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Recent advancement in impurity profiling


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Recent advancement in impurity profiling

  1. 1. A seminar on<br />Recent advancement in impurity profiling<br />Presented by<br />Sudipkumar modh<br />1<br />
  2. 2. Agenda<br />2<br />1. Definition of ICH<br />2. Importance<br />3. History<br />4. Systemic approach<br />5.Isolation And Identification<br />6.Case study<br />7.Methodology<br /> a)Classical approaches<br /> b)Modern approaches<br />8.Conclusion<br />
  3. 3. Definition of ICH (Q6A specifications)<br /><ul><li>Impurity:</li></ul>“Any component of the new drug substance that is not the chemical entity defined as the new drug substance“<br />“Any component of the drug product which is not the chemical entity defined as the drug substance or an Excipient in the drug product.”<br />Impurity profiling:<br /><ul><li>Analytical activities, aim of …
  4. 4. Detection, identification/structureelucidation
  5. 5. Quantitative determination
  6. 6. Of organic and inorganic impurities and residual solvents in bulk drugs & pharmaceutical formulations</li></ul>3<br />
  7. 7. Importance<br />all phases of synthetic drug research and production from the gram scale preparation of new compounds <br />For pharmacological screening up to the scaling up procedure and finally the production of bulk drugs<br />Differentiate between synthesis-related<br /> impurities and degradation products<br />4<br />
  8. 8. HISTORY of instrumental analysis<br />Period following world war II,rapid development of pharmaceutical industry<br />Instrumental analysis was developed with military technology<br />Qualitative analysis was met by molecular spectroscopy but with complexity<br />Spectroscopic method were generally complex, and there was a need of expert spectroscopist <br />Separations techniques came, at the end of 19th century revolution came by development of TLC <br />After world war II,GC developed and HPLC after two decades<br />The fully Instrumental technique science arrived<br />5<br />
  9. 9. Systemic approach<br />According to ICH Q3A(R2) <br />6<br />Maximum Reporting Identification Qualification<br />daily dose threshold threshold threshold<br />(g/day) (%)<br />
  10. 10. Systemic approach<br />7<br />
  11. 11. Systemic approach<br /><ul><li>What to be done after separation ??</li></ul>Purified and sample should be split into aliquots for MS,NMR, and vibrational spectroscopy<br />Repetition of MS of sample is required <br /><ul><li>1st confirm that molecular weight of sample corresponds to initial LC/MS data
  12. 12. 2nd determine fragmentation pathway from MS/MS data </li></ul>Empirical formula of HRMS is useful, if impurity is unknown and unrelated with drug <br />In this case, by FT-IR,FT-Raman or both, functional groups are identified<br />Structure is eluted by MS and NMR (Homo and hetero nuclear direct and long range chemical shift correlation experiments)<br />8<br />
  13. 13. Isolation And Identification<br />Preparative chromatography<br />In reversed phase chromatography good isolation can be achieved, by using stationary non polar phase and polar mobile phase with polarity modifiers, pairing ions, buffers<br />Due to diverse nature of impurity, not only RP-HPLC , but also <br /><ul><li>Normal phase chromatography
  14. 14. Supercritical fluid chromatography
  15. 15. Gas chromatography
  16. 16. Capillary electrophoresis</li></ul>May be a good choice<br /><ul><li>Isolation</li></ul>9<br />
  17. 17. Isolation And Identification<br /><ul><li>Isolation</li></ul>After isolation sample may contain artifacts from the isolation process, this constituents are referred as “chemical noise”<br />This may affect particularly to high sensitivity NMR probes(1.7 mm) <br />10<br />
  18. 18. Isolation And Identification<br /><ul><li>Identification
  19. 19. MS</li></ul>Molecular weight changes show gain or loss of some neutral species exp is CO<br />Formation of adduct ions can be useful for preliminary identification of molecular ion<br />Exp adduct ions like sodium,pottasium,acetonitrile at +23,+39,+43<br />Isotope patterns also useful like of Cl, Br <br />HRMS is useful for unrelated impurity<br />MS/MS provides site of chemical modification and type of modification<br />Not useful for detection of positional isomers<br /><ul><li>NMR</li></ul>Besides of convectional NMR ,<br /><ul><li> 1 D NMR
  20. 20. DEPT
  21. 21. INEPT
  22. 22. 2d NMR like
  23. 23. COSY
  24. 24. TOCSY
  25. 25. HSQC
