Raman spectrocopy of mannitol

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Mannitol is a polyol, a molecule similar to sugar but is unable to form a ring structure as it only contains hydroxyl functional groups. It is widely used as an artificial sweetener and in the pharmaceutical industry it is widely used as a cryoprotectant to preserve the functionality of proteins during freeze drying or sub zero storage. In order to understand how cryoprotectants protect the active ingredients it is important to understand their physical behaviour during the processes the formulation are subjected to. Often a range of cryoprotectants are tested and the ‘best’ is chosen based on the extent to which the active ingredient has retained its functionality. Ideally it would be preferable to understand the process in order to allow design of the formulations, reaching a more rapid optimisation.

Mannitol is an unusual cryoprotectant in that it forms polymorphic crystals during freezing, in contrast to other common cryoprotectafrozen drop mannitol solution annealing crystallisationnts such as sucrose and trehalose. Anhydrous mannitol can form a number of different crystalline structures, alpha, beta and delta (gamma was found to be a mixture of polymoprhs, not a true one) as well as amorphous, and there is the added complication of a hemi hydrate form too.

For mannitol it was observed that frozen solutions of mannitol formed an opaque ring round the edge of the frozen drop (Fig 1). The samples were assessed using X-Ray crystallographic techniques and found that the samples were crystallising the mannitol one specific polymorph, beta. The issue with the X-ray methods were that it was slow (unable to determine any kinetic changes during annealing) and could not be used to indicate the localisation of the crystalline material.

Raman spectra Mannitol polymorphism alpha beta delta amorphous molten solution

Raman spectroscopy was chosen to shed further light on this issue as it is sensitive to the physical structure of the sample, including crystalline packing and conformational changes within the individual molecules. For this study only the CH an OH stretching regions were analysed as these contained sufficient information to discriminate the polymorphs and to provide insight into the changes in ice structure associated with the annealing. The Raman signals for each of the mannitol polymorphs is unique, as is the signal from amorphous and hemi-hydrate (Figure 2).  In fact the amorphous signal contains some structure, with a doublet of bands at the high and low wavenumber sides of the CH region, dependant on the temperature of the sample. All amorphous manitol share a dominant broad bell shaped curve (labelled as amorphous/common). At high temperatures a doublet of peaks appears at the low wavenumber side of the amorphous curve, with the modes arising from the hydroxyls shifting to a very high position. The position of OH stretching modes is inversely related to the strength of the hydrogen bonding, suggesting that in the molten mannitol the hydrogen bonding between the molecules is very weak. In contrast the OH modes in the solution phase amorphic mannitol exhibit a shift towards a lower wavenumber, and a considerable increase in intensity. This would suggest stronger hydrogen bond with an increase in OH modes involved - i.e. the formation of a solvation shell.

Principal component analysis of the annealing dataset revealed two main principal components associated with the mannitol, as shown in Figure 3. The main mannitol PC was dominated by the beta form, but also showed significant contribution from the delta form. The next PC was equivalent to the subtraction of the signal arising from the beta polymorph from that of the delta polymorph, revealing that PC measured the balance of these two polymorphs. The other polymorphic forms were present in much smaller quantities. Figure 4 shows the trends with annealing time for these two PCs. It was known from calorimetry that some kind of change of physical structure change was occurring over the first 40 minutes (see bottom trends on Figure 4), which then tapered off. The Raman results unequivocally demonstrate that this is not due to crystallisation from amorphous mannitol, but that the mannitol spontaneously crystallises, and does so predominantly in the delta form (PC4 centre data). The thermal events observed in the DSC experiments do in fact arise from a delta to beta polymorphic transition.Raman spectroscopy mannitol polymorphs beta delta DSC annealingRaman spectroscopy mannitol polymorph beta delta principal component analysis

Curiously, the edge trend differs significantly from the centre of the drop, with almost all the mannitol crystallising as beta form initially, then some delta creeps in, presumably as delta crystals from the interior are pushed outwards. Ultimately though, all the mannitol gradually converts to the beta form (based on overnight annealing, data not shown).

The top row in Figure 5 shows an animation of the distribution changes of the crystalline mannitol (green) in the frRaman spectroscopy distribution map mannitol annealing beta delta crystallisation annealingozen drop as it is annealed. At the edge there is a clear directionality in the trend, whereas in the centre the changes are more chaotic and are non-directional as would be anticipated from random diffusion.   The second row highlights the conversion from delta to beta and it is readily appreciable that in the first frame (03 min) the edge is a mixture of red and green (delta and beta), while the centre is almost exclusively delta, in agreement with figure 4 above. Over time the delta converts to beta, reflected in a conversion from red to green in Figure 5 second row.

This data demonstrated the first direct measurement of mannitol crystallisation behaviour in a frozen solution, incorporating assessment of time dependency, spatial distribution, diffusion and polymorphic transitioning. In addition to the insights into the mannitol the Raman data also unveiled key discoveries regarding the role of the solvation shell in the process

Further Reading

1.            Beattie, J. R., et al., European Journal of Pharmaceutics and Biopharmaceutics 2007, 67, 569-578.

2.            Campbell Roberts, S. N., et al., Journal of Pharmaceutical and Biomedical Analysis 2002, 28, (6), 1135-1147.

3.            Burger, A., et al., J. Pharm. Sci. 2000, 89, (4), 457-468.

4.            Reinhard, V., Applied Spectroscopy 2005, 59, (3), 286-292.

5.            Telang, C., et al., Pharmaceutical Research 2003, 20, (12), 1939-1945.

6.            Okumura, T.; Otsuka, M., Pharmaceutical Research 2005, 22, (8), 1350-1357.

7.            Vehring, R., Applied Spectroscopy 2005, 59, (3), 286-292.

8.            Roy, A., et al., Free Radical Research 2004, 38, (2), 139-146.

9.            Auer, M. E., et al., Journal of Molecular Structure 2003, 661, 307-317.

10.          Roberts, S. N. C., et al., Journal of Pharmaceutical and Biomedical Analysis 2002, 28, (6), 1149-1159.

11.          Braun, D. E., et al., International Journal of Pharmaceutics 2010, 385, (1-2), 29-36.

12.          Hulse, W. L., et al., Drug Development and Industrial Pharmacy 2009, 35, (6), 712-718.

13.          Ye, P.; Byron, T., American Laboratory 2008, 40, (14), 24-27.


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