LC-MS/MS

A 1290 Infinity series UHPLC system (Agilent Technologies, Santa Clara, CA, USA) with a Kinetex C18 column (2.1 * 100 mm, 1.7 μm; Phenomenex, Torrance, CA, USA) was used in LC-MS/MS analysis. The mobile phase A consisted of 40% water, 60% acetonitrile, and 10 mmol/L ammonium formate. The mobile phase B consisted of 10% acetonitrile and 90% isopropanol, with 50 mL of 10 mmol/L ammonium formate per liter of mixed solvent. UHPLC runs were carried out based on the following elution gradient scheme: within the initial 12 min, 40–100% of mobile phase B; 12–13.5 min, 100% of mobile phase B; 13.5–13.7 min, 100–40% of mobile phase B; and 13.7–18 min, 40% of mobile phase B. Additional parameters were: column temperature, 45 °C; auto-sampler temperature, 4 °C; injection volume, 6 μL (POS) or 6 μL (NEG), respectively.

A triple time-of-flight mass spectrometer was used to generate MS/MS spectra on an IDA. Analyst TF 1.7 software (AB Sciex, Framingham, MA, USA) was used for a continuous evaluation of full MS scans as well as for collecting and acquiring MS data and MS/MS spectra based on preselected criteria. In each cycle, the 12 precursor ions with an intensity above 100 were chosen for MS/MS using 45 eV as the collision energy (12 MS/MS events with 50 msec of accumulation time each). Electrospray ionization source conditions were as follows: gas 1, 60 psi; gas 2, 60 psi; curtain gas, 30 psi; source temperature, 600 °C; declustering potential, 100 V; ion spray voltage floating, 5000 V or -4500 V in the positive or negative mode, respectively.