Exionlc infinity series uhplc system

The ExionLC Infinity series UHPLC System (AB Sciex) with the Kinetex C18 column (2.1 × 100 mm, 1.7 μm; Phenomen) was used for LC/MS analyses. The mobile phase A consisted of 40% water and 60% acetonitrile with 10 mmol/L ammonium formate, and the mobile phase B consisted of 10% acetonitrile and 90% isopropanol, with 10 mmol/L ammonium formate. The elution gradient was set to 0–12.0 min, 40–100% B; 12.0–13.5 min, 100% B; 13.5–13.7 min, 100–40% B; 13.7–18.0 min, 40% B. The column temperature was set to 45 ℃. The injection volume was 2 μL (pos) and 6 μL (neg), respectively.
The TripleTOF 5600 Mass Spectrometer was used to acquire the MS/MS spectra on an information-dependent basis (IDA) with the acquisition software (Analyst TF 1.7, AB Sciex) to evaluate the full scan survey MS data. The most intensive 12 precursor ions with intensity > 100 were selected for MS/MS at collision energy (CE) of 45 eV. The ESI source conditions were set as follows: Gas 1 as 60 psi, Gas 2 as 60 psi, Curtain Gas as 30 psi, source temperature as 600℃, declustering potential as 100 V, and ion spray voltage floating (ISVF) as 5000 V or − 3800 V in positive or negative modes, respectively^{16 (link)}.

Ultra (high) performance liquid chromatography (UHPLC) separation was carried out using an ExionLC Infinity series UHPLC System (AB Sciex), equipped with a UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm, Waters). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide ( CNW Technologies) in water (pH = 9.75) (A) and acetonitrile (B). The analysis was carried out with an elution gradient. The column temperature was 25℃. The autosampler temperature was 4 ℃, and the injection volume was 2 μL (pos) or 2 μL (neg), respectively.
TripleTOF 5600 mass spectrometry (AB Sciex) was used for its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during LC/MS experiments. In this mode, the acquisition software (Analyst TF 1.7, AB Sciex) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria. In each cycle, the most intensive 12 precursor ions with intensity above 100 were chosen for MS/MS at a collision energy (CE) of 30 eV. In the detection process, standard products and blank controls were strictly used for quality control, and data quality control was also carried out. The UHPLC-QTOF-MS analysis was performed at Biomarker Technologies Corporation, Beijing, China (https://international.biocloud.net/).