Varian hplc system

Culture samples were primarily analyzed by TLC on Silica gel 60 F₂₅₄ precoated plates (Merck, Germany). All plates were run in a closed TLC chamber and developed using standard visualization techniques and agents: UV fluorescence (254 nm) or by staining with 10 % (v/v) H₂SO₄ and subsequent charring. The mobile phase for detecting the various flavonols and corresponding glycosides consisted of an ethyl acetate:acetic acid:formic acid:water (100:11:11:27 v/v) mixture [63 (link)]. Product spot intensities of other flavonol glycosides were processed and quantified using ImageJ [64 ]. HPLC quantification of sucrose, fructose and glucose was performed using an X-bridge Amide column (35 μm, Waters, USA) as described previously [45 ]. Quercetin, hyperoside, quercitrin and isoquercitrin were detected with the method described by Pandey et al. [41 (link)] using a Varian HPLC system (Agilent technologies, California). Mass spectrometry for determination of the various flavonol glycosides was performed with a Micromass Quattro LC (McKinley Scientific, USA). Detection was performed in negative mode ESI-224 MS with a capillary voltage of 2.53 kV, a cone voltage of 20 V, cone and desolvation gas flows of 93 and 420 L/h, and source and cone temperatures of 150 and 350 °C, respectively.

The residue of DDHR in tomato fruit was investigated as follows: Surface-sterilized tomato fruit were wounded using a sterile nail (one wound per tomato fruit; 3 mm deep and 3 mm wide). Each wound was then treated with 5 μL of DDHR (1000 mg L⁻¹). All treatments were performed in three replicates. At 0, 6, 12, 24, and 48 h after the treatment, the tomato fruit was extracted with methanol. Then, two-fold-diluted methanol extracts were analyzed using the Varian HPLC system (Agilent Technologies) equipped with a J′sphere ODS-H80 column (150 × 4.6 mm i.d., 4 µm) (YMC). Chromatography was performed using a linear gradient elution system (20%–100% acetonitrile in H₂O) for 10 min, followed by isocratic elution (100% acetonitrile) for 5 min at a flow rate of 1 mL min⁻¹. DDHR was monitored at an absorbance of 330 nm. The residue of the commercial fungicide fludioxonil in the tomato fruit was investigated as described above. Fludioxonil was monitored at an absorbance of 254 nm. Each extract was analyzed in three replicates, calculating the average sample area.

The quantification of apigenin, catechin, cyanidin, delphinidin, flavanone, hesperidin, and isorhamnetin in the ethanolic extracts was achieved using flavonoid analytical standards (Sigma-Aldrich) as reported by Morreeuw et al. [40 (link)].
Reverse phase high-performance liquid chromatography (RP-HPLC) analysis was performed according to Mendez-Flores et al. [83 ] on a Varian HPLC system (Agilent Technologies, Santa Clara, CA, USA), including an autosampler (Varian ProStar 410, Agilent Technologies), a ternary pump (Varian ProStar 230I), and a photodiode array detector (PDA, Varian ProStar 330, USA). Briefly, samples (5 µL) were injected onto a Denali^® C18 column (150 mm × 2.1 mm, 3 µm, Grace, Williamsburg, MI, USA) maintained at 30 °C. The mobile phase consisted of formic acid (0.2 %, v/v; solvent A) and acetonitrile (solvent B).
The following gradient was applied: initial, 3% B; 0–5 min, 9% B linear; 5–15 min, 16% B linear; and 15–45 min, 50% B linear. The column was then washed and reconditioned. The detection of released compounds was performed through the PDA detector at 280 and 360 nm. The UV spectra were analyzed using Chromatography Workstation Star Toolbar (version 6.30) software from Agilent for the Varian equipment.