Effect of Cryogrinding on Chemical Stability Table II Comparison of the Impurity Levels Formed During Cryomilling of Furosemide Analyte label Relative retention Furosemide time 3227 imp. 1 imp. 2 imp. 3 imp. 4 imp. 5 Furosemide imp. 6 imp. 7 imp. 8 imp. 9 imp. 10 0.26 0.35 0.76 0.85 0.92 1.00 1.10 1.40 1.42 1.49 1.64 Crystalline Cryomilled for 30 min Cryomilled for 60 min Cryomilled for 120 min Cryomilled for 180 min Amount of analyte [%] 0.05 0.70 – – – – – – – – 99.95 99.20 – – – – – 0.10 – – – – Total impurities Total impurities 0.05 0.80 1.68 0.06 – – 0.06 97.82 – 0.06 0.23 – 0.09 Total impurities 2.18 4.50 0.06 – 0.13 0.13 94.02 – 0.17 0.58 0.10 0.31 Total impurities 5.98 5.13 0.07 – 0.14 0.14 93.19 – 0.21 0.66 0.12 0.34 Total impurities 6.81 were recorded (Fig. 4, respective chromatograms as indicated by arrows). It can be seen that with increasing milling time, significant increment of the unknown Imp.1 assigned as the main degradation product was observed, and several minor peaks arise as well. A comparison of impurity profiles of the crystalline furosemide and that cryomilled for different periods of time is given in Table II and Fig. 5. Impurities below 0.05% are not reported. The quantity of Imp.1 was closely related to the duration of the cryomilling process, and when the substance was milled for 180 min it reached a level of 5.13%, while the total content of all impurities formed during this time was 6.81%. To the best of the authors’ knowledge, this particular behaviour of furosemide is the first evidence directly showing that cryomilling can be responsible for chemical degradation of the drug. Nevertheless, to investigate this phenomenon in more detail, it is necessary to perform additional studies. From the literature, it is known that by preparing solid dispersion it is possible to improve physical and chemical stability of amorphous systems (34). One of the methods which can be used to prepare solid dispersion is co-grinding with specially selected excipients such as polyvinylpyrroli- done (PVP) or polysaccharides. Polymer carriers or saccha- ride derivatives are likely to form amorphous solid solutions. Moreover, they usually have high melting and glass transition points so that their addition to the solid dispersion mixture increases its overall glass transition temperature Tg. Consequently, a considerable decrease in molecular mobility responsible for crystallization tendency and chemical reactivity of amorphous systems can be attained. As believed, the improvement of stability of amorphous mixtures resulted from specific interaction excipient—drug and formation of molecular alloys. Besides, more than 20 years ago, Doherty and Perk revealed that PVP tends to form hydrogen bond with furosemide in solid dispersion, which may account for its stabilization (35,36). Hence, in order to check whether it is possible to prevent chemical degradation of furosemide upon cryomilling, co- grinding has been performed. The mixtures of furosemide- PVP and furosemide-inulin (in weight ratios of 1:1 each) were submitted to mechanical treatment at liquid nitrogen conditions. After 60 min of cryomilling, a complete amorphization of both mixtures was proven by X-ray Fig. 5 Comparison of furosemide impurity profiles in relation to the duration of the cryomilling process.