A method for simultaneous detection of four cytokinins in litchi using UPLC-MS / MS

Abstract

The present invention relates to the technical field of plant hormones, and in particular to a method for simultaneously detecting four cytokinins in litchi using UPLC-MS / MS. The method adopts ultra-high performance liquid chromatography (UPLC), which further improves the separation degree and analysis speed on the basis of HPLC, and mass spectrometry MS ionizes atoms or molecules according to the mass-to-charge ratio of atoms or molecules, thereby realizing the separation and detection of compounds, and has exclusive identification capabilities. MS has the characteristics of high selectivity and high sensitivity, which effectively makes up for the defects of insufficient selectivity and sensitivity of HPLC, avoids the problem that HPLC cannot effectively separate multiple compounds with similar structures and polarities due to co-elution, and lays a foundation for the subsequent simultaneous detection of four cytokinins with similar structures in litchi.

Description

Technical Field

[0001] The invention relates to the technical field of plant hormone detection, and in particular to a method for simultaneously detecting four cytokinins in litchi by using UPLC-MS / MS. Background Art

[0002] Currently, enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC) are commonly used to determine cytokinins in litchi.

[0003] However, the current detection method has shortcomings: the ELISA method uses the specific reaction of antigen and antibody to establish a relationship between the analyte and the enzyme, and then produces a color reaction between the enzyme and the substrate for quantitative determination. However, since a class of hormones is often composed of multiple substances with similar structures, there is a possibility of misidentification when the antigen and antibody combine, and it is also easily affected by the external environment and errors occur, and the specificity and sensitivity need to be improved.

[0004] HPLC separates and purifies samples in a chromatographic column by utilizing the different interactions between sample components and two different phases (stationary phase and mobile phase). Since HPLC is fast and relies on the different polarities of compounds, two compounds with similar structures and polarities can leave the chromatograph at the same time or almost at the same time, resulting in co-elution. Co-elution makes it impossible to accurately determine at what point the mixture elutes. HPLC has strong separation capabilities, but lacks selectivity and sensitivity. Summary of the invention

[0005] The object of the present invention is to avoid the shortcomings of the prior art and provide a method for simultaneously detecting four cytokinins in litchi using UPLC-MS / MS. The method can accurately and simultaneously detect the four cytokinins in litchi and has the advantages of easy operation, short detection cycle, good reproducibility, high sensitivity and low cost.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for simultaneously detecting four cytokinins in litchi by UPLC-MS / MS is provided, comprising the following steps:

[0008] Step 1, extraction and purification, which includes:

[0009] S11, obtaining litchi sample tissue, grinding it under liquid nitrogen conditions to obtain sample powder;

[0010] S12, placing the sample powder in a centrifuge tube, adding acetonitrile solution, extracting at 3-4°C for 6-10 hours, shaking regularly during the extraction, centrifuging the centrifuge tube at 3-4°C, collecting the supernatant and residue,

[0011] Wherein, 5 to 11 mL of acetonitrile solution is added to every 1 g of sample powder, and the concentration of the acetonitrile solution is 60 to 85%;

[0012] S13, repeating step S12 with the residue, recovering the supernatant obtained from the residue and combining it with the supernatant obtained in step S12 to obtain a crude sample extract;

[0013] S14, using the crude sample extract to activate and balance the PCX column with methanol and 2% formic acid aqueous solution in sequence, then eluting with an eluent, and then eluting with an eluent, collecting the eluent,

[0014] Wherein, the eluent is composed of methanol solution and formic acid, and the content of formic acid is 0.05-0.2%.

[0015] The eluent is composed of methanol solution and ammonia water, the content of the ammonia water is 1-3%, and 15-21 mL of the eluent is added for every 1 g of sample powder;

[0016] S15, concentrating the eluted liquid nitrogen, and then adding methanol aqueous solution to redissolve it to obtain a reconstituted solution, wherein the concentration of the methanol aqueous solution is 10% to 20%;

[0017] Step 2: UPLC-MS / MS detection and analysis, which includes:

[0018] S21, subjecting the reconstituted solution to UPLC-MS / MS detection and analysis,

[0019] The liquid phase conditions are as follows: mobile phase B is methanol, mobile phase A is 5mmol / L ammonium formate aqueous solution, and elution is performed by gradient elution. The elution program is shown in Table 1.

