A cottonyphenol solution standard substance and its preparation method and detection method

Abstract

The invention discloses a cottonyphenol solution standard substance and a preparation method and a detection method thereof, and is particularly related to the field of chemical detection. The preparation method comprises dissolving a cottonyphenol standard substance in acetone to obtain a cottonyphenol acetone solution; adding phosphoric acid to the cottonyphenol acetone solution, adjusting the pH value to 3.0-5.5, and ultrasonically treating to prepare a uniform cottonyphenol acetone phosphoric acid solution; diluting the cottonyphenol acetone phosphoric acid solution with anhydrous ethanol solution to obtain a required cottonyphenol solution standard substance. The cottonyphenol solution standard substance of the present invention is prepared by adding phosphoric acid to adjust the pH in the preparation of the cottonyphenol solution standard solution, and adding a small amount of acetone as a stabilizer to ensure the stability of cottonyphenol; so the cottonyphenol solution standard substance prepared by the present invention has the characteristics of good stability, convenient transportation and detection. The cottonyphenol solution standard substance of the present invention has good uniformity, is stored in a dark and refrigerated state, has little change in value when left standing for a long time, and has a stable period of up to 2 years.

Description

Technical Field

[0001] The invention relates to the field of chemical detection, and in particular to a cottonyphenol solution standard substance and a preparation method and a detection method thereof. Background Art

[0002] Cottonpol is a polyphenolic hydroxybinaphthaldehyde compound, chemically named 2,2'-bis-8-methylacid-1,6,7-trihydroxy-5-isopropyl-3-methylnaphthalene, with a structure as follows: Figure 1 As shown. Cottonpol is mainly found in the roots, stems, leaves and seeds of the mallow plant cotton, and has a certain resistance to many cotton diseases and pests.

[0003] The structure of gossypol is as follows:

[0004]

[0005] From the chemical structure of cottonpol, it can be seen that cottonpol has six phenolic hydroxyl groups and two phenolic aldehyde groups, so it has strong biological and chemical activity. Its phenolic hydroxyl groups make it weakly acidic, and the phenolic hydroxyl groups and aldehyde groups can react or combine with other groups or substances to denature. The structure of cottonpol leads to its own instability, light and heat instability, and easy oxidation and decomposition under different reaction conditions. In the laboratory test process, in order to avoid the loss of cottonpol and ensure the accuracy of the experimental test results, the standard substances used in the experiment include those on the machine and those spiked, which need to be configured at the same time and stored under the same conditions, and operate in the dark during the entire experiment. However, the cottonpol solution standard substances currently on the market are easy to change in value during the storage period and have poor stability, especially low-concentration standard substances, which must be prepared on the spot. With the increase in public attention to food safety, the frequency of determination of free cottonpol in plant-based foods has increased rapidly, and cottonpol solution standard substances have been in short supply. Therefore, it is imperative to prepare standard substances that can be stable for more than one year. Summary of the invention

[0006] To this end, the present invention provides a cottonyphenol solution standard substance and a preparation method and a detection method thereof, so as to solve the problems of easy discoloration during storage and poor stability at low concentration of the existing cottonyphenol solution standard substance.

[0007] The invention finds that cottonpol has good stability under acidic conditions and the action of a stabilizer. Phosphoric acid is added to adjust the pH value in the preparation of a cottonpol solution standard solution, and a small amount of acetone is added as a stabilizer to ensure the stability of cottonpol.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] According to a first aspect of the present invention, there is provided a method for preparing a cottonyphenol solution standard substance, comprising:

[0010] Dissolve the cottonpol standard in acetone to prepare a cottonpol acetone solution;

[0011] adding a phosphoric acid solution to the cottonyphenol acetone solution, adjusting the pH value to 3.0-5.5, and ultrasonically treating the solution to prepare a uniform cottonyphenol acetone phosphoric acid solution;

[0012] Dilute the high concentration gossypol acetone phosphate aqueous solution with anhydrous ethanol solution to obtain the required gossypol solution standard substance.

