A rapid method for determining the content of active ingredients in fresh toad venom juice using HLB solid-phase extraction-UV method.

Abstract

This invention provides a method for rapidly determining the content of active ingredients in fresh toad venom extract using HLB solid-phase extraction-UV. This invention further provides the application of the above-mentioned method for rapidly determining the content of active ingredients in fresh toad venom extract using HLB solid-phase extraction-UV in the analysis of the quality of fresh toad venom extract. This invention also provides an analytical method for the quality of fresh toad venom extract. The method for rapidly determining the content of active ingredients in fresh toad venom extract using HLB solid-phase extraction-UV provided by this invention can purify and separate fresh toad venom extract using an HLB solid-phase extraction column to obtain a mixture of three components: bufotoxin, bufotoxin base, and bufotoxin ligand. The content of these components can be directly detected by ultraviolet spectrophotometer, which can rapidly characterize the quality of toad venom medicinal materials and provide a basis for the quality control of fresh toad venom.

Description

Technical Field

[0001] This invention belongs to the technical field of traditional Chinese medicine component detection, and relates to a method for rapid detection of the content of effective components in fresh toad venom juice using HLB solid-phase extraction-UV method. Specifically, it relates to a method for rapid detection of the total content of bufotoxin, bufotoxin base, and bufotoxin ligand in fresh toad venom juice using HLB solid-phase extraction-UV method. Background Technology

[0002] Toad venom is the dried secretion of the Chinese giant toad or the black-rimmed toad, both belonging to the Bufonidae family. Toads are mostly captured in summer and autumn, washed, and the white fluid from the parotid and skin glands is extracted, processed, and dried. The chemical composition of toad venom is very complex, including bufotalin, indole alkaloids, and sterols. Among these, bufotalin is a very important active ingredient in toad venom. To date, nearly 100 bufotalidide compounds have been reported in toad venom. The total content of bufotalin, bufotalin, and bufotalin lignans in toad venom is an important indicator for evaluating its quality. The 2020 edition of the Chinese Pharmacopoeia stipulates that, calculated on a dried basis, it contains bufotalin (C... 24 H 34 O4), bufotoxin (C 26 H 34 06) and bufotoxin ligand (C 24 H 32 The total amount of O4 must not be less than 7.0%.

[0003] In recent years, with the continuous rise in toad venom prices and the increasing demand from enterprises, the toad venom content has become crucial to the quality of the finished product of Musk Heart-Protecting Pills and the procurement of toad venom. The 2020 edition of the Chinese Pharmacopoeia requires the processing, drying, pulverizing, extraction, and HPLC analysis of fresh toad venom pulp to determine its content. This method is time-consuming and requires expensive analytical instruments. Currently, there is no rapid method for detecting the total content of bufotoxin, bufotoxin base, and bufotoxin ligand in fresh toad venom pulp. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for rapid detection of the content of effective components in fresh toad venom using HLB solid-phase extraction-UV method, which can rapidly and accurately detect the total content of bufotoxin, bufotoxin base, and bufotoxin ligand in fresh toad venom, and rapidly characterize the quality of toad venom medicinal materials.

[0005] To achieve the above and other related objectives, the first aspect of this invention provides a method for rapid detection of the content of active ingredients in fresh toad venom using HLB solid-phase extraction-UV method, comprising the following steps:

[0006] 1) Dissolve the fresh toad venom sample in the first solvent, then add the second solvent for ultrasonic extraction, take the supernatant, mix well, and cool to obtain the extract;

[0007] 2) The extract obtained in step 1) is loaded onto an HLB solid-phase extraction column, washed with a third solvent, eluted with a fourth solvent, and diluted to a final volume to obtain the test solution.

[0008] 3) Dissolve the reference standard of bufotoxin in the second solvent and dilute to volume to obtain the reference standard solution;

[0009] 4) The test solution obtained in step 2) and the reference solution obtained in step 3) were determined by ultraviolet spectrophotometry (UV) and quantified by standard curve method to determine the total content of bufotoxin, bufotoxin group and bufotoxin group in the test solution.

[0010] The second aspect of this invention provides the application of the above-mentioned method for rapid detection of the content of effective components in fresh toad venom juice using HLB solid-phase extraction-UV method in the analysis of the quality of fresh toad venom juice.

