CN115598232A - Gossypol solution standard substance and preparation method and detection method thereof - Google Patents
Gossypol solution standard substance and preparation method and detection method thereof Download PDFInfo
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- CN115598232A CN115598232A CN202210994972.1A CN202210994972A CN115598232A CN 115598232 A CN115598232 A CN 115598232A CN 202210994972 A CN202210994972 A CN 202210994972A CN 115598232 A CN115598232 A CN 115598232A
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- gossypol
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- acetone
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- QBKSWRVVCFFDOT-UHFFFAOYSA-N gossypol Chemical compound CC(C)C1=C(O)C(O)=C(C=O)C2=C(O)C(C=3C(O)=C4C(C=O)=C(O)C(O)=C(C4=CC=3C)C(C)C)=C(C)C=C21 QBKSWRVVCFFDOT-UHFFFAOYSA-N 0.000 title claims abstract description 306
- QHOPXUFELLHKAS-UHFFFAOYSA-N Thespesin Natural products CC(C)c1c(O)c(O)c2C(O)Oc3c(c(C)cc1c23)-c1c2OC(O)c3c(O)c(O)c(C(C)C)c(cc1C)c23 QHOPXUFELLHKAS-UHFFFAOYSA-N 0.000 title claims abstract description 153
- 229930000755 gossypol Natural products 0.000 title claims abstract description 153
- 229950005277 gossypol Drugs 0.000 title claims abstract description 153
- 239000000126 substance Substances 0.000 title claims abstract description 75
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000000243 solution Substances 0.000 claims abstract description 105
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 82
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 30
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 29
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 18
- ZPLUZNXSYCCJOE-UHFFFAOYSA-N phosphoric acid;propan-2-one Chemical compound CC(C)=O.OP(O)(O)=O ZPLUZNXSYCCJOE-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000012086 standard solution Substances 0.000 claims abstract description 12
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 8
- 238000007865 diluting Methods 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 5
- 238000004811 liquid chromatography Methods 0.000 claims description 3
- MSZYQCNHWFXSAK-UHFFFAOYSA-N oxolane;phosphoric acid Chemical compound C1CCOC1.OP(O)(O)=O MSZYQCNHWFXSAK-UHFFFAOYSA-N 0.000 claims description 3
- 239000003381 stabilizer Substances 0.000 abstract description 4
- 230000008859 change Effects 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 17
- 239000012071 phase Substances 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 235000019441 ethanol Nutrition 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 235000013824 polyphenols Nutrition 0.000 description 5
- 238000013112 stability test Methods 0.000 description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 240000000982 Malva neglecta Species 0.000 description 1
- 235000000060 Malva neglecta Nutrition 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- -1 polyphenol hydroxy binaphthyl aldehyde compound Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000000528 statistical test Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention discloses a gossypol solution standard substance, a preparation method and a detection method thereof, and particularly relates to the field of chemical detection. The preparation method comprises the steps of dissolving a gossypol standard substance in acetone to prepare a gossypol acetone solution; adding phosphoric acid into the gossypol acetone solution, adjusting the pH value to 3.0-5.5, and performing ultrasonic treatment to prepare a uniform gossypol acetone phosphoric acid solution; diluting the gossypol acetone phosphoric acid solution into the required gossypol solution standard substance by using an anhydrous ethanol solution. According to the gossypol solution standard substance, phosphoric acid is added in the preparation of the gossypol solution standard solution to adjust the pH value, and a small amount of acetone is added to serve as a stabilizer to ensure the stability of gossypol; therefore, the gossypol solution standard substance prepared by the method has the characteristics of good stability and convenience in transportation and detection. The gossypol solution standard substance has good uniformity, is refrigerated and stored in a dark place, has small change of quantity value when standing for a long time, and has a stable period of 2 years.
Description
Technical Field
The invention relates to the field of chemical detection, in particular to a gossypol solution standard substance and a preparation method and a detection method thereof.
Background
Gossypol is a polyphenol hydroxy binaphthyl aldehyde compound with the chemical name of 2, 2' -bi-8-methyl acid-1, 6, 7-trihydroxy-5-isopropyl-3-methylnaphthalene, and the structure of the compound is shown in figure 1. Gossypol is mainly present in the roots, stems, leaves and seeds of mallow cotton and has certain resistance to a plurality of cotton diseases and pests.
