Biopharma compass 3

HPLC-ESI-MS analysis was performed on Impact II QqTOF high-resolution mass spectrometer (Bruker Daltonik, Germany) with Elute UHPLC (Bruker Daltonik, Germany) on Intensity Solo 1.8 C18-2 2.1 × 100 mm 1.8 μm 90 Å reverse-phase column (Bruker Daltonik, Germany) with the following conditions: column flow 0.25 ml/min, gradient elution from 5 to 70% B in 25 min (A: 0.1% formic acid in water, B: 0.1% formic acid in AcN), column temperature 40°C, injection volume 5 μl, ESI source in positive mode, HV capillary at 4.5 kV, spray gas–nitrogen at 2.1 bar, dry gas–nitrogen at 8 l/min 220°C, scan range m/z 50–2200, 2-Hz scan rate for full scan, automatic MS/MS mode (CID) with dynamic scan rate 2–8 Hz, nitrogen as collision gas, collision energy from 23 eV at m/z 300 to 65 eV at m/z >1300, and automatic internal calibration with ESI-L low concentration tuning mix (Agilent Technologies, USA). Spectra were processed with BioPharma Compass 3.1.1 (Bruker Daltonik, Germany).

High-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) analysis was performed with the Impact II QqTOF high-resolution mass-spectrometer (Bruker Daltonik, Germany), the UHPLC (Bruker Daltonik), and the Nucleodur C4 300-5 ec 4.6*150 mm column (Macherey-Nagel, Germany). Data acquisition conditions were as follows: flow 1 ml/min, split 1:10, gradient elution from 2% to 98% B in 40 min (A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile), column temperature 40 °C, 30–50 µg of sample per injection, ESI source in positive mode, HV capillary at 4.5 kV, spray gas—nitrogen at 1.8 bar, dry gas—nitrogen at 8 L/min 220 °C. Spectra were processed with BioPharma Compass 3.1.1 (Bruker Daltonik, Germany).

HPLC-MS/MS analysis was performed on Impact II QqTOF high-resolution mass-spectrometer (Bruker Daltonik, Germany) equipped with Elute UHPLC (Bruker Daltonik, Germany) on Intensity Solo 1.8 C18-2 2.1*100 mm 1.8 µm 90 Å reverse-phase column (Bruker Daltonik, Germany) with following conditions: column flow 0.25 mL/min, gradient elution from 30% to 100% B in 60 min (A: 0.1% (v/v) formic acid in water, B: 0.1% (v/v) formic acid in ACN), column temperature 40°C, injection volume 15 μL (1 µg/µL of dry sample in 60% (v/v) ACN), ESI source in positive mode, HV capillary at 4.5 kV, spray gas – nitrogen at 2.1 bar, dry gas – nitrogen at 8 L/min 220°C, scan range m/z 50-2200, 2 Hz scan rate for full scan, automatic MS/MS mode (CID) with dynamic scan rate 2-8 Hz, nitrogen as collision gas, collision energy from 23 eV at m/z 300 to 65 eV at m/z >1300, automatic internal calibration with ESI-L low concentration tuning mix (Agilent Technologies, USA). Spectra were processed with BioPharma Compass 3.1.1 (Bruker Daltonik, Germany).