CN109856290B - Method for determining polyamine content of cotton flowers based on ultra-high liquid chromatography - Google Patents
Method for determining polyamine content of cotton flowers based on ultra-high liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method for determining polyamine content in cotton flowers based on ultra-high liquid chromatography, which comprises the following steps: (1) adding liquid nitrogen into cotton flowers, grinding the cotton flowers into powder, adding pectinase, cellulase and perchloric acid solution, leaching, adding pvpp for adsorption treatment after leaching, sequentially performing ultrasonic treatment and centrifugal treatment after adsorption treatment, taking supernate obtained after centrifugal treatment, adding benzoyl chloride and NaoH solution for acylation and fixation, adding saturated Nacl solution after acylation and fixation, mixing, adding diethyl ether for extraction, then performing centrifugal treatment, taking diethyl ether phase obtained after centrifugal treatment, drying, performing elution treatment by using methanol, and filtering eluent obtained after elution treatment to obtain a sample to be detected; (2) and detecting the sample to be detected by using the ultra-high liquid chromatography. The method has high polyamine extraction efficiency and more accurate measurement result.
Description
Technical Field
The invention relates to the field of substance content detection, in particular to a method for determining the polyamine content of cotton flowers based on ultra-high liquid chromatography.
Background
The polyamine is a kind of aliphatic nitrogenous base micromolecule substance with strong biological activity widely existing in organisms. Higher plant polyamines are of a wide variety, the main type being Putrescine (Put); spermidine (Spd); spermine (Spm). Since polyamines are protonated at normal cellular pH values and their ability to initially bind to anionic macromolecules (dnas, rnas, chromatin, and proteins) is related to biological functional analysis using infrared spectroscopy, polyamines act as regulatory molecules in many fundamental cellular processes in addition to stabilizing macromolecular structures. The polyamine has the effects on the growth and development, flowering, fruiting, aging, photomorphogenesis and other processes of higher plants. Meanwhile, the polyamine can participate in drought resistance, saline-alkali resistance, disease resistance, high temperature resistance and other biotic and abiotic stresses and in regulation of endogenous hormones. Therefore, the method for simply, quickly and accurately detecting the polyamine content of the cotton is explored, and has theoretical significance and practical significance for researching the relation between the polyamine content of the cotton and the biotic and abiotic stress and yield of the cotton.
The cotton journal 2014,26(2) 138-144 disclose a cotton polyamine HPLC determination method, which comprises an extraction process and a detection process, wherein the extraction process comprises the steps of leaching by perchloric acid, then performing benzoyl chloride derivatization reaction, and finally performing extraction, and the detection is performed by a standard curve determination method by HPLC. Although convenient, this method has low polyamine extraction efficiency, resulting in inaccurate assay results.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for measuring the polyamine content of cotton flowers based on ultra-high liquid chromatography, which has high polyamine extraction efficiency and more accurate measurement.
In order to solve the technical problems, the invention adopts the following technical scheme: a method for measuring polyamine content of cotton flowers based on ultra-high liquid chromatography comprises the following steps:
(1) preparation of test article
Adding liquid nitrogen into cotton flowers, grinding the cotton flowers into powder, adding pectinase, cellulase and perchloric acid solution, leaching, adding pvpp for adsorption treatment after leaching, sequentially performing ultrasonic treatment and centrifugal treatment after adsorption treatment, taking supernate obtained after centrifugal treatment, adding benzoyl chloride and NaoH solution for acylation and fixation, adding saturated Nacl solution after acylation and fixation, mixing, adding diethyl ether for extraction, then performing centrifugal treatment, taking diethyl ether phase obtained after centrifugal treatment, drying, performing elution treatment by using methanol, and filtering eluent obtained after elution treatment to obtain a sample to be detected;
(2) detection of test article
And detecting the sample to be detected by using the ultra-high liquid chromatography.
