Abstract
Raman spectroscopy is an established analytical method in the pharmaceutical industry. Micro-spectroscopic setups utilizing the backscattered signal are common in the analysis of solid samples such as tablets. However, backscattering arrangements typically share the disadvantage of collecting signal from a very small spot and thus potentially missing important information. Transmission Raman spectroscopy has been proposed as a solution to this problem. The present article introduces the concept of transmission Raman spectroscopy and discusses its applications in pharmaceutical analysis.
Background
Many pharmaceutical products are comprised of a rather small amount of the active ingredient and a number of excipients, which may act as stabilizers/protectors, fillers, or therapeutic enhancers. Due to the potentially small amount of the pharmaceutically active compound (PhAC), its distribution in the overall formulation may not be homogeneous. For example, a tablet could contain micrometersized aggregates of the PhAC embedded in a filler matrix. This poses a challenge to the chemical analysis of the product.
In many analytical applications in engineering and science, high spatial resolution is an important requirement. Therefore, methods are developed to obtain information from a very small area or volume. For example, spectroscopic techniques such as infrared, near-infrared, fluorescence, and Raman spectroscopy are capable of providing detailed chemical information. They are pushed towards high resolution so that objects and structures at the micrometer and sub-micron level can be resolved. Larger areas and volumes are scanned stepwise in order to characterize entire objects. This scanning, however, takes time and thus is not suitable for rapid screening of products. In order to overcome this problem, spectroscopic approaches based on transmission are very promising and have been developed for pharmaceutical analysis. Amongst the four techniques mentioned above, near-infrared (NIR) absorption and Raman scattering spectroscopy are typically the methods of choice for the application in transmission mode. Transmission (mid-)infrared spectroscopy is difficult to apply to products like tablets as the strong absorption in the mid-IR would call for additional sample preparation, e.g. to prepare thin pellets with a thickness significantly smaller than a millimeter. Fluorescence spectroscopy on the other hand usually uses ultraviolet light, which may not be able to penetrate the sample due to either strong absorption or scattering. NIR and Raman spectroscopy are better suited. In the last couple of years, however, there was a strong push for the development of transmission Raman methods as the Raman signal is more specific than the NIR spectrum. Moreover, Raman is advantageous in aqueous environments such as solutions, emulsions, and suspensions, which are common types of pharmaceutical products as well. In summary, transmission Raman spectroscopy (TRS) is a favorable method for pharmaceutical analysis. In the following, the concept of TRS for pharmaceuticals, which was pioneered by Matousek and Parker about a decade ago,1 will be introduced and discussed.
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Method
Raman spectroscopy is a means of vibrational spectroscopy, in which monochromatic light, typically from a laser source, is scattered by the molecules of a sample. The majority of the scattered photons exhibit the same wavelength as the incident light, but a small fraction is scattered inelastically, i.e. the Raman scattering. This means that an energy transfer takes place during the process and, consequently, the Raman signal is frequency shifted. Usually, the molecules are initially in the vibrational ground state and are transferred into a vibrationally excited state via a virtual intermediate state. The energy of the scattered photon is therefore reduced by the energy difference of the two vibrational states involved. As these energy differences are highly specific for any molecule, the Raman spectrum represents a molecular fingerprint. It can be employed to determine the chemical composition and quantify the individual components.
The common Raman micro-spectroscopy approach that utilizes the backscattered signal is illustrated in Figure 1a. A laser beam is focused onto the sample surface by a lens, e.g. a microscope objective, and the scattered signal is collected and collimated by the same lens. This arrangement is usually referred to as confocal microscopy. It enables recording spectra from a tiny measurement spot of about 1 μm diameter when appropriate spatial filtering is applied. In order to separate the laser and signal paths, a dichroic mirror reflecting the laser wavelength and transmitting the signal can be used. However, as mentioned above, a high spatial resolution or a pure surface measurement is not desirable for a rapid quality control during pharmaceutical production as it may miss important information. The transmission Raman approach illustrated in Figure 1 is a suitable alternative. In TRS, the laser travels through the entire sample, giving rise to Raman scattering in an extended volume. The signal emitted in direction of the laser beam is collected and analyzed. Sufficiently blocking the laser radiation is of special interest here in order to avoid severe interference. This can be achieved by using suitable dichroic mirrors and Notch filters. The wavelength of the laser needs to be selected carefully, because the sample must be transmissive for both the laser and the signal. Near-infrared radiation has been used in most TRS applications to date as it also reduces the likelihood of photodamage and fluorescence interference.