[0011] A third aspect of the present invention provides a method for analyzing the quality of fresh toad venom slurry, comprising the following steps:

[0012] a) The absorbance values ​​corresponding to the total content of bufotoxin, bufotoxin group and bufotoxin group in the test sample solution were obtained by using the above-mentioned HLB solid phase extraction-UV method for rapid detection of the content of effective components in fresh toad venom juice.

[0013] b) The moisture content of the fresh toad venom slurry sample was determined using a rapid moisture analyzer;

[0014] c) Substitute the absorbance value from step a) and the moisture value from step b) into formula (1) to calculate the total dry weight content of bufotoxin, bufotoxin base, and bufotoxin base in the fresh toad venom sample.

[0015] The formula (1) is: ,

[0016] In the formula, A is the absorbance value of the test solution; m is the weight of the fresh toad venom sample, in mg; and the moisture content is the moisture value measured by a rapid moisture meter.

[0017] d) Compare the dry weight total content of bufotoxin, bufotoxin, and bufotoxin ligand in the toad venom pulp sample obtained in c) with the specified dry weight total content value to determine the quality of the toad venom pulp sample.

[0018] As described above, the method for rapid determination of the content of effective components in fresh toad venom slurry using HLB solid-phase extraction-UV method provided by the present invention has the following beneficial effects:

[0019] (1) The present invention provides a method for rapid detection of effective components in fresh toad venom using HLB solid phase extraction-UV method. It can purify and separate fresh toad venom using Oasis PRiME HLB solid phase extraction column to obtain a mixture of three components: bufotoxin, bufotoxin base and bufotoxin ligand. The sample processing method is simple and fast, uses less organic solvent, and has a fast analysis speed. The sample can be directly loaded after pretreatment without activation and equilibration.

[0020] (2) The present invention provides a method for rapid detection of the content of effective components in fresh toad venom using HLB solid phase extraction-UV method. The content is directly detected by ultraviolet spectrophotometer. The detection results are rapid and accurate. The detection instrument is inexpensive and portable. The purpose of detecting the content can be achieved at toad breeding bases or fresh toad venom suppliers.

[0021] (3) The present invention provides a method for rapid detection of the content of effective components in fresh toad venom juice by HLB solid phase extraction-UV method. By combining it with rapid moisture meter determination, it can rapidly characterize the quality of toad venom medicinal materials, comprehensively improve the quality control system of fresh toad venom juice, and provide a basis for the quality control of fresh toad venom juice. Attached Figure Description

[0022] Figure 1 shows the ultraviolet absorption spectra of the reference standard and the test sample of the present invention. Figure 1a , 1b ,in, Figure 1a This is the ultraviolet absorption spectrum of the reference standard. Figure 1b This is the ultraviolet absorption spectrum of the test sample.

[0023] Figure 2 The graph shown is a linear curve of the toad venom base of the present invention. Detailed Implementation

[0024] The first aspect of this invention provides a method for rapid determination of the content of effective components in fresh toad venom slurry using HLB solid-phase extraction-UV method, comprising the following steps:

[0025] 1) Dissolve the fresh toad venom sample in the first solvent, then add the second solvent for ultrasonic extraction, take the supernatant, mix well, and cool to obtain the extract;

[0026] 2) The extract obtained in step 1) is loaded onto an HLB solid-phase extraction column, washed with a third solvent, eluted with a fourth solvent, and diluted to a final volume to obtain the test solution.

[0027] 3) Dissolve the reference standard of bufotoxin in the second solvent and dilute to volume to obtain the reference standard solution;

[0028] 4) The test solution obtained in step 2) and the reference solution obtained in step 3) were determined by ultraviolet spectrophotometry (UV) and quantified by standard curve method to determine the total content of bufotoxin, bufotoxin group and bufotoxin group in the test solution.

[0029] Preferably, in step 1), the first solvent is an inorganic solvent, including but not limited to water, and preferably water.

[0030] Preferably, in step 1), the ratio of the mass g of the toad venom fresh sample added to the volume mL of the first solvent added is 0.2:5-50, specifically such as 0.2:5-12.5, 0.2:12.5-25, 0.2:25-50, and preferably 0.2:12.5.

