Effect of Cryogrinding on Chemical Stability have found that within just the first 960 min (~16 h), the degradation rate of the cryomilled material was roughly about two times higher than that of the crystalline one. As a matter of fact, the cryomilled furosemide undergoes more rapidly acid hydrolysis, and its chemical reactivity is certainly greater than that of the crystalline substance. However, this argument cannot be considered as the dominant reason for the chemical degradation of the cryoground material. Moreover, if degradation of furose- mide were only the result of amorphization (i.e. the amorphous form of cryomilled furosemide were so meta- stable that it decomposed immediately after being formed), after the complete transformation crystalline to amorphous solid, we shouldn’t observe any differences in the total impurities amount for all the cryomilled and amorphous samples. At this point, it should also be noted that up to the present, there are no reports in the pharmaceutical literature clearly showing that cryomilling, as a mechanical process of particle size reduction, might activate and aid chemical degradation of pharmaceuticals. However, the fact that the prolonged high-energy milling can trigger not only structural changes, but also can activate and signifi- cantly accelerate chemical process is nothing new. This type of reaction is well-known and recognized as mechanochem- ical processes. Mechanochemical reactions have very interesting applications, especially in inorganic synthesis (39,40). For example, by ball milling, transition metals such as Ni or Co and their oxides can be synthesized. Furthermore, mechanically activated chemical reactions allowed production of semiconductors, alloys or spinels with no need of high temperature conditions required to initiate them otherwise. Originally, chemical processes taking place during mechanical treatment were ascribed to the mechanical energy generated and converted into local heat. However, the resulting products of melting and mechanically initiated reactions might be completely different (40). For this reason, chemical processes occurring in the mill cannot be identified only with an increase in temperature even up to the melting point. Interestingly, in organic chemistry these types of reactions are less likely to proceed and are practically absent. Some signs of chemical transformations caused by mechanical mill- ing were reported for the drug piroxicam (18). In that case, proton transfer (tautomerisation) accompanied solid- state amorphization. In newly designed high-energy mills the impact generated during the grinding process might be orders of magnitude greater than that achieved in traditional devices. Thus, obviously not only particles sizes can be reduced much faster and easier, but also some chemical reactions which probably would never be accessible by traditional milling can be activated therein. Mechanical milling facilitates chemical 3233 reactions due to the randomness of the flattening process. This provides (as grain size decreases) repeatedly new reaction interface. Moreover, at cryogenic temperature, pharmaceuti- cal materials become hard and brittle. Since mechanical treatment of a substance in the mill creates a great amount of stress, such material is obviously more susceptible to any physical and chemical changes. Bearing in mind the above, it is obvious that the milling device must be treated in any circumstances as a chemical reactor where under favourable conditions chemical processes can be mechanically provoked. It is also worth mentioning that in contrast to typical thermochemical reactions, for chemical reactions initiated and promoted by high-impact mechanical treatment, a considerably lower activation energy is required. For exam- ple, the activation energy of thermochemical reaction of iron oxide formation was found to be 54 kJ/mol, while the same reaction caused by mechanical treatment required only 0.7 kJ/mol (40). Rarely have any thermally activated chemical reactions taken place at a very low temperature; if so, it involves mostly inorganic compounds. The rate of thermochemical reaction strongly depends on the temperature and usually increases with increasing temperature. Thus, in the case of cryogenic grinding of furosemide, thermally activated chemical decomposition is simply impossible. On the other hand, the energy required for releasing the mechanochem- ical reactions is applied to the solid state through mechanical process, not thermally. This would explain why decomposition occurs during high-energy milling even at liquid nitrogen temperature. Furthermore, the degradation pathway of crystalline and amorphous furosemide in solid state as well as in aqueous solution involves formation of CSA. Conversion of furose- mide to CSA proceeds by cleavage of the C-N bond (see Scheme 1). As reported by Beyers et al., the experimentally measured activation energy for thermal decomposition of furosemide is 47.7 kcal/mol, while typically the energy barrier for C-N bond cleavage is about 59 kcal/mol (38). As they speculated in the case of furosemide, the weakening of the C-N bond is the consequence of negative inductive effect of the furan ring on the one hand and delocalization Scheme 1 Degradation pathway of the cryomilled furosemide.