Gossypol has the following structure:
according to the chemical structure of gossypol, gossypol has six phenolic hydroxyl groups and two phenolic aldehyde groups, so that the gossypol has strong biological and chemical activities, the phenolic hydroxyl groups make the gossypol weakly acidic, and the phenolic hydroxyl groups and the aldehyde groups can react with or be combined with other groups or substances to be denatured. The mechanism of gossypol causes its own instability, is not light and heat resistant, and is easily oxidized and decomposed under different reaction conditions. In the laboratory testing process, in order to avoid the loss of gossypol, ensure the accuracy of experiment testing result, the standard substance that the experiment was used including the machine of going up with add the mark, need dispose simultaneously and preserve under the same condition, the operation of keeping out of the sun in whole experimentation. However, the gossypol solution standard substance on the market at present has variable storage period and poor stability, and particularly, the standard substance with low concentration must be prepared at present. With the increase of public concern on food safety, the frequency of measuring free gossypol in vegetable food is rapidly increased, and the standard substance of gossypol solution is not provided for. Therefore, it is urgent to prepare a standard substance that can be stabilized for one year or more.
Disclosure of Invention
Therefore, the invention provides a gossypol solution standard substance, a preparation method and a detection method thereof, which are used for solving the problems that the existing gossypol solution standard substance is easy to discolor during storage, has poor low-concentration stability and the like.
The invention finds that the gossypol has better stability under the acidic condition and the action of the stabilizer, the pH is adjusted by adding phosphoric acid in the preparation of the standard solution of the gossypol solution, and a small amount of acetone is added to serve as the stabilizer to ensure the stability of the gossypol.
In order to achieve the above purpose, the invention provides the following technical scheme:
according to a first aspect of the present invention, there is provided a method for preparing a gossypol solution standard substance, comprising:
dissolving a gossypol standard product in acetone to prepare a gossypol acetone solution;
adding phosphoric acid solution into gossypol acetone solution, adjusting pH value to 3.0-5.5, and performing ultrasonic treatment to obtain uniform gossypol acetone phosphoric acid solution;
diluting the high-concentration gossypol acetone phosphoric acid aqueous solution into the required gossypol solution standard substance by using an absolute ethanol solution.
Further, the purity of the gossypol standard substance is more than or equal to 98.5%.
Further, the phosphoric acid purity is chromatographic purity
Further, the acetone purity is chromatographic purity.
Further, the volume fraction of the acetone solution is 0.1%.
Further, the concentration of the gossypol solution standard substance is 1000 mug/mL.
Further, the method comprises:
1.000g of gossypol, 1.0-2mL of phosphoric acid and 1mL of acetone with the mass percent of 100 percent according to the purity are weighed;
adding absolute ethyl alcohol to a volumetric flask with the volume of 1000mL to obtain a gossypol standard solution with the concentration of 1000 mug/mL and the pH value of 3.0-5.5.
According to a second aspect of the present invention, there is provided a gossypol solution standard substance prepared by the method as described above.
According to a third aspect of the invention, a method for detecting a gossypol solution is provided, wherein the detection is carried out by adopting a liquid chromatography-diode array detector/ultraviolet detector combined method.
Further, the conditions of the liquid chromatography are that
A chromatographic column: c18 250mm × 4.6mm × 0.5 μm;
and/or the mobile phase is mobile phase A, mobile phase B = 12;
wherein, the mobile phase A is 2% phosphoric acid tetrahydrofuran = 5; the mobile phase B is methanol;
and/or, detecting wavelength: 235nm;
and/or, flow rate: 1.0ml/min;
and/or, sample size: 10 μ L.
The invention has the following advantages:
the raw materials of the invention adopt gossypol purity standard substance with purity of 98.5% -100%, and acetone solution is added, thus greatly improving the stability of the product;
according to the gossypol solution standard substance, phosphoric acid is added in the preparation of the gossypol solution standard substance to adjust the pH value, and a small amount of acetone is added to serve as a stabilizer to ensure the stability of gossypol; therefore, the gossypol solution standard substance prepared by the method has the characteristics of good stability and convenience in transportation and detection.
The gossypol solution standard substance has good uniformity, is stored in a refrigerated and light-proof manner, has small change of magnitude when standing for a long time, and has a stabilization period of 2 years.
The invention adopts a liquid chromatogram-diode array detector/ultraviolet detector to detect; compared with a national standard method GB 5009.148-2014, the method adopts the high performance liquid chromatography for detection, not only reduces the complexity of preparing the standard solution, but also widens the detection environment.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other implementation drawings can be derived from the drawings provided by those of ordinary skill in the art without any creative effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the scope of the present invention.
