1) (Aldrich, Steinheim, Germany), diluted in ethanol (VWR, Fontenay-sous-Bois, France). Fig. 1 Chemical structures of a busulfan (1,4-butanediol

dimethanesulphonate), b diethyldithiocarbamate, and c dibromopentane The high-performance liquid chromatography (HPLC) system consisted of a quaternary pump (Merck Hitachi® L7100), an automatic injector (Merck Hitachi® L2200), an ultraviolet (UV) visible detector EPZ-6438 in vivo (Waters® 2487), and Multi HSM Manager software (Merck Hitachi®). The analysis was run using an Agilent Zorbax® SB C18 column (5 μm, 150 × 4.6 mm) (Agilent Technologies). The column was thermostatically controlled at 40 °C during use and then rinsed with a water (H2O)/methanol (50/50, v/v) mixture. Busulfan was detected by absorbance at 281 nm. In GDC-0973 solubility dmso isocratic mode, a mobile phase consisting of acetonitrile (ACN), H2O, and trifluoroacetic acid (TFA) (proportions: 650/350/1, v/v/v) was run through the system at a flow rate of 2 mL/min. 2.2 Sample Preparation,

Storage, and Processing Busulfan preparations were produced by diluting the product Busilvex® in 0.9 % NaCl to obtain a final concentration of 0.55 mg/mL (therapeutic concentration). The containers used for the preparations were PP syringes (Becton Dickinson, Franklin Lakes, NJ, USA; 50 mL, ref: 300865), EasyFlex® PVC bags (MacoPharma, Tourcoing, France) and 250-mL glass bottles containing 0.9 % NaCl (CDM Lavoisier, Paris, France). Busulfan solutions were then aliquoted into smaller containers so that the solutions remained under

the defined storage conditions throughout the evaluation period. These containers were 3-mL PP syringes (Becton Dickinson; ref: 300910), 50-mL EasyFlex® PVC bags (MacoPharma) and 2-mL glass bottles (Schott, St Gallen, Switzerland; ref: VCDIN2R) of borosilicate pharmaceutical Phospholipase D1 type I glass adapted for injections, fitted with chlorobutyl stoppers. The storage conditions for each of the containers were 2–8 °C, 13–15 °C (thermostatically controlled chamber), and, finally, RT (20 ± 5 °C). For each of the conditions (container and storage temperature), a sample was processed and analysed by HPLC-UV either every 6 h or every 3 h. Samples were processed in glass tubes because of the use of dimethylacetamide (DMA). To 0.5 mL of aliquot, we added 0.5 mL of DMA, followed by 0.1 mL of IS. After stirring, 1.0 mL of DMA and 0.5 mL of derivatization agent were added for a final volume of 2.6 mL. The solution was stirred a second time before being left to stand for 1 h at RT, the time required for derivatization prior to injection. A 30-μL test sample was then injected into the chromatographic system. 2.3 Transfer and Validation of Method We adapted the analytical method registered by Pierre Fabre Laboratories in the marketing authorization application. The validation of this method was conducted at our laboratory according to International Conference on Harmonisation (ICH) topic Q2R guidelines [14].