[0020] Table 1 Gradient elution program

[0021]

[0022] The mass spectrometry conditions were as follows: electrospray ion source, detection mode was multiple reaction detection mode, ion source temperature was 150°C, capillary voltage was 0.35 kV, desolvation temperature was 500°C, nebulizer gas flow rate was 1000 L / Hr, cone gas flow rate was 50 L / Hr; collision argon gas was 0.17 L / h. The relevant mass spectrometry parameters of the four cytokinins are shown in Table 2.

[0023] Table 2 Related mass spectrometry parameters of four cytokinins

[0024]

[0025] In some embodiments, in S12, the shaking is performed 1 to 3 times.

[0026] In some embodiments, in S12, the centrifugal speed is 9000-11000 r / min, and the centrifugal time is 7-15 min.

[0027] In some embodiments, in S21, the reconstituted solution is vortex mixed and then passed through a 0.22 um organic filter membrane before being subjected to UPLC-MS / MS detection and analysis.

[0028] The beneficial effects of the method of the present invention for simultaneously detecting four cytokinins in litchi by using UPLC-MS / MS are as follows:

[0029] The method for simultaneously detecting four kinds of cytokinins in litchi by liquid phase secondary mass spectrometry (UPLC-MS / MS) of the present invention adopts ultra-high performance liquid chromatography (UPLC), UPLC further improves separation and analysis speed on the basis of HPLC, mass spectrum MS ionizes atoms or molecules according to the mass-to-charge ratio of atoms or molecules, thereby realizing separation and detection of compounds, having exclusive identification capability, MS having the characteristics of high selectivity and high sensitivity, effectively making up for the defect that HPLC selectivity and sensitivity are not enough, avoiding the problem that HPLC cannot effectively separate multiple compounds with similar structures and polarities due to co-elution, and laying the foundation for subsequent simultaneous detection of four kinds of cytokinins with similar structures in litchi. UPLC-MS / MS then combines the advantages of HPLC and MS, having the characteristics of high separation and high speed of UPLC, and having the characteristics of high selectivity and high sensitivity of MS, the invention has the advantages of easy operation of pre-treatment, short detection cycle, good reproducibility, high sensitivity, low cost, etc., the final result is reliable, suitable for rapid screening and quantitative determination of cytokinins in litchi, and can be applied in practical work. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The present invention is a flowchart of a method for simultaneously detecting four cytokinins in litchi by using UPLC-MS / MS according to a specific embodiment of the present invention. DETAILED DESCRIPTION

[0031] The preferred embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the preferred embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0032] Example

[0033] See also Figure 1 This embodiment discloses a method for simultaneously detecting four cytokinins in litchi using UPLC-MS / MS, comprising the following steps:

[0034] Step 1, extraction and purification, which includes:

[0035] S11, obtaining litchi sample tissue, grinding it under liquid nitrogen conditions to obtain sample powder;

[0036] Grinding was performed under liquid nitrogen protection to avoid oxidation of the sample.

[0037] S12, placing the sample powder in a centrifuge tube, adding acetonitrile solution (ACN), extracting at 3-4°C for 6-10 hours, shaking regularly during the extraction, centrifuging the centrifuge tube at 3-4°C, collecting the supernatant and residue,

[0038] Wherein, 5 to 11 mL of acetonitrile solution is added to every 1 g of sample powder, and the concentration of the acetonitrile solution is 60 to 85%;

[0039] The cytokinins in the sample powder are collected into the acetonitrile solution through the leaching action of the acetonitrile solution. The shaking method can effectively improve the leaching effect.

[0040] S13, repeating step S12 with the residue, recovering the supernatant obtained from the residue and combining it with the supernatant obtained in step S12 to obtain a crude sample extract;

[0041] The residue is repeatedly extracted to achieve the maximum possible recovery of the cytokinins in the residue.

[0042] S14, using methanol (MEOH) and 2% formic acid aqueous solution (Foxmic acidwater) to activate and balance the PCX column with the crude sample extract, then eluting with an eluent, and then eluting with an eluent, collecting the eluent,

[0043] The eluent is composed of methanol (MEOH) solution and formic acid (Foxmic acid), and the content of formic acid is 0.05-0.2%.

[0044] The eluent is composed of methanol solution and ammonia water, the content of the ammonia water is 1-3%, and 15-21 mL of the eluent is added to every 1 g of sample powder;

[0045] Purification is carried out through the steps of activation, equilibrium, washing and elution.

