Chemical and reagents
Gibberellin A1(GA1), GA3, GA4, GA5, GA7, GA8, GA9, GA20, GA51, IAA, ABA, and JA were purchased from OlChemIm Ltd. (Olomouc, Czech Republic). [2H2]GA1, [2H2]GA4, [2H2]GA9, [2H6]ABA, [2H5]JA, and [2H2]IAA were purchased from OlChemIm Ltd. (Olomouc, Czech Republic) and used as internal standards (ISTDs). EDC (>98.0%) was purchased from TCI Development Co., Ltd., (Shanghai, China). Methanol (≥ 99.9%) and acetonitrile (≥ 99.9%) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, USA). The matrix dispersion sorbents for experiments included kieselguhr, C18-bonded silica (C18), primary secondary amine (PSA), alumina-N, and florisil were purchased from CNW Technologies (Shanghai, China). Ti3AlC2 powder (> 98 wt% purity) was obtained from 11 Technology Co., Ltd, China. Ultrapure water (resistivity ≥ 18.25 MΩ/cm) obtained from WaterPro water system (ULUPURE, China) was used in all experiments. Individual stock standard solutions (1000 mg/L) of each compound were prepared using methanol and stored in the refrigerator at -20 °C. The mixed standard solutions were stored at 4 °C. EDC solutions were prepared by freshly dissolving EDC powder in the methanol. All the reagents were of analytical grades (least 98% purity).
Preparation of magnetic nano-composite Fe3O4@Ti3C2@β-CD
Fig.4 illustrates the method of preparation of Fe3O4@Ti3C2@β-CD hybrid. 2 g of the Ti3AlC2 powder was immersed in 20 mL of hydrofluoric acid (HF) solutions by stirring for 24 h at 60 °C for complete removal of the Al layers. Then, the solution was centrifuged and rinsed several times using deionized water to remove the HF and until the pH attained 5-6. The obtained Ti3C2 powder was then vacuum-dried at 80 °C for 24 h.
Fe3O4 nanoparticles were synthesized according to the hydrothermal method [38]. 100 mg of Ti3C2 power and 50 mg of Fe3O4 nanoparticle were ultrasonically dispersed in 80 mL and 20 mL of deionized water for 30 min, respectively. Both were mixed by ultrasonification for 120 min and argon shield. Then the suspension was filtrated to obtain the Fe3O4@Ti3C2 hybrid and followed drying in a vacuum at 50 °C for 24 h.
Fe3O4@Ti3C2 composite loaded with β-CD, 100 mg of Fe3O4@Ti3C2, and 2.0 g of β-CD were dispersed into 60 mL deoxygenated water by ultrasonification for 20 min. Then, the reaction system was kept in an oil bath at 60 °C for 4 h and argon shield. Finally, the obtained Fe3O4@Ti3C2@β-CD was washed with deionized water and then vacuum-dried at 50 °C for 24 h.
Plant materials
Seeds of Brassica napus (rapeseed), Sesamum indicum (sesame), Glycine max (bean), and Arachis hypogaea (peanut) were obtained from Hunan Branch of National Center of Oilseed Crops Improvement (Changsha, China). The different seeds of oil crops were harvested, frozen in liquid nitrogen, and stored at -80 °C until further analyzed.
Sample preparation and in situ derivatization
Single fresh rapeseed seed was mixed with 2 mg clean-up sorbents and beads into a 2.0 mL microcentrifuge tube. The mixture was immediately ground with one cold ZrO2 mill ball by ball milling (TissueLyser II, QIAGEN, Germany) at 20 Hz for 4 min. Then, the internal standards (IS) were added into the tube. The ground sample was extracted with 200 μL of cold methanol and vortexed at 4 °C for 30 S, then left to stand at 4 °C for 12 h. The supernatant was collected by centrifugation at 10000 ×g for 5 min and transferred to a 1.5 mL microcentrifuge tube. The supernatant was added with 5 mg magnetic nanoparticles and 50 μL of derivatizing agent (20 mM EDC), then incubated at 40 °C for 90 min. Both the extraction and derivatization were completed at one step. The nanoparticles were collected from the suspension by magnetic separation and re-dispersed by 50 μL of 5 % menthol. After desorption for 20 min, the eluent was collected and determined by UHPLC-ESI-MS/MS. The general process of sample pre-treatment is illustrated in Fig.4.
Instruments and analytical conditions
The UPLC-MS/MS was equipped with an Agilent 1290 series (Agilent Technologies, USA) and Agilent 6460 triple quadruple mass spectrometer (Agilent Technologies, USA). The analytes were separated on a Waters ACQUITY UPLC HSS T3 column (100 mm × 2.1 μm). The optimized separation conditions were as follows: the column oven temperature was kept at 40 °C, and the sample injection volume was 10 μL, the flow rate of the mobile phase was 0.3 mL/min. The elution gradient program of the positive ion mode was performed, as depicted in Table S1.
The multiple reaction monitoring (MRM) was employed for the quantitative analysis of the targeted compounds. Nitrogen gas was used as the drying and collision gas. The ionization source conditions were as follows: the flow rate of the nebulizer gas was 8 L/min, the source temperature of the mass spectrometer was 300 °C, the nebulizer pressure was 50 psi, and the capillary voltage was 3500 V. The details of the EDC-derived phytohormones and their optimized MRM parameters are listed in Table 4. The MRM chromatograms of the target EDC-derived phytohormones were shown in Fig. 5. The MRM chromatograms with subsection of target phytohormone derivates in a single rapeseed were shown in Fig. 6.
Method validation
To validate the developed method, the linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, and matrix effect (ME) were investigated. Calibration samples at 6 concentrations (0.01, 0.05, 0.10, 0.50, 1.0, 10 ng/mL of GAs derivates, 0.10, 0.50, 1.0, 5.0, 10, 100 ng/mL of IAA, ABA, JA derivates) with a fixed concentration of IS ([2H2] GA1 0.1 ng/g, [2H2] GA4 0.1 ng/g, [2H2] GA9 0.1 ng/g, [2H2] IAA 0.2 ng/g, [2H6] ABA 1 ng/g, and [2H6] JA 0.1 ng/g for each). The LOD values were calculated as 3 times of the standard deviation of 11 replicates for rapeseed samples spiked at the 0.01 ng/mL level for GAs, 1 ng/mL level for IAA, ABA, and JA, respectively. LOQ values were calculated as 10 times of the standard deviation of 11 replicates for rapeseed samples spiked at the same level as used in calculating the LOD values.
Accuracy was evaluated by the recovery of each target analyte at low, medium, and high levels, respectively. The precision was investigated by the intra-day precision (repeatability) and inter-day precision (reproducibility). The intra-day precision was evaluated by analyzing 7 replicates on the same day while the inter-day precision was carried out for 3 consecutive days (7 replicates per day). During the trace analyses with a complex matrix, the ME usually occurs and its percentage can be calculated as per the equation below:
where, Aextract stands for the slope of matrix-match calibration curves, Asolvent stands for the slope of solvent calibration curves.