Zorbax extend c18

The HPLC‐ELSD conditions were used for the determination of the alkaloids, and an LC‐20AT Shimadzu high‐performance liquid chromatography system (Kyoto, Japan) equipped with an Alltech 2000 evaporative light‐scattering detector (Grace,) was used. The chromatographic separations were performed over a ZORBAX Extend‐C18 (250 mm × 4.6 mm, 5 µm; Agilent) column at a column temperature of 25℃. The column was eluted with a mixture of acetonitrile (mobile phase A) and water‐ammonium bicarbonate (PH = 10.10, mobile phase B) at a flow rate of 1.0 ml/min. The elution conditions were as follows: 0–10 min, 75%A to 60% A; 10–20 min, 60% A; 20–30 min, 60% A to 30% A; 30–45 min, 30% A to 10% A; 45–55 min, 10% A; 55–60 min, 10% A to 75% A; 60–65 min, 75% A. The drift tube temperature of the ELSD was set at 115℃, and using nitrogen as the carrier gas at a flow rate set at 3.0 L/min, the gain value was 2, and the injection volume was 20 µl for analysis. The concentration of each compound was calculated based on external standard method, using the calibration curves of each compound.

Chromatographic separation was performed on a Zorbax Extend C₁₈ UHPLC column (2.1 mm × 100 mm, 1.8 µm, Agilent, Palo Alto, CA, USA). The column temperature was set at 25 °C. Flow rate was 0.3 mL/min and the sample injection volume was 3 μL. The mobile phase consisted of solvent A (water) and solvent B (acetonitrile). The optimized UHPLC gradient elution program was as follows: 0–20 min, 5–70% B; 20–24 min, 70–100% B; and 24–26 min, 100% B.

The HPLC analysis was performed on an Agilent Series 1200 chromatographic system (Agilent Technologies, Santa Clara, CA, USA) coupled with a photodiode array detector (PAD) (Agilent Technologies, Santa Clara, CA, USA) and using a Zorbax Extend C18 (4.6 × 250 mm, 5 μm i.d.) chromatographic column (Agilent Technologies, USA). The elution was performed in a gradient with a mobile phase consisting of (A) 1% (v/v) formic acid solution in water and (B) 1% (v/v) formic acid solution in acetonitrile. The regimen was as follows: 0 min—7% B; 5 min—7% B; 45 min—40% B; 47 min—70%; 52 min—70% B. The flow was 1 mL min⁻¹, the injection volume 5 μL and the column temperature 25 °C. The chromatograms were recorded at 278 and 320 nm. The analysis for all samples was performed in duplicate. All samples were filtered through a 0.45 μm membrane filter (Nylon Membranes, Supelco, Bellefonte, PA, USA) prior to the analysis. Identification of individual phenolic compounds and caffeine was performed by comparing the retention times and the characteristic absorption spectrum (190–400 nm) with commercially available standards, while quantification was enabled by establishing calibration curves (20–100 µg mL⁻¹).

An LC instruments set (Agilent Technologies 1260 Infinity II, Santa Clara, CA, USA) was connected to a time-of-flight mass spectrometer (TOFMS; Agilent Technologies G6230B) with an electrospray ionization source (Dual Agilent Jet Stream ESI). TOFMS detection was operated in negative ionization mode using the following scan source parameters: capillary voltage 3.5 kV, ion source gas temperature 300 °C, drying gas flow 10 L/min, and sheath gas temperature 350 °C with sheath gas flow 11 L/min. The column used was ZORBAX Extend-C18 (2.1 × 50 mm, particle size 1.8 μm; Agilent Technologies, P/N: 727700-902), and the column temperature was set at 45 °C. Column equilibration was carried out 1 day before actual measurement by passing mobile phases A and B at the starting ratio according to each measurement method.

The content of rosmarinic acid was determined on an Agilent Series 1200 chromatographic system (Agilent Technologies, Santa Clara, CA, USA) using a Zorbax Extend C18 (4.6 × 250 mm, 5 μm i.d.) chromatographic column (Agilent Technologies, Santa Clara, CA, USA) and coupled with a Photodiode Array Detector (PAD) (Agilent Technologies, Santa Clara, CA, USA) (Table S1, Figure S1). The gradient elution was performed with a two-component mobile phase consisting of 1% (v/v) formic acid solution in water and 1% (v/v) formic acid solution in acetonitrile, as described by Šeremet et al. [26 (link)].

For ZEA degradation, ZHDCP, ZHD101 and carboxypeptidase enzymes had chosen. The ZEA degradation test (in a total volume of 1 mL) was started by adding concentrated purified protein, where ZEA 5 ppm, 100-μg enzyme, 900-μL reaction buffer (150 mM NaCl, 25 mM Tris-HCl, pH: 9.5). The reaction was completed by heating the reaction mixture at 98 °C for 2 min. For identification of ZEA and its metabolite, the reaction mixture was filtered and analyzed by UV-HPLC (274 nm). The optimum pH was investigated at 30 °C in different buffers in the pH range 4-11. The optimum temperature was studied at pH 7.5 using temperatures ranging from 20 to 45 °C. The UV-HPLC running parameter deployed are as follows: Agilent Zorbax Extend-C18, 3 × 150 mm, 3.5 μm particle size); mobile phase: methanol/water/formic acid (10/90/0.1, v/v/v), flow rate: 0.2 mL/min; nitrogen drying gas: 10 L/min, nebulizer pressure: 25 psi, drying gas temperature: 350 °C, capillary voltage: 4 kV.

The lipophilicity of synthesized compound was evaluated using Agilent 1200 Series HPLC instrument (Agilent technologies, Waldbron, Germany) controlled through the Chemstation software (version B.04.03). The separation was performed on ZORBAX Extend-C18 (3 × 150 mm, 3.5 µm) column (Agilent Technologies, Waldbron, Germany). Methanol (constituent A) and purified water (constituent B) were used as a mobile phase. Total flow rate was set up to 0.4 mL/min; the injection volume was 1 µL; and the column temperature was maintained at 25 °C. The detection wavelength of 210 nm was used. Retention factors of the compounds were measured under five different isocratic conditions in the 65:35–80:20 range (A:B; v/v), in duplicate. The log k_w values (indexes of lipophilicity) were estimated by linear regression analysis based on the following equation,

where logk represents a logarithm of an individual isocratic retention factor, Φ is the organic phase concentration and S is a constant derived from linear regression analysis.