1200 series high performance liquid chromatograph

The HPLC-MS/MS system consisted of an Agilent 1200 series high performance liquid chromatograph (HPLC) and an Agilent 6410 triple quadruple mass spectrometer equipped with an electrospray ionization (ESI) source (Agilent Technologies, Santa Clara, CA, USA). Data were analyzed by Mass Hunter software (Agilent Corporation, Santa Clara, CA, USA). A Waters XSELECT™ HSS T₃ column (100 mm × 2.1 mm, i.d., 2.5 μm) was used for chromatographic separation. The mobile phase was consisted of acetonitrile and 0.1% formic acid in water (65:35, v/v). The column was equilibrated and eluted at a constant flow rate of 0.3 mL · min⁻¹, maintained at 35°C. The sample injection volume was 10 μL, and the run time was 3.0 min. Data acquisition was performed using multiple reaction monitoring (MRM) of bufalin with the corresponding IS. Transitions were monitored at m/z 387.3 → 255.3 for bufalin, and at m/z 443.2 → 365.1 for the IS (Supplementary Table 1, Supplementary Figure 2). The detection parameters were optimized as follows: drying gas temperature, 325°C; drying gas flow rate, 10.0 L · min⁻¹; nebulizer pressure, 40 psi; capillary voltage, 4,000 V.

An Agilent 1200 series high-performance liquid chromatograph coupled to an ELSD detector was used. The reversed-phase HPLC measurements were carried out on a Zorbax® RX-C18 analytical column (4.6 × 250 mm, 5 μm). The mobile phase was 50 mM ammonium acetate and HPLC grade acetonitrile as organic modifier (pH 7.0) or TFA 0.1% buffer aqueous solution and 0.1% TFA HPLC grade acetonitrile (pH 2). The mobile phase flow rate was ranging from 0.1–1.0 mL min⁻¹. The dead time (t₀) was measured by injecting sodium nitrate together with the sample at a concentration of 10 mM. The samples were dissolved at 1 mg/1 mL in water. Few drops of acetonitrile were added when the dissolution of the peptide was incomplete. The logarithm of retention factor k (log k) values were obtained according to the eqn (1): where t_R is the retention time of the compound and t₀ the retention time of the unretained sodium nitrate solute.
The log k values were plotted as a function of the acetonitrile concentration. The slope (S) and the intercept (log k_w) values were calculated from at least three concentrations of acetonitrile. The correlation coefficients of the linear fit were higher than 0.99. The isocratic hydrophobicity index φ₀ was calculated according to the eqn (2): where log k_w is the log k value extrapolated to the 0% acetonitrile concentration.