3222 authors suggested that this effect was related to the preparation method rather than to degradation (19). The aim of this study is to examine the effect of cryogrinding on the physical and chemical stability of the popular diuretic agent, furosemide. The crystalline furose- mide is sparingly soluble in aqueous systems, especially the acidic ones. Experimental water solubility of crystalline furosemide (0.006 mg/mL (20)) as well as its bioavailability (20–60% (21)) allow for classifying the drug as a class IV compound in BCS. From the literature it is also known that furosemide appears to be very unstable at lower pHs and undergoes hydrolysis and photochemical degradation in aqueous solution (22). Moreover, it decomposes at a melting point (Tm = 206°C) (23). Consequently, it is impossible to prepare its amorphous form in a conventional way. For this reason, furosemide appeared to be one of the challenging molecules for pharmaceuticals researchers. In the past, some attempts had been made to improve its stability in aqueous systems as well as its dissolution profile (24,25). For example, a remarkable enhancement of the dissolution rate was reported for a solid dispersion of furosemide-PVP (26) or for ball milling of furosemide with crospovidone (27). Interestingly, previous researchers didn’t report about any signs of degradation of the drug caused by milling. In this study, we have tested the effect of cryogrinding of pure furosemide on its stability. The samples cryomilled for 30, 60, 120 and 180 min were then analyzed with the use of several techniques, namely X-Ray Powder Diffraction (XRD), Mass Spectrometry (MS), Nuclear Magnetic Res- onance (NMR) and Ultra-Performance Liquid Chromatog- raphy (UPLC). The latter is known to be the most sensitive analytical method, allowing detection of any trace impurity. As will be shown in this paper, the milling of furosemide in liquid nitrogen is actually not such a safe method of solid- state amorphization as expected. Briefly, our result leads to the conclusion that during cryomilling (milling carried out at a liquid nitrogen temperature) chemical degradation of furosemide induced by mechanical treatment occurs. For this reason, it seems that the effect of cryomilling on the physicochemical properties of APIs ought to be more carefully studied. MATERIALS AND METHODS Materials Furosemide (4-chloro-2-(furan-2-ylmethylamino)-5- sulfamoylbenzoic acid, C12H11ClN2O5S, is a white crystal- line powder. It was supplied by the Polpharma Pharma- ceutical Works Poland and used without further purification. The purity of the drug was declared by Adrjanowicz et al. supplier as being greater than 99%. The chemical structure of the drug is shown in the inset in Fig. 1. For cogrinding, polyvinylpyrrolidone (PVP) K30 (average molecular weight 40,000) and inulin (purity 99%) were used. Both materials were supplied by the Polpharma Pharmaceutical Works Poland. Preparation Technique—Cryogrinding of the Crystalline Material The grinding was performed using a cryogenic impact mill (6750 freezer/mill SPEX CertiPrep, Inc., USA) consisting of a stainless steel vessel immersed in liquid nitrogen, within which a stainless steel rod was vibrated by means of magnetic coil. Before grinding, a 10-min precooling time was programmed. Then, the mill was set at an impact frequency of 15 cycles per second for 6-min grinding periods separated by 3-min cool-down periods. The mass of each sample was 1 g. Total milling times of furosemide were 30, 60, 120 and 180 min. The powdered mixtures of furosemide-PVP and furosemide-inulin were prepared at 1:1 mass ratios and cryomilled for 60 min. After milling, the grinding vial was transferred to a vacuum oven and allowed to warm to room temperature. The freshly prepared cryomilled furosemide is a grey powder with a slightly brown hue, intensified upon increasing the milling time. Both furosemide-PVP and furosemide-inulin mixtures after 60 min of cryomilling are yellow powders. Expected humidity of the cryomilled Fig. 1 Powder x-ray diffraction patterns (XRDP) starting from crystalline furosemide (a), furosemide after 240-milling in a traditional ball mill (pattern b) and after cryogrinding for 30, 60 and 120 min (respectively, patterns c–e).