[0031] Preferably, in step 1), the dissolution is performed by vortexing until the toad venom paste sample is free of lumps.

[0032] Preferably, in step 1), the second solvent is an organic solvent, including but not limited to methanol, ethanol, acetone, etc., and preferably methanol.

[0033] Preferably, in step 1), the ratio of the mass g of the toad venom fresh sample added to the volume mL of the second solvent added is 0.2:5-50, specifically such as 0.2:5-12.5, 0.2:12.5-25, 0.2:25-50, and most preferably 0.2:12.5.

[0034] Preferably, in step 1), the ultrasonic extraction time is 10-50 min, specifically 10-30 min, 30-50 min, and preferably 30 min.

[0035] Preferably, in step 1), the supernatant is taken after ultrasonic extraction.

[0036] Preferably, in step 1), the cooling process involves bringing the temperature to room temperature. The room temperature is 20-30°C.

[0037] Preferably, in step 2), the HLB solid-phase extraction column is an Oasis PRiME HLB solid-phase extraction column (3cc / 150mg, Waters).

[0038] The PRi ME HLB solid-phase extraction column requires no activation or equilibration steps; it can effectively remove interfering substances such as proteins, phospholipids, and pigments after direct filtration, thus purifying the sample.

[0039] Preferably, in step 2), the amount of the extract loaded onto the HLB solid-phase extraction column is 0.5-2 mL, specifically 0.5-1.0 mL, 1.0-2 mL, and preferably 1.0 mL.

[0040] Preferably, in step 2), the third solvent is an acetonitrile aqueous solution with a volume percentage concentration of 28-30%, specifically 28-28.5%, 28.5-29%, or 29-30%, preferably 29%. The third solvent is used as a cleaning agent and is discarded after use.

[0041] Preferably, in step 2), the volume ratio of the extract to the third solvent is 0.5-2:4.5-5.5, specifically such as 0.5-1:4.5-5.0, 0.5-1:5.0-5.5, 1.0-2:4.5-5.0, 1.0-2:5.0-5.5, and most preferably 1:5.

[0042] Preferably, in step 2), the fourth solvent is an aqueous solution of acetonitrile with a volume percentage concentration of 90-98%, specifically 90-95%, 95-98%, and preferably 95%.

[0043] Preferably, in step 2), the volume ratio of the extract to the fourth solvent is 0.5-2:5.5-6.5, specifically such as 0.5-1:5.5-6.0, 0.5-1:6.0-6.5, 1.0-2:5.5-6.0, 1.0-2:6.0-6.5, and most preferably 1:6.

[0044] Preferably, in step 2), the elution rate is 5-30 s (s / mL) per milliliter of flow rate, specifically 5-15 s / mL or 15-30 s / mL.

[0045] Preferably, in step 2), the volume is adjusted to 8-20 mL, specifically 8-10 mL, 10-20 mL, and more preferably 10 mL.

[0046] Preferably, in step 3), the reference solution is prepared by first dissolving the reference standard of bufotoxin in a second solvent to prepare a reference standard stock solution, and then gradually diluting the reference standard stock solution with a fourth solvent.

[0047] Preferably, the concentration of the reference stock solution is 99-101 μg / mL, specifically 99-100 μg / mL, 100-100.1 μg / mL, or 100.1-101 μg / mL.

[0048] Preferably, in step 3), the concentration range of the reference solution is 15-66 μg / mL, and more preferably 15.015-65.065 μg / mL.

[0049] Preferably, in step 4), the detection wavelength of the ultraviolet spectrophotometer (UV) is 294-296 nm, specifically 294.5-295.5 nm, and more preferably 295 nm.

[0050] Preferably, in step 4), the standard curve method includes the following steps:

[0051] A) Prepare a series of reference solutions of different concentrations according to step 3), perform UV detection on each solution, obtain the linear relationship between the absorbance value of the reference solution and the corresponding concentration, plot the corresponding standard working curve, and calculate the regression equation of the standard working curve.

[0052] B) Perform UV detection on the test solution, and substitute the absorbance value of the test solution into the regression equation of the corresponding standard working curve in step A) to calculate the total content of bufotoxin, bufotoxin group and bufotoxin group in the test solution.