Furthermore, the addition amount of the pectinase is 0.18-0.22 percent of the weight of the cotton flowers, the addition amount of the cellulase is 0.12-0.18 percent of the weight of the cotton flowers, the concentration of the perchloric acid solution is 4.5-5.5 percent, and the material-liquid ratio of the leaching treatment is 1: 12-1: 18. The amount of the enzyme is required to meet the requirement of being capable of carrying out enzymolysis on cell walls, the experimental result is ensured not to be influenced, the amount of the enzyme is less, the cell walls cannot be sufficiently enzymolyzed, the pH value is influenced by the excessive amount of the enzyme, and the experimental result is influenced by adsorbing enzymolysis substances. In the process of implementing the invention, the inventor finds that the addition amount of the enzyme is most appropriate, the cell wall can be guaranteed to be enzymolyzed, the experimental result cannot be influenced, the feed-liquid ratio is also the optimal feed-liquid ratio, and the liquid amount can be saved on the basis of guaranteeing the extraction rate; perchloric acid solution can promote enzymolysis.
Further, the condition of the leaching treatment is pH5.2-5.8, the temperature is 42-48 ℃, and the time is 5 h. In the implementation process of the invention, the inventor finds that the pH value of 5.2-5.8 and the temperature of 42-48 ℃ are most suitable for the enzymolysis process of the compound enzyme group of pectinase and cellulase, the enzymolysis activity of the double enzymes is maximized, the polyamine extraction efficiency is improved, and the quick and effective extraction is ensured.
Further, the ultrasonic treatment conditions are that the power is 120 w-180 w and the time is 60 min. In the process of implementing the invention, the inventor finds that the ultrasonic treatment condition is most appropriate, can effectively promote the extraction and ensure the detection accuracy.
Further, the amount of benzoyl chloride added was 3% of the volume of the supernatant, the concentration of the NaoH solution was 2mol/l, the amount added was 2 times of the volume of the supernatant, and the amount of the saturated Nacl solution was 4 times of the volume of the supernatant. In the process of implementing the invention, the inventor finds that the above conditions are most beneficial to fully benzoylating the polyamine of the cotton flower tissue, and are beneficial to fixing and eluting the polyamine at the later stage, so that the polyamine extraction efficiency is increased.
Further, the acylation fixing condition is that the temperature is 35-40 ℃ and the time is 40 min. In the course of carrying out the present invention, the inventors have found that the reaction efficiency is low below this temperature; above this temperature the benzoyl chloride starts to decompose and the higher the temperature the faster the decomposition speed, affecting the experimental results.
Further, drying is carried out using N2And (5) drying. N2 is dried by blowing, so that the temperature in the drying process can be ensured to be lower, the influence on experiments is minimum, and the temperature of other drying methods is higher, the stability of polyamine is influenced, and the extraction rate of polyamine is reduced.
Further, the conditions of the first centrifugal treatment are 6000r/min and 25min, and the conditions of the second centrifugal treatment are 5000r/min and 5 min. Fully separating the solid substance of the sample from the cell exudate by centrifugation for the first time, and taking the supernatant for research; the second time, after addition of ether, was centrifuged again to dissolve the polyamine in ether in the supernatant treatment, and the ether phase was taken for study.
Further, the chromatographic conditions adopted for detection are as follows: a chromatographic column Agilent XDB-C18 reverse phase column with the size of 4.6 multiplied by 250 mm; the volume ratio of the mobile phase methanol to the water is 70: 30. The Agilent XDB-C18 reverse phase column is a high-strength silica gel column, can resist high pressure, can provide excellent peak patterns of analytes, and has good separation effect on polyamine in cotton flowers. The length of the chromatographic column used in the invention is longer than that of the prior art, and the pressure is higher due to the design, but in the process of implementing the invention, the inventor finds that the higher the pressure is, the better the separation effect is;
in addition, the ratio of the mobile phase used in the present invention is different from the prior art, and in the process of carrying out the present invention, the inventors found that methanol to water (70: 30) provides better polyamine separation and can increase elution capacity, shorten the retention time of the components in the column, and reduce the analysis time.
Further, the chromatographic conditions adopted for detection are as follows: the flow rate is 1 mL/min; detecting the column temperature at 30 ℃; the detection wavelength is 230 nm; retention time 20 min. By adopting the condition, the chromatogram peak shape of the UPLC is stable, the peak emergence time is short, the separation degree is good, and the efficiency is high.