  26. 26. HMBC</li></ul>Are used now a days<br />11<br />HSQC (Heteronuclear Single Quantum Coherence) <br /> HMBC (Heteronuclear Multiple Bond Coherence) <br />Distortion less Enhancement by Polarization Transfer(DEPT)<br />Insensitive Nuclei Enhanced by Polarization Transfer (INEPT)<br />
  27. 27. Isolation And Identification<br /><ul><li>Vibrational spectroscopy</li></ul>With advancement in 2d NMR,vibrational spectroscopy like FT-IR and FT-RAMAN is somewhat not acquired <br />But this types of spectroscopy is essential in functional group detection,parallely with MS and NMR<br />Carbonyl function groups are detected efficiently in vibrational spectroscopy<br />But it is transparent in COSY and HSQC<br />In HMBC long correlations are observed with carbonyl moiety<br /><ul><li>Data integrations</li></ul>12<br />
  28. 28. Case study<br />Isolation And Identification<br />Characterization of an unstable process impurity in the protease inhibitor Tipranavir<br /> Colored contaminant was being formed in final step of the synthetic process<br /> Formed due to coupling of aniline-like derivative with pyridine being used<br /> as acid scavenger<br />Isolated by methanol stripping of silica gel chromatography<br />methanol solution containing the colored impurity was bright red <br /> half life of about 18 h in methanol and <10 min in acetone<br />By 1.7-mm 600-MHz SMIDG probe, sample was prepared and data set consisting of a proton reference spectrum, TOCSY, HSQC, and 10-Hz optimized HMBC spectra was acquired<br />13<br />SMIDG=sub micro-inverse-detection gradient <br />
  29. 29. Characterization of an unstable process impurity in the protease inhibitor Tipranavir<br />Case study<br />Isolation And Identification<br />drug<br />Formed colored impurity<br />14<br />Starting material<br />
  30. 30. in addition to the resonances normally observed for the drug, showed three new well-resolved signals, <br /><ul><li>8.25 (d)
  31. 31. 7.82(t)
  32. 32. 6.27(t)</li></ul>The doublet (8.25) and one of the triplets (6.27 ppm) , twice the intensity of triplet (7.82 ppm), which had an integrated intensity corresponding to half of that of a one proton signal in the drug molecule.<br />Conclusion: impurity contained two molecules of drug and that the new resonances were present in a ratio of 2 : 2 : 1<br />The TOCSY spectrum confirmed the coupling of the new resonances in the aromatic region<br />Isolation And Identification<br />Case study<br />Characterization of an unstable process impurity in the protease inhibitor Tipranavir<br />15<br />
  33. 33. A HRMS spectrum gave exact mass of 848.47287Da (less the ionizing proton) corresponding to the molecular weight of two of the aniline precursor molecules and a C5H5 fragment<br />consistent with the integration of the new aromatic resonances in proton spectrum.<br />GHSQC data established the following direct proton–<br />carbon correlation: 8.25/152.7, 7.82/125.8, and 6.27/106.2 ppm<br /> HMBC correlations linked at 8.25/152.7 ppm resonant pair to aromatic quaternary carbon bearing aniline group resonating at 142.6 ppm. <br />other correlations were internal to five-carbon fragment<br />Isolation And Identification<br />Case study<br />Characterization of an unstable process impurity in the protease inhibitor Tipranavir<br />16<br />
  34. 34. Isolation And Identification<br />HSQC spectrum<br />Case study<br />Characterization of an unstable process impurity in the protease inhibitor Tipranavir<br />17<br />
  35. 35. Isolation And Identification<br />HMBC spectrum<br />Case study<br />Characterization of an unstable process impurity in the protease inhibitor Tipranavir<br />18<br />
  36. 36. Isolation And Identification<br /><ul><li>Data from the NMR data were supported by vibrational data</li></ul>Included increase in intensity of the band at<br />1636 cm–1 suggesting increased double bond<br />1547 cm–1 consistent with C=N stretching that is not present in Tipranavir<br />1170 cm–1 region of C–O stretching and C–OH bending that can be accounted for by the dimeric nature of the impurity of Tipranavir<br />The five carbon fragment linking the two aniline moieties arose from the pyridine ring coming apart during the reaction<br />Case study<br />Characterization of an unstable process impurity in the protease inhibitor Tipranavir<br />19<br />
  37. 37. Methodology<br /><ul><li>Classical approach
  38. 38. separation and determination of impurities with known structure
  39. 39. Isolation of the impurity and off-line structure elucidation
  40. 40. Modern approach
  41. 41. Using on-line hyphenated separation/spectroscopic methods</li></ul>20<br />
  42. 42. Classical approach<br />Methodology….<br />separation and determination of impurities with known structure<br />HPLC<br />TLC<br /><ul><li>Electrophoretic and Related Methods