[0013] Furthermore, the purity of the cottonpol standard is ≥98.5%.

[0014] Further, the phosphoric acid purity is chromatographically pure

[0015] Furthermore, the acetone is chromatographically pure.

[0016] Furthermore, the volume fraction of the acetone solution is 0.1%.

[0017] Furthermore, the concentration of the cottonpol solution standard substance is 1000 μg / mL.

[0018] Furthermore, the method comprises:

[0019] Weigh 1.000 g of cotton phenol converted to 100% purity, 1.0-2 mL of phosphoric acid, and 1 mL of acetone;

[0020] Add anhydrous ethanol to a 1000mL volumetric flask to obtain a cottonyphenol standard solution with a concentration of 1000μg / mL and a pH value of 3.0-5.5.

[0021] According to a second aspect of the present invention, a cottonypol solution standard substance is provided, which is prepared by the method described above.

[0022] According to a third aspect of the present invention, a method for detecting a cottonyphenol solution is provided, which adopts a liquid chromatography-diode array detector / ultraviolet detector combination method for detection.

[0023] Further, the conditions of the liquid chromatography are

[0024] Chromatographic column: C18, 250 mm × 4.6 mm × 0.5 μm;

[0025] And / or, the mobile phase is mobile phase A:mobile phase B=12:88;

[0026] Wherein, mobile phase A is 2% phosphoric acid: tetrahydrofuran = 5:1; mobile phase B is methanol;

[0027] and / or, detection wavelength: 235 nm;

[0028] and / or, flow rate: 1.0 ml / min;

[0029] And / or, injection volume: 10 μL.

[0030] The present invention has the following advantages:

[0031] The raw material of the present invention is a cotton phenol purity standard product with a purity of 98.5%-100%, and an acetone solution is added, which greatly improves the stability of the product;

[0032] The cottonpol solution standard substance of the present invention is prepared by adding phosphoric acid to adjust the pH value of the cottonpol solution standard solution, and adding a small amount of acetone as a stabilizer to ensure the stability of the cottonpol; therefore, the cottonpol solution standard substance prepared by the present invention has the characteristics of good stability and convenient transportation and detection.

[0033] The cottonphenol solution standard substance of the invention has good uniformity, can be stored in a refrigerated and dark place, has little change in value when left standing for a long time, and has a stable period of up to 2 years.

[0034] The present invention adopts liquid chromatography-diode array detector / ultraviolet detector for detection; compared with the national standard method GB5009.148-2014 which uses high performance liquid chromatography for detection, it not only reduces the complexity of preparing standard solutions, but also broadens its detection environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0036] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0037] Figure 1 HPLC spectrum of the cottonpol solution standard substance of Example 2 provided by the present invention;

[0038] Figure 2 Provides a stability test diagram of the cottonpol solution standard substance of Comparative Example 1 of the present invention;

[0039] Figure 3Provides a stability test diagram of the cottonpol solution standard substance of Comparative Example 2 of the present invention;

[0040] Figure 4 Provides a stability test diagram of the cottonpol solution standard substance of Comparative Example 3 of the present invention;

[0041] Figure 5 Provide a standard curve diagram of a cottonysol solution standard substance for the present invention;

[0042] Figure 6 The present invention provides a long-term stability diagram of a cottonyphenol solution standard substance. DETAILED DESCRIPTION

[0043] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1 Preparation of cottonpol solution standard substance

[0045] This embodiment provides a method for preparing a cottonyphenol solution standard substance.

[0046] Step 1, weigh 1.0000 g of cottonypol converted to 100% by mass according to purity, dissolve it in 1 mL of acetone solution, add 1 mL of phosphoric acid, adjust the pH to 5.5, and prepare a cottonypol acetone phosphoric acid solution.

[0047] The purity of the cottonpol is 98.5%-100%. It can be directly purchased or prepared by itself through reaction.

[0048] Step 2: Ultrasonicate the cottonpol acetone phosphate solution and transfer it into a 1000 mL volumetric flask.