An important advantage of this approach is the large probe volume. Not only does this ensure sufficient sampling, which is helpful in analyzing heterogeneous samples, but also enhances the sensitivity of the measurement resulting in an improved limit of detection. The large probe volume contains a higher number of target molecules, which is proportional to the signal intensity. If sensitivity is not an issue, the measurement time can be reduced instead, which is beneficial for rapid screening of products. A detailed comparison between the backscattering and transmission approaches can be found in reference1.
TRS spectra can be evaluated with the traditional univariate and multivariate tools. In simple cases, it may be sufficient to calibrate the intensity of a given spectral signature against the concentration of a target compound. Complex matrices may be dealt with using chemometrics in terms of regression approaches such as partial linear least squares regression (PLSR), support vector machines (SVM), and artificial neural networks (ANN). The method of choice can be selected based on the complexity of the samples to be analyzed and the availability of reliable data for training the algorithm: the more complex the sample the larger the required training data set.
Applications
Transmission Raman spectroscopy has already found many applications in the pharmaceutical sector during the past couple of years. The majority of them has been summarized in several recent review articles.2-5 To give an idea, the list of applications includes:
- Monitoring of the settling dynamics of pharmaceutical suspensions.
- Quantification of the pharmaceutically active compound in model and commercial tablet and capsule formulations.
- Determination of the state in terms of polymorphs, crystalline and amorphous content, and co-crystals inside tablets and capsules.
- At-line quality control of content uniformity of pharmaceutical tablets.
- Process monitoring, e.g. during the growth of PhAC crystals.
The data evaluation in virtually all practical applications has been achieved using chemometrics because of the complexity of the data.
Conclusion
In conclusion, this paper described transmission Raman spectroscopy, which is an interesting approach for at-line and inline applications in the pharmaceutical and chemical industries. A key advantage over traditional Raman techniques is the large probe volume, which allows compensation for inhomogeneous distributions of the target compound as well as low concentrations. It also minimizes contributions from thin surface layers and capsule shells. Moreover, it is worth mentioning that Raman spectroscopy is a non-destructive method and, hence, tested specimen do not need to be disposed. With the first TRS instruments already on the market, the number of real world applications will rapidly grow in the foreseeable future.
Acknowledgment
The author gratefully acknowledges financial support from Deutsche Forschungsgemeinschaft (DFG) through grant KI1396/4-1.
References
- P. Matousek, A.P. Parker, Bulk Raman analysis of pharmaceutical tablets, Applied Spectroscopy 60 (2006) 1353-1357.
- J.A. Griffen, A.W. Owen, D. Andrews, P. Matousek, Recent advances in pharmaceutical analysis using transmission Raman spectroscopy, Spectroscopy 32 (2017) 37-43.
- K.A. Esmonde-White, M. Cuellar, C. Uerpmann, B. Lenain, I.R. Lewis, Raman spectroscopy as a process analytical technology for pharmaceutical manufacturing and bioprocessing, Analytical and Bioanalytical Chemistry 409 (2017) 637-649.
- K. Buckley, P. Matousek, Recent advances in the application of transmission Raman spectroscopy to pharmaceutical analysis, Journal of Pharmaceutical and Biomedical Analysis, Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 645-652.
- C.C. Corredor, C. Vikstrom, A. Persson, X. Bu, D. Both, Development and robustness verification of an atline transmission Raman method for pharmaceutical tablets using quality by design (QbD) principles, Journal of Pharmaceutical Innovation 13 (2018) 287-300.
Author Biography
Prof. Dr. Johannes Kiefer is Chair Professor and Head of the division Technische Thermodynamik at the University of Bremen, Germany. In addition, he is an Honorary Professor at the University of Aberdeen, Scotland, and he holds a guest professorship of the Erlangen Graduate School in Advanced Optical Technologies (SAOT) at the University Erlangen-Nuremberg, Germany. His research interests are the areas of developing and applying spectroscopic techniques for the characterization of advanced materials and processes.