1. 10/11/2005 1 ENGINEERING RESEARCH CENTER FOR S TRUCTURED O RGANIC P ARTICULATE S YSTEMS RUTGERS UNIVERSITY PURDUE UNIVERSITY NEW JERSEY INSTITUTE OF TECHNOLOGY UNIVERSITY OF PUERTO RICO AT MAYAGÜEZ Vibrational Spectroscopy for Pharmaceutical Analysis VIII. Applications of Raman Spectroscopy Rodolfo J. Romañach, Ph.D.

2. 2 An Investigation of Solvent-Mediated Polymorphic Transformation of Progesterone Using in Situ Raman Spectroscopy F. Wang, J.A. Wachter, F.J. Antosz, K.A. Berglund, Organic Process Research and Development, 2000, 4, 391-395. Progesterone has five known polymorphs Form I – melting point of 129.1 o C From II – melting point of 121.2 o C Immersible fiber optic probe and Raman spectroscopy used to monitor the transformation from form II to form I.

3. 3 Raman Spectra of Form I and II

4. 4 Crystallization Progesterone 2 grams in 25 mL of an organic solvent were added to 500 mL of distilled water, kept at constant temperature. Following completion of addition the solution was stirred at isothermal conditions for several hours. During this period the transition from Form II to Form I was monitored with Raman spectroscopy. Takes advantage of water being a poor Raman scatterer. F. Wang, J.A. Wachter, F.J. Antosz, K.A. Berglund, Organic Process Research and Development, 2000, 4, 391-395.

5. 5 Change in Spectra During Phase Transformation Bold spectra obtained at the beginning and end of crystallization.

6. 6 Determination of Drug Content in Tablets Using Raman Spectroscopy The progress of this application has been slowed by the very small size of the sampling area interrogated by Raman beam (making representative measurements difficult). Sample may be rotated to increase area in contact with beam ( J. Johansson, S. Pettersson, S. Folestad, J. Pharm. Biomed. Anal. 39 (2005) 510.

7. 7 Research Efforts M. Kim, H. Chung, Y. Woo, M. Kemper “ New reliable Raman collection system using the wide area illumination (WAI) scheme combined with the synchronous intensity correction standard for the analysis of pharmaceutical tablets ”, Analytica Chimica Acta, 2006, 579, 209 – 216. H. Wikstr ö m, S. Romero-Torres, S. Wongweragiat, J. A. Stuart Williams, E.R. Grant, and L.S. Taylor, “ On-Line Content Uniformity Determination of Tablets Using Low-Resolution Raman Spectroscopy ”, Appl. Spectrosc., 2006, 60(6), 672 – 681.

8. 8 Representative Measurement - Area P h AT System 6 mm Diameter Traditional Dispersive and FT-Raman 2-80- 500 microns 10 mm P h AT System 3 mm Diameter Slide – Courtesy Kaiser Optics.

9. 9 Monitoring of Lyophilization with Non-Invasive Raman Spectroscopy Lyophilization enhances product stability by removing water without submitting formulation to high temperatures that can cause drug degradation. Removal of water is important since water may serve as solvent for drug degradation reactions, or re-crystallization, and also participate in hydrolysis reactions. S. Romero-Torres, H. Wikström, E.R. Grant, L.S. Taylor, “Monitoring of Mannitol Phase Behavior during Freeze-Drying Using Non-Invasive Raman Spectroscopy, PDA Journal of Pharmaceutical Science & Technology, 2007, 61(2), 131 – 145.

10. 10 Experimental Replaced door of lyophilizer with an in-house built door that included a circular quartz window with a diameter of 4.5 cm to allow monitoring of a vial in the top sample shelf Raman spectra collected every five minutes, with total exposure time of 2 minutes. Used a small spot Raman instrument with beam diameter of 150 μm and a second with 6 mm beam diameter. All sample vials contained 2 mL of 10% (w/v) mannitol solution. Paper describes manner in which three different mannitol polymorphs and its hemihydrate were obtained.

11. 11 Freeze Drying Monitoring Set-Up Raman Probe Quartz Window BELL Quartz Window Freeze-Drier Shelf Timely measurements Intensity Units Raman Shift [cm -1 ] PhAT System

12. 12 Raman Shift [cm -1 ]  Mannitol is a common excipient  Has three reported anhydrate polymorphs (beta, alpha and delta)  Outcome will depend on the freeze drying history and concentration  High resolution Raman spectroscopy has proven to discriminate between mannitol polymorphs Two Theta [˚] Intensity Why Mannitol?

13. 13 Results Before freezing the mannitol solution provided a spectrum similar to that of amorphous mannitol. As freezing started and progressed the intensity of the mannitol peaks decreased until a minimum was reached. Minimum in mannitol peaks corresponded to ice crystallization (confirmed by monitoring product temperature). Mannitol peaks corresponding to crystalline mannitol were then observed, and their intensity increase. Comparison of spectra obtained during freeze drying to reference spectra of the polymorphs, showed that the β form crystallizes during lyophilization, and the hemihydrate in some instances.

14. 14 On-Line Slow Freezing Bands associated to mannitol solution disappear (black arrows) Hemihydrate mannitol Raman features emerge (red arrows) After Freezing 937 cm -1 967 cm -1 1054 cm -1 1044 cm -1 894 cm -1 873 cm -1 1019 cm -1 1024 cm -1 Raman Shift [cm -1 ] Before Freezing

15. 15 Reaction Monitoring Raman Spectroscopy May be Used to Monitor the Progress of a Reaction in the Synthesis of an Active Pharmaceutical Ingredient. R. Wethman, C. Ray, J. Wasylyk, “Development and Implementation of an In-Line Quantitative Raman Method for In-Process Pharmaceutical Monitoring”, American Pharmaceutical Review, 2005, 5(6), 57 – 63. Immersible Fiber Optic probe used to monitor progress of reaction.

