Zorbax sb c18 4.6 250 mm

The major phenylpropanoids were extracted from the exocarp of tomato and the 0.2 g skin samples were ground to powder in liquid nitrogen and extracted overnight at −20 °C in 2 mL methanol with 100% chromatographic grade purity (Sigma-Aldrich, Saint Louis, MO, USA). The first 2 h were shaken and mixed every 15 min for full extraction. The sample was centrifuged at 4 °C and 4000 g for 20 min. The supernatant was filtered with 0.22 µm microporous membrane and stored in dark at −20 °C. The phenylpropanoids were quantified by HPLC (high-performance liquid chromatography, Agilent Technologies 1200 series, Beijing, China) with a chromatographic column (Agilent Technologies ZORBAX SB-C18 4.6 × 250 mm). Aqueous phase A was 0.1% acetic acid solution; organic phase B was pure methanol. The gradient as follow: t = 0.0 min, 20% B; t = 10 min, 30% B; t = 25 min, 90% B; t = 27 min, 90% B; t =28 min, 20% B; t = 32 min, 20% B, each sample was injected 10 µL and the flow rate was 1 mL/min. Detection by ultraviolet (UV) chromatograms was recorded at 325 nm and the column temperature was 35 °C [30 (link)]. All phenylpropanoid standards, rutin, naringenin chalcone and kaempferol rutinoside were obtained from Sigma-Aldrich.

The major flavonols in tomato fruits of Micro-Tom, CSl09-03 and Sheng Nv-Guo were extracted from freeze-dried tomato test specimens using 70% methanol from Sigma (dry-weight basis), and the major flavonols from the ten tomato cultivars numbered 1 to ten were extracted from skin samples (0.2 mg) using 2 ml of 100% methanol from Sigma (http://www.sigmaaldrich.com/) (fresh-weight basis). The flavonols were quantified by HPLC (high-performance liquid chromatography, Agilent Technologies 1200 series) with acolumn (Agilent Technologies ZORBAX SB-C18 4.6*250 mm). A gradient elution was performed with solvent A consisting of 3% acetonitrile and 10% formic acid and solvent B consisting of acetonitrile (50%) and formic acid (10%), with the following elution program: 0 min 4% B, 20 min 20% B, 35 min 40% B, 40 min 60% B, 45 min 90% B, 55 min 4% B, flow rate of 1 mL/min^{7 (link)}. Detection by ultraviolet (UV) chromatograms was recorded at 325 nm. All flavonol standards, rutin, kaempferolrutinoside and naringenin chalcone were obtained from either Sigma-Aldrich or Extrasynthèse (Genay, France).

The HPLC 1260 Infinity (Agilent Technologies, Santa Clara, CA, USA) was used for the determination. The method by Gómez-Estaca et al. [14 (link)] was used, with some modifications. The 1% phosphoric acid (A) and acetonitrile (B) were used as a mobile phase as follows: 80% A and 20% B for 20 min, 70% A and 30% B for 20 to 25 min, 60% A and 40% B for 25 to 40 min. The separation was performed on Zorbax SB-C18 4.6 × 250 mm (Agilent Technologies, Santa Clara, CA, US. The flow rate was 1 mL/min, the injection volume was 10 μL, and the DAD setting was 324.5 nm. Each sample was analyzed in triplicate.