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Protocol for An UHPLC-MS/MS Method for Target Profiling of Stress-Related Phytohormones

To obtain accurate and reliable results in UHPLC-MS/MS analysis, it is crucial to optimize the sample preparation process. The use of 10% methanol–water for extraction is highly recommended as it ensures solubility of compounds of interest and minimizes sample contamination from lipophilic pigments or other interfering compounds. The sample purification process also plays a critical role in determining the sensitivity of the final analysis. It is important to carefully select and validate the sample purification methods to ensure their efficacy in removing interfering compounds and achieving desired sensitivity. Thorough validation of the method is essential and involves evaluating at least seven critical parameters such as method selectivity, sensitivity, extraction recovery, matrix effects, linearity, accuracy, and precision. By optimizing the sample preparation and validation processes, researchers can obtain reliable and robust results in UHPLC-MS/MS analysis of plant metabolites.

1 Sampling for Phytohormone Profiling

(a) Grind the frozen plant material into fine powder using a mortar and a pestle under the liquid nitrogen.

(b) Transfer individually amounts of 20–25 mg of fresh weight into 2.0 mL safe-lock microcentrifuge tubes (at least three replicates per sample).

(c) Freeze the plant tissues immediately in liquid nitrogen and store them at -80 ℃ for no longer than several weeks before sample extraction.

2 Extraction of Stress-Related Plant Hormones

(a) Place frozen samples from the -80 ℃ in a crushed-ice bath.

(b) Add 1.0 mL of ice-cold extraction solution into each microcentrifuge tube.

(c) Place 2 mm ceria-stabilized zirconium oxide beads (4 in total) into each tube together with 10 μL of IS mixture.

(d) Place microcentrifuge tubes in a precooled cassette of a vibration bead mill and homogenize at a frequency of 27 Hz for 3 min.

(e) Sonicate homogenized samples in a precooled ultrasonic bath at 37 kHz for 3 min.

(f) Extract samples for 30 min on the benchtop laboratory rotator at 4 ℃.

(g) Centrifuge samples at 14,000 × g for 10 min at 4 ℃.

(h) Collect supernatants of sample extracts into new 2.0 mL microcentrifuge tubes and place them in a crushed-ice bath.

Keep the remaining pellet on ice for further re-extraction.

(i) Re-extract samples by dissolving the pellet in 1.0 mL of ice-cold extraction solution, vortex for 10 s, and centrifuge as before (step g).

(j) Pool the supernatants of one sample in a new 2.0 mL microcentrifuge tube.

3 Sample Purification by Nonselective SPE

(a) Prior to sample loading onto SPE Oasis® HLB columns, perform sorbent conditioning with 1 mL of 100% methanol and subsequent equilibration with 1 mL of 0.1% formic acid–water (v/v).

(b) Apply the sample extract (~2 mL) onto SPE column. Discard flow-through fraction.

(c) Remove interfering compounds by washing with 1 mL of extraction solution. Discard flow-through fraction.

(d) Insert clean glass tubes into the manifold rack, and then elute the sample from the column with 2 mL of 80% methanol (v/v).

(e) Evaporate the collected fractions to dryness under a gentle stream of nitrogen.

(f) Store dried sample extracts at -20 ℃ until LC–MS/MS analysis.

4 Phytohormonal Metabolite Profiling

(a) Dissolve the samples in 40 μL of 10% methanol, and place the vials in an autosampler with a temperature controlled system set at 4 ℃.

(b) Separate and detect JA, its biosynthetic precursors and amino acid conjugates, SA, ABA, IAA, and its metabolites by UHPLC-MS/MS.

(c) Inject 10 μL of each sample onto an ACQUITY UPLC® CSH C18 column (1.7 μm, 2.1 × 100 mm).

(d) Elute the samples using an 28-min gradient composed of eluent (A) acetonitrile and eluent (B) 10 mM formic acid in water, at a flow rate of 0.4 mL/min, column temperature of 36 ℃, and a binary linear gradient: 0 min, 15:85 (A:B); 5.0 min, 15:85 (A:B); 15.0 min, 45:55 (A:B); 22.5 min, 47:53 (A:B).

(e) At the end of the gradient, wash the column with 100% methanol for 1.5 min, and readjustment to initial conditions for 4.0 min.

(f) Determine stress-related phytohormones by multiple reaction monitoring (MRM) of the precursor [M+H]+ and the appropriate product ions according to retention times of the analytes..

(g) Run all calibration solutions and construct calibration curves for each analyte.

(h) Process all data using appropriate mass spectrometry software, and determine the concentrations of each phytohormone using the stable isotope dilution method.


  1. António, C. (Ed.). (2018). Plant metabolomics: Methods and protocols. Humana Press.
* For Research Use Only. Not for use in diagnostic procedures.
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