Nucleosil ods reversed phase column

For the hormonal analysis, fresh material was frozen in liquid N, ground and freeze-dried. Fresh tissue (0.5 g) was immediately homogenized in 2.5 mL of ultrapure water, and 100 ng mL^-1 of a mixture of internal standards [(²H₆-ABA (to quantify ABA), ²H₄-SA (to quantify SA), dihydrojasmonic acid [to quantify OPDA, JA, and JA-Ile) and propylparaben (to quantify ferulic acid), all acquired from Sigma–Aldrich] were added prior to extraction (Flors et al., 2008 (link)). After extraction, a 20-μL aliquot was injected directly into a ultra-high-performance liquid chromatography (UPLC) system. Hormone analyses were carried out in a Waters Alliance 2690 UPLC system (Milford, MA, United States) inside a nucleosil ODS reversed-phase column (100 mm × 2 mm i.d.; 5 μm; Scharlab, Barcelona, Spain⁵). The chromatographic system was interfaced to a Quattro LC (quadrupole–hexapole–quadrupole) mass spectrometer (Micromass⁶). Version 4.1 of the MASSLYNX NT software (Micromass), was used to process the quantitative data from the calibration standards and plant samples. The concentrations of hormones and metabolites were determined in each sample by normalizing the chromatographic area for each compound with the fresh weight of the corresponding sample.

Fifty mg of freeze dried leaves were harvested in parallel of microarray samples and homogenized after addition of a mixture of internal standards containing 100 ng dihydro-JA and D₆-SA [39] (link). After centrifugation the supernatant was purified by organic partitioning, as previously described [40] (link), [41] (link). A dried aliquot was re-suspendended in 1 ml of MeOH: H₂O (10∶90) and injected into the HPLC system. Analyses were carried out using a Waters Alliance 2690 HPLC system (Milford, MA, USA) with nucleosil ODS reversed-phase column (100×2 mm i.d.; 5 lm; Scharlab, Barcelona, Spain) coupled to a Quatro LC (quadrupole-hexapole-quadrupole) mass spectrometer (Micromass). Data was quantified after comparative analysis with standards using Mass Lynx (v 1.4, Mycromass) software.

For hormonal analysis, fresh material was frozen in liquid N, ground, and freeze dried. Fresh tissue (0.5g) was immediately homogenized in 2.5ml of ultrapure water, and a mixture of internal standards ([²H₆]ABA, [²H₄]SA, dihydrojasmonic acid, and propylparaben) was added at 100ng ml^–1 prior to extraction. After extraction, a 20 μl aliquot was injected directly into the high-performance liquid chromatography (HPLC) system. For PA analysis, fresh material was frozen in liquid N. Before extraction, according to the method described by Sánchez-López et al. (2009) (link), a mixture of internal standards containing [¹³C₄]putrescine and 1,7-diamineheptane was added. A 20 μl aliquot of this solution was directly injected into the HPLC system.
Analyses of hormone and PA samples were carried out using a Waters Alliance 2690 HPLC system (Milford, MA, USA) with a nucleosil ODS reversed-phase column (100 mm×2mm, i.d. 5 μm; Scharlab, Barcelona, Spain; http://www.scharlab.com). The chromatographic system was interfaced to a Quatro LC (quadrupole–hexapole–quadrupole) mass spectrometer (Micromass; http://www.micromass.co.uk). The MASSLYNX NT software version 4.1 (Micromass) was used to process the quantitative data from calibration standards and plant samples.