16. 16 Reasons for Choosing Raman Spectroscopy for In-Line Reaction Monitoring Wethman and collaborators provide the following reasons: 1.Relative insensitivity of Raman spectroscopy to the water used in reactions. 2.The use of a fiber optic line between the probe and the spectrometer allowed the spectrometer to be located outside of the processing center and eliminated the need for it to be explosion proof. 3.The ability to insert the probe directly into the reaction chamber, avoided the need for developing a sampling system.

17. 10/11/2005 17 ENGINEERING RESEARCH CENTER FOR S TRUCTURED O RGANIC P ARTICULATE S YSTEMS RUTGERS UNIVERSITY PURDUE UNIVERSITY NEW JERSEY INSTITUTE OF TECHNOLOGY UNIVERSITY OF PUERTO RICO AT MAYAGÜEZ Raman Imaging & Mapping

18. 18 Imaging and Mapping Arise from the use of microscopy with Raman spectrometers. E. Smith and G. Dent, “Modern Raman Spectroscopy A Practical Approach”, John Wiley & Sons Ltd; (Chichester, United Kingdom), 2005, pages 47 – 50.

19. 19 Imaging A set of filters may be used, so that only radiation of the frequency range of interest passes through to the detector.

20. 20 Mapping Motorized samples stages (XYZ device) are used to move the sample. A spectrum is obtained from a small area, then the sample is moved to place a new area under microscope objective and a second spectrum is obtained. This is done repeatedly until spectra from a selected area are obtained. After collecting the samples, a spectral band is selected and a map of the intensity variation for that vibration is plotted. E. Smith and G. Dent, “Modern Raman Spectroscopy A Practical Approach”, John Wiley & Sons Ltd; (Chichester, United Kingdom), 2005, pages 47 – 50.

21. 21 Raman intensity ratio map of deposits from stages 3 (left) and 5 (right) of Andersen Cascade Impactor. Ratio of intensity of 1610 cm -1 (salbutamol) and 1662 cm -1 to BDP (beclometasone dipropionate). Orange light components relate to salbutamol and and black components to BDP. Scale bars represent 10 μm. The AAPS Journal, 2004, 6(4), article 32

22. 22 D. Fraser Steele, P.M. Young, R. Price, T. Smith, S. Edge, and D. Lewis, “The Potential Use of Raman Mapping to Investigate In Vitro Deposition of Combination Pressurized Metered- Dose Inhalers, The AAPS Journal, 2004, 6(4), article 32

23. 23 Advantages & Disadvantages Imaging vs. Mapping Imaging is rapid but only a particular region of the spectra is examined at one time and the resolution is limited. Mapping stores the entire spectrum, but is slow (time consuming).

24. 24 Acquisition Time & Area Sampled Images of approximately 2 x 2 mm 2 were acquired with a spatial resolution of 25 μm, with a typical acquisition time of 21 hours for Raman mapping and 3 hours in a similar NIR system. S. Ŝaŝiĉ, Appl. Spectrosc., 2007, 61(3), 239 -250.

25. 25 Further Reading Imaging & Mapping S. Ŝaŝiĉ, “An In-Depth Analysis of Raman and Near- Infrared Chemical Images of Common Pharmaceutical Tablets”, Appl. Spectrosc., 2007, 61(3), 239 -250.

26. 26 Confocal Raman Spectroscopy The microscope allows a change in the beam focus in the Z direction. The confocal arrangement consists of a pin hole in the focal plane. The pinhole avoids the collection of the majority of the other radiation that is not focused sharply in the plane of the pinhole. E. Smith and G. Dent, “Modern Raman Spectroscopy A Practical Approach”, John Wiley & Sons Ltd; (Chichester, United Kingdom), 2005, pages 45 - 47.

27. 27 Solid Dispersions of Drugs There is significant interest in formulations that increase the bioavailability of insoluble drugs. Some studies have indicated that solid dispersions of drugs increase the bioavailability of these compounds. Researchers have applied both NIR and Raman spectroscopy to these systems, since both allow their study without sample preparation. Interested in learning about drug distribution within the suspension, and the crystalline form of the drug.

28. 28 J. Breitenbach, W. Schrof, and J. Neumann, “Confocal Raman Spectroscopy: Analytical Approach to Solid Dispersions and Mapping of Drugs. Pharmaceutical Research”, 1999, 16(7), 1109- 1113.

29. 29 Authors found that ibuprofen band shifts to 1613 cm -1 in the PVP extrudates This band is observed a lower Raman shift in crystalline (lower energy) state. This spectral band was also observed at 1613 cm -1 when ibuprofen was dissolved. Band at 1613 cm-1 indicates that ibuprofen in the amorphous state, as confirmed by other analytical methods. J. Breitenbach, W. Schrof, and J. Neumann, Pharmaceutical Research, 1999, 16(7), 1109- 1113.

30. 30 Additional Information A He:Ne laser at 633 nm, was used without any fluorescence problems. Recorded ratio of 1613 cm -1 band from ibuprofen and 1673 cm -1 band from PVP in area of 45 x 25 micrometers (200 measurements) to evaluate homogeneity. Each measurement was performed with a resolution of 2 μm 3. The method did not show reveal ibuprofen crystalline aggregates in the extruded material. J. Breitenbach, W. Schrof, and J. Neumann, “Confocal Raman Spectroscopy: Analytical Approach to Solid Dispersions and Mapping of Drugs. Pharmaceutical Research”, 1999, 16(7), 1109- 1113.