Spd m20a dad detector

The plant material was obtained from winter oilseed rape plants at BBCH growth stages 12-13, which corresponded with plant stage used for experiments on aphid development and probing behavior. Plant samples were stored under -80 °C for 24 h and lyophilized. Glucosinolates were isolated by the enzymatic desulfation procedure according to the Commission Regulation (EC) No 1864/90 (Commission of the European Communities 1990). In total, 200 mg of freeze-dried material was transferred to test tubes and heated at 75 °C in a hot water bath for 1 min. Next, glucosinolates were extracted three times with 2 ml of boiling methanol (70%), stirring occasionally with the UltraTurrax IKA T-25 homogenizer (Jankel & Kunkel, Germany). The supernatants were centrifuged, combined and filled up to 10 ml. Glucotropaeolin was used as an internal standard. Desulfoglucosinolates were analyzed by HPLC according to the method described by Ciska et al. (2008) . The separation was performed in an HPLC system with an autosampler (LC-20) and the SPD-M20A DAD detector (Shimadzu, Japan) using the LiChrospher ® 100 RP-18 (5 µm, 250 × 4 mm) column (Merck, Darmstadt, Germany) with a flow rate of 1.2 mL/ min. Desulfo-glucosinolates were separated in a gradient of water and 20% acetonitrile as previously described (Ciska et al. 2008) .

The individual phenolic compounds of the cherry laurel sample were determined by the HPLC system coupled to a diode array (HPLC-DAD, Shimadzu Corp., Kyoto, Japan). HPLC system consisted of an LC-20AD pump, an SPDM20A DAD detector, a SIL-20A HT autosampler, a CTO-10ASVP column oven, a DGU-20A5R degasser, and a CMB-20A communications bus module (Shimadzu Corp., Kyoto, Japan). A reversed-phase column (Intersil ® ODS C-18, GL Sciences, Tokyo, Japan) with a 250 mm × 4.6 mm length, 5 µm particle size was used to perform separation of phenolic compounds at 40 • C. The mobile phases consisted of solvent A (distilled water with 0.1% (v/v) acetic acid) and solvent B (acetonitrile with 0.1% (v/v) acetic acid). Gradient elution was applied at a flow rate of 1 mL/min and it was: 10% B (0 to 2 min), 10% to 30% B (2 to 27 min), 30% to 90% B (27 to 50 min) and 90% to 100% B (51 to 60 min). Chromatograms were obtained at 254-356 nm. Identification and quantification analysis were conducted based on standard curves and retention times and. The detection of phenolic compounds was performed at 260 nm (epicatechin), 280 nm (gallic, syringic, protocatechuic, caffeic, cinnamic, p-hydroxybenzoic acid and catechin), 320 nm (chlorogenic, vanillic, p-coumaric, ferulic acid) and 360 nm (rutin, quercetin). The concentration of each phenolic compound was expressed as mg per 100 g of sample (DM) [12] (link).