Abstract :【Objective】A method has been developed to simultaneously measure four types of cytokinins (tZR, IPR, DHZ, IP) in litchi using ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-MS/MS).【Method】The experiment was conducted using litchi pericarp 50 days after flowering. The XSelect HSS T3 chromatographic column was selected. Methanol and 5 mmol·L-1 ammonium formate aqueous solution were used as the mobile phase with a flow rate of 0.3 mL·min-1. A gradient elution was conducted for 7 minutes. Positive electrospray ionization (ESI+) and the multiple reaction monitoring (MRM) mode were set for mass spectrometry detection. Under these conditions [The ion source temperature was maintained at 150℃, the capillary voltage at 0.35 kV, the desolvent gas temperature at 500℃, the atomization gas flow rate at 1000 L·hr-1, the cone hole gas flow at 50 L·hr-1, and the collision gas (argon gas) at 0.17 L·h-1], quantification of cytokinins was achieved.The effects of different extraction solvents (80% methanol, 80% acetone, 80% acetonitrile), different extraction times (4 h, 8 h, 12 h, 16 h), different purification methods (PCX column, C18 column, HLB column, no column), different concentrations of ammonium-methanol eluent (0.5%, 1%, 2.5%, 5%), and different aqueous phases when methanol is the organic phase (0.05% formic acid solution, 0.1% formic acid solution, 1 mmol·L-1 ammonium formate solution, 5 mmol·L-1 ammonium formate solution) were separately examined for their impact on cytokinin extraction, enrichment, and separation efficiency.Compared to other plant endogenous hormones, cytokinins are unique in that they have a purine ring and are alkaline. The PCX column is a strong cation exchange column filled with polymer cation exchange resin, which concentrates alkaline analytes and thereby enhances the detection sensitivity of alkaline compounds. Cytokinins exist as cations in the acidified extraction solution (pH≈2~3). They are tightly adsorbed by the filler during passage through the PCX column, and then rinsed with a 0.1% formic acid methanol solution (pH≈5~6). At this point, most acidic and neutral compounds are removed, leaving the target substance on the column to be eluted with a 2.5% ammonia-methanol solution (pH≈11.3). Target substances flow out with the eluent and are collected ultimately. In addition, cytokinins are also polar substances which can be separated according to the principles of C18 column and HLB column separation, namely the principle of similar solubility. The stationary phase of C18 and HLB reverse phase columns is a non-polar stationary phase. When the polarity of the mobile phase is greater than that of the stationary phase, the target substance is eluted with the polar mobile phase.【Results】As for the extraction conditions, the extraction effect is better when the extraction solvent is 80% acetonitrile and the extraction time is 8 hours, resulting in smooth, sharp peaks and high response values. Regarding the purification conditions, solid phase extraction with PCX is chosen, and the concentration of the ammonia methanol eluent is 2.5%; under this condition, chromatogram has less interference, offering the best enrichment separation effect. For the mobile phase conditions, when methanol is the organic phase, a 5 mmol·L-1 aqueous solution of ammonium formate is most effective, with which the response intensity of the target substance is high and the peak time is appropriate.Take fresh litchi samples and grind them finely in a mortar with liquid nitrogen. Accurately weigh 0.25 g of the sample on a one-thousandth balance into a 15 mL plastic centrifuge tube, and add 2.5 mL of 80% acetonitrile pre-cooled at 4℃. Extract it at 4℃ for 8 hours, shaking it twice during this period. After extraction, centrifuge it at a speed of 10,000 r·min-1 for 10 minutes at 4℃, and collect the supernatant. Add an equal volume of 80% acetonitrile to the residue, shake for 10 minutes, and centrifuge at a speed of 10,000 r·min-1 for 10 minutes at 4℃. Combine the two extracts, add formic acid to adjust the pH≈2-3, mix well to obtain the crude extract of the sample. Then consecutively use 2.5 mL methanol and an equal volume of 2% aqueous solution of formic acid for activation and balancing. The crude extract is passed through the column at a rate of 1-2 drops per second. Afterwards, rinse with an equal volume of 0.1% formic acid methanol solution, and finally wash twice with an equal volume of 2.5% ammonia-water methanol solution. Collect the eluent in a test tube, blow it almost dry with nitrogen, and add 0.25 mL of a 15% methanol solution to re-dissolve. After vortex mixing, filter it through a 0.22 µm organic membrane for testing.Methodological validation of this method: the detection and quantification limits for the four cytokinins were both below 18.12 and 60.39 pg·g-1, respectively, showing a good linear relationship within the concentration range of 0.05~50 ng·mL-1, with a correlation coefficient (r2) greater than 0.999. At three spiking levels of high, medium, and low concentrations (0.4, 2, and 20 ng·mL-1), the average recovery rates of the four cytokinins were between 80.0%~108.2%, with a standard deviation ranging from 0.8%~15.5%. The established method was used to measure the endogenous cytokinins in litchi fruit pericarp, pulp, seeds, young fruit, leaves, and detachment zones, and all four cytokinins could be detected.【Conclusion】This method has the advantages of easy preprocessing, short detection cycle, good reproducibility, high sensitivity, and low cost. The final results are reliable, making it suitable for the rapid screening and quantitative detection of cytokinins in various parts of the litchi. The method is of great practical value.
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