Agilent sb c18 column

The qualitative and quantitative analysis of C3S in samples was carried out by LCMS-8050 liquid chromatography-mass spectrometry [25 (link)]. An Agilent SB-C18 column (4.6 × 150 mm, 2.7 μm; Agilent Technologies Inc., Palo Alto, CA, USA) was used to separate the anthocyanin samples. The flow rate and injection volume were 1 mL/min and 20 μL, respectively. The column temperature was 35 °C. The chromatographic column passed through phase A (5% formic acid in water) and phase B (5% formic acid in acetonitrile). The elution gradient was as follows: 5% B (0–5 min); 5–10% B (5–10 min); 10–27.5% B (10–40 min).
The acquisition mode of MS was automatic ESI-MS/MS in positive ion mode, under the following conditions: atomizing gas flow rate, 3 L/min; heating gas flow rate, 10 L/min; interface temperature, 350 °C; DL temperature, 250 °C; heating module temperature, 400 °C; dry air flow rate, 10 L/min; fragmentation voltage, 4 KV; scan mode, Q3 Scan; scan range, 100–1000 m/z.

10 μL sample was injected onto an Agilent SB-C18 column (2.1 mm × 50.0 mm, Agilent) and eluted at a flow rate of 400 μL/min. Mobile phase A was H₂O containing 0.1% formic acid. Mobile phase B was acetonitrile containing 0.1% formic acid. Elution started with 10% mobile phase B for 3 min, which then raised to 100% over 21 min, stayed at 100% for 2 min, reduced to 10% over 0.1 min, and stayed at 10% for 3.9 min. The column temperature and UV detection wavelength were 30°C and 210 nm, respectively. Mass spectrometric analysis was performed on a Waters XEVO-G2 QTOF of a quadrupole and orthogonal acceleration time-of-flight tandem mass spectrometer. The scan range was m/z 50–600. For positive-ion mode, the capillary and cone voltage were set at 3.5 kV and 40 V, respectively. The desolvation gas was set to 600 L/h at a temperature of 450°C. The ion source temperature was set to 110°C.All data were acquired and processed using Waters MassLynxv4.1 software.

Chromatographic separation was conducted on an Agilent SB-C18 column (1.8 μm; 2.1 mm × 100 mm; Agilent Technologies, Santa Clara, CA, United States) at 40°C. The mobile phase consisted of solvent A [0.1% (v/v) formic acid in pure water] and solvent B (acetonitrile). The linear gradient program for elution was as follows: (a) from 0.00 to 9.00 min, phase B was in the range of 5–95% and maintained at 95% for 1 min; (b) from 10.00–11.10 min, the proportion of phase B was reduced to 5% and held for 14 min. The flow rate was 0.35 mL/min, and the injection volume was 4 μL. The effluent was alternately connected to an electrospray ionization-triple quadrupole-linear ion trap. The main conditions of the mass spectrometry were as follows: electrospray ionization temperature, 550°C; MS voltage, 5,500 V; curtain gas pressure, 30 psi; and collision-activated dissociation parameter, high. In triple quadruple pole (QQQ) mode, each ion pair was scanned according to the optimized declustering potential and collision energy (Chen et al., 2013 (link)).

Melatonin was extracted from cabbge seeds and seedlings according to the method described in our previous work (Zhang H. J. et al., 2014 (link)). Approximately 1 g frozen samples were ground into powder with liquid nitrogen and homogenized with 10 mL methanol. After centrifugation at 11,417 g at 4°C for 15 min, the supernatants were collected and dried using nitrogen gas. The extracts were then dissolved in 5% methanol and purified using a C18 solid phase extraction (SPE) cartridge (Waters, Milford, MA, USA). The cartridge was next washed with 10 mL 5% methanol, and melatonin was eluted finally at a natural flow rate with 2 mL 80% methanol. The extract was subsequently filtered through a 0.22 μm PTFE syringe filter before UHPLC-ESI-MS/ MS analysis. Melatonin determination and quantification was analyzed using a UHPLC-ESI-MS/MS (UHPLC-1290 Series and a 6460 QqQ-MS/MS; Agilent Technologies, Waldbronn, Germany) with an Agilent SB-C18 column (4.6 9 50 mm; 1.8 l m; Agilent Technologies, Santa Clara, CA, USA).

To evaluate the sample extracts, a UPLC‒ESI‒MS/MS system was used (UPLC, SHIMADZU Nexera X2; MS, Applied Biosystems 4500 Q TRAP). The analytical conditions were as follows: UPLC, Agilent SB-C18 column (1.8 µm, 2.1 mm*100 mm); mobile phase, solvent A (pure water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid); gradient elution, 95%-5% A at 0-9 min, 5% A at 9-10 min, 5%-95% A at 10 min-11.1 min, and 95% A at 11.1 min-14 min; flow rate, 0.35 mL per minute; temperature, 40 °C; and injection volume, 4 μL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS [51 (link)].

Corn powder (0.1 g) was dissolved in hydrochloric acid (6 mol/L, 8 mL) and bubbled with nitrogen gas, followed by hydrolysis at 120 °C for 2 h. Sodium hydroxide (10 mol/L, 4.8 mL) was then added to the previously mentioned solution [31 ]. The solution was filtered and diluted to 25 mL. The content of amino acids in the supernatant was determined using an Agilent-1200 HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a quaternary pump, online degasser, auto-sampler, and column heater–cooler. Separation was performed on an Agilent SB-C18 column (250 × 4.6 mm, particle size 5 mm; Agilent Technologies, Newport, DE, USA). Chromatographic analyses were carried out using gradient elution with mobile phase A, consisting of 0.05 mol/L sodium acetate buffer solution (pH = 6.50), and mobile phase B, consisting of an acetonitrile-water solution (v/v = 1:1). Gradient elution was initiated with 30% B, which was held for 5 min. Thereafter, it was increased linearly up to 100% B in 35.0 min. This composition was held for another 2.0 min before being reduced to 16% B in 7.0 min, followed by a re-equilibration time of 6.0 min, yielding a total run time of 55.0 min. The flow rate was set at 1.0 mL/min, and column temperature was maintained at 23 °C. The detection wavelength was 360 nm. Aliquots of 10 μL of the sample extract were injected into the chromatographic system.