Agilent 1290 infinity liquid chromatograph

The C. racemosa chloroform extract was analyzed using a LCMS on Agilent 1290 infinity liquid chromatograph (Agilent Technologies, Wilminton, DE, USA), coupled with an Agilent 6520 Accurate-Mass Q-ToF Mass Spectrometer with dual ESI source. Separation of compound was achieved using reverse-phase HPLC with an Agilent Zorbax Eclipse XDB-C18 column, Narrow-Bore 2.1 × 150 mm, of particle size 3.5 µm at 25 °C, and equilibrated with solvent A (0.1% formic acid in Milli-Q water) and solvent B (0.1% formic acid in acetonitrile). The flow rate was set at 0.5 mL per min. The total run time was 30 min, including a 25 min run time, and a 5 min post-run time. The ESI-TOF/MS conditions were optimized as follows: Drying gas temperature, 300 °C; drying gas flow, 10 L/min; nebulizer gas pressure, 45 psi; capillary voltage, 3500 V for positive ion mass spectra and 4000 V for negative ion mass spectra; fragmentation voltage, 125 V; and skimmer, 65 V. The mass spectrum was scanned from m/z 100 to m/z 3200 in both the positive and negative ionization modes. Calibration reference solutions obtained from Agilent were used to calibrate the mass spectrometer daily. Reference solution was used and the two ions with m/z of 119.03632 and 966.0097 were selected for mass calibration in order to eliminate systematic errors.

Centipede haemolymph was tested for further chemical identity. Haemolymph was subjected for LC–MS analysis on Agilent 1290 infinity liquid chromatograph (Agilent Technologies, Wilmington, DE), coupled with an Agilent 6520 Accurate-Mass quadrupole-time of flight (Q-TOF) mass spectrometer with dual electrospray ionization source (ESI). Reverse-phase high performance liquid chromatography was used for separation of compounds, with an agilent Zorbax Eclipse XDB-C18, Narrow-Bore 2.1 × 150 mm, 3.5-micron column at 25 °C, and equilibrated with solvent A (0.1% formic acid in Milli-Q water) and solvent B (0.1% formic acid in Acetonitrile). 0.5 mL/min flow rate with a linear gradient was used as follows: 5% solvent B for 5 min, 100% solvent B for 20 min, and 100% solvent B for 25 min. The total run time was 30 min. The compounds were ionized using dual ESI + Accurate-Mass Q-TOF mass spectrometer. The ion source parameters were as follows: capillary voltage at 4000 V for positive and 3000 V for negative ion polarity. Flow rate of drying gas was 10 L/min with a fragmentor voltage of 125 V and gas temperature of 300 °C. Pressure of nebulizer gas was set at 45 psi with Quadrupole-TOF detector, while 50% MeOH + 50% Milli-Q water was used as blank after processing each sample.