S3

51. September 2006PQRI Training Course51 Expanded ESI+ TOF Mass Spectrum of Irganox 1010 [M+NH4]+ [M+Na]+

52. September 2006PQRI Training Course52 Elemental Composition Report for Irganox 1010 from ESI+ TOF-MS Single Mass Analysis Tolerance = 5.0 PPM / DBE: min = -0.5, max = 40.0 Monoisotopic Mass, Odd and Even Electron Ions 289500 formula(e) evaluated with 3 results within limits (all results (up to 1000) for each mass) Minimum: -0.5 Maximum: 2.0 5.0 40.0 Mass Calc. Mass mDa PPM DBE Formula 1199.7727 1199.7738 -1.1 -1.0 19.5 12C73 1H108 16O12 23Na 1199.7763 -3.6 -3.0 22.5 12C75 1H107 16O12 1199.7763 -3.6 -3.0 22.5 12C75 1H107 16O12 1199.7680 4.7 3.9 28.5 12C80 1H104 16O7 23Na 1199.7680 4.7 3.9 28.5 12C80 1H104 16O7 23Na

53. September 2006PQRI Training Course53 My Philosophy with LC/MS ► For CMC impurity profiling studies use APCI as a primary technique. ► Reproduce the UV chromatogram. ► Use every trick in the book to make APCI work (mobile phase additives, alternate acquisition techniques, etc.). ► Use electrospray to complement and confirm APCI.

54. September 2006PQRI Training Course54 IPAC-RS Proposed Identification Categories Table 2. Identification Categories for Structure Elucidation of Extractables and Leachables by GC/MS and LC/MS CategoryIdentification Data AMass spectrometric fragmentation behavior BConfirmation of molecular weight CConfirmation of elemental composition D Mass spectrum matches automated library or literature spectrum E Mass spectrum and chromatographic retention index match authentic specimen

55. September 2006PQRI Training Course55 Identification Categories ► A Confirmed identification means that identification categories A, B (or C), and D (or E) have been fulfilled. ► A Confident identification means that sufficient data to preclude all but the most closely related structures have been obtained. ► A Tentative identification means that data have been obtained that are consistent with a class of molecule only.