The invention has the beneficial effects that:
compared with the prior art, the pectinase and cellulase are added during leaching, so that cell wall enzymolysis of cotton tissue can be realized, the polyamine extraction efficiency is improved, pigments in the cotton tissue enzymolysis can be adsorbed by virtue of the absorption of the pvpp (cross-linked polyvinylpyrrolidone), the interference of the pigments on polyamine determination is reduced, the stability of an ultra-high liquid chromatography (UPLC) chromatogram and the stability of repeated tests are facilitated, in addition, the polyamine content extraction efficiency can be further improved by virtue of ultrasonic treatment, and the methanol is further used for elution treatment and is used for UPLC sample injection analysis, so that the separation effect is better, the polyamine extraction efficiency is high, and the determination result is more accurate.
Drawings
FIG. 1 is a standard liquid chromatogram of putrescine, the separation degree of the putrescine and the internal standard (1, 6-hexanediamine) peak is better, the peak-out time is 4.0min for putrescine, and the standard is 6.25 min.
FIG. 2 is a standard liquid chromatogram of spermidine, wherein the separation degree of spermidine and internal standard (1, 6-hexanediamine) peaks is good, the peak-off time is 5.5min for putrescine, and the standard sample is 6.25 min.
FIG. 3 is a standard liquid chromatogram of spermine, showing that the separation of spermine and internal standard (1, 6-hexanediamine) peaks is good, the time of appearance of the peaks is 7.0min for putrescine, and the time of appearance of the peaks is 6.25min for the standard.
FIG. 4 is a standard liquid chromatogram of polyamines, showing better separation of the peaks of putrescine, spermidine, spermine and internal standard (1, 6-hexanediamine).
FIG. 5 is a standard liquid chromatogram of cotton tissue processed by a double-enzyme method, and the separation degrees of the putrescine, spermidine, spermine and internal standard (1, 6-hexanediamine) peaks are better and consistent with the peak emergence time of a standard product.
FIG. 6 is a standard liquid chromatogram of control (not treated with the two-enzyme method) cotton tissue.
FIG. 7 is a comparison of polyamine extraction efficiency for several treatments.
Detailed Description
In the following detailed description, for the purposes of promoting an understanding of the principles of the invention, reference will now be made to certain exemplary embodiments of the invention, which are illustrated by way of example only. Needless to say, a person skilled in the art realizes that the described embodiments can be modified in various different ways without departing from the spirit and scope of the present invention.
Before the embodiment, a standard curve is drawn, and the specific steps are as follows:
(1) the putrescine, spermidine and spermine are respectively weighed to be 100mg, 5.5% perchloric acid is used for respectively metering to 3 volumetric flasks with 10ml, and the obtained concentration is 10 mg/ml.
(2) By using a gradient dilution method, 50, 100, 250, 500 and 1000 μ g/ml of standard solution 3 were prepared, and the volume was respectively determined to 10 ml.
(3) Taking 10mg of internal standard (1, 6-hexanediamine), metering to 10ml of volumetric flask, preparing 1mg/ml of internal standard storage solution, taking 5ml of internal standard storage solution, metering to 10ml of volumetric flask by using 5.5% perchloric acid, and preparing 500 mu g/ml of internal standard working solution.
(4) 50. mu.l of each of the diluted 50, 100, 250, 500 and 1000. mu.g/ml 3 standard solutions were taken and added to 10ml of an EP tube, and 10. mu.l of an internal standard (1, 6-hexanediamine), 2.5ml of 5.5% perchloric acid, 2ml of 2mol/l sodium hydroxide and 1.5. mu.l benzoyl chloride were added thereto.
(5) The liquid in the EP tube was vortexed and mixed, and placed in a 37 ℃ water bath for 40min to benzoylate the three polyamines.
(6) The pH was adjusted to 5.5 using 0.2mol/l phosphate buffer (pH 5.0).
(7) And (3) adding 2ml of diethyl ether into the solutions respectively, centrifuging the obtained mixed solution for 5min under the condition of 5000r/min, and then putting 1ml of diethyl ether phase obtained by centrifugation into a 2ml centrifuge tube.
(8) By using N2After blow-drying, 200 mul of methanol is used for elution, and then the eluent obtained by elution is filtered by a 0.22 mu m needle filter, so that the sample to be tested is obtained.
(9) Taking 10 mu l of sample to be detected, and detecting by using an ultra-high liquid chromatography, wherein the chromatographic conditions adopted by the detection are as follows: a chromatographic column Agilent XDB-C18 reverse phase column with the size of 4.6 multiplied by 250 mm; mobile phase methanol and water, wherein the volume ratio of the methanol to the water is 70: 30; the flow rate is 1 mL/min; detecting the column temperature at 30 ℃; the detection wavelength is 230 nm; retention time 20 min.