[0049] Step 3: dilute the cotton phenol acetone phosphate obtained in step 2 to 1000 mL with anhydrous ethanol.

[0050] A cottonyphenol standard solution with a concentration of 1000 μg / mL and a pH value of 5.5 was obtained.

[0051] The above standard substances were stored in a frozen state (-18°C) and protected from light.

[0052] Example 2 Preparation of cottonpol solution standard substance

[0053] This embodiment provides a method for preparing a cottonyphenol solution standard substance.

[0054] Step 1: weigh 1.0000 g of cottonypol converted to 100% by mass in terms of purity, dissolve it in 1 mL of acetone solution, add 2 mL of phosphoric acid, adjust the pH to 3.0, and prepare a cottonypol acetone phosphoric acid solution.

[0055] The purity of the cottonpol is 98.5%-100%. It can be directly purchased or prepared by itself through reaction.

[0056] Step 2: Ultrasonicate the cottonpol acetone phosphate solution and transfer it into a 1000 mL volumetric flask.

[0057] Step 3: dilute the cottonyphenol acetone phosphate obtained in step 2 to 1000 mL with anhydrous ethanol to obtain a cottonyphenol standard solution with a concentration of 1000 μg / mL and a pH value of 3.0.

[0058] The above standard substances were stored in a frozen state (-18°C) and protected from light.

[0059] Example 3 Cottonphenol solution detection method

[0060] This embodiment provides a method for detecting a cottonycin solution:

[0061] This example uses Agilent 1200 high performance liquid chromatograph for detection.

[0062] The conditions of the liquid chromatography are

[0063] Chromatographic column: C18, 250 mm × 4.6 mm × 0.5 μm;

[0064] And / or, the mobile phase is mobile phase A:mobile phase B=12:88;

[0065] Wherein, mobile phase A is 2% phosphoric acid: tetrahydrofuran = 5:1; mobile phase B is methanol;

[0066] and / or, detection wavelength: 235 nm;

[0067] and / or, flow rate: 1.0 ml / min;

[0068] And / or, injection volume: 10 μL.

[0069] Methanol and tetrahydrofuran were filtered through an organic phase filter membrane, and the phosphoric acid aqueous solution was filtered through an aqueous phase filter membrane.

[0070] Comparative Example 1 Preparation of Cotton Phenol Solution Standard Material

[0071] This comparative example provides a method for preparing a cottonyphenol solution standard substance.

[0072] Step 1: weigh 1.0000 g of cottonypol converted to 100% by mass in terms of purity, and dissolve it in 1 mL of acetone solution to prepare a cottonypol acetone solution.

[0073] The purity of the cottonpol is 98.5%-100%. It can be directly purchased or prepared by itself through reaction.

[0074] Step 2: Ultrasonicate the cottonpol acetone solution and transfer it into a 1000 mL volumetric flask.

[0075] Step 3: dilute the cotton phenol acetone obtained in step 2 to 1000 mL with anhydrous ethanol.

[0076] A standard cottonyphenol solution with a concentration of 1000 μg / mL and a pH value of 3.0 was obtained.

[0077] The above standard substances were stored in a frozen state (-18°C) and protected from light.

[0078] Comparative Example 2 Preparation of Cottonphenol Solution Standard Material

[0079] This comparative example provides a method for preparing a cottonyphenol solution standard substance.

[0080] Step 1: weigh 1.0000 g of cottonypol converted to 100% by mass in terms of purity, and dissolve it in 1 mL of acetone solution to prepare a cottonypol acetone solution.

[0081] The purity of the cottonpol is 98.5%-100%. It can be directly purchased or prepared by itself through reaction.

[0082] Step 2: Ultrasonicate the cottonpol acetone solution and transfer it into a 1000 mL volumetric flask.

[0083] Step 3: dilute the cotton phenol acetone obtained in step 2 to 1000 mL with anhydrous ethanol.

[0084] A standard cottonyphenol solution with a concentration of 1000 μg / mL and a pH value of 7.0 was obtained.

[0085] The above standard substances were stored in a frozen state (-18°C) and protected from light.

