, USA. Sterile Water for Injection was supplied by Nirma Ltd., India. Methanol and acetonitrile solvents were of HPLC grade and were

procured from Merck. The reverse phase HPLC method was chosen for the quantitative determination GDC-0199 manufacturer of the PM181104 in the formulations. Standard and formulation samples were diluted with acetonitrile: methanol (1:1; v/v) to obtain a final concentration of 0.1 mg mL−1 and then injected a 10 µL injection volume directly to HPLC system. Agilent 1200 HPLC system (Agilent, USA) with a Kromasil 100 C18 analytical column (150×4.6 mm2, particle size 3.5 µm) was used for the studies. The mobile phase was acetonitrile–water mixture (50:50, v/v). The flow rate was 1.0 mL min−1 and the detection wavelength was set to 309 nm. Percentage assay calculated with respective to the chromatograms of standard Afatinib price and sample area. PM181104 nanoparticles were prepared by anti-solvent precipitation technique, using water for injection (WFI) as the anti-solvent [12]. By using this method nanoparticles can be manufactured in the absence of mechanical forces which can have influence on peptide stability

[13]. For this, the specified amount of T-80 was thoroughly mixed with the specified amount of PEG 400 under vortex followed by sonication, to form the excipient mixture. The prepared excipient mixture was used to dissolve the required amount of PM181104 using sonication carried out with intermittent cooling (to maintain the temperature below 40 °C) until a turbid free solution clear of any undissolved particulate matter was obtained. The resultant clear, colorless and viscous drug excipient

mixture was then injected slowly and continuously through drop wise addition using a buret to the anti-solvent under rapid mixing (1000 rpm, 4��8C magnetic stirrer). Precipitation of the solid drug particles were occurred immediately upon contact with the anti-solvent. The resulting formulation suspension was sterilized by filtering through 0.2 µm filter assembly connected to vacuum. A total of eight formulations were made, and divided into two sets based on their excipient composition. The first set consisted of formulations, made with a reduced concentration of T-80. The second set consisted of reduced concentration of PEG 400. The optimization of the excipient composition in the first set of formulations (F1−F5) was carried out using ternary compositions containing water for injection (WFI), PEG 400 8% (w/v) and a decreasing amounts of T-80 (8–0.05%) while maintaining the final concentration of PM181104 at 0.25 mg mL−1. In the second set, another three formulations (F6–F8) were prepared using ternary compositions containing WFI, T-80 8% (w/v) and a decreasing amounts of PEG 400 (6–0.5%) while retaining the final concentration of the PM181104 at 0.5 mg mL−1. The concentration of the drug in the described formulation was reconfirmed using HPLC analysis.