Nova pak c18

Unless otherwise noted, the materials were obtained from commercial suppliers and used without any purification. D₂O was obtained from Sigma-Aldrich. 2′,5′-Diacetyl-3′-deoxy-3′-xylobromoinosine 7 was synthesized as described in [18 ]. Recombinant E.coli PNP was obtained as described in [15 (link)].
Analytical HPLC was performed on the Waters system (Waters 1525, Waters 2489, Breeze 2), column Nova Pak C₁₈, 4.6 × 150 mm, 5 µm, flow rate 0.5 mL/min. Method (I): gradient H₂O → 25% MeOH/H₂O, 20 min. Method (II): gradient H₂O → 50% MeOH/H₂O, 10 min.
NMR spectra were recorded on Bruker Avance II 700 spectrometers (Bruker BioSpin, Rheinstetten, Germany) in DMSO-d6 at 30 °C. Chemical shifts in ppm (δ) were measured relative to the residual solvent signals as internal standards (2.50). Coupling constants (J) were measured in Hz.
Liquid chromatography-mass spectrometry was performed on an Agilent 6210 TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA).
UV spectra were recorded on Hitachi U-2900 spectrophotometer (Tokyo, Japan).

Cherry fruits and leaves of transgenic and wild-type plants were collected to detect PA content. The steps were as follows: grind the material to a powder using liquid nitrogen and weigh 0.5 g of the sample in a 10 mL centrifuge tube; add 1.5 mL of pre-cooled perchloric acid, shake well and mix, then put it into the refrigerator at 4 °C for 1 h; then centrifuge at 4 °C, 15,000 rpm for 30 min; take 1 mL of supernatant and add 10 μL of benzoyl chloride and 1 mL of 2 mol/L sodium hydroxide and mix well, then put it into the water bath at 37 °C for 30 min; add 2 mL of saturated sodium chloride and 2 mL of ether and mix well and centrifuge at 6000 rpm for 5 min; take 2 mL of ether phase, blow dry with a nitrogen blowing instrument; add 2 mL of ether, shake well, and centrifuge for 20 s, then blow dry with nitrogen; add 1000 μL of 60% methanol and shake centrifuge for 30 s; and filter with a 0.45 μm organic filter membrane and fix the volume in a 1.5 mL brown volumetric flask for testing. The determination of polyamines was performed by a high-performance liquid chromatograph (Agilent 1260, Agilent, American) on a chromatographic column (Nova-Pak C18, WATERS, American) with the mobile phase of methanol: water (60:40) at a flow rate of 1 mL·min⁻¹, column temperature of 30 °C, a wavelength of 254 nm, and experiments were set up with three biological replicates.

HPLC separation was performed on the instrument already cited in Section 2.5. A Waters (Milford, MA, USA) reversed-phase column Nova-Pak^® C18 (3.9 mm × 300 mm; 4 μm), thermostated at 40 °C, was used. Mobile phases were A (25 mM acetate buffer pH = 5.65) and B (80:20 mixture of acetonitrile and methanol). Flow rate: 1.1 mL/min. HPLC gradient, for solvent A was: 0 min, 100%; 7 min, 96%; 18 min, 94%; 23 min, 92%; 25 min, 92%; 28 min, 85%; 50 min, 77%; 60 min, 55%; 65 min, 40%; 67 min, 20% and 70 min, 100%.
Detection was performed at 280 nm while quantification was based upon calibration curves obtained by plotting peak areas vs. concentration of solutions of standard amino acids and amines at known concentration.

The analysis of target compounds was carried via HPLC (Agilent 1100 Series; Agilent Technologies, Santa Clara, CA, USA) that was coupled with a diode array detector (DAD, G1315A, Agilent Series). Measurements were done at wavelength of 285 and 240 nm for 3,5-DCP and 2,4-DCA, respectively. A Nova-Pak C18 (3.9 mm × 150 mm, 4 µm, Waters Corporation, Milford, MA, USA) reversed-phase column was used. The mobile phase consisted of 70% methanol and 30% ultrapure water. The flow rate and temperature of the column were set as 1.0 mL/min and 25°C, respectively. The injection volume was 100 µL. Analytical methods were validated for a concentration range of 0.0625–2 mg/L. Six-point calibration curves showed good linearity (R²) with correlation coefficients of 0.9998 and 0.9996 obtained for 3,5-DCP and 2,4-DCA, respectively. Detection and quantification limits were established as 0.06 and 0.21 mg/L for 3,5-DCP and 0.03 and 0.12 mg/L for 2,4-DCA, respectively. An Orion (USA) 720A+ model pH-meter was used for all pH measurements. A Jenway 6300 model spectrophotometer (Staffordshire, UK) was employed for colorimetric, residual PS analyses [53]. Fe and Al releases were monitored on a Perkin-Elmer ICP-MS (PerkinElmer, Inc., Waltham, MA, USA) [54].

The HPLC system (Beckman-Coulter) consisted of a 126 solvent module, 166 detector, and IBM personal computer. Data acquisition and processing were carried out using 32 Karat 7.0 version software (Beckman-Coulter). Samples (20 μL) were injected in a Nova Pak C18, 300 × 3.9 mm i.d., and 4 μm reversed-phase column (Waters), and elution was carried out at 0.9 mL/min using a 25 mM glacial acetic acid/acetonitrile binary gradient as shown in Table 1. Mobile phases were filtered through a 0.45 μm membrane filter. The column was maintained at 18°C.