(10) Standard curve: and (3) taking the concentration of the standard substance as a horizontal coordinate, taking the peak area of the standard substance with the corresponding concentration and the peak area ratio of the internal standard (1, 6-hexamethylene diamine) as a vertical coordinate, and obtaining a linear regression equation by using a weighted least square method. Putrescine linear equation Y ═ 0.0133x +0.0066 (R)20.9914), spermidine linear equation Y0.0141 x +0.0226 (R)20.9918), linear equation Y of spermine 0.0190x +0.0337 (R)20.9985). R of three polyamines2Values were all greater than 0.99, indicating that the standard curves for the three polyamines are well linear.
Example 1
Determination of polyamine content in cotton flowers
The method comprises the following steps:
(1) taking complete cotton flowers, grinding the cotton flowers into powder in liquid nitrogen, and then weighing 1.0g of cotton flower powder;
(2) adding 5ml of 5% perchloric acid into cotton flower powder, adding 2mg of pectinase and 1.5mg of cellulase, adding water to adjust the material-liquid ratio to 1:15, adjusting the pH to 5.5, leaching at 45 ℃ for 5h, adding 0.05g of cross-linked polyvinylpyrrolidone, and treating for 1 h;
(3) carrying out ultrasonic treatment on the mixed solution obtained in the step (2) for 60min under the condition of 160w power by adopting an ultrasonic cleaning instrument, and then centrifuging for 25min under the condition of 6000 r/min;
(4) adding 500 mu l of supernate obtained by centrifugation in the step (3) into a 10ml centrifuge tube, adding 15 mu l of benzoyl chloride, adding 1ml of 2mol/l NaoH solution, performing vortex for 30s, performing water bath at 37 ℃ for 40min, adding 2ml of saturated Nacl, mixing, and adding 2ml of diethyl ether for extraction;
(5) centrifuging the mixed solution obtained in the step (4) for 5min under the condition of 5000r/min, then taking 1ml of ether phase obtained by centrifugation in a 2ml centrifuge tube, and utilizing N2After blow-drying, eluting with 200 μ l methanol, and filtering the eluate with 0.22 μm needle filter to obtain sample to be tested;
(6) taking 10 mu l of sample to be detected, and detecting by using an ultra-high liquid chromatography, wherein the chromatographic conditions adopted by the detection are as follows: a chromatographic column Agilent XDB-C18 reverse phase column with the size of 4.6 multiplied by 250 mm; mobile phase methanol and water, wherein the volume ratio of the methanol to the water is 70: 30; the flow rate is 1 mL/min; detecting the column temperature at 30 ℃; the detection wavelength is 230 nm; the retention time is 20min, and the polyamine content of cotton flowers can be calculated by substituting the detection value into the standard curve.
Example 2
Determination of polyamine content in cotton flowers
The method comprises the following steps:
(1) taking complete cotton flowers, grinding the cotton flowers into powder in liquid nitrogen, and then weighing 1.0g of cotton flower powder;
(2) adding 5ml of perchloric acid with the concentration of 4.5% into cotton flower powder, adding 1.8mg of pectinase and 1.8mg of cellulase, adding water to adjust the material-liquid ratio to 1:18, adjusting the pH to 5.2, leaching for 5 hours at the temperature of 42 ℃, adding 0.05g of cross-linked polyvinylpyrrolidone, and treating for 1 hour;
(3) carrying out ultrasonic treatment on the mixed solution obtained in the step (2) for 60min under the condition of 120w power by adopting an ultrasonic cleaning instrument, and then centrifuging for 25min under the condition of 6000 r/min;
(4) adding 500 mu l of supernate obtained by centrifugation in the step (3) into a 10ml centrifuge tube, adding 15 mu l of benzoyl chloride, adding 1ml of 2mol/l NaoH solution, performing vortex for 30s, performing water bath at 35 ℃ for 40min, adding 2ml of saturated Nacl, mixing, and adding 2ml of diethyl ether for extraction;
(5) centrifuging the mixed solution obtained in the step (4) for 5min under the condition of 5000r/min, then taking 1ml of ether phase obtained by centrifugation in a 2ml centrifuge tube, and utilizing N2After drying, elution was carried out with 200. mu.l of methanol, followed by a 0.22 μm needleFiltering and eluting the obtained eluent by using a filter to obtain a sample to be detected;
(6) taking 10 mu l of sample to be detected, and detecting by using an ultra-high liquid chromatography, wherein the chromatographic conditions adopted by the detection are as follows: a chromatographic column Agilent XDB-C18 reverse phase column with the size of 4.6 multiplied by 250 mm; mobile phase methanol and water, wherein the volume ratio of the methanol to the water is 70: 30; the flow rate is 1 mL/min; detecting the column temperature at 30 ℃; the detection wavelength is 230 nm; the retention time is 20min, and the polyamine content of cotton flowers can be calculated by substituting the detection value into the standard curve.
