Zorbax rrhd eclipse xdb c18 column

UPLC-MS/MS analyses were conducted on an Agilent UPLC-MS/MS system consisting of 1290 UPLC-system coupled with an Agilent 6470 triple-quadrupole mass spectrometer (Agilent Technologies). For analysis, 3 μL of the extract were injected. Chromatographic separation was achieved on an Agilent ZORBAX RRHD Eclipse XDB C18 column (2.1 × 100 mm, 1.8 μm particles) using a flow rate of 0.659 mL/min at 45 °C during a 13 min gradient (0–12 min from 68% A to 20% A, 12–13 min 5%A), while using the solvents A, water containing 0.005% formic acid, and B, acetonitrile containing 0.005% formic acid. Electrospray ionization was performed in the negative ion mode using N2 at a pressure of 30 psi for the nebulizer with a flow of 10 L/min and a temperature of 300 °C, respectively. The sheath gas temperature was 350 °C with a flow rate of 11 L/min. The capillary was set at 3500 V and the nozzle voltage was 500 V. Multiple reaction monitoring (MRM) has been used for quantification of screening fragment ions.
Data preprocessing: Peak determination and peak area integration were performed with MassHunter Workstation software (Version B.08.00, Agilent Technologies) while auto-integration was manually inspected and corrected if necessary. The obtained peak areas of targets were corrected by appropriate IS and calculated response ratios were used throughout the analysis.

Liquid fungal cultures (50 ml) including fungal tissue and medium were frozen using a dry ice-acetone bath and lyophilized. The lyophilized residues were extracted with 20 ml of ethyl acetate-methanol (9:1) for 1.5 h with vigorous stirring. Extracts were filtered over cotton, evaporated to dryness, and stored in 4-ml vials. Crude extracts were suspended in 0.5 ml of methanol and centrifuged to remove insoluble materials, and the supernatant was subjected to UHPLC-HRMS analysis. An Agilent Zorbax RRHD Eclipse XDB-C₁₈ column (2.1 by 100 mm, 1.8-µm particle diameter) was used with acetonitrile (organic phase) and 0.1% formic acid in water (aqueous phase) as solvents at a flow rate of 0.5 ml/min. A solvent gradient scheme was used, starting at 2% organic for 1 min, followed by a linear increase to 100% organic over 14 min, holding at 100% organic for 2.5 min, decreasing back to 2% organic for 0.1 min, and holding at 2% organic for the final 1.4 min, for a total of 18 min.

Samples analyzed by mass spectrometry were resolved using a Zorbax Eclipse XDB-C18 RRHD column (2.1 × 50 mm, 1.8 μm, room temperature, Agilent Technologies part # 981757-902) fitted with a guard column (Zorbax Eclipse XDB-C18, 2.1 × 5 mm 1.8 μm, Agilent part # 821725-903) using a 1290 Infinity II UHPLC (G7120AR, Agilent). The mobile phases used were (A) 0.1% formic acid in water; and (B) 0.1% formic acid in 100% acetonitrile. This 11.5-min method used a flow rate of 0.7 ml/min and began with Mobile Phase B held at 5% for 2 min, followed by a linear gradient from 5 to 95% B over 7.5 min, then finally B held at 95% for 2 min. The following parameters were used: fragmentor voltage of 175 V, gas temperature of 300ºC, gas flow of 12 l/min, sheath gas temperature of 350ºC, sheath gas flow of 12 l/min, nebulizer pressure of 35 psi, skimmer voltage of 65 V, Vcap of 3500 V, and collection rate of 3 spectra/s. Expected exact masses of puromycin–phenylalanine–caproic acid–biotin (m/z: 958.4604) and leucine enkephalin (m/z: 556.2771) were calculated using ChemDraw 19.0 and extracted from the total ion chromatograms ±100 ppm by LC–HRMS with an Agilent 6530 Q-TOF AJS-ESI (G6530BAR).
The yield of C-pmn-pcb in the fragment reactions was determined by calculating it using the equation below:

Before the UPLC analysis, the ZEA in the growth medium was extracted and cleaned up using an IAC-SEP^® ZEA-specific immunoaffinity column (Clover Technology Group, Beijing, China) according to the manufacturer’s instructions. The resulting sample was transferred to an autosampler vial, and 100 μL was injected into the UPLC column for analysis.
The ZEA content was tested following the Chinese standard method GB 5009.209-2016 [35 ]. Quantitative analysis was performed on a Dionex Ultimate 3000 UPLC system (Thermo Fisher Scientific, Waltham, MA, USA) consisting of an Ultimate 3000 RS autosampler, pump, column oven, and fluorescence detector, and controlled by Chromeleon 7.2 software. Separation was performed on a Zorbax Eclipse XDB-C18 RRHD column (100 × 2.1 mm, 1.8 μm; Agilent). The isocratic mobile phase was acetonitrile/water/methanol (46:46:8, v/v) with a flow rate of 1.0 mL/min. The fluorescence detection excitation wavelength was 274 nm, and the emission wavelength was 440 nm.
The ZEA was identified and quantified via comparison with the retention time and the chromatographic peak area of an external standard. The standard curve was obtained by preparing standard solutions at six ZEA concentrations (5, 10, 50, 100, 200, and 500 ng/mL), and each concentration was analyzed in triplicate. The resulting regression equation for ZEA was as follows: y = 2912.3x − 6081.6 (R² = 0.9998).