Simultaneous determination of four cytokinins in litchi by ultra-high performance liquid chromatography triple quadrupole mass spectrometry

Abstract: 【Objective】A method was developed to simultaneously measure four types of cytokinins (tZR, IPR, DHZ and IP) in litchi using ultra- high performance liquid chromatography- tandem triple quadrupole mass spectrometry (UPLC-MS/MS).【Methods】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 [ion source temperature 150 ℃, capillary voltage 0.35 kV, dissolvent gas temperature 500 ℃, atomization gas flow rate 1000 L·h^-1 , cone hole gas flow 50 L·h^-1 , and collision gas (argon gas) 0.17 L·h^-1 ], quantification of cytokinins was achieved. The effects of different extraction solvents (80% methanol, 80% acetone, and 80% acetonitrile), different extraction times (4 h, 8 h, 12 h, and 16 h), different purification methods (PCX column, C18 column, HLB column, and no column), different concentrations of ammonium-methanol eluent (0.5%, 1.0%, 2.5%, and 5.0% ), and different aqueous phaseswhen methanol is the organic phase (0.05% formic acid solution, 0.1% formic acid solution, 1 mmol·L-1 ammonium formate solution, and 5 mmol·L^{- 1} ammonium formate solution) were separately examined for their impact on cytokinin extraction, enrichment, and separation efficiency. Compared to other 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 substances on the column to be eluted with a 2.5% ammonia-methanol solution (pH≈11.3). Target substances flow out with the eluent 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 non- polar. 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 was better with extraction solvent of 80% acetonitrile than the other extraction solutions and the extraction time was 8 hours, resulting in smooth, sharp peaks and high response values. Regarding the purification conditions, solid phase extraction with PCX was chosen, and the concentration of the ammonia methanol eluent was 2.5%. Under this condition, chromatogram had less interference, offering the best enrichment separation effect. For the mobile phase conditions, when methanol was used as the organic phase, a 5 mmol·L- 1 aqueous solution of ammonium formate was most effective, with which the response intensity of the target substance was high and the peak time was appropriate. Fresh litchi samples were taken and ground into fine powder in a mortar with liquid nitrogen. 0.25 g of the sample was put into a 15 mL plastic centrifuge tube, and added with 2.5 mL of 80% acetonitrile pre-cooled at 4 ℃. Cytokinins were extracted at 4 ℃ for 8 hours, during which the sample was shaken twice. After extraction, the extract was centrifuged at a speed of 10 000 r·min-1 for 10 minutes at 4 ℃, and the supernatant was collected. An equal volume of 80% acetonitrile was added to the residue, shaken for 10 minutes, and centrifuged at a speed of 10 000 r·min- 1 for 10 minutes at 4 ℃. The supernatant was combined, added with formic acid to adjust the pH value to 2-3, and mixed well to obtain the crude extract. Consecutively, 2.5 mL methanol and an equal volume of 2% aqueous solution of formic acid were added for activation and balancing. The crude extract was passed through the column at a rate of 1-2 drops per second, rinsed with an equal volume of 0.1% formic acid methanol solution, and finally washed twice with an equal volume of 2.5% ammonia-water methanol solution. The eluent was collected in a test tube, blown to almost dryness with nitrogen, and added with 0.25 mL 15% methanol solution to re-dissolve. After vortex mixing, the eluent was filtered through a 0.22 µm organic membrane for testing. The limits of detection and quantification for the four cytokinins were 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 (r 2 ) 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 pericarp, pulp, seed, young fruit, leaves, and the abscission zone, 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 rapid screening and quantitative detection of cytokinins in various parts of litchi. The method is of great practical value.