Poroshell 120 sb c18

The positive ion mode is adjusted as follows: Agilent Poroshell 120 SB-C18 (100 mm × 4.6 mm, 2.7 μm) is used; the mobile phase consists of 0.1% formic acid water (A)–acetonitrile (B); gradient elution conditions are 0–25 min, 5–40% B; 25–35 min, 40–75% B; 35–40 min, and 75–100% B; the flow rate is 0.8 ml min⁻¹; the column temperature is 30°C; the injection volume is 1 μl; the electrospray ion source (ESI) is detected in the positive ion mode; the drying gas flow is 13 L/min; the drying gas temperature is 350°C; the capillary voltage (Vcap) is 4,000 V; the neutralizer pressure is 45 psig; the fragmentor voltage is 125 V; the skimmer voltage is 65 V; the mass scanning range is 50–1,000 m/z; and the secondary MS collision voltage is 40 eV.
The negative ion mode is set as follows: Agilent Poroshell 120 SB-C18 (100 mm × 4.6 mm, 2.7 μm) is used; the mobile phase consists of water (A)–acetonitrile (B); gradient elution conditions are 0–30 min, 5–100% B; the flow rate is 0.8 ml min⁻¹; the column temperature is 30°C; the injection volume is 5 μl; the drying gas flow is 11 L/min; the drying gas temperature is 250°C; the Vcap is 3,500 V; the neutralizer pressure is 45 psig; the fragmentor voltage is 125 V; the skimmer voltage is 65 V; the mass scanning range is 50–1,000 m/z; and the secondary MS collision voltage is 40 eV (Wang et al., 2021 (link)).

GCE, eGCE and tannic acid were analyzed by high performance liquid chromatography coupled to electrospray ionization-time of flight-mass spectrometry (HPLC-ESI-TOF-MS) [19 (link)], a tandem system. The Agilent 1200 series HPLC consisted of a model G1315D diode array detector (DAD), a G1312B binary Pump, a G1379B vacuum degasser, a G1367C auto-sampler and a G1316B column heater. The concentration of the testing solution was 1mg/mL, and the injection volume was 5 μL. Gradient elution HPLC was applied at a flow rate of 0.4 mL/min with detection at 270 nm. Two solvents were used for the mobile phase: (A) 0.1% formic acid and 10 mM ammonium formate (pH3.0), (B) acetonitrile. Compounds were separated using the following gradient: 0-5 min, 5% B; 5-8 min, 20% B; 20-30 min, 30% B; 30-40 min, 20% B; 40.0-40.1 min, 5% B. The separation of components in GCE was performed on an Agilent column (Poroshell 120 SB-C18, 150mm × 4.6mm, particle size 2.7μm) at 40 °C.
The time of flight (TOF) mass detector (G1969A, Agilent, US) was equipped with electrospray ionization (ESI) interface. The ESI voltage was 3.5 kV, and a mass range of 50-3000 m/z was scanned in negative full scan mode. Data processing was performed on Agilent Mass Hunter (v.B.01.04) software.

ABA and JA concentrations were determined according to the method described by You et al. [52 (link)] with little modification. First, 2 mL of extracted sap sample were placed into a microcentrifuge tube containing 5 mL extraction buffer composed of isopropanol/hydrochloric acid and 8 μL 1 μg mL⁻¹ deuterated internal standard solutions. Then, 10 mL dichloromethane were added and sap samples were shaken at 4 °C for 30 min and then centrifuged at 13,000× g for 5 min at 4 °C. The supernatant was carefully removed and the lower organic phase was dried by N₂ in shade conditions and dissolved in 400 μL methanol (containing 0.1% methane acid), then filtered with a 0.22-mm filter membrane. The purified sample was then subjected to high-performance liquid chromatography‒tandem mass spectrometry (HPLC-MS/MS), fitted with a POROSHELL120 SB-C18 (Agilent Technologies Inc., Santa Clara, CA, USA) column (2.1 mm × 150 mm; 2.7 mm), at 30 °C. The solvent gradient used was 100% A (99.9% methanol: 0.1% CHOOH) to 100% B (99.9% H₂O: 0.1% CHOOH) over 15 min. The injection volume was 2 μL. MS conditions were as follows: the spray voltage was 4500 V; the pressure of the air curtain, nebulizer, and aux gas were 15, 65, and 70 psi, respectively; and the atomizing temperature was 400 °C. The ABA and JA concentrations were calculated with reference to the peak area of the deuterated internal standard.