Uhplc q exactive plus orbitrap ms

KGY (1 g) was placed in a round-bottomed flask. Methanol aqueous solution (1:1, v/v) (50 mL) was added and weighed. After refluxing for 1 h, the lost weight was made up with methanol aqueous solution (1:1, v/v), mixed, and filtered for use.
The 2 μL KGY sample was detected by UHPLC-Q-Exactive Plus Orbitrap MS (Thermo Fisher Scientific, USA). The flow rate was set at 300 μL/min. The mobile phase gradients (A: acetonitrile, 0.1% formic acid; B: H2O, 0.1% formic acid) were as follows: 2% A at 0–2 min; 2–15% A at 2–3 min; 15–25% A at 3–8 min; 25–40% A at 8–9 min; 40–98% A at 9–11 min; 98% A at 11–15 min; 98–2% A at 15–18 min and 2% A at 18–20 min. The heated electrospray ion source (HESI) was used for simultaneous detection in positive and negative ion modes, with high purity N₂ as the nebulizing gas and high purity He as the collision gas. The sheath gas flow rate was 35 arb; the auxiliary gas flow rate was 10 arb; the spray voltage was 3500 V/2500 V (±); the capillary temperature was 320 °C; the heater temperature was 350 °C; the scanning mode was Full MS/dd-MS²; the dd-MS² resolution was 17500; the Full MS resolution was 70,000; the scanning range: 100–1500 m/z; the step collision energy: 20 V, 40 V, 60 V.