Quantifying the amorphous phase with nearinfrared and Raman spectroscopic techniques Marja Savolainen Department of Pharmaceutics and Analytical Chemistry Dias 1 Physical Pharmacy Symposium 29.1.2009
Outline Introduction Case study I Building quantification models for different solid-state forms of indomethacin II Evaluation of assay errors III Dissolution behaviour of amorphous IMC in waterethanol solution (50%(w/w)) Conclusions Dias 2 Physical Pharmacy Symposium 29.1.2009
Vibrational spectroscopy Virtual energy states MIR NIR v = 4 v = 3 v = 2 v = 1 v = 0 Rayleigh Stokes Anti-Stokes Raman MIR and NIR spectroscopy Absorbance techniques Change in dipole moment Raman spectroscopy Scattering technique Change in polarizability Dias 3 Physical Pharmacy Symposium 29.1.2009
NIR and Raman spectrosocpy Aspect Spectral region Selection rule Typical vibrations Spectral intensity Resolved bands Typical sampling volume Sensitivity to water Sensitivity to hydrogen bonding Interfering phenomena Near-Infrared 12300-4000 cm -1 dipole moment Overtones, combination bands weak no large strong very strong specular reflection Raman 4000-200 cm -1 polarisability Fundamental intramolecular vibrations weak some small very weak medium fluorescence, heating Fiber optics Problematic samples long (~300 m) f (watery) long (~100 m) g coloured Adapted from Jørgensen et al., J. Pharm. Sci. submitted Dias 4 Physical Pharmacy Symposium 29.1.2009
Amorphous vs. crystalline solid Drug molecule Water, solvent Polymorph I Polymorph II Hydrate, solvate Amorphous In amorphous material more variation in the intermolecular interactions Intramolecular interactions are also affected Dias 5 Physical Pharmacy Symposium 29.1.2009
NIR and Raman spectra of indomethacin Amorphous form Absorbance Intensity Metastable (α) form Stable (γ) form 1500 1600 1700 1800 1900 Wavelength / nm 1000 1200 1400 1600 Raman shift / cm -1 Spectral differences are more vague than between crystalline forms Merging, broadening, and decrease in intensity of peaks Multivariate techniques, e.g. PLS regression analysis The complete spectra instead of just few peaks can be used for the analysis Dias 6 Physical Pharmacy Symposium 29.1.2009
Building quantification models for different solidstate forms of indomethacin Dias 7 Physical Pharmacy Symposium 29.1.2009
Materials and methods Indomethacin (IMC) γ-imc α-imc Amorphous IMC prepared by quench cooling Ternary mixtures in triplicate in various concentrations NIR and Raman spectroscopy Each samples was analyzed three times Quantification model using PLS regression analysis The spectra were SNV corrected and mean centered before PLS analysis Dias 8 Physical Pharmacy Symposium 29.1.2009
Building up the PLS model NIR Raman Spectral region Preprocessing None SNV MSC 1 st derivative 2 nd derivative Scaling None Unit variance Mean centering Calibration model (2/3 data) Testing of model (1/3 data) Dias 9 Physical Pharmacy Symposium 29.1.2009
NIR spectroscopy R 2 Y = 98.9% Q 2 Y = 98.9% RMSEC = 2.7 (amorphous) 3.2 (α-imc) 3.8 (γ-imc) RMSEP= 4.4 (amorphous) 3.2 (α-imc) 3.6 (γ-imc) Heinz et al., Eur. J. Pharm. Sci. 32(3): 182-192, 2007 Dias 10 Physical Pharmacy Symposium 29.1.2009
Raman spectroscopy R 2 Y = 96.5% Q 2 Y = 96.4% RMSEC = 5.0 (amorphous) 5.9 (α-imc) 5.3 (γ-imc) RMSEP= 6.2 (amorphous) 5.5 (α-imc) 6.2 (γ-imc) Heinz et al., Eur. J. Pharm. Sci. 32(3): 182-192, 2007 Dias 11 Physical Pharmacy Symposium 29.1.2009
Evaluation of assay errors Dias 12 Physical Pharmacy Symposium 29.1.2009