Example 3
Determination of polyamine content in cotton flowers
The method comprises the following steps:
(1) taking complete cotton flowers, grinding the cotton flowers into powder in liquid nitrogen, and then weighing 1.0g of cotton flower powder;
(2) adding 5ml of 5.5% perchloric acid into cotton flower powder, adding 2.2mg of pectinase and 1.2mg of cellulase, adding water to adjust the material-liquid ratio to 1:12, adjusting the pH to 5.8, leaching at 48 ℃ for 5h, adding 0.05g of cross-linked polyvinylpyrrolidone, and treating for 1 h;
(3) carrying out ultrasonic treatment on the mixed solution obtained in the step (2) for 60min under the condition of 180w power by adopting an ultrasonic cleaning instrument, and then centrifuging for 25min under the condition of 6000 r/min;
(4) adding 500 mu l of supernate obtained by centrifugation in the step (3) into a 10ml centrifuge tube, adding 15 mu l of benzoyl chloride, adding 1ml of 2mol/l NaoH solution, performing water bath at 40 ℃ for 40min after swirling for 30s, adding 2ml of saturated Nacl, mixing, and adding 2ml of diethyl ether for extraction;
(5) centrifuging the mixed solution obtained in the step (4) for 5min under the condition of 5000r/min, then taking 1ml of ether phase obtained by centrifugation in a 2ml centrifuge tube, and utilizing N2After blow-drying, eluting with 200 μ l methanol, and filtering the eluate with 0.22 μm needle filter to obtain sample to be tested;
(6) taking 10 mu l of sample to be detected, and detecting by using an ultra-high liquid chromatography, wherein the chromatographic conditions adopted by the detection are as follows: a chromatographic column Agilent XDB-C18 reverse phase column with the size of 4.6 multiplied by 250 mm; mobile phase methanol and water, wherein the volume ratio of the methanol to the water is 70: 30; the flow rate is 1 mL/min; detecting the column temperature at 30 ℃; the detection wavelength is 230 nm; the retention time is 20min, and the polyamine content of cotton flowers can be calculated by substituting the detection value into the standard curve.
Example 4
Effect test of pectinase and cellulase on polyamine extraction efficiency of Cotton flowers
On the basis of example 1, the same ground cotton flower powder was used as the raw material, and pectinase and cellulase were omitted, and ultrasonic treatment and pvpp treatment were omitted from the leaching, as a control group 1; during leaching, pectinase and cellulase and ultrasonic treatment are omitted, and the obtained product is used as a control group 2; ultrasonic treatment and pvpp treatment were omitted during leaching as control group 3; during leaching, pectinase and cellulase are omitted, and pvpp treatment is omitted and used as a control group 4; example 1 served as control group 5. The standard liquid chromatogram obtained from the control group 5 is shown in fig. 5, the standard liquid chromatogram obtained from the control group 1 is shown in fig. 6, the standard liquid chromatograms of putrescine, spermidine, spermine and polyamine are respectively shown in fig. 1 to fig. 4, and the final detection result of each control group is shown in fig. 7.
