3200 q trap

An Agilent 1200 chromatograph (Agilent Technologies, Palo Alto, CA, USA) coupled to a 3200 QTRAP^® (Applied Biosystems, AB Sciex, Foster City, CA, USA) with Turbo Ion Spray (ESI) electrospray ionization was employed for the determination. The instrumental parameters were fixed as previously indicated [31 (link)].
The chromatographic separation was performed with a Gemini-NX column C18 (Phenomenex, 150 mm × 4.6 mm, 5 particle size) preceded by a guard column. Mobile phases consisted of: 5 mM ammonium formate, 0.1% formic acid water (A) and 5 mM ammonium formate, 0.1% formic acid methanol (B). The gradient program started with a proportion of 0% for eluent B; increasing to 100% in 10 min, then decreasing to 80% in 5 min, and finally to 70% in 2 min. Then, the column was cleaned and readjusted to initial conditions in the next 6 min, and equilibrated during 7 min. The instrumental parameters were fixed as follows: injection volume of 20 µL, flow rate at 0.25 mL/min and oven temperature at 40 °C. The Turbo Ion Spray operated in positive ionization mode (ESI+). Nitrogen served as a nebulizer and collision gas. Ion spray voltage was fixed at 5500 V, the curtain gas was set at 20 (arbitrary units), the nebulizer (GS1) and TIS (GS2) gases at 50 and 50 psi, respectively, and the probe temperature (TEM) was set at 450 °C.

Analysis for physiological phenolics in EFAA was performed using the 3200 QTRAP with a hybrid triple quadrupole-linear ion mass spectrometer (Applied Biosystems, Foster City, CA, USA), and C₁₈ column (250 × 4.6 mm, 5.0 μm, ProntoSIL, BISCHOFF Chromatography, Germany) was used. The eluent solvents were used to A (0.1% formic acid in distilled water) and B (0.1% formic acid in acetonitrile), and a gradient condition was applied as follows (min, %B): (0, 20), (20, 60), (30, 90). The flow rate was 0.5 mL/min with a 20 μL injection volume, column oven temperature of 30°C, and all the analyses were carried out using a TurboIonSpray ionization source, and ESI-MS conditions were as follows: negative-ion mode, curtain gas (N₂) 20 (arbitrary units), drying gas (N₂) heated to 650°C, and a variety of collision energies.
Analysis for contents of phenolics in EFAA was performed using the high performance liquid chromatography (HPLC) with a photodiode array UV-Vis detector system (Shimadzu Corporation, Kyoto, Japan), and C₁₈ column (250 × 4.6 mm, 5.0 μm, ProntoSIL, BISCHOFF Chromatography, Germany) was used. The mobile phase was used to acetonitrile: 10 mM KH₂PO₄ (10 : 90, v/v), isocratic, and monitored for 30 min (wavelength 205 nm). The flow rate was 1.0 mL/min with a 10 μL injection volume and column oven temperature of 30°C.

Frozen samples (100 mg FW) were homogenized with liquid nitrogen in a mortar and pestle. The phytohormones were extracted with a cold (−20 °C) methanol/water/formic acid mixture (15/4/1, v/v), as described in [84 ]. Internal isotope-labeled standards (10 pmol per sample) were added for hormone analysis: ¹³C₆-IAA (Cambridge Isotope Laboratories, Tewksbury, MA, USA); ²H₄-SA (Sigma-Aldrich, St. Louis, MO, USA); ²H₃-PA, ²H₃-DPA, ²H₅-ABA-GE (NRC-PBI, Saskatoon, SK, Canada); and ²H₆-ABA, ²H₅-JA, and others (Olchemim, Olomouc, Czech Republic). The extracts were passed through reversed-phase cation exchange SPE columns (Oasis-MCX, Waters, Milford, MA, USA) in a mixed mode (mixed phase–cation exchange). The hormone fraction containing ABA, IAA, SA, and JA was eluted with methanol. Hormone metabolites were analyzed using HPLC (Ultimate 3000, Dionex, Sunnyvale, CA, USA) coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer (3200 Q TRAP, Applied Biosystems, Waltham, MA, USA). The quantification of hormones was carried out using the isotope dilution method with multilevel calibration curves (R² > 0.99). Data processing was carried out with Analyst 1.5 software (Applied Biosystems).

7-day-old Arabidopsis WT, npc1-1, NPC1-OE seedlings were grown on agar plates and used for hormone analyses. The plants were kept in HS (42°C) or in control conditions for 45 min. After treatment, 150 mg of fresh weight was frozen in liquid nitrogen. The determination of plant hormones included extraction and purification on a mixed mode reversed phase – cation exchange SPE column (Oasis-MCX, Waters). The purified fractions were analyzed as in Dobrev and Vankova (2012) (link) on an HPLC (Ultimate 3000, Dionex) coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer (3200 Q TRAP, Applied Biosystems) set in selected reaction monitoring mode. Quantification of hormones was performed using an isotope dilution method with multilevel calibration curves (r²> 0.99). Data processing was carried out with Analyst 1.5 software (Applied Biosystems).