3224 LC/MS Analysis applied. After 10 min the composition of the mobile phase was changed gradually up to 80% acetonitrile: 20% acetic acid, which was maintained for the last 10 min. LC/MS system consisted of an Agilent 1100 series LC system (Agilent Technologies, Santa Clara, CA, USA), interfaced with MS-Bruker Daltonics HCTultra with a turbo electrospray ionization detector (ESI). C18, Phe- nomenex C18 Luna column (150 × 4.6 I.D. particle size 5 μm; 100 Å) was used to determine sample degradation products. Furosemide samples were dissolved in acetoni- trile, and the solution was introduced into the ESI source by continuous infusion using a syringe pump at a flow rate of 0.5 ml/min. The ESI source was operated at 4 kV (in positive and negative polarization), with the capillary heater at 350°C and sheath gas pressure of 30 psi. Mass spectra in the negative and positive ion modes were acquired over the range m/z 50–500. The LC experi- ments were carried out in the same conditions as by HPLC method. Preparative Separation by High Performance Liquid Chromatography (HPLC) The separation was carried out with Kontron Instruments (Milan, Italy) using a thermostated (25°C) Phenomenex C18 Columbus column (250×10.00 I.D. particle size 5 μm; 100 Å) to separate impurity from waste fractions. Detection wavelength was 230 nm. The elution conditions were a linear gradient of acetonitrile and 0.1% trifluoroacetic acid (5–40% of acetonitrile) in 60 min with a flow rate of 4.0 ml/min. After 60 min the composition of the mobile phase was changed gradually up to 90% acetonitrile for the last 15 min. Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF-MS) MS Mass Spectrometry The MALDI-TOF mass spectra ware recorded on a Bruker Biflex III (MA, USA) instrument in a reflector mode. The ion source was set to 19.0 kV with the lens and reflector voltage of 9.6 kV and 20.0 kV, respectively. For the analysis, the DHB (2,5-dihydrobezoic acid) matrix was used. Laser excitation was at 337.1 nm with 27% attenuation. Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy The freeze-dried sample, collected after preparative sepa- ration by means of HPLC, was diluted in DMSO solvent. 1H NMR spectrum was registered on a Varian Mercury Adrjanowicz et al. 400 MHz spectrometer. The acquisition time and delay times were 2 and 1 s, respectively. RESULTS AND DISCUSSION Physical Characterization of Cryomilled Furosemide Figure 1a shows the X-ray diffraction pattern of the intact, non-milled furosemide, which was taken at room temper- ature and used as a reference. The sharp and highly intense Bragg’s peaks confirmed crystalline nature of the drug. The diffraction patterns presented in Fig. 1c–e correspond respectively to the samples after 30, 60 and 120 min of cryogrinding. In those samples, a strong reduction of crystallinity proceeds with increasing milling time, resulting in a considerable decrease of diffraction peaks. As illustrat- ed in Fig. 1c, significant broadening and decrease of peak intensities appeared just after 30 min of cryomilling. For this sample, the degree of crystallinity (calculated as a ratio of X-ray diffraction peaks areas for partially crystalline sample to that being 100% crystalline) was found to be 12%. After 60 min of cryomilling, furosemide becomes almost completely amorphous (degree of crystallinity 2%). Complete conversion crystalline-amorphous furosemide was noticed after 70 min (data not shown). The diffraction patterns recorded at room temperature for samples cryomilled for 120 and 180 min revealed absence of Bragg’s peaks, which were replaced by halo patterns (calculated degree of crystallinity 0%), being a characteristic feature of materials having no long-range three- dimensional molecular order, i.e amorphous one. As frequently reported in the literature, transforma- tion of a crystalline material to a completely disordered one caused by mechanical milling occurs only at temper- atures much lower than the glass transition temperature, Tg, of corresponding supercooled liquid (11). This explains why the crystalline-amorphous powder transfor- mation takes place more readily at a lower temperature. It is worth pointing out that the reduction of the milling temperature brings the same effect as the increase in milling intensity. Hence, by cryomilling it is possible to reduce time required for considerable particle size reduction and to avoid extremely long milling times to get the same effect as in a traditional ambient-temperature device. To illustrate the lower efficiency in production of amorphous materials by mechanical milling carried out at room temperature, the grinding process of FUR was also performed in a traditional ball mill (milling intensity Ω = 400 rpm). In Fig. 1b the XRD patterns of a sample ground for 240 min at ambient conditions are shown. Certainly, reduction of peak intensities indicating a decrease in grain size and distortion of crystalline lattice