From the above, it can be concluded that the peak-off time of putrescine, spermidine and spermine is 4.1min, 5.4min and 6.9min, the peak-off time of the internal standard is 6.2min, the peak separation of each standard is good, and the extraction efficiency of example 1 is improved a lot compared with that of omitting pectinase and cellulase, the extraction efficiency can be improved by ultrasonic treatment and pvpp treatment, and compared with the method for measuring cotton polyamine HPLC disclosed in "journal of cotton" 2014,26(2) 138-144, the comprehensive extraction rate of polyamine in control 1 is 1.47 times of that of the method; control 2 polyamine extraction was 3.72 times that of the method; the comprehensive extraction rate of the contrast 3 is 2.45 times that of the method; the comprehensive extraction rate of contrast 4 is 3.75 times that of the method; compared with the method, the comprehensive extraction rate of the control 5 is 4.87 times that of the method, and the extraction efficiency of the polyamine can be obviously improved.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the present disclosure, and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this disclosure.
Claims (6)
1. A method for measuring polyamine content of cotton flowers based on ultra-high liquid chromatography is characterized by comprising the following steps: the method comprises the following steps:
(1) preparation of test article
Taking cotton flowers, adding liquid nitrogen, grinding into powder, adding pectinase, cellulase and perchloric acid solution for leaching, wherein the addition amount of the pectinase is 0.18-0.22 percent of the weight of the cotton flowers, the addition amount of the cellulase is 0.12-0.18 percent of the weight of the cotton flowers, the concentration of the perchloric acid solution is 4.5-5.5 percent, the material-liquid ratio of the leaching is 1: 12-1: 18, the condition of the leaching is pH5.2-5.8, the temperature is 42-48 ℃, the time is 5 hours, after the leaching, adding pvpp for adsorption treatment, after the adsorption treatment, sequentially carrying out ultrasonic treatment and centrifugal treatment, then taking supernatant obtained after the centrifugal treatment, adding benzoyl chloride and NaOH solution for acylation and fixation, wherein the addition amount of the benzoyl chloride is 3 percent of the volume of the supernatant, the concentration of the NaOH solution is 2mol/l, the addition amount is 2 times of the volume of the supernatant, and the addition amount of the saturated solution is 4 times of the volume of the NaCl, acylating and fixing at 35-40 deg.c for 40min, adding saturated NaCl solution, mixing, adding ether for extraction, centrifuging to obtain ether phase, drying, eluting with methanol to obtain eluent, and filtering to obtain the sample to be tested;
(2) detection of test article
And detecting the sample to be detected by using the ultra-high liquid chromatography.
2. The method for determining polyamine content of cotton flowers based on ultra-high liquid chromatography as claimed in claim 1, wherein: the ultrasonic treatment is carried out under the conditions of 120 w-180 w of power and 60min of time.
3. The method for determining polyamine content in cotton flowers based on ultra-high liquid chromatography as claimed in claim 1 or 2, wherein: drying with N2And (5) drying.
4. The method for determining polyamine content in cotton flowers based on ultra-high liquid chromatography as claimed in claim 1 or 2, wherein: the first centrifugation treatment is carried out at 6000r/min for 25min, and the second centrifugation treatment is carried out at 5000r/min for 5 min.
5. The method for determining polyamine content in cotton flowers based on ultra-high liquid chromatography as claimed in claim 1 or 2, wherein: the chromatographic conditions adopted for detection are as follows: a chromatographic column Agilent XDB-C18 reverse phase column with the size of 4.6 multiplied by 250 mm; the volume ratio of the mobile phase methanol to the water is 70: 30.
6. The method for determining polyamine content in cotton flowers based on ultra-high liquid chromatography as claimed in claim 1 or 2, wherein: the chromatographic conditions adopted for detection are as follows: the flow rate is 1 mL/min; detecting the column temperature at 30 ℃; the detection wavelength is 230 nm; retention time 20 min.
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| CN103575826A (en) * | 2013-11-05 | 2014-02-12 | 四川农业大学 | Efficient liquid-phase chromatographic detection method for content of polyamines in goose tissue as well as application thereof |
| CN105424823A (en) * | 2015-11-02 | 2016-03-23 | 贵州省烟草科学研究院 | Detection method of polyamine substances in tobacco roots, stems and leaves |
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| CN103575826A (en) * | 2013-11-05 | 2014-02-12 | 四川农业大学 | Efficient liquid-phase chromatographic detection method for content of polyamines in goose tissue as well as application thereof |
| CN105424823A (en) * | 2015-11-02 | 2016-03-23 | 贵州省烟草科学研究院 | Detection method of polyamine substances in tobacco roots, stems and leaves |
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