C18 reversed phase analytical column

Each sample was separated by HPLC liquid phase system EasynLC with nano-liter flow rate. The peptides were loaded onto a reverse phase trap column (100 μm × 2 cm; Thermo Fisher Scientific, Waltham, MA), connected to the C18-reversed phase analytical column (length: 10 cm, inner diameter: 75 μm) in buffer A (0.1% formic acid), and then separated with a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min controlled by IntelliFlow technology (Cholewa et al., 2014 (link)). After chromatographic separation, the samples were analyzed by Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA). The mass spectrometer was operated in the form of positive ions, with the scan range of the precursor ion 300 to 1,800 m/z and the related parameters as follow: automatic gain control target: 3e6, the maximum inject time: 10 ms, dynamic exclusion duration: 40.0 s, resolution of survey scans: 70,000 at m/z 200, resolution for HCD spectra: 17,500 at m/z 200, isolation width: 2 m/z, normalized collision energy: 30 eV, and underfelt ratio (specifies the minimum percentage of the target value likely to be reached at maximum fill time) of 0.1%. The peptide recognition mode of the instrument was selected, and 20 fragments was collected after each full scan.

To study the effects of the prepared solid dispersions on the solubility of VPN, the equilibrium solubility was determined as previously reported.6 (link) An excess amount of either pure drug or the prepared solid dispersions was added to screw-capped vials containing 10 mL of distilled water. The vials were placed in a thermostatically controlled shaking water bath (Model 1031; GFL Corporation, Burgwedel, Germany) at 25°C±0.5°C for 48 hours. Samples from the vials were taken and analyzed for VPN concentrations every day until equilibrium was reached (the drug solubility values on two consecutive days did not vary by more than 5%). Aliquots withdrawn were filtered through a 0.22 µm pore size Millipore filter and assayed for drug content using high-performance liquid chromatography (HPLC) method mentioned earlier by Ding et al,36 except for slight modifications. An isocratic HPLC method was performed using Agilent 1200 series equipped with UV diode array detector. Reversed phase C18 analytical column (4×250 mm, 5 µm; Thermo Fisher Scientific, Waltham, MA, USA) was used. The mobile phase composed of a mixture of methanol and 0.05 ammonium acetate buffer of pH 5.5, 80:20 (v/v). The flow rate of the mobile phase was 1 mL/min, the injection volume was 20 µL, and the detection wavelength was 273 nm. Each experiment was performed in triplicate.