[0053] Preferably, in the standard working curve, the absorbance value is used as the vertical axis (Y-axis), and the corresponding concentration (i.e., content) is used as the horizontal axis (X-axis).

[0054] Because bufotalin, bufotoxin, and bufotalin have similar properties, they can all be detected at the same specific detection wavelength using ultraviolet spectrophotometry. In existing detection methods, such as high-performance liquid chromatography (HPLC), bufotalin is often used as a control to calculate the total content of bufotalin, bufotalin, and bufotalin. For details, please refer to the "Content Determination" section on page 401 of the Toad Venom section in the 2020 edition of the Chinese Pharmacopoeia, Volume 1, Medicinal Materials and Processed Pieces. Therefore, this application also uses bufotalin as a control, plotting a corresponding standard working curve. The absorbance values ​​of bufotalin, bufotalin, and bufotalin in the test solution detected by UV at the same specific detection wavelength are substituted into the regression equation of the standard working curve to calculate the total content of bufotalin, bufotalin, and bufotalin in the test solution.

[0055] The second aspect of this invention provides the application of the above-mentioned method for rapid detection of the content of effective components in fresh toad venom juice using HLB solid-phase extraction-UV method in the analysis of the quality of fresh toad venom juice.

[0056] A third aspect of the present invention provides a method for analyzing the quality of fresh toad venom slurry, comprising the following steps:

[0057] a) The absorbance values ​​corresponding to the total content of bufotoxin, bufotoxin group and bufotoxin group in the test sample solution were obtained by using the above-mentioned HLB solid phase extraction-UV method for rapid detection of the content of effective components in fresh toad venom juice.

[0058] b) The moisture content of the fresh toad venom slurry sample was determined using a rapid moisture analyzer;

[0059] c) Substitute the absorbance value from step a) and the moisture value from step b) into formula (1) to calculate the total dry weight content of bufotoxin, bufotoxin base, and bufotoxin base in the fresh toad venom sample.

[0060] The formula (1) is: ,

[0061] In the formula, A is the absorbance value of the test solution; m is the weight of the fresh toad venom sample, in mg; and the moisture content is the moisture value measured by a rapid moisture meter.

[0062] d) Compare the dry weight total content of bufotoxin, bufotoxin, and bufotoxin ligand in the toad venom pulp sample obtained in c) with the specified dry weight total content value to determine the quality of the toad venom pulp sample.

[0063] Preferably, in step a), the absorbance value is obtained by ultraviolet spectrophotometry (UV).

[0064] Preferably, in step b), the detection temperature of the rapid moisture analyzer is 110-130℃, more preferably 120℃.

[0065] Preferably, in step b), the sample detection amount of the rapid moisture analyzer is 0.5-2.0g, more preferably 1.0g.

[0066] Preferably, in step d), the specified total dry content value is not less than 7.0%, and more preferably 7.0%.

[0067] The specified total dry content value is based on the 2020 edition of the Chinese Pharmacopoeia, which stipulates that the total amount of bufotoxin, bufotoxin base, and bufotoxin ligand in fresh toad venom juice shall not be less than 7.0% when calculated on a dried basis. Therefore, this value is used as the specified total dry content value.

[0068] Preferably, in step d), when the total dry weight content is not less than the specified total dry weight content value, the quality of the toad venom paste meets the requirements; when the total dry weight content is less than the specified total dry weight content value, the quality of the toad venom paste does not meet the requirements.

[0069] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0070] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0071] The reagents and instruments used in the following examples are as follows:

[0072] 1. Reagents

[0073] The toad venom extract samples were produced by Hutchison Whampoa Pharmaceuticals. The specific sample batch numbers and place of origin information are shown in Table 1.

[0074] Table 1 Sample Batch Number and Origin Information

[0075] serial number batch number Place of origin 1 202205-1 Wanzhou, Chongqing 2 202206-2 Yangzhou, Jiangsu 3 202206-3 Nanyang, Henan 4 202206-4 Rizhao, Shandong 5 202206-5 Nantong, Jiangsu 6 202206-6 Xuancheng, Anhui 7 202206-7 Jining, Shandong 8 202206-8 Anqing, Anhui

[0076] Toad venom base (batch number 110803-201807, purity: 99.60%) was purchased from the China National Institutes for Food and Drug Control; methanol (analytical grade, Sinopharm Group); acetonitrile (chromatographic grade, Thermo); ultrapure water was prepared by the Milli-Q pure water system.