FIG. 1 is an HPLC chromatogram of a gossypol solution standard substance of example 2 provided by the present invention;
FIG. 2 is a graph showing the stability test of a gossypol solution standard substance according to the present invention in comparative example 1;
FIG. 3 is a graph showing the stability test of a gossypol solution standard according to the present invention in comparative example 2;
FIG. 4 is a graph showing the stability test of a gossypol solution standard according to the present invention in comparative example 3;
FIG. 5 is a graph of a standard curve of a gossypol solution standard according to the present invention;
FIG. 6 is a graph showing the long-term stability of a standard substance of a gossypol solution provided by the present invention.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 preparation of gossypol solution Standard substance
This example provides a method for preparing a gossypol solution standard.
Step one, 1.0000g of gossypol with the mass percent of 100 percent by purity is weighed and dissolved in 1mL of acetone solution, 1mL of phosphoric acid is added, and the pH value is adjusted to 5.5 to prepare the gossypol acetone phosphoric acid solution.
Wherein the purity of the gossypol is 98.5% -100%. Can be directly obtained by commercial purchase or can be prepared by reaction.
And step two, carrying out ultrasonic treatment on the gossypol acetone phosphoric acid solution, and transferring the gossypol acetone phosphoric acid solution into a 1000mL volumetric flask.
Step three, diluting the gossypol acetone phosphoric acid obtained in the step two to 1000mL by adopting absolute ethyl alcohol,
the gossypol standard solution with the concentration of 1000 mu g/mL and the pH value of 5.5 is obtained.
The above standard substance was stored in a frozen state (-18 ℃) protected from light.
EXAMPLE 2 preparation of gossypol solution Standard substance
This example provides a method for preparing a gossypol solution standard.
Step one, 1.0000g of gossypol with the mass percent of 100 percent by purity is weighed and dissolved in 1mL of acetone solution, 2mL of phosphoric acid is added, and the pH value is adjusted to 3.0 to prepare the gossypol acetone phosphoric acid solution.
Wherein the purity of the gossypol is 98.5% -100%. Can be directly obtained by commercial purchase or can be prepared by reaction.
And step two, carrying out ultrasonic treatment on the gossypol acetone phosphoric acid solution, and transferring the gossypol acetone phosphoric acid solution into a 1000mL volumetric flask.
And step three, diluting the gossypol acetone phosphoric acid obtained in the step two to 1000mL by adopting absolute ethyl alcohol, and obtaining a gossypol standard solution with the concentration of 1000 mu g/mL and the pH value of 3.0.
The above standard substance was stored in a frozen state (-18 ℃) protected from light.
EXAMPLE 3 gossypol solution detection method
The embodiment provides a method for detecting a gossypol solution, which comprises the following steps:
in the embodiment, agilent1200 high performance liquid chromatograph is adopted for detection.
The conditions of the liquid chromatography are that
A chromatographic column: c18 250 mm. Times.4.6 mm. Times.0.5 μm;
and/or the mobile phase is mobile phase A, mobile phase B = 12;
wherein, the mobile phase A is 2% phosphoric acid tetrahydrofuran = 5; the mobile phase B is methanol;
and/or, detecting wavelength: 235nm;
and/or, flow rate: 1.0ml/min;
and/or, sample size: 10 μ L.
Filtering methanol and tetrahydrofuran with organic phase filter membrane, and filtering phosphoric acid water solution with water phase filter membrane.
Comparative example 1 preparation of gossypol solution Standard substance
This comparative example provides a method of preparing a gossypol solution standard.
Step one, 1.0000g of gossypol with the mass percent of 100 percent by purity is weighed and dissolved in 1mL of acetone solution to prepare the gossypol acetone solution.
Wherein the purity of the gossypol is 98.5% -100%. Can be directly obtained by commercial purchase or can be prepared by reaction.
And step two, carrying out ultrasonic treatment on the gossypol acetone solution, and transferring the gossypol acetone solution into a 1000mL volumetric flask.
Step three, diluting the gossypol acetone obtained in the step two to a constant volume of 1000mL by adopting absolute ethyl alcohol,
the gossypol standard solution with the concentration of 1000 mu g/mL and the pH value of 3.0 is obtained.
The above standard substance was stored in a frozen state (-18 ℃ C.) in the dark.
Comparative example 2 preparation of gossypol solution Standard substance
This comparative example provides a method of preparing a gossypol solution standard.
Step one, 1.0000g of gossypol with the purity of 100 percent by mass is weighed and dissolved in 1mL of acetone solution to prepare gossypol acetone solution.
Wherein the purity of the gossypol is 98.5% -100%. Can be directly obtained by commercial purchase or can be prepared by reaction.
And step two, carrying out ultrasonic treatment on the gossypol acetone solution, and transferring the gossypol acetone solution into a 1000mL volumetric flask.
