Hplc kinetex c18 analytical column

Samples stored at −80 °C were freeze dried and powdered for subsequent analysis. Thirty milligrams of freeze dried powder were extracted at 4 °C with 1 ml of H2O:MeOH (90:10) containing 100 ng/ml of internal standards. After 20 min of incubation, samples were centrifuged at full speed for 15 min at 4 °C. The supernatant was recovered and adjusted to pH 2.8 with 6% acetic acid, and subsequently partitioned twice against diethylether. The fractions were pooled and dried in a speed vacuum and resuspended in H2O:MeOH (90:10). A 20 μl aliquot was injected into an Acquity ultra-performance liquid chromatography system (UPLC) (Waters, Mildford, MA, USA) interfaced to a triple quadrupole mass spectrometer (TQD, Waters, Manchester, UK). The LC separation was performed by HPLC Kinetex C18 analytical column 5 μm particle size, 2.1 × 100 mm (Phenomenex). The chromatographic conditions and mass spectrometry were performed as described previously74 (link).

Hormone extraction was performed from freeze-dried powdered plant leaves as described in Sanchez-Bel et al. (2016) (link). Six independent biological replicates per treatment were analyzed. Briefly, 30 mg of plant dry tissue was extracted with 1 ml of H₂O:MeOH (9:1) containing 0.001% of HCOOH and 100 ng/ml of internal standards. After different centrifugations and resuspensions, an aliquot of the extract was injected into an Acquity Ultra Performance Liquid Chromatography system (UPLC) (Waters, Mildford, MA, USA). Hormones were chromatographically separated using an HPLC Kinetex C18 analytical column (Phenomenex) connected to a triple quadrupole mass spectrometer (TQD, Waters, Manchester, UK). The chromatographic and mass spectrometry conditions were those used by Gamir et al. (2012) (link). Hormone quantification (ng/g dry weight) was performed using calibration curves with each pure chemical standard. The plant hormones abscisic acid (ABA), indolacetic acid (IAA), jasmonic acid (JA), its precursor (+)-12-oxo-phytodienoic acid (OPDA) and salicylic acid (SA) were determined.