Ammonia hydroxide

The UHPLC separation was conducted using a 1,290 Infinity series UHPLC System (Agilent Technologies, MA, United States) coupled to a UPLC BEH amide column (1.7 µm, 2.1 × 100 mm; Waters, Milford, United States). The mobile phase was composed of ammonium acetate (25 mmol/L, CNW Technologies, Shanghai, China) and ammonia hydroxide (25 mmol/L, CNW Technologies), which were added to water (pH = 9.75) (A) and acetonitrile (B), respectively. The following elution gradient was used for analysis: 0–0.5 min, 95% B; 0.5–7.0 min, 95%–65% B; 7.0–8.0 min, 65%–40% B; 8.0–9.0 min, 40% B; 9.0–9.1 min, 40%–95% B, and 9.1–12.0 min, 95% B. The injection volume, auto-sampler temperature, and column temperature were 2 µl (both positive and negative modes), 4°C, and 25°C, respectively (19 (link)).
MS/MS data acquisition and analysis were conducted using the Analyst TF v1.7 (AB Sciex, MA, United States) according to a preset standard. In each cycle, a collision energy (CE) of 30 eV and cycle time of 0.56 s was used to select the densest 12 precursor ions with an intensity >100 for MS/MS. The following were set as the electrospray ionization (ESI) source conditions: curtain gas (35 psi), gas 1 (60 psi), gas 2 (60 psi), ion spray voltage floating (5,000 V in the positive mode and −4,000 V in the negative mode), declustering potential (60 V), and source temperature (600°C) (19 (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/).