  43. 43. Capillary Electrophoresis
  44. 44. Electrophoresis-related Chromatographic</li></ul> Techniques<br />Methods not Requiring Separation <br /><ul><li>Polarography & UV</li></ul>21<br />
  45. 45. HPLC<br />HPLC is often used with UV<br />Chemometric methods used for separation optimisation but also for improving quantitative analysis<br />Dimension 150-250 x 4.0-4.6 mm<br />RP-stationary phase with particle size of 3.5-5.0 μm <br />Rt in range of 10-20 min<br />Ultra-performance liquid chromatography (UPLC) uses short and narrow-bore columns with small particle size packing<br />Exp Impurities of primaquine phosphate <br />dimensions of the column were 50 x 2.1mm <br /> C18 silica packing and 1.7 μm<br />separation was achieved within 2 minutes<br />Classical approach….<br />22<br />
  46. 46. A new development in RP-HPLC is the introduction of oil-in-water emulsion into the mobile phase<br /> This method, named micro emulsion liquid chromatography (MELC)<br />special application in field of impurity profiling by HPLC is high-speed counter-current chromatography (HSCCC)<br />(semi)preparative Analysis by HPLC-DAD to determine relative purities of each fraction in course of the preparative isolation of drugs from e.g. medicinal plant matrices<br />23<br />HPLC<br />Classical approach….<br />
  47. 47. TLC<br /><ul><li>It has good ease of use, cost-effectiveness, good sensitivity, speed of separation</li></ul>capacity to analyze multiple samples simultaneously<br />complementary technique to HPLC.<br />HPTLC method is suitable for quantitation of impurities of alprazolam<br />Over pressured liquid chromatography (OPLC) a forced flow<br /> variant of HPTLC for determination of impurities of norethisterone by fluorimetric scanning after sulphuric acid spray<br />24<br />Classical approach….<br />
  48. 48. Electrophoresis and Related Methods<br /><ul><li>Capillary Electrophoresis</li></ul>CE is primarily suitable separation and quantification of charged impurities<br />example for use detection of ammonium ion<br />Useful for ammonium salts as impurities not contributed by residue of ignition and not detected by TLC or HPLC methods<br /><ul><li>Electrophoresis-related Chromatographic Techniques</li></ul>micro emulsion electro kinetic chromatography (MEEKC) and capillary electro chromatography (CEC).<br />Exp determination of impurities in ketorolac by MEEKC<br />Determination of the extremely toxic N-methyl-4-phenyl- 1,2,3,6 tetrahydropyridine impurity in pethidine<br />Performances of HPLC, CE and MEKC compared<br />Shortest run time (less than 5 min) was achieved by CE<br />Best sensitivity by MEKC<br />25<br />Classical approach….<br />
  49. 49. Classical approach….<br />Methods not Requiring Separation<br /><ul><li>Electro analytical methods never played important role in drug analysis due to limited selectivity and sensitivity
  50. 50. An example is the polarographic and UV methods
  51. 51. Exp Determination of benzophenone impurity in phenytoin impurities
  52. 52. and low concentration of phenytoin do not allow direct use of UV spectrophotometry in drug impurity profiling
  53. 53. UV Derivative spectrophotometry is advantageous
  54. 54. Used in drug degradation studies where the relative concentration of the degradants is in per cent range
  55. 55. Exp Determination of omeprazole sulphone in omeprazole
  56. 56. monitoring of the photo degradation of amlodipine to it pyridine degradation product </li></ul>26<br />
  57. 57. Methodology<br />Isolation of the impurity and off-line structure elucidation<br /><ul><li>HPLC-UV Studies
  58. 58. HPLC-MS Studies
  59. 59. GC-MS Studies
  60. 60. TLC-MS Studies
  61. 61. CE-MS Studies
  62. 62. MEKC-MS and CEC-MS Studies
  63. 63. HPLC-NMR Studies</li></ul>27<br />
  64. 64. Methodology<br />Isolation of the impurity and off-line structure elucidation<br /><ul><li>HPLC-UV Studies
  65. 65. HPLC-MS Studies
  66. 66. GC-MS Studies
  67. 67. TLC-MS Studies
  68. 68. CE-MS Studies
  69. 69. MEKC-MS and CEC-MS Studies
  70. 70. HPLC-NMR Studies</li></ul>28<br />
  71. 71. Modern approach<br /><ul><li>LC-MS</li></ul>Very often the molecular mass of the impurities obtainable by using the standard soft ionisation techniques<br />tandem mass spectrometer is available (HPLCMS/ MS) so, fragmentation of impurity can be used for the structure elucidation<br />Group exchanged on-line hydrogen to deuterium by using D2O as HPLC mobile phase component<br />powerful tool for identifying active hydrogen atoms (hydroxyl, thiol, amine, acid groups)<br />Useful for distinguishing between isomeric structures, difficult by product ion spectral data or accurate mass data<br />Separation power and usefulness of HPLC-MS as a tool for rapid identification of several impurities with different polarities can be improved by using the “heart-cut” technique<br />29<br />Methodology….<br />
