Effect of Cryogrinding on Chemical Stability results of our studies are in a complete opposition to Doherty and Perk’s findings, who claimed that no chemical degradation was found in any of their solid solutions (36). However, to prepare amorphous solid dispersions they have chosen a solvent method, in which amorphization mecha- nism is completely different than that of milling. It is also worth pointing out that they have prepared their solid dispersions in very mild conditions where no degradation occurred (T= 50°C). On the other hand, by carrying out the cryomilling experiment, we have exposed furosemide on extreme conditions and provided at the same time high- energy to the system. In addition, application of mechanical force might lower activation barrier for the chemical reaction to occur. This issue will be discussed later in this paper. The worsening of chemical stability of furosemide when ground with PVP and inulin as compared to when it was milled on its own is another quite surprising result. Our preliminary measurements indicate that greater chemical instability of cryomilled furosemide-PVP and furosemide- inulin solid solutions might be related to the formation of a hydrogen bonding between furosemide and excipient creating the possibility of proton migration across hydrogen bond. Interestingly, application of non-hydrogen bonding matrices improves significantly the chemical stability of cryomilled furosemide, but more detailed studies will be given elsewhere. To identify the main degradation product formed upon cryomilling (Imp.1), the mass spectrometry and Nuclear Magnetic Resonance techniques have been used. First, the liquid chromatography/mass spectroscopy technique (LC/ MS) was chosen, since it enables not only physical separation of individual impurities, but also can provide valuable information about the molecular weight, structure and/or quantity of a sample. The chromatogram was registered using a PDA detector and a mass spectrometer. The first detection showed the presence of Imp.1 in the eight minute of HPLC analysis (data not shown), but in the mass spectrum the corresponding peak was missing. This could be due to poor ionizability of the unidentified Imp.1 by means of Electrospray Ionization (ESI) technique (see Fig. 7a). Nevertheless, from the recorded mass spectrum we can initially accept the molecular weight of the unknown degradation product formed upon cryomilling as 250.1 daltons. This is confirmed by the presence of [M+H]+ ion of m/z 251.1 (Fig. 7b). However, to be sure that 250.1 is the exact mass of the unknown Imp. 1, another ionization technique was used. At first, preparative separation of it from the waste fractions was carried out using LC technique. Thus, a fraction containing Imp.1 was isolated and lyophilized. Next, the Matrix Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI- TOF-MS) was used to establish the mass weight of the 3229 impurity. This technique was employed mainly owing to its slightly different ionization mechanism. As a MALDI matrix, 2,5-dihydroxybenzoic acid (DHB) was used. In Fig. 7c, a positive-ion MALDI-TOF mass spectrum of the main degradation product formed during cryomilling is presented. The single peak with a m/z ratio of 176.9 corresponds to a signal from the DHB matrix with an Na+ adduct. Two very well-shaped signals with m/z ratios of 273.0 and 288.3 correspond respectively to the Na+ and K+ adducts of the unknown impurity of molecular weight 250 u. Consequently, the MS analysis has provided reliable information about the most probable molecular weight of the unidentified impurity. However, to avoid any specula- tion concerning determination of the chemical structure of Imp.1, the 1H NMR spectroscopy was employed. A 400- MHz 1H NMR spectrum of Imp.1 recorded at 30°C covering the range of 0.5 to 9.5 ppm is shown in Fig. 8. As seen in the 1H spectrum, except the peak of the DMSO solvent (a singlet at 2.5 ppm, labeled as A), the other three peaks appear at 6.9 ppm (B), 7.2 ppm (C), 8.3 ppm (D). These 1H chemical shifts were compared with those in the 1H NMR spectrum already known for crystalline furose- mide (37). Single and sharp signals at 6.9 ppm and 8.3 ppm were assigned to two aromatic protons, while the single and broad peak at 7.2 ppm was ascribed to sulfonamide NH2. As being distinct from the spectrum of crystalline furose- mide, in that of the unknown Imp. 1, there is no response from protons of the furan ring (6.35 ppm, 6.4 ppm and 7.6 ppm (37), aromatic -NH proton (8.6 ppm (37)), and that arising from the CH2 group (4.5 ppm (37)). Based on these results, we have concluded that the most probable degradation product formed upon cryomilling of furose- mide is 4-chloro-5-sulfamoylanthranilic acid (CSA) with molecular weight equal to 250 u. It should also be noted here that an identical 1HNMR spectrum has been reported for a thermal decomposition product of furosemide (38). The chemical structure of CSA is drawn in the inset in Fig. 8b. The fact that the main degradation product of high- impact cryomilling of furosemide is exactly the same compound as that formed due to thermal decomposition suggests that during extensive mechanical cryomilling, the temperature locally increases in an uncontrollable way so that amorphization occurs due to thermal melting followed by quenching. However, between each cycle, cool-down periods were programmed so that the heat of reaction was repeatedly removed and a low temperature environment was maintained. Still more so, the melting point of furosemide is >200°C, while the milling was carried out at a liquid nitrogen temperature (−196°C). For this reason, local growth of temperature caused by mechanical treat- ment and resulting in melting with decomposition of furosemide is actually unlikely to happen. Moreover,