While, plant hormones such as ABA has been measured individually in crude extracts,  no one method has been published that allows simultaneous simple, rapid and accurate measurement of the three acidic hormones, JA, SA and ABA via LC/MS. We therefore developed a method with an extraction solvent that allowed the reproducible and stable extraction of the analytes of interest from relatively small amounts of starting material as well as the ability to inject directly relatively large volumes of the sample whilst retaining good peak shapes. While here we report characterization of this method on Arabidopsis thaliana leaves, this method is equally applicable to other plant species such as tomato (M. Grant unpublished).
Plants were grown for four to five weeks in a controlled environment chamber under short days (10 h), 70% humidity as previously described .
Pathogen or abiotic stressed plant material was harvested into liquid nitrogen and freeze dried. Samples were next placed in a 2 ml microfuge tube and ground in a bead beater (Qiagen or equivalent) with 3 mm tungsten beads at 25 Hz/s for 3 min. Ten milligram of powdered tissue (~110 mg fresh weight, or equivalent to approximately two fully expanded Arabidopsis leaves) was weighed into a new 2 ml microfuge tube and extracted with 400 μl of 10% methanol containing 1% acetic acid to which internal standards had been added (1 ng of 2H6 ABA, 10 ng of 2H2 JA and 13.8 ng 2H4 SA). Each treatment also included an extraction control containing no plant material. A 3 mm tungsten bead was placed in each microfuge tube and samples were extracted in the bead beater for 2 min at 25 Hz/s, placed on ice for 30 min then centrifuged at 13,000 g for 10 min at 4°C. The supernatant was carefully removed and the pellet re-extracted with 400 μl of 10% methanol containing 1% acetic acid. Following a further 30 min incubation on ice the extract was centrifuged and the supernatants pooled. The two extractions resulted in 90–95% recovery of the targeted analytes.
Samples (50 μl) were then analysed by HPLC-electrospray ionisation/MS-MS using an Agilent 1100 HPLC coupled to an Applied Biosystems Q-TRAP 2000 (Applied Biosystems, California, USA). Chromatographic separation was carried out on a Phenomenex Luna 3 μm C18(2) 100 mm × 2.0 mm column, at 35°C. The solvent gradient used was 100%A (94.9% H2O: 5% CH3CN: 0.1% CHOOH) to 100%B (5% H2O: 94.9% CH3CN: 0.1% CHOOH) over 20 min. Solvent B was held at 100% for 5 min then the solvent returned to 100% A for 10 min equilibration prior to the next injection. The solvent flow rate was 200 μl/min. To reduce contamination of the MS, the first 2 min of the run was directed to waste using the inbuilt Valco valve.
Analysis of the compounds was based on appropriate Multiple Reaction Monitoring (MRM) of ion pairs for labelled and endogenous JA, SA and ABA using the following mass transitions; 2H2-JA 211 > 61, JA 209 > 59, 2H4 SA 141 > 97, SA 137 > 93, 2H6 ABA 269 > 159, ABA 263 > 153, SA-glyc 299 > 93.
The MS was operated in the negative mode using Turbo-Ionspray™ as the ion source. Optimal conditions were determined using the Quantitative Optimisation feature of the Analyst software both by infusing standards into the MS by syringe pump and injecting standards into a 200 μl/min flow of 50% Solvent A/50% Solvent B.
The optimised conditions were as follows: Temperature 400°C, Ion source gas 1 50 psi, Ion source gas 2 60 psi, Ion spray voltage -4500 V, curtain gas 40 psi, CAD gas setting 2; the DP (-25 V), EP (-9) and CEP (-2) were held constant for all transitions. Collision energies (CE) and dwell times (DT) were specific for each compound/internal standard pair, the parameters used were JA CE-25, DT 100 ms, ABA CE-17, DT 250 ms and SA CE-38, DT 50 ms. Data were acquired and analysed using Analyst 1.4.2 software (Applied Biosystems).
Hormones were determined in three independent samples for each treatment or timepoint.