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23 October 2019

Agata Papa, Lab Manager

Polymorphs discrimination using Morphologically-Directed Raman Spectroscopy

Several pharmaceutical ingredients (API) tend to crystallize in different crystalline (polymorphic) packings. In a pharmaceutical formulation, the identity or the possible conversion of the API could alter the characteristics and the efficacy of the pharmaceutical form, in particular leading to a different bioavailability. Established analytical techniques are often unable to provide both separation and identification of species in a mixed system, as they are used to probe chemistry (bulk spectroscopy), or individual particle characteristics (optical and electron microscopy). Morphologically Directed Raman Spectroscopy or MDRS is a powerful technique that combines automated particle imaging with Raman spectroscopy to provide particle size, shape and chemical identification in a single analysis.Combining techniques capable of characterize particulates with absolute chemical specificity provides a new method into describing the complicated mixtures common in today’s competitive pharmaceutical market.

In this work we show how polymorphism can be successfully recognized and characterized by using MDRS (Morphologically-Directed Raman Spectroscopy).

We have analyzed in Alfatestlab two polymorphic mixtures of an API provided by our customer (Sample 1 and Sample 2, synthesized with different protocols ) using MDRS technique (Morphologi 4ID – Malvern Panalytical) to characterize them in terms of particle size, shape and chemical identity. The polymorphic mixture consist of the Type A and Type B polymorphs.

Morphological distributions of the two samples are shown in Figure 1. Results show that samples have different particle sizes, both in number and in volume. No differences were found in the shape as can be seen in the “aspect ratio” distribution, which indicates the ratio between particle width and length.

Figure 1. Size and shape parameter distributions for  Sample 1 (blue) and Sample 2 (orange) particles. Left: Number size distribution; Center: Volume size distribution: Right: Aspect ratio distribution

Chemical analysis

Raman spectra of individual particles were collected for each sample and compared to a library of Reference spectra. Reference spectra for Raman analysis were obtained from pure materials. Spectra were collected with a 785 nm laser (20 mW) illuminating a 2 μm spot size at 50x magnification. The results of correlation against the library of pure polymorph spectra revealed the presence of the two polymorphs in both samples and allowed us to obtain their proportions as number percentages (Figure 2).


Figure 2. Classification based on chemistry and quantification of the polymorphs A and B proportions in the two samples.

Sample 1 shows about 35% of Polymorph A and 65% of B, while Sample 2 shows about 14% of Type A and 86% of B. A correlation score of greater than 0.8 was required for a positive identification as either Polymorph A or Polymorph B.

Examples of particles with different morphology analyzed by Raman are shown in Figure 3.


Figure 3: Raman spectra collected from example particles and correlation score obtained by spectra comparison with reference spectra used to create the library


Morphologically Directed Raman Spectroscopy MDRS provided a suitable solution to chemically identify multiple polymorphs in a sample and determine the relative proportions of each particle type.  Moreover, a morphological description of each chemically-identified component was then generated in terms of size and shape distributions, using the data from the automated image analysis.

 These results confirms that the two different synthesis protocols have led to different proportions of polymorphisms in the sample and could generate different behaviors of the API in the final formulation.

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