Johannes Kiefer, Hesamodin Hosseini Ghahi, and Claudia C. Rullich - University of Bremen, Engineering Thermodynamics and MAPEX Center for Materials and Processes
Vibrational Spectroscopy in terms of Raman and infrared spectroscopy is a powerful toolbox for the analysis of solutions containing Active Pharmaceutical Ingredients (API). In this article, we provide a brief introduction to selected techniques that are capable of quantifying the API content, of shedding light at the physicochemical dissolution mechanisms, and of distinguishing API enantiomers.
Solutions in which an active pharmaceutical compound is dissolved in a liquid are very common throughout the entire life cycle of a pharmaceutical product. When the API is produced in a chemical or biotechnological process there are most likely a number of steps during which it occurs as a solute either in water or in an organic solvent. Producing pharmaceuticals from algae in an aqueous environment and utilizing the anti-solvent precipitation concept are prominent examples. In some cases, the pharmaceutical product is sold as a solution or as a powder, which has to be dissolved prior to injection. And last but not least, API dissolution in body fluids is an important feature with respect to drug delivery and excretion of the metabolic waste as well. Therefore, it is needless to say that the analysis of dissolved pharmaceutical compounds is an important task and selecting the appropriate analytical method in a given application is the key to get the desired answers.
While chromatographic and mass spectrometric approaches are still very common in the field of pharmaceutical analysis, the various methods of the vibrational spectroscopy toolbox have become more and more popular. This is particularly true for FTIR and Raman spectroscopy. Both methods provide a molecular fingerprint of the sample by probing the vibrations of the covalent bonds. This fingerprint contains a multitude of information including the qualitative and quantitative chemical composition of the sample, but also details about intermolecular interactions, e.g. between a solvent and a solute. Therefore, these methods are perfectly suited for analyzing API solutions
Although both FTIR and Raman spectroscopy are vibrational methods, their spectra look different as the underlying physical principles are different: absorption in FTIR and scattering in Raman. In absorption, light (in this case infrared light) is sent to the sample, which then absorbs certain wavelengths and transmits others. The transmitted light is spectrally analyzed and reveals the fingerprint signatures of the sample. By contrast, in Raman scattering a laser is pointed to the sample and the scattered light is analyzed in a spectrometer. This scattered light carries the same information but through a frequency difference between the laser wavelength and the molecular vibrations of the sample. Figure 1 illustrates both effects in an energy level diagram. It becomes clear, that both methods probe the same molecular transitions but take different paths. As a consequence, the spectra appear similar but exhibit distinct differences, as there are transitions that are exclusively IR-active or Raman-active depending on the dipole and polarizability properties, respectively. For detailed descriptions of the methods, the reader is referred to the common text books, see for instance.1-4
FTIR and Raman spectroscopy have a number of great features for pharmaceutical analysis. Depending on the specific technique applied and on the instrument employed, they offer a rapid and nondestructive full chemical analysis of the sample. In many cases, neither sampling nor sample preparation is necessary. In order to illustrate how vibrational spectra of a typical API dissolved in a common organic solvent look like, Figure 2 shows the FTIR and Raman spectra of the nonsteroidal anti-inflammatory drug Naproxen (racemic mixture) dissolved in dimethyl sulfoxide (DMSO). The solution spectra are, of course, dominated by the signatures of the solvent. Nevertheless, the features of Naproxen are clearly visible.
Identifying the individual compounds in the vibrational spectra of a solution can be rather straightforward. For instance, the spectra can be compared to library data to find out which components are contributing to the signal. Alternatively, the signatures can be assigned to their corresponding functional groups to ultimately yield the compounds. In complicated cases, chemometrics, sophisticated cross-correlation techniques, and artificial intelligence approaches can help to analyze the data. For completeness, we would like to highlight that recent developments even added features like enantiomeric discrimination, for example by the enantioselective Raman (esR) method;5 the esR technique is particularly interesting for solution analysis.
When the compounds in the solution are known and the task is to determine their concentration, a variety of methods can be used for data analysis. In simple systems comprising only a small number of components, each compound may exhibit a clearly distinguishable spectral signature, the intensity of which increases with the concentration in a predictable manner. In more complex systems, where many compounds are present and/or their spectral signatures heavily overlap with each other, more sophisticated regression techniques can be employed to obtain the composition details. Again, the enantioselective Raman method is interesting in this context as it offers determination of the enantiomeric ratio and enantiomeric excess.6
While qualitative and quantitative analysis by FTIR and Raman spectroscopy have been state-of-the-art for quite some time, their capability of analyzing intermolecular interactions is not often utilized in non-academic labs. Nevertheless, the information is in the data and can be evaluated to better understand the physicochemical behavior like, e.g., the molecular dissolution mechanism. The formation of intermolecular interactions like hydrogen bonds lead to small modifications of the covalent bonds (due to a charge transfer) and hence affect their vibrational frequencies. Such effects can be observed as peak shifts in the FTIR and Raman spectra. A detailed data analysis allows the identification of interaction sites as well as the assessment of the interaction strength. Such information can then be used to optimize the solutions stability and/or the dissolution process, e.g. by using a different solvent or maybe adding a co-solvent or by selecting more appropriate process conditions.
Conclusion
In this article, we have introduced FTIR and Raman spectroscopy for the analysis of active pharmaceutical compounds dissolved in a liquid. The two vibrational spectroscopic methods offer great features for qualitative and quantitative analyses in the pharmaceutical industry. They represent nondestructive and fast methods that can provide a comprehensive chemical characterization of a sample. Recent developments have even added enantioselective capabilities.
References
- P. Vandenabeele, Practical Raman Spectroscopy, Wiley, Chichester, UK, 2013.P.R. Griffiths, J.A. De Haseth, Fourier Transform Infrared Spectrometry, 2nd ed., Wiley, Chichester, UK, 2007.
- P.J. Larkin, Infrared and Raman Spectroscopy: Principles and Spectral Interpretation, Elsevier, Amsterdam, NL, 2011.
- K. A. Bakeev, Process analytical technology: spectroscopic tools and implementation strategies for the chemical and pharmaceutical industries. John Wiley & Sons: 2010.
- C.C. Rullich, J. Kiefer, Principal component analysis to enhance enantioselective Raman spectroscopy, Analyst 144 (2019) 2080-2086.
- C.C. Rullich, J. Kiefer, Chemometric analysis of enantioselective Raman spectroscopy data enables enantiomeric ratio determination, Analyst 144 (2019) 5368-5372
Author Biographies
Johannes Kiefer is professor of engineering thermodynamics. His research interests include the development of optical spectroscopic methods for engineering and life science applications.
Hesamodin Hosseini Ghahi is a PhD student at the University of Bremen and develops enantioselective Raman techniques for pharmaceutical analysis.
Claudia C. Rullich is a postdoctoral fellow at the University of Bremen working in the field of analytical chemistry for pharmaceutical applications.
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