Effect of Cryogrinding on Chemical Stability samples was less than 4%. The ICP-AES analysis showed that the iron content of a sample cryomilled for 180 min sample was at a level matching that of a tap water. The ground samples prepared in such a way were then analyzed by the methods indicated below. For the ball milling of furosemide at ambient tempera- ture, a Planetary Ball Mill (Retsch) was used. A zirconium jar was filled with 10 g of furosemide, and 6 balls (20 mm in diameter) of the same material were added. The rotation speed of 400 rpm was set. Each milling cycle lasted 15 min and was followed by a 5-min break. The total milling times were 240 min (4 h) and 1440 min (24 h). X-ray Diffraction (XRD) The X-ray diffraction experiment was performed at ambient temperature on a Rigaku-Denki D/MAX RAPID II-R diffractometr with a rotating anode AgKα tube (λ= 0.5608 Å), an incident beam (002) graphite monochromator and an image plate in the Debye-Scherrer geometry. The pixel size was 100× 100 μm. All samples were placed inside Lindemann glass capillaries (2 mm in diameter). Measure- ments were run with the sample filled and empty capillaries, and the intensity of the empty capillary was then subtracted. The beam width at the sample was 0.1 mm. The two-dimensional diffraction patterns were converted into the one-dimensional intensity data using a suitable software. Differential Scanning Calorimetry (DSC) DSC measurements were carried out using a Perkin Elmer Pyris 1 DSC instrument (PerkinElmer Life And Analytical Sciences, Massachusetts, USA) with liquid nitrogen cooling. The instrument was calibrated for temperature and heat flow using high-purity standards of indium. The sample (m =0.00756 g) was placed in a sealed aluminium pan. The Table I Chromatographic Conditions (UPLC) 3223 thermogram was collected during heating of amorphous furosemide prepared by cryomilling at a rate of 10°C/min from −20°C to 190°C. Test of Purity of Cryomilled Furosemide and Chemical Stability in Solid State and Solutions Ultra-Performance Liquid Chromatography (UPLC) For the analysis of Furosemide and its degradation products (impurities) a simple and sensitive chromatographic method has been developed. The same method was used for examination of the stability of the drug in the solid state and solutions. The analyses were performed using the Waters Acquity UPLC system (Millford, MA, USA), equipped with a Binary Solvent Manager, Sample Manager, Column Manager, and Photodiode Array eλ Detector. Data acquisition and evalua- tion were carried out using the Empower Pro 2 software (Waters, Millford, MA, USA). A summary of the chromato- graphic conditions is given in Table I. The LC separation was achieved within a run time of 10 min. Separation and Detection of Main Degradation Product Formed During Cryomilling of Furosemide High Performance Liquid Chromatography HPLC The analysis was performed on an HPLC Beckman Gold System (Beckman Coulter, Brea, CA, USA) instrument using a thermostatted (30°C) Phenomenex C18 Luna column (150 ×4.6 I.D. particle size 5 μm; 100Å). The eluate was monitored at 230 nm. Total run time was 30 min with a flow rate of 0.5 ml/min. Acetonitrile and acetic acid (0.1%) were filtered through a 0.45 μm membrane filter and used as a mobile phase. First, an isocratic flow (acetonitrile: acetic acid 20:80 v/v) was Column: Mobile phase A: Mobile phase B: Gradient program: Flow rate: Column temp: Injection volume: Injection mode: Detection wavelength: Data collection rate: ACQUITY UPLC BEH C18 1.7 μm 2.1×150 mm (Waters, Wexford, Ireland) 0.1% formic acid in water, pH adjusted to 5.6 with triethylamine acetonitrile start with 10% of mobile phase B at 9.0 min 50% of mobile phase B at 10.0 min 70% of mobile phase B 0.3 ml/min 30°C 1.0 μl Partial Loop with Needle Overfill 270 nm 40 points/s