Liquid chromatography was carried out using a UFLC with an autosampler (Shimadzu Corporation, Kyoto, Japan). A Waters Atlantis T3 (Waters Corporation) column (2.1 × 150 cm, 3 μm) was used at ambient temperature. The injected volume of sample was 10 μL. The elution gradient was carried out with binary solvent system consisting of 0.02% acetic acid in H2O (solvent A) and 0.02% acetic acid in MeCN (solvent B) at a constant flow rate of 250 μL/min. A linear gradient profile with the following proportions (v/v) of solvent B was applied: gradient profile 0 to 5 min and 0% of B, 5 to 8 min and 0% to 16% of B, 8 to 20 min and 16% to 100% of B, 20 to 25 min and 100% of B, 25 to 28 min and 100% to 0% of B, and 28 to 32 min with 4 min for re-equilibration and 0% of B.
To diagnose the hormone precursor-to-product ion transitions, mixtures of 150 ng/mL of the standard compounds dissolved in 50% MeCN were directly infused into a hybrid triple quadrupole/linear ion trap mass spectrometer (ABI 4000 Q-Trap, Applied Biosystems, Foster City, CA, USA) outfitted with an electrospray ion source. The analysis parameters were optimized for the production of characteristic precursor-to-product ion transitions in negative or positive ionization modes. ABA, IAA, IAA-Asp, JA, JA-Ile, SA, sakuranetin, naringenin, and their internal standards were scanned in the negative mode, whereas momilactone A and MeJA were analyzed in the positive mode. The mixtures of standard compounds were separated by reversed-phase UFLC and analyzed by ESI-MS/MS in the MRM mode with 50 ms dwell time, 5 ms of pause time between mass ranges, and 700 ms of settle time for switching polarities. The identities of phytohormones and metabolites in the crude plant extracts were confirmed by analysis of product ion fragments obtained by the hybrid triple quadrupole/linear ion trap mass spectrometer, operating in the IDA mode, with a source voltage of 4.5 kV and source temperature of 550. In the ''Enhanced Product Ion" scan mode, precursor ions were fragmented with collision energy +25 kV or -25 kV and products in the range of 50 to 500 m/z were detected.
UFLC-ESI-MS/MS assays were repeated twice biologically, with each repetition having three replicates. Similar results were obtained in repeated experiments; only the result in one repetition was presented.
To diagnose the hormone precursor-to-product ion transitions, mixtures of 150 ng/mL of the standard compounds dissolved in 50% MeCN were directly infused into a hybrid triple quadrupole/linear ion trap mass spectrometer (ABI 4000 Q-Trap, Applied Biosystems, Foster City, CA, USA) outfitted with an electrospray ion source. The analysis parameters were optimized for the production of characteristic precursor-to-product ion transitions in negative or positive ionization modes. ABA, IAA, IAA-Asp, JA, JA-Ile, SA, sakuranetin, naringenin, and their internal standards were scanned in the negative mode, whereas momilactone A and MeJA were analyzed in the positive mode. The mixtures of standard compounds were separated by reversed-phase UFLC and analyzed by ESI-MS/MS in the MRM mode with 50 ms dwell time, 5 ms of pause time between mass ranges, and 700 ms of settle time for switching polarities. The identities of phytohormones and metabolites in the crude plant extracts were confirmed by analysis of product ion fragments obtained by the hybrid triple quadrupole/linear ion trap mass spectrometer, operating in the IDA mode, with a source voltage of 4.5 kV and source temperature of 550. In the ''Enhanced Product Ion" scan mode, precursor ions were fragmented with collision energy +25 kV or -25 kV and products in the range of 50 to 500 m/z were detected.
UFLC-ESI-MS/MS assays were repeated twice biologically, with each repetition having three replicates. Similar results were obtained in repeated experiments; only the result in one repetition was presented.
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