[0086] Comparative Example 3 Preparation of Cotton Phenol Solution Standard Material

[0087] This embodiment provides a method for preparing a cottonyphenol solution standard substance.

[0088] Step 1: weigh 1.0000 g of cottonpol converted to 100% by mass in terms of purity, and dissolve it in 10 mL of anhydrous ethanol solution to prepare a cottonpol ethanol solution.

[0089] The purity of the cottonpol is 98.5%-100%. It can be directly purchased or prepared by itself through reaction.

[0090] Step 2: Ultrasonicate the cottonpol ethanol solution and transfer it into a 1000 mL volumetric flask.

[0091] Step 3: dilute the cotton phenol ethanol obtained in step 2 to 1000 mL with anhydrous ethanol.

[0092] A standard cottonyphenol solution with a concentration of 1000 μg / mL and a pH value of 7.0 was obtained.

[0093] The above standard substances were stored in a frozen state (-18°C) and protected from light.

[0094] Test Example 1

[0095] The method of Example 3 was used to detect the cottonphenol content in Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3. The test results are as follows:

[0096] The HPLC spectrogram of Example 2 is as follows Figure 1 As shown, it can be seen that the separation degree of acetone and gossypol is greater than 1.5 in the spectrum with acetone added, and acetone has no interference in the detection of gossypol. The detection baseline is stable, and the peak shape of the gossypol chromatographic peak is symmetrical.

[0097] The HPLC spectrogram of Comparative Example 1 is as follows Figure 2 As shown, it can be seen that the separation degree of acetone and gossypol is greater than 1.5 in the spectrum with acetone added, and acetone has no interference with the detection of gossypol. The baseline of the detection without adding phosphoric acid is not stable, the gossypol chromatographic peak is split into two chromatographic peaks, the chromatographic peak shape is asymmetric, and the separation degree is poor. The detection result of gossypol is calculated by the peak area of ​​the chromatographic peak, and the peak shape difference of the chromatographic peak will affect the integration result of the peak area, resulting in low accuracy of the detection result.

[0098] The HPLC spectrogram of Comparative Example 2 is as follows Figure 3 As shown, it can be seen that in the spectrum without adding acetone, the detection baseline is stable and the peak shape of the gossypol chromatographic peak is symmetrical.

[0099] The HPLC spectrogram of Comparative Example 3 is as follows Figure 4 As shown, it can be seen that the baseline of the test without adding phosphoric acid and acetone is not stable, the gossypol chromatographic peak is split into two chromatographic peaks, the chromatographic peak shape is asymmetric, and the separation is poor. The detection result of gossypol is calculated by the peak area of ​​the chromatographic peak. The peak shape difference of the chromatographic peak will affect the integration result of the peak area, resulting in low accuracy of the detection result.

[0100] Therefore, the comparative products with poor peak shapes were no longer subjected to stability testing, and only comparative examples 1 and 3 with good peak shapes were compared with embodiment 2.

[0101] In summary, phosphoric acid in the gossypol standard solution not only affects the stability of gossypol, but also affects the chromatographic peak shape during the detection of the gossypol solution, which directly affects the accuracy of gossypol detection.

[0102] Test Example 2 Establishment of Standard Material Curve of Cottonpol Solution

[0103] This test example provides a method for establishing a standard substance curve of a cottonyphenol solution.

[0104] The cottonypol standard solution of Example 2 was diluted 200 times, 100 times, 50 times, 25 times, and 20 times with anhydrous ethanol to obtain cottonypol solution standard substances with nominal values ​​of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 50 μg / mL.

[0105] The cotton phenol solution standard substances were injected into the liquid chromatograph respectively, and the peak areas were recorded using the chromatographic conditions described in Example 3. A linear regression was performed with the concentration of the cotton phenol solution standard substance as the ordinate and the peak area as the abscissa to obtain a standard curve. Figure 5 .

[0106] Test Example 3 Stability of Cottonpol Solution Standard Material

[0107] The method of Example 3 was used to detect the cottonpol content of Example 1, Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3 after being placed for different times. The results are shown in Table 1.

