1290 infinity lc system

500 ng of genomic DNA was incubated with 5 U of DNA Degradase Plus (Zymo Research) at 37 °C for 3 h. The resulting mixture of 2′-deoxynucleosides was analysed on a Triple Quad 6500 mass spectrometer (AB Sciex) fitted with an Infinity 1290 LC system (Agilent) and an Acquity UPLC HSS T3 column (Waters), using a gradient of water and acetonitrile with 0.1% formic acid. External calibration was performed using synthetic standards, and for accurate quantification, all samples and standards were spiked with isotopically labelled nucleosides. 5mC and 5hmC levels are expressed as a percentage of total cytosines.

500 ng of genomic DNA was incubated with 5 U of DNA Degradase Plus (Zymo Research) at 37 °C for 3 h. The resulting mixture of 2’-deoxynucleosides was analysed on a Triple Quad 6500 mass spectrometer (AB Sciex) fitted with an Infinity 1290 LC system (Agilent) and an Acquity UPLC HSS T3 column (Waters), using a gradient of water and acetonitrile with 0.1% formic acid. External calibration was performed using synthetic standards, and for accurate quantification, all samples and standards were spiked with isotopically labeled nucleosides. HmC levels are expressed as a percentage of total cytosines.

The detection of phenolic compounds (other than ANC) was conducted by LC-MS/MS using an Agilent 6460C triple quadrupole mass spectrometer equipped with infinity 1290 LC system (Agilent Technologies, Santa Clara, CA, USA). The conditions for separation was similar to the HPLC-DAD as mentioned above. The method was modified from Zhao, et al. [25 (link)]. Samples were analysed in negative mode with a gas temperature of 250 °C, gas flow rate of 10 L/min, and Vcap 3500 V. Quantifications were carried out by standard curves of each analytical standard.

LC-MS was performed on an Agilent Technologies
(Santa Clara, CA) Infinity 1290 LC system coupled via a dual-source
AJS electrospray interface to an Agilent Technologies 6560B ESI Ion
Mobility Q-TOF. Glycopeptide standards were analyzed with a SeQuant
ZIC-HILIC column (20 × 2.1 mm², 3.5 μm particles;
Merck, Darmstadt, Germany), with 0.1% (v/v) formic acid as eluent
A and ACN as eluent B, using a linear gradient from 90–50%
B over 5 min and maintaining 50% B for 8 min. MS was performed in
positive ion mode for glycans 1 and 3 and
negative ion mode for glycans 2 and 4. O-glycans, released from BSM oxidatively or by β-elimination,
were separated with a ZIC-HILIC column (150 × 2.1 mm², 3 μm particles) with a ZIC-HILIC guard column (20 ×
2.1 mm², 3 μm particles; Merck, Darmstadt, Germany)
using 5 mM ammonium formate (pH 6.5) as eluent A and ACN as eluent
B. Chromatographic separation was achieved using 85% B for 5 min,
followed by a linear gradient to 50% B over 25 min at 0.2 mL/min. O-glycans released by β-elimination were additionally
analyzed with the same gradient using 10 mM ammonium bicarbonate (pH
7.8) as eluent A to preserve sulfated moieties better. MS analysis
was performed in negative ion mode with a capillary voltage of 3500
V, nozzle voltage of 2000 V, nebulizer pressure of 40 psi, drying
gas flow rate of 300 °C at 8 L/min, and sheath gas temperature
of 300 °C at 11 L/min.

Samples were prepared using a modification of the method reported by Liu et al. [57 (link)]. Tobacco leaves were ground to a powder in liquid nitrogen. One hundred milligrams of powder were dissolved into 1.0 mL of extraction solvent (CH₃OH/H₂O, 80/20, v/v, with internal standards ABA-d₆ at 20 ng/mL). The mixture was sonicated for 30 min and then centrifuged at 4 °C for 5 min at 8000 rpm. The supernatant was then filtered through a 0.22-μm filter and evaluated with HPLC-MS/MS analysis.
A 1290 Infinity LC system coupled to a 6490 Triple Quad mass spectrometer (Agilent) was employed in the ABA determination. The chromatographic separation was completed using an Agilent SB-C₁₈ column (2.1 mm × 100 mm, 1.8 μm) held at 50 °C, with a sample injection volume of 5 μL. Mobile phase A (MA) was 0.001% formic acid in water and mobile phase B (MB) was acetonitrile. The flow rate was 200 μL/min. The gradient elution method and mass spectrometer instrumental parameters were the same as in [47 (link)].

Samples (2 µL) were analyzed by HPLC/UV (1290 Infinity LC System, Agilent Technologies, Cernusco sul Naviglio (Milano), Italy) using a reverse phase column (Zorbax Extend-C18, 1.8 µm, 50 × 3.0 mm i.d.; Agilent Technologies, cat. n. 727975-302) and a UV diode array detector (190–500 nm). Solvents A and B were water containing 0.1% trifluoroacetic acid (TFA) and acetonitrile, respectively. The gradient for B was as follows: 10% for 0.5 min, then from 10% to 100% in 3.5 min, 100% for 1 min; the flow rate was 0.6 mL/min and the column compartment was maintained at 35 °C. Eluate was preferentially monitored at 286, 300, and 320 nm (corresponding to absorbance maxima of the internal standard, derivatives/metabolites, and pterostilbene, respectively).