Step three, diluting the gossypol acetone obtained in the step two to a constant volume of 1000mL by adopting absolute ethyl alcohol,
the gossypol standard solution with the concentration of 1000 mu g/mL and the pH value of 7.0 is obtained.
The above standard substance was stored in a frozen state (-18 ℃) protected from light.
Comparative example 3 preparation of gossypol solution Standard substance
This example provides a method for preparing a gossypol solution standard.
Step one, 1.0000g of gossypol with the purity of 100 percent by mass is weighed and dissolved in 10mL of anhydrous ethanol solution to prepare gossypol ethanol solution.
Wherein the purity of the gossypol is 98.5% -100%. Can be directly obtained by commercial purchase or can be prepared by reaction.
And step two, carrying out ultrasonic treatment on the gossypol ethanol solution, and transferring the gossypol ethanol solution into a 1000mL volumetric flask.
Step three, adopting absolute ethyl alcohol to dilute the gossypol ethyl alcohol obtained in the step two to a constant volume of 1000mL,
the gossypol standard solution with the concentration of 1000 mu g/mL and the pH value of 7.0 is obtained.
The above standard substance was stored in a frozen state (-18 ℃) protected from light.
Test example 1
The gossypol content in example 2, comparative example 1, comparative example 2 and comparative example 3 was determined by the method of example 3, and the results were as follows:
the HPLC spectrum of example 2 is shown in FIG. 1, from which it can be seen that the acetone added spectrum has a separation degree of acetone from gossypol of more than 1.5, and acetone does not interfere with the detection of gossypol. The detected baseline is stable, and the peak shape of the gossypol chromatographic peak is symmetrical.
The HPLC spectrum of comparative example 1 is shown in fig. 2, from which it can be seen that the acetone added spectrum, the degree of separation of acetone from gossypol is greater than 1.5, and acetone does not interfere with the detection of gossypol. The baseline of the detection without adding phosphoric acid is not stable, the gossypol chromatographic peak is split into 2 chromatographic peaks, the chromatographic peak has asymmetric peak shape, and the separation degree is poor. The detection result of the gossypol is calculated by the peak area of a chromatographic peak, and the peak type difference of the chromatographic peak influences the integral result of the peak area, so that the accuracy of the detection result is low.
The HPLC chromatogram of comparative example 2 is shown in FIG. 3, from which it can be seen that the chromatogram without acetone addition has a smooth baseline and a symmetrical peak shape of the gossypol chromatogram peak.
The HPLC profile of comparative example 3 is shown in fig. 4, from which it can be seen that the baseline of the detection without adding phosphoric acid and acetone is not smooth, the gossypol chromatographic peak is split into 2 chromatographic peaks, the peak shape of the chromatographic peak is asymmetric, and the degree of separation is poor. The detection result of the gossypol is calculated by the peak area of a chromatographic peak, and the peak type difference of the chromatographic peak influences the integral result of the peak area, so that the accuracy of the detection result is low.
Therefore, the comparative products having poor peak patterns were not subjected to the stability test again, and only comparative examples 1, 3 and example 2 having good peak patterns were compared.
In conclusion, phosphoric acid in the gossypol standard solution not only affects the stability of gossypol, but also affects the chromatographic peak shape in the detection process of the gossypol solution, and directly affects the accuracy of gossypol detection.
Test example 2 creation of a gossypol solution Standard substance Curve
The test example provides a method for establishing a standard substance curve of a gossypol solution.
The gossypol standard solution of example 2 was diluted 200-fold, 100-fold, 50-fold, 25-fold, and 20-fold with absolute ethanol to obtain gossypol solution standard substances with nominal values of 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, and 50. Mu.g/mL.
The gossypol solution standard substance was injected into a liquid chromatograph, and the peak area was recorded under the chromatographic conditions described in example 3. Taking the concentration of the gossypol solution standard substance as the ordinate and the peak area as the abscissa to perform linear regression, and obtaining a standard curve shown in figure 5.
Test example 3 stability of gossypol solution as standard substance
The gossypol content of examples 1, 2, 1, 2 and 3 was measured by the method of example 3 for various periods of time, and the results are shown in table 1.
TABLE 1
Stability tests As can be seen from the data in Table 1, the gossypol solution standard substances in the invention in the examples 1 and 2 have very good stability, and the values can be stabilized for 24 months under the condition of normal-temperature storage;
the gossypol standard substance without phosphoric acid in comparative example 1 can be stabilized for 6 months, and then the value rapidly declines, so that the product stability is poor.
The standard substance without acetone solution can be stabilized for only 12 months, and then the value is in a descending trend, and the product stability is poor.