[0046] S15, concentrating the eluted liquid nitrogen, and then adding methanol aqueous solution to redissolve it to obtain a reconstituted solution, wherein the concentration of the methanol aqueous solution is 10-20%;

[0047] After purification and concentration, the solution is reconstituted to a specific volume.

[0048] Step 2: UPLC-MS / MS detection and analysis, which includes:

[0049] S22, subjecting the reconstituted solution to UPLC-MS / MS detection and analysis,

[0050] The liquid phase conditions are as follows: mobile phase B is methanol, mobile phase A is 5mmol / L ammonium formate aqueous solution, and elution is performed by gradient elution. The elution program is shown in Table 1.

[0051] Table 1 Gradient elution program

[0052]

[0053] The mass spectrometry conditions were as follows: electrospray ion source, detection mode was multiple reaction detection mode, ion source temperature was 150°C, capillary voltage was 0.35 kV, desolvation temperature was 500°C, nebulizer gas flow rate was 1000 L / Hr, cone gas flow rate was 50 L / Hr; collision argon gas was 0.17 L / h. The relevant mass spectrometry parameters of the four cytokinins are shown in Table 2.

[0054] Table 2 Related mass spectrometry parameters of four cytokinins

[0055]

[0056] After the sample is separated by liquid chromatography, each component enters the mass spectrometer detector in turn. Each component is ionized in the ion source to produce ions with a certain charge and different mass numbers. Different ions have different movement behaviors in the electromagnetic field. The mass analyzer is used to separate the ions according to different mass-to-charge ratios (m / z) to obtain a mass spectrum arranged in the order of mass-to-charge ratio. The qualitative and quantitative results of the sample are obtained by analyzing and processing the mass spectrum.

[0057] In this embodiment, in S12, the shaking is performed 1 to 3 times.

[0058] In this embodiment, in S12, the centrifugal speed is 9000-11000 r / min, and the centrifugal time is 7-15 min.

[0059] In this embodiment, in S21, the reconstituted solution is vortex mixed and then passed through a 0.22 um organic filter membrane before being subjected to UPLC-MS / MS detection and analysis.

[0060] Impurities are removed through organic filter membranes to improve detection accuracy.

[0061] Experimental example

[0062] In order to verify the effect of the present invention on detecting four cytokinins in litchi, the following experiment was performed, comprising the steps of:

[0063] Extraction and purification: Take 0.25 g of liquid nitrogen-ground litchi sample into a 15 mL plastic centrifuge tube, add 2.5 mL of 80% acetonitrile solution, and extract at 4°C for 8 h, shaking twice during the extraction.

[0064] After taking out, centrifuge at 10000 r / min at 4°C for 10 min, collect the supernatant and obtain the residue.

[0065] 2.5 mL of 80% acetonitrile was added to the residue, shaken for 10 min, and centrifuged at 10000 r / min at 4°C for 10 min. The two extracts were combined to obtain a crude sample extract.

[0066] Activate and balance with methanol and 2% formic acid aqueous solution in turn, rinse with methanol solution (containing 0.1% formic acid), and finally elute twice with 2.5mL2% ammonia-methanol solution. Collect the eluate in a test tube, blow nitrogen to nearly dryness, add 0.25mL15% methanol aqueous solution to dissolve, vortex mix, and pass through a 0.22um organic filter membrane for testing.

[0067] Liquid phase and mass spectrometry conditions: In the liquid phase conditions, methanol was selected as mobile phase B and 5 mmol / L ammonium formate aqueous solution was selected as mobile phase A. The elution gradient was as shown in Table 1 above.

[0068] The mass spectrometry conditions were electrospray ionization source (ESI+), and the detection mode was multiple reaction monitoring (MRM) mode. The ion source temperature was 150°C, the capillary voltage was 0.35 kV, the desolvation temperature was 500°C, the nebulizer gas flow rate was 1000 L / Hr, the cone gas flow rate was 50 L / Hr; the collision gas (argon) was 0.17 L / h. Other mass spectrometry parameters are shown in Table 2 above.

[0069] Result analysis:

[0070] As shown in Tables 3 and 4, the detection limits (3 times signal-to-noise ratio) and quantification limits (10 times signal-to-noise ratio) of the four cytokinins (TZR, IPR, DHZ, IP) were lower than 9.35 and 31.15 pg / g, respectively, and showed a good linear relationship in the concentration range of 0.05-50 ng / mL, with a linear correlation coefficient (r2) greater than 0.999. At three spiked levels of high, medium and low concentrations (0.4, 2 and 20 ng / mL), the average recoveries of the four cytokinins were 80.0%-108.2%, and the standard deviations were between 0.8% and 15.5%.

