Positive ion calibration solution

For non-targeted metabolomic analysis, the tandem hybrid mass spectrometer Q Exactive worked in the positive full scan mode with resolving power (PR) = 70,000 (FWHM) at 200 m/z in the range 50 to 750 m/z and in the centroid mode. Before the start of each acquisition series, the mass spectrometer was externally calibrated to the mass accuracy with a positive ion calibration solution and a negative ion calibration solution (both Thermo Fisher Scientific (Massachusetts, USA). The “lock-mass” calibration was set to the molecular mass of [M+Na]⁺ = 64.01577 g mol⁻¹ and [M₂+H]⁺ = 83.06037 g mol⁻¹ for the acetonitrile ion, and was run continuously during the acquisition. The mass spectrometer setting was as follows: sheath gas flow rate 30 (unit), aux gas flow rate 5 (unit), spray voltage 4.5 kV, capillary temperature 320 °C, heater temperature 180 °C, S-lens RF level 50, AGC target of 6 × 10⁶ and a maximum injection time of 200 ms. The whole LC-(HR)MS system was controlled and the obtained data were stored and processed using Xcalibur 3.1 software.

For MS/MS spectra collection, each sample was dissolved in DMSO (final concentration: 10 mM) and, after further dilution in acetonitrile (final concentration 100 nM), it was analyzed by flow injection mass spectrometry (FIA-MS). Briefly, five microliters of sample were injected into an eluent flow containing 0.1% formic acid in acetonitrile, generated by a Vanquish UHPLC system (Thermo Fisher Scientific, San Jose, CA, USA). The flow rate was 100 µL/min. The eluent was directly sent to a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a heated electrospray ion source (HESI-II). Prior to each series of acquisitions, the mass spectrometer was externally calibrated with Positive Ion Calibration Solution (Thermo Fisher Scientific, San Jose, CA, USA). The same MS operating parameters as LC-MS analysis were applied. Full scan data were processed with Xcalibur version 4.1 (Thermo Fisher Scientific, San Jose, CA, USA). High-resolution mass spectra, ranging from 100 to 600 m/z, were acquired in positive ion mode.