The standard substance of phosphoric acid and acetone solution is not added, the quantity value of the standard substance of the gossypol solution is in a rapid decline trend, and the product stability is poor.
As can be seen from Table 1, the gossypol solution standard substance prepared by the invention has good stability; the gossypol standard substance can be stored under refrigeration (-18 ℃) for 2 years with stable value.
Test example 4 Gossypol solution Standard substance homogeneity
According to the rules of JJG 1006-1994 first-class standard substance technical specification and JJF1343-2012 general principle and statistical principle of standard substance quantitative determination, the statistical treatment of the standard substance uniformity detection result usually adopts a variance analysis method, the method adopts the comparison of variance between groups and variance within groups, and if the ratio of the two is less than the critical value of the statistical test, the sample is considered to be uniform. The number of units extracted depends on the number of units of the total sample and knowledge of the degree of sample homogeneity. A total of 15 samples were randomly withdrawn before, during and after the production sequence, and each sample was measured 3 times to observe the uniformity of the gossypol solution having a concentration of 1000. Mu.g/mL in example 1.
Through calculation, the statistic F of the gossypol solution standard substance in the ethanol is smaller than the critical value Falpha, no obvious difference is found among groups in the group, and the sample uniformity is good. Specific data are shown in table 2.
TABLE 2 examination of the homogeneity of the gossypol solution standard in ethanol
Test example 5 Long-term stability of gossypol solution as a standard substance
The long-term stability is to be assessed periodically over a longer period of time for the stability of the predetermined characteristic value of the standard substance under defined storage conditions, and to examine the ability of the characteristic value of the standard substance to remain within a defined range, which is the standard substance. If necessary, the short-term stability of the standard substance under specific conditions should be examined by simulating the temperature, the placing mode and the like under transportation and severe conditions. Sampling according to the principle that time is dense and then sparse, and taking 5 samples at intervals of 0, 1, 4, 8, 12, 18 and 24 months every time. The results of the test conducted under the test conditions of example 3 in example 1 are shown in FIG. 6.
It can be seen from fig. 6 that none of the solution standards exhibited a significant tendency to rise or fall over the observation time, indicating that the sample was stable over the observation time.
Although the invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. A preparation method of a gossypol solution standard substance is characterized by comprising the following steps:
dissolving a gossypol standard product in acetone to prepare a gossypol acetone solution; adding phosphoric acid into the gossypol acetone solution, adjusting the pH value to 3.0-5.5, and performing ultrasonic treatment to prepare a uniform gossypol acetone phosphoric acid solution; diluting the gossypol acetone phosphoric acid solution into the required gossypol solution standard substance by using an anhydrous ethanol solution.
2. The method for preparing the gossypol solution standard substance according to claim 1, wherein the purity of the gossypol standard substance is more than or equal to 98.5%.
3. The method for preparing a gossypol solution standard substance according to claim 1, wherein the phosphoric acid is chromatographically pure.
4. The method of claim 1, wherein the acetone is chromatographically pure.
5. The method of claim 1, wherein the acetone is present in an amount of 0.1% by volume.
6. The method for preparing a gossypol solution standard substance according to claim 1, wherein the method comprises:
1.0000g of gossypol, 1.0-2.0mL of phosphoric acid and 1.0mL of acetone with the mass percent of 100 percent according to the purity are weighed;
adding absolute ethanol to a volumetric flask with the volume of 1000mL to obtain a gossypol standard solution with the concentration of 1.00mg/mL and the pH value of 3.0-5.5.
7. A gossypol solution standard substance, characterized in that it is prepared by the method of any one of claims 1-6.
8. The method for detecting the gossypol solution is characterized by adopting a liquid chromatography-diode array detector/ultraviolet detector combined method for detection.
9. The method for detecting the gossypol solution according to claim 8, wherein the conditions of the liquid chromatography are that the chromatographic column: c18 250mm × 4.6mm × 0.5 μm;
and/or the mobile phase is mobile phase A, mobile phase B = 12;
wherein, the mobile phase A is 2% phosphoric acid tetrahydrofuran = 5; the mobile phase B is methanol;
and/or, detecting wavelength: 235nm;
and/or, flow rate: 1.0ml/min;
and/or, sample size: 10 μ L.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20120321715A1 (en) * | 2011-05-05 | 2012-12-20 | Wisconsin Alumni Research Foundation | Micelles for the solubilization of gossypol |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20120321715A1 (en) * | 2011-05-05 | 2012-12-20 | Wisconsin Alumni Research Foundation | Micelles for the solubilization of gossypol |
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