Uv6000 pda

Starting from the analytical conditions reported in literature,^{19 (link)} the column length was decreased from 250 mm to 150 mm (Hypersil BDS C18, 5 μm, 150×4.6 mm, Thermo Scientific, MA, USA) in order to improve tailing factors of the two active ingredients. A Thermo Fisher HPLC system equipped with a P4000 pump and an AS3500 autosampler were employed (Thermo Fisher Scientific Inc., Waltham, MA, USA). A Thermo Scientific UV6000 PDA was set at 250 nm for SX and 238 nm for FP, while the retention time was 3.0 and 6.7 min for SX and FP, respectively. LOQ was about 0.043 μg/mL for salmeterol and 0.035 μg/mL for fluticasone propionate. The linearity of response of salmeterol and fluticasone propionate was evaluated on standard solutions in the concentration range 0.04–13 μg/mL and 0.04–90 μg/mL, respectively, with a correlation coefficient R²: ≥0.99 in both of the cases.

Chromatographic quantifications of formoterol fumarate and beclomethasone dipropionate were carried out using a liquid chromatography equipped with Thermo Fisher P4000 pump and Thermo Fisher AS3500 autosampler (Thermo Fisher Scientific Inc). A Thermo Scientific UV6000 PDA operating at 223 nm for FF and 238 nm for BDP was employed. The injection volume was set at 50 μL and a Sinergy Fusion column C18, 4 μm, 50×4.6 mm (Phenomenex, Castel Maggiore, Bologna, IT) was used as stationary phase.
A gradient programme using a solution containing a water phase of 0.02 M of NaH₂PO₄, adjusted at the pH of 3.0, and acetonitrile was employed in order to elute first FF and then BDP. The column temperature was maintained at 40°C and the flow rate was set at 1 mL/min. The retention time for was 3.1 and 10 min for FF and BDP, respectively. LOQ was about 0.019 μg/mL for FF and 0.049 μg/mL for BDP. The linearity of response of FF and BDP was evaluated on standard solutions in the concentration range 0.02–2.2 μg/mL and 0.05–36 μg/mL, respectively, with a correlation coefficient R²: ≥0.99 in both of the cases.