[0108] Table 1

[0109]

[0110] Stability Test As shown in Table 1, the cottonphenol solution standard substances of Examples 1 and 2 of the present invention have very good stability. Under room temperature storage conditions, the value can be stable for 24 months.

[0111] The cotton phenol standard substance without adding phosphoric acid in Comparative Example 1 was only stable for 6 months, after which the value showed a rapid downward trend, indicating poor product stability.

[0112] The standard substance without the addition of acetone solution can only be stable for 12 months, after which the value shows a downward trend, and the product stability is poor.

[0113] Without adding phosphoric acid and acetone solution, the value of cottonyphenol solution standard substance showed a rapid downward trend, and the product stability was poor.

[0114] As can be seen from Table 1, the cottonyphenol solution standard substance prepared by the present invention has good stability; the cottonyphenol standard substance can be stored under refrigeration (-18° C.) and the value is stable for 2 years.

[0115] Test Example 4: Uniformity of Cottonpol Solution Standard Material

[0116] According to JJG 1006-1994 "Technical Specifications for Primary Standard Materials" and JJF1343-2012 "General Principles and Statistical Principles for the Determination of Standard Materials", the statistical processing of the uniformity test results of standard materials usually adopts variance analysis, which is a method of comparing the variance between groups and the variance within groups. 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 to be drawn depends on the number of units in the overall sample and the understanding of the uniformity of the sample. A total of 15 pieces were randomly drawn before, during and after the production order, and each piece was measured 3 times to observe the uniformity of the cottonphenol solution with a concentration value of 1000μg / mL in Example 1.

[0117] The statistics F of the standard substances of cotton phenol solution in ethanol were calculated to be less than the critical value Fα, and no significant difference was found between groups, indicating that the samples had good uniformity. The specific data are shown in Table 2.

[0118] Table 2 Homogeneity test of standard substances of cotton phenol solution in ethanol

[0119]

[0120] Test Example 5 Long-term stability of cottonyphenol solution standard material

[0121] Long-term stability should be evaluated regularly for the stability of the characteristic values ​​of the standard substance under the specified storage conditions over a long period of time to examine the ability of the characteristic values ​​of the standard substance to remain within the specified range. This is the stability of the standard substance. If necessary, the short-term stability of the standard substance under specific conditions should be examined by simulating the temperature and placement methods under transportation and harsh conditions. Sampling should be carried out according to the principle of dense sampling first and sparse sampling later, with 5 samples taken each time at intervals of 0, 1, 4, 8, 12, 18, and 24 months. Example 1 was tested according to the test conditions of Example 3, and the results are as follows: Figure 6 shown.

[0122] from Figure 6 It can be seen that the solution standard substances did not show an obvious upward or downward trend during the observation time, indicating that the samples were stable during the observation time.

[0123] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for preparing a cottonyphenol solution standard substance, characterized in that: include: The cotton phenol standard was dissolved in acetone to prepare a cotton phenol acetone solution, specifically: 1.0000 g of cotton phenol converted to 100% by mass in terms of purity was weighed and dissolved in 1.0 mL of acetone to prepare a cotton phenol acetone solution; Add 1.0-2.0 mL of phosphoric acid to the cottonyphenol acetone solution, adjust the pH value to 3.0-5.5, and perform ultrasonic treatment to obtain a cottonyphenol acetone phosphoric acid solution; The cottonpol acetone phosphate solution was diluted to 1000 mL with anhydrous ethanol to obtain the cottonpol solution standard substance.

2. The method for preparing a cottonpol solution standard substance according to claim 1, characterized in that: The purity of the cottonpol standard is ≥98.5%.

3. The method for preparing a cottonyphenol solution standard substance according to claim 1, characterized in that: The phosphoric acid is chromatographically pure.

4. The method for preparing a cottonyphenol solution standard substance according to claim 1, characterized in that: The acetone is chromatographically pure.

5. A cottonyphenol solution standard substance, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 4.

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