Hplc lc 20ad xr analytical system

Anthocyanin and stilbene content analysis was performed by the method HPLC-MS as described [45 (link),47 (link),48 (link)]. Briefly, for anthocyanins 100 mg fresh cells tissue were subsequently homogenized using a mortar and a pestle in 1 mL of 1% (v/v) hydrochloric acid in methanol. Then, shredded tissue was extracted for 1 d at 4 °C. For stilbenes 100 mg of the dried shredded cells tissue were extracted for 2 h at 60 °C in 3 mL of methanol. Then, anthocyanin and stilbene extracts were filtered through a 0.25-um nylon membrane for further analysis. Next, samples were separated on Shim-pack GIST C18 column (150 mm, 2.1-nm i.d., 3-_m part size; Shimadzu, Japan) the on HPLC LC-20AD XR analytical system (Shimadzu, Japan), equipped with an SPD-M20A photodiode array detector. Liquid chromatography-high-resolution mass spectrometry for qualification of all components was performed using a 1260 Infinity analytical system (Agilent Technologies, Santa Clara, CA, USA) as described [24 (link),44 (link)].
The commercial standard cyanidin chloride, petunidin chloride, delphinidin chloride, malvidin chloride, t-resveratrol, t-piceid, t-piceatannol, ε-viniferin were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used as the control.

The content of stilbenes was measured by HPLC as described [43 (link),59 (link)]. Briefly, the extracts were separated on Zorbax C18 column (column temperature 40 °C, 150 mm, 2.1-mm i.d., 3.5-lm part size, Agilent Technologies, Santa Clara, CA, USA) the on HPLC LC-20AD XR analytical system (Shimadzu, Kyoto, Japan). The mobile phase consisted of a gradient elution of 0.1% aqueous acetic acid (A) and acetonitrile (B). The gradient profile with a flow rate of 0.2 mL/min was: 0 min 0% B; 35 min 40% B; 40 min 50% B; 50 min 100% B and then eluent B until 60 min. The injected volume was 5 µL. Liquid chromatography-high-resolution mass spectrometry for quantification of all components was performed using a 1260 Infinity analytical system (Agilent Technologies, USA) as described [20 (link),59 (link)]. HPLC for quantification of all components was performed using LC-20 analytical HPLC system (Shimadzu, Japan) equipped with an SPD-M20A photodiode array detector, LC-20ADXR pump, Shim-pack XR-ODS II column and SIL-20ACXR auto sampler as described [46 (link)].

The frozen A. thaliana rosettes that underwent treatment were homogenized using a mortar and pestle. Shredded tissue was weighed and then subjected to extraction in 1 mL of 1% (v/v) hydrochloric acid and methanol for 24 h at 4 °C. Afterwards, the mixture was centrifuged at 13,200 rpm for 15 min. To prepare samples for HPLC–MS analysis, a 0.45-um nylon filter was used for sample filtration. All anthocyanins present were identified using an Agilent Technologies 1260 Infinity analytical HPLC system (Santa Clara, CA, USA) coupled with Bruker HCT ultra PTM Discovery System (Bruker Daltonik GmbH, Bremen, Germany) that featured an electrospray ionization (ESI) source, according to a protocol described previously [20 (link)]. A Shimadzu HPLC LC-20AD XR analytical system (Kyoto, Japan) equipped with diode array detection (HPLC–DAD) was employed for quantification of all anthocyanins following previously described procedures [20 (link)]. The anthocyanin contents were determined using an external standard method with the four-point regression calibration curves constructed using available standards. As a control, the commercial standard cyanidin chloride, obtained from Sigma-Aldrich (St. Louis, MO, USA), was used.