A Thermo Scientific Dionex Ultimate 3000 UHPLC system, hyphenated with a Thermo high resolution Q-Exactive focus mass spectrometer (Thermo, Bremen, Germany) was used for the analysis. The chromatographic system was coupled to the MS with a heated electrospray ionization source II (HESI II). Nitrogen (purity > 99.999%) obtained from a Genius NM32LA nitrogen generator (Peak Scientific, Billerica, MA, USA) was employed to produce MS fragmentation. Mass calibration for the Orbitrap spectrometer and HESI parameters were explained in detail and precisely in previous reports [5 (link),24 (link)].
Solvent delivery was performed at 1 mL/min using ultrapure water supplemented with 1% formic acid (A) and acetonitrile with 1% acid formic (B) and a program starting with 5% B at zero time, then maintained 5% B for 5 min, then changing to 30% B within 10 min, then maintaining 30% B for 15 min, then going to 70% B for 5 min, then maintaining 70% B for 10 min, and finally returning to 5% B in 10 min and keeping this condition for twelve additional minutes to achieve column stabilization before the next injection of 20 µL [5 (link),24 (link)].
Solvent delivery was performed at 1 mL/min using ultrapure water supplemented with 1% formic acid (A) and acetonitrile with 1% acid formic (B) and a program starting with 5% B at zero time, then maintained 5% B for 5 min, then changing to 30% B within 10 min, then maintaining 30% B for 15 min, then going to 70% B for 5 min, then maintaining 70% B for 10 min, and finally returning to 5% B in 10 min and keeping this condition for twelve additional minutes to achieve column stabilization before the next injection of 20 µL [5 (link),24 (link)].
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