Jet stream nebulizer

All samples were analyzed with an Agilent 1290 series HPLC system coupled with an Agilent 6530 quadrupole-time of flight (qTOF) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Twenty micro liters of sample were injected into the column using an autosampler at 5 °C. An InfinityLab Poroshell 120 EC-C18 column (3.0 mm × 50 mm, 2.7 μm) (Agilent Technologies, Palo Alto, CA, US) held at 35 °C was used to separate compounds at a flow rate of 600 μl min⁻¹. Compound separation was achieved using gradient of solvent, with water (A) and acetonitrile (B), both with 0.1% formic acid during a total run time of 30 min.
An Agilent Jet Stream nebulizer was used as electrospray ionization (ESI) source. Samples ran twice, one in positive and one in negative ESI mode, with mass ranges of 60–1000 and 100–970 m z⁻¹ for positive and negative, respectively. It was operated at 250 °C with ionization set at 3000(+)/4000(−) V and fragmentor voltage at 130 V. Nebulizer gas pressure, temperature and drying gas flow rate were set at 45 psi, 400 °C and 10 l min⁻¹. Mass measurements were recalibrated using Agilent reference masses m/z 121.0508 and 922.0098 in positive mode, and m/z 119.0363 and 966.0007 in negative mode. In both cases, all ions MS/MS was performed at collision energies of 0 and 15 V. Mass spectra were acquired at a scan rate of 2 spectra/s.

EBC samples were directly lyophilized and the obtained dried extract was reconstituted in mobile phase (5% acetonitrile in water) to obtain a concentration factor of 20. Samples were analyzed using an Agilent 1290 series HPLC system coupled with an Agilent 6530 quadrupole -time of flight (qTOF) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). 20 μL of each sample were injected through an InfinityLab Poroshell 120 EC-C18 column (2.7 μm, 3.0 mm × 50 mm; Agilent Technologies, Palo Alto, CA, USA). The mobile phases consisted of water (A) and acetonitrile (B), both with 0.1% formic acid. The solvent flow rate was set to 0.6 ml min⁻¹, the column temperature to 35 °C and the autosampler to 8 °C to increase sample stability. An electrospray ionization (ESI) source with an Agilent Jet Stream nebulizer was used in positive and negative mode with the following operating parameters: capillary voltage, 4000(−)/3500(+) V; nebulizer pressure, 25 psi; drying gas, 10 L min⁻¹; gas temperature, 250 °C; fragment voltage, 130 V. Mass spectra were acquired at MS resolution level at a scan rate of 2 spectra/s over a range of m/z 100-950.