  72. 72. Modern approach<br />LC-MS<br /><ul><li>HPLC-APCI-MS</li></ul> (HPLC – atmospheric pressure chemical ionisation – MS)<br /> for the identification of impurities co-eluting with the main component (acids, bases and zwitter-ions) not detectable by UV-DAD detector<br />30<br />
  73. 73. COMET <br />31<br />Modern approach<br />fully automated Comprehensive Orthogonal Method Evaluation Technology (COMET)<br />A Hewlett-Packard 1100 liquid chromatography system<br />(Agilent Technologies, Palo Alto, CA)<br /> equipped with<br /><ul><li>vacuum degasser quaternary pump
  74. 74. auto-sampler
  75. 75. UV diode array detector
  76. 76. HP-MSD modelD mass spectrometer
  77. 77. thermo stated column selector
  78. 78. six-position Cheminert valve is inserted on “D” line of quaternary pump to extend mobile phase selection
  79. 79. Both column switcher and valve are connected through contact closures
  80. 80. controlled electronically through the ChemStation software</li></li></ul><li>The mobile phase components are placed in the reservoirs<br />as follows: line A contains tetrahydrofuran<br /> line B contains acetonitrile<br /> line C contains methanol <br /> line D contains the aqueous solutions connected to the six-position Cheminert valve.<br /> The mobile phase valve positions 1–6 contain the aqueous solutions water, 0.1% formic acid (pH ∼2.1), 0.1% glacial acetic acid (pH ∼3.5) , 10mM ammonium acetate (pH 7.0), 0.1% trifluoroacetic acid<br />Extensive evaluation of 32 HPLC columns and 18 mobile<br />phase combinations were performed to provide a series of MS compatible methods, each with unique selectivity<br />Peak tracking with DAD<br />Manual peak tracking with mass spectra<br />32<br />COMET instrumentation<br />
  81. 81. 33<br />Modern approach<br />COMET instrumentation<br />fully automated Comprehensive Orthogonal Method Evaluation Technology (COMET)<br />
  82. 82. 34<br />COMET instrumentation<br />software<br />
  83. 83. 35<br />
  84. 84. 36<br />
  85. 85. 37<br />Modern approach<br />COMET instrumentation<br />
  86. 86. <ul><li>GC-MS </li></ul>Advantages<br /><ul><li>high-resolution separations
  87. 87. highly selective
  88. 88. sensitive detection
  89. 89. wide ionisation capabilities</li></ul>Anise oil as starting material for the synthesis of 4-methoxy amphetamine was screened by GC-MS for impurities<br />38<br />Modern approach<br />GC-MS<br />
  90. 90. TLC-MS<br />Separated spots on the plate could be subjected<br /> to direct matrix-assisted-laser-desorption <br /> ionization<br />Time-of-flight mass spectrometry (TLC-MALDI TOF<br /> -MS) <br /> no need to remove the spots from plate<br />Only wetting spot with methanol required to transfer <br /> the analyte from inside the silica gel to the surface<br />thus enhancing the MALDI-TOF-MS signal<br />39<br />Modern approach<br />TLC-MS<br />MEOH<br />TLC PLATE<br />
  91. 91. 40<br />Modern approach<br />Approaches for detection<br />MS<br />
  92. 92. majority of applications are from the fields of metabolite and natural product analyses<br />stopped flow HPLC-NMR technique (together with HPLC-MS) in identifying impurities in acarbose<br />41<br />Modern approach<br />LC-NMR<br />
  93. 93. conclusion<br />Impurity profiling is very important in the field of pharmaceutical analysis<br />Unidentified and potentially toxic impurities are health hazards and in order to increase safety, impurity should be identified<br />That’s all!!!<br />42<br />
  94. 94. references<br />Analysis of drug impurities Edited by Richard J. smith and michael L .Webb Blackwell publishing<br />ICH guidelines<br />Handbook of modern pharmaceutical analysis by satinder ahuja and stephan scypinski Volume III of SEPARATION SCIENCE AND TECHNOLOGY<br />Recent Advances in the Impurity Profiling of Drugs Dorottya Bartos and Sándor Görög* Current Pharmaceutical Analysis, 2008, 4, 215-230<br />Characterization of a trace by‐product of the synthesis of the protease inhibitor tipranavir GE Martin, RH Robins, FW Crow… - Journal of …, 1999 - Wiley Online Library<br /><br /><br /><br /><br />Automated peak tracking for comprehensive impurity profiling in orthogonal liquid chromatographic separation using mass spectrometric detection Gang Xue∗, Anne D. Bendick Journal of Chromatography A, 1050 (2004) 159–171<br />43<br />
  95. 95. 44<br />
  96. 96. 45<br />HYDERABAD<br />Thank you<br />