The production yield was calculated as the mass of powder collected after spray drying divided by the total mass of solid input. To measure the drug content in the co-spray dried powder, weighed powder was dissolved in ultra-pure water to prepare a 5 mL solution. The dissolved sample was filtered through a 0.45-µm nylon membrane filter and quantified with high-performance liquid chromatography (HPLC, Agilent 1260 Infinity, Agilent Technologies, Santa Clara, USA). The contents of capreomycin, peptide and mannitol were measured separately and were calculated as the measured amount with respect to the powder mass. The HPLC method for quantification of capreomycin was adopted and modified according to a previous study [14 (link)]. The mobile phase consisted of 0.1% TFA aqueous solution (pH = 2) and acetonitrile (95:5, v/v) was run at a rate of 1 mL/min for 10 min. Each sample containing 50 μL solution was injected and passed through the C18 column (5 μm, 250 mm, Agilent, USA) at room temperature, and the capreomycin was detected by UV at wavelength 268 nm. The capreomycin IIA/IIB eluted at approximately 4.6 min and capreomycin IA/IB at around 5.4 min. Linearity for total capreomycin was demonstrated between 5.0 μg/mL and 200 μg/mL (R2 = 0.9999). D-LAK peptides were quantified using an established HPLC method [9 (link)]. The mobile phase consisted of acetonitrile and ultra-pure water with 0.1% TFA. A linear gradient from 20 to 80% acetonitrile was applied over 20 min at 1 mL/min. Each sample containing 100 μL of solution was injected by an auto-sampler and passed through a C18 column (250 mm × 4.6 mm, 5 μm, VydacTM GraceTM, IL, USA) at ambient temperature. The peptide was detected by UV at wavelength 220 nm. The retention time of D-LAK peptides was around 9.85 min. Linearity for D-LAK peptides was demonstrated between 8.0 μg/mL and 200 μg/mL (R2 = 0.9998). The HPLC method for mannitol measurement was detailed in Sect. “Aerosol performance and quantification of mannitol ”.
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