[0071] It can be seen that the present invention can efficiently and simultaneously detect four cytokinins in litchi.

[0072] Table 3 Spiked recovery and relative standard deviation of four cytokinins in litchi peel (%)

[0073]

[0074] Table 4 Linear equations, linear ranges, correlation coefficients and detection limits of four cytokinins

[0075]

[0076] The above analysis results effectively illustrate the feasibility of the method of the present invention for simultaneously detecting four cytokinins in litchi using UPLC-MS / MS.

[0077] The above technical scheme uses UPLC-MS / MS to simultaneously determine the four cytokinins in litchi, namely trans-zeatin riboside (TZR), dihydrozeatin (DHZ), isopentenyl adenine (IP), and isopentenyl adenine riboside (IPR). 0.25g of litchi sample was extracted with acetonitrile-water (80:20, v / v) solution for 8h, purified by Bond Elut Plexa PCX solid phase extraction column (PCX column), eluted with 2.5% ammonia methanol and then detected by 0.22um organic filter membrane. XSelect HSS T3 chromatographic column was selected, and methanol and 5mmol / L ammonium formate aqueous solution were used as mobile phase for 7min gradient elution (Table 1). Electrospray positive ion (ESI+) mode was used for ionization, and the selected reaction monitoring (MRM) mode was used for quantification of cytokinins, so as to achieve simultaneous detection and analysis of four cytokinins in litchi with similar structures.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting four cytokinins in litchi using UPLC-MS / MS, characterized in that: The following steps are included: Step 1, extraction and purification, which includes: S11, obtaining litchi sample tissue, grinding it under liquid nitrogen conditions to obtain sample powder; S12, placing the sample powder in a centrifuge tube, adding acetonitrile solution, extracting at 3-4°C for 6-10 hours, shaking regularly during the extraction, centrifuging the centrifuge tube at 3-4°C, collecting the supernatant and residue, Wherein, 5 to 11 mL of acetonitrile solution is added to every 1 g of sample powder, and the concentration of the acetonitrile solution is 60 to 85%; S13, repeating step S12 with the residue, recovering the supernatant obtained from the residue and combining it with the supernatant obtained in step S12 to obtain a crude sample extract; S14, using the crude sample extract to activate and balance the PCX column with methanol and 2% formic acid aqueous solution in sequence, then eluting with an eluent, and then eluting with an eluent, collecting the eluent, Wherein, the eluent is composed of methanol solution and formic acid, and the content of formic acid is 0.05-0.2%. The eluent is composed of methanol solution and ammonia water, the content of ammonia water is 1-3%, and 15-21 mL of eluent is added for every 1 g of sample powder; S15, concentrating the eluted liquid nitrogen, and then adding methanol aqueous solution to redissolve it to obtain a reconstituted solution, wherein the concentration of the methanol aqueous solution is 10-20%; Step 2: UPLC-MS / MS detection and analysis, which includes: S21, subjecting the reconstituted solution to UPLC-MS / MS detection and analysis, The liquid phase conditions are as follows: mobile phase B is methanol, mobile phase A is 5mmol / L ammonium formate aqueous solution, and elution is performed by gradient elution. The elution program is shown in Table 1. Table 1 Gradient elution program The mass spectrometry conditions were as follows: electrospray ion source, detection mode was multiple reaction detection mode, ion source temperature was 150°C, capillary voltage was 0.35 kV, desolvation temperature was 500°C, nebulizer gas flow rate was 1000 L / Hr, cone gas flow rate was 50 L / Hr; collision argon gas was 0.17 L / h. The relevant mass spectrometry parameters of the four cytokinins are shown in Table 2. Table 2 Related mass spectrometry parameters of four cytokinins 2. The method for simultaneously detecting four cytokinins in litchi by UPLC-MS / MS according to claim 1, characterized in that: In S12, shake 1 to 3 times.

3. The method for simultaneously detecting four cytokinins in litchi by UPLC-MS / MS according to claim 1, characterized in that: In S12, the centrifugal speed is 9000-11000 r / min, and the centrifugal time is 7-15 min.

4. The method for simultaneously detecting four cytokinins in litchi by UPLC-MS / MS according to claim 1, characterized in that: In S21, the reconstituted solution is vortex mixed and then passed through a 0.22 um organic filter membrane before being subjected to UPLC-MS / MS detection and analysis.

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