Acclaim rslc 120 c18 column

To extract SA, 50 mg of flash frozen plant tissue was ground in liquid nitrogen and extracted at 4 °C with 10% mM methanol and 1% acetic acid containing deuterated SA standard. Extracts were centrifuge twice and 20 µl of the clear supernatant was analyzed using a Dionex Ultimate 3000 binary RSLC system coupled to Thermo Q-Exactive Focus mass spectrometer with a heated electro spray ionization source. Sample separation was performed using a AcclaimTM RSLC 120 C18 column (100 × 2.1 mm, particle size 2.2 µM; Thermo Scientific 068982). Gradient elution was acetonitrile containing 0.1% formic acid followed with water containing 0.1% formic acid at a flow rate of 200 µL/min. The column temperature was maintained at 35 °C. Mass spectra were acquired in negative mode. Retention time and mass transitions was monitored on purified standard and plant matrixes. For relative quantitation, peak area for each compound (MS2; Thermo Trace Finder Software) was normalized to the initial fresh weight mass.

Spectrum acquisition was performed by an ultrahigh performance liquid chromatography system (Dionex UltiMate 3000 System, Thermo Scientific, USA) and screened with electrospray ionization-mass spectrometry (ESI-MS). The chromatographic separation was performed on an Acclaim TM RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific, USA). The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (acetonitrile) with a gradient elution (0-1 min, 98–84% A; 1–15 min, 84–81% A; 15–17 min, 81–2% A; 17–20 min, 2–2% A). The column was set at 40°C with a flow rate of 0.3 ml/min, and the temperature of the sample manager was set at 4°C.
Mass spectrometry analysis was performed using quadrupole time of flight mass spectrometry (Q-TOF/MS; maXis HD, Bruker, Germany) with an ESI source. Full scans were applied to both positive and negative modes. The mass data were acquired in centroid storage mode using a 50 to 1500 m/z scan range with a 1.0 Hz scan rate over the entire analysis. The following parameters were employed: the pressure of nebulizer gas, 2.0 Bar; drying gas (N₂) flow rate, 8 L/min; drying gas temperature, 350°C; capillary voltage, 3.5 kV and 3.2 kV (in positive and negative mode, respectively).

Analysis was performed on a Dionex UltiMate 3000 UHPLC system coupled to a QExactive Quadrupole-Orbitrap mass spectrometer from Thermo Fisher Scientific (Waltham, MA, United States). Peptides were separated on an AcclaimTM RSLC 120 C18 column (2.2 µM, 150 mm × 2.1) from Thermo Scientific (Sunnyvale, CA, United States). The mobile phase consisted of (A) 0.1% FA in Milli-Q water containing 5% ACN and (B) 0.1% FA in ACN containing 5% Milli-Q water. The column temperature was 40°C. The gradient of the LC method for the untargeted and confirmation analysis of the commercial proteins and the initial screening of the treated samples from mice was performed as follows: 0–2 min, 5% B; 2–35 min, 5–50% B; 35–35.1 min, 50–95% B; 35.1–41 min, 95% B; 41–41.1 min, 95–5% B; 41.1–45 min, 5% B. Quantification of the detected adducted peptides was performed with a shorter gradient: 0–20 min, 50% B; 20–23 min, 50–95% B; 23–27 min, 95% B; 27–27.01 min, 5% B; 27.01–30 min, 5% B.

Separation was performed by the Dionex UltiMate 3000 UPLC system (Thermo Scientific, USA) and screened with ESI-Q-TOF/MS. The LC analysis was performed on an Acclaim^TM RSLC 120 C₁₈ column (2.2 µm, 2.1 × 100 mm; Thermo Scientific, USA) at 40 °C. The mobile phase was composed of solvent A (0.1% formic acid-water) and solvent B (acetonitrile) with a gradient elution (0–2 min, 95–30% A; 2–5 min, 30–15% A; 5–8 min, 15–2% A). The sample manager temperature was set at 4 °C and the flow rate was 0.3 mL/min.
The MS analysis was performed on a maXis HD Q-TOF/MS (Bruker, Germany) using an ESI source. The capillary voltage was 3200 V and 3500 V in negative and positive mode, respectively. The scanning mass range (m/z) was 50–1500 and spectra rate was 1.00 Hz. The nebulizer pressure, dry gas temperature and continuous dry gas flow rate were set at 2.0 Bar, 230 °C, and 8 L/min, respectively.

The optimized extract of D. esculentum was re-dissolved in dimethyl sulfoxide (DMSO) and filtered through a 0.2 µm nylon filter. Prior to injection, the extract was diluted with type I water to a final concentration of DMSO at 0.1%. A 2 µL solution (without acid hydrolysis) was loaded on an AcclaimTM RSLC 120 C18 column (100 mm × 2.1 mm, 2.2 µm particle size, pore size 120 Å, Thermo Fisher Scientific, Waltham, MA, USA), which was connected to HPLC-qTOF-MS (TripleTOF^® 6600 ± quadrupole time-of-flight mass analyzer, AB SciEx, Framingham, MA, USA). A gradient mobile phase consisting of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) with a 0.4 mL/min flow rate for 30 min was used for the separation as follows: 0.00–1.00 min, 1–1% B; 1.00–21.00 min, 1–80% B; 21.00–21.10 min, 80–95% B; 21.10–25.00 min, 95–95% B; 25.00–25.10 min, 95–1% B; 25.10–30.00 min, 1–1% B. Molecular mass analysis was evaluated in both positive ion and negative ion full scan mode with a mass range of 100–1000 m/z. To identify potential chemicals, the MS spectra were aligned with a database of the natural products high-resolution MS/MS Spectral Library on SCIEX OS (AB SciEx, Framingham, MA, USA) and the National Institute of Standards and Technology (NIST). Tentative compounds with a possibility score (library score) of more than 90% were reported.

Samples were analyzed using the UPLC Dionex Ultimate 3000 RS LC (Dionex Suftron, GmbH, Thermo Fischer Scientific, Germening, Germany) coupled to the QTOF Bruker Maxis Impact HD (Bruker Daltonik, Bremen, Germany), equipped with an Enclosure services interface operating in negative ion mode. It had a mass range of m/z 50–1000, the capillary voltage was 2500 V, dry N₂ gas flow of 8.0 L/min (200 °C), nebulizer pressure 2.0 bars, end plate offset 500 V, collision energy 25 eV, and an acquisition time factor of 1 s.
Chromatographic separation was carried out using an Acclaim RSLC 120 C18 column (2.2 µm, 120 Å, 2.1 × 100 mm) (Dionex, Thermo Fischer Scientific, Sunnyvale, CA, USA). The mobile phase consisted of 90% methanol with 5 mM ammonium acetate and 50% methanol with 5 mM ammonium acetate. Injection volume was 1.0 µL. Mass calibration was performed using 1 mM sodium formate/acetate in 50% isopropanol with 0.2% formic acid, HCOO(NaCOOH)₁ (m/z 112.9856), Ac(NaAc)₁ (m/z 141.0169), and Ac(NaF)₁ (m/z 127.0013).
Data analysis and calculations were performed using the following software: Data Analysis 4.1 (SmartFormula, SmartFormula 3D, Isotope Pattern, and Fragmentation Explorer), Profile Analysis 2.1 (PCA and Bucket Statistic Plot), Metabolite Detect 2.0 from Bruker Daltonik, Bremen, Germany, MetFrag (version 2010) [32 ], Metlin [33 ], and MassBank [34 ].