Evaluated errors Source of error Instrument variability Instrument reproducibility Intra-day reproducibility Inter-day reproducibility Sample effects Sample mixing Sample packing Sample positioning Particle size Fluorescence Overall method error Evaluation Five consecutive measurements were performed on a sample. A sample was measured once every hour for a period of five hours. A single measurement was performed on a sample each day over five days. A sample was divided into five sub-samples and each of them measured once. A sample was measured five times and repacked after each measurement. Five consecutive measurements were performed on a sample that was placed on a stationary sample holder and repositioned after each measurement. A second sample of a particle size range form 125 µm to 250 µm was prepared and analyzed by acquiring five consecutive spectra Five consecutive Raman spectra of a sample were recorded without initially performing photochemical bleaching. Four independent mixtures of the same composition (1/3 : 1/3 : 1/3) were prepared and analyzed by performing five measurements on each sample over one day. Dias 13 Physical Pharmacy Symposium 29.1.2009
Materials and methods Centerpoint mixture 1/3 amorphous IMC, 1/3 α-imc,1/3 γ-imc Particle size <125 µm NIR spectroscopy Rotating sample holder Raman spectroscopy Rotating sample holder To minimize the effect of fluorescence the samples were irradiated with the laser for 1 h before measurements Dias 14 Physical Pharmacy Symposium 29.1.2009
Results Source of error a Instrument response Instrument reproducibility Intra-day variability Inter-day variability Sample effects Sample positioning Sample packing Sample mixing Particle size Fluorescence Overall method error NIR spectroscopy (RSD%) α-imc 1.5 4.8 7.8 5.5 2.3 6.2 3.9 NA 6.7 γ-imc 0.7 6.0 12.4 4.5 0.8 6.9 5.0 NA 8.4 a n=5, except for overall method error n=20. Amorphous IMC 2.2 3.6 8.0 10.7 3.2 5.1 5.2 NA 13.0 Raman spectroscopy (RSD%) α-imc 0.7 5.0 2.8 11.4 1.4 3.3 1.3 5.2 6.3 γ-imc 0.8 4.6 7.3 8.4 2.3 2.7 0.4 6.8 8.8 Amorphous IMC 1.0 1.8 7.4 19.1 3.3 2.7 0.8 2.5 12.0 Heinz et al., Eur. J. Pharm. Sci. 32(3): 182-192, 2007 Dias 15 Physical Pharmacy Symposium 29.1.2009
Dissolution behaviour of amorphous IMC in water-ethanol solution (50%(w/w)) Dias 16 Physical Pharmacy Symposium 29.1.2009
Solid-state analysis during dissolution Complete picture of the dissolution process Solid phase analysis with Raman spectroscopy Liquid phase analysis with UV/Vis spectroscopy Solid sample Molten IMC Dissolution medium Water-ethanol solution (50% (w/w)) Savolainen et al., Eur. J. Pharm. Biopharm. 71: 71-79, 2009 Dias 17 Physical Pharmacy Symposium 29.1.2009
Model transfer The multivarate calibration model for Raman spectrocopy did not work as such: The calibration samples differed from the melted, glassy dissolution test samples. Amorphous samples prepared by melting were included in the model. Dias 18 Physical Pharmacy Symposium 29.1.2009
Dissolution of amorphous IMC in water-ethanol solution (50% (w/w)) Dias 19 Physical Pharmacy Symposium 29.1.2009
Conclusions Both NIR and Raman spectroscopy can be used to quantify the amorphous phase. The method of choice depends on e.g. sample properties, environmental conditions, whether the quantification is done in process environment. PLS regression analysis can be used for quantification, when there are no well resolved peaks in the spectra. When building quantification models major source of error comes from inhomogeneous mixing of the samples. Increasing the samples volume will improve the model performance. When a quantification model is used to analyze several different processes, some model adjustment might be needed. Dias 20 Physical Pharmacy Symposium 29.1.2009
Acknowledgements Dr. Andrea Heinz Dr. Clare Strachan Dr. Jaakko Aaltonen Doc. Leena Peltonen Prof. Thomas Rades Prof. Jouko Yliruusi Dias 21 Physical Pharmacy Symposium 29.1.2009