Peak 2

The Shimadzu UPLC system (Shimadzu, Kyoto, Japan) consists of pumps (LC-30AD), an online degasser (DGU-20A^5R), an auto-sampler (SIL-30AC), and a column oven (CTO-30aHE). Chromatographic separation was performed on a Thermo Hypercarb analytical column (2.1 mm × 100 mm, 5 μm) at 40 °C. The mobile phase consisted of an aqueous phase (2 mmol/L ammonium acetate solution containing 0.06% diethylamine, pH 11) and an organic phase (acetonitrile containing 2 mmol/L ammonium acetate and 0.06% diethylamine). The linear gradient elution was 2%~15%~40%~60% organic phase at 0~4~7.5~10 min. The constant flow rate was 0.6 mL/min. The sample solution of 2 µL was injected into the UPLC system by the auto-sampler.
Time-of-flight mass spectrometry measurements were performed on a 4600 Q-TOF mass spectrometer (AB Sciex, Concord, CA, USA) equipped with an electrospray ionization source. The Q-TOF/MS analysis was performed in negative mode with the following parameters: Ion source gas 1 (GS1) (N₂) at 50 psi, ion source gas 1 (GS1) at 50 psi, curtain gas at 35 psi, a temperature of 500 °C, and an ionspray voltage floating at −4500 V. The mass range was set to m/z 100–1000. The system was operated under Analyst 1.6 and Peak 2.0 (AB Sciex, Concord, CA, USA) and used an APCI negative calibration solution to calibrate the instrument’s mass accuracy in real-time.

The Shimadzu UPLC ((Shimadzu, Kyoto, Japan) system consists of an online degasser (DGU-20A5R), an auto-sampler (SIL-30AC), two pumps (LC-30AD), and a column oven (CTO-30aHE). Chromatographic separation was performed on a Waters BEHC18 analytical column (2.1 × 100 mm, 1.7 μm, Waters, Milford, MA, USA) at 40 °C. The mobile phase consisted of 0.2% formic acid and acetonitrile. The linear gradient elution with a constant flow rate of 0.2 mL/min was 10%~10%~40%~95%~10% acetonitrile at 0~1~10~13~15 min. The sample solution and mixed working solutions of 5 µL were injected into the UPLC system by the auto-sampler.
TOF/MS measurements in negative ion mode were performed on a 4600 Q-TOF mass spectrometer (AB Sciex, Concord, CA, USA) equipped with an electrospray ionization (ESI) source with the following parameters: Ion source gas 1 (GS1) at 50 psi, ion source gas 1 (GS1) (N₂) at 50 psi, curtain gas at 35 psi, temperature at 500 °C, and ionspray voltage floating at −4500 V. The mass range was set to m/z 100–800. The system was operated under Analyst 1.6 and Peak 2.0 (AB Sciex, Concord, CA, USA) and used an APCI negative calibration solution to calibrate the instrument’s mass accuracy in real-time.