[0077] 2. Instruments

[0078] TU-1901plus UV-Vis spectrophotometer (Shanghai Yuropo Scientific Instruments Co., Ltd.); 204E analytical balance (Mettler Toledo, Switzerland); KQ-800DE ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); Milli-Q pure water system (Millipore, USA); 1000μL pipette (Plander, Germany); Oasis PRiME HLBCartridge solid phase extraction column (Waters, 3cc / 150mg); vortex mixer (Thermo Scientific, USA); HE83 rapid moisture analyzer (Mettler-Toledo Instruments (Shanghai) Co., Ltd.).

[0079] Example 1

[0080] Accurately weigh 200 mg of fresh toad venom extract and place it in a 50 mL centrifuge tube. Accurately add 12.5 mL of water and vortex until the extract is dissolved and free of lumps. Then add 12.5 mL of methanol and sonicate for 30 min to mix well. Cool to obtain the extract. Then, accurately measure 1 mL of the extract and load it onto an Oasis PRiME HLB (Waters, 3 cc / 150 mg) solid-phase extraction column. Wash with 5 mL of 29% acetonitrile aqueous solution and discard the washing solution. Elute with 6 mL of 95% acetonitrile aqueous solution. Transfer the eluent to a 10 mL volumetric flask and dilute to volume with 95% acetonitrile aqueous solution to obtain test solution 1#.

[0081] Example 2

[0082] Accurately weigh 200 mg of fresh toad venom extract and place it in a 50 mL centrifuge tube. Accurately add 15 mL of water and vortex until the extract is dissolved and free of lumps. Then add 15 mL of methanol and sonicate for 40 min to mix well. Cool to obtain the extract. Then, accurately measure 1 mL of the extract and load it onto an Oasis PRiME HLB (Waters, 3 cc / 150 mg) solid-phase extraction column. Wash with 5.5 mL of 28.5% acetonitrile aqueous solution and discard the washing solution. Then elute with 5.5 mL of 96% acetonitrile aqueous solution. Transfer the eluent to a 10 mL volumetric flask and dilute to volume with 96% acetonitrile aqueous solution to obtain test solution #2.

[0083] Example 3

[0084] Prepare a reference solution by dissolving bufotoxin in methanol to a concentration of 100-100.1 μg / mL. Then, dilute the reference solution with 95% acetonitrile aqueous solution to prepare the reference solution. The concentration range of the reference solution is 15-66 μg / mL.

[0085] Example 4

[0086] The test solution 1# obtained in Example 1 and the reference solution obtained in Example 3 were determined by ultraviolet spectrophotometry (UV) and quantified using the standard curve method to determine the total content of bufotoxin, bufotalin, and bufotalin in the test solution. The detection wavelength of ultraviolet spectrophotometry (UV) was 295 nm.

[0087] Specifically, a series of reference solutions of different concentrations were prepared according to Example 3. UV detection was performed on each solution to obtain the linear relationship between the absorbance value and the corresponding concentration, and a corresponding standard working curve was plotted. The regression equation of the standard working curve was calculated. Then, the test solution was subjected to UV detection. The absorbance value of the test solution was substituted into the regression equation of the corresponding standard working curve to calculate the total content of bufotoxin, bufotalin, and bufotalin in the test solution. The UV absorption spectra of the reference solution and the test solution are shown below. Figure 1a , Figure 1b .

[0088] Example 5

[0089] Accurately pipette 100.1 μg / mL of the bufotoxin reference stock solution and dilute with 95% acetonitrile aqueous solution to 15.015, 20.02, 30.03, 35.035, 40.04, 45.045, 50.05, 55.055, 60.06, and 65.065 μg / mL, respectively, to prepare a series of reference solutions. Using 95% acetonitrile aqueous solution as a blank control, measure the absorbance at a wavelength of 295 nm using a UV spectrophotometer. Figure 2 As shown, a standard curve was plotted with the concentration (X) of the reference solution on the x-axis and absorbance (Y) on the y-axis. The linear regression equation obtained was Y = 0.0125X + 0.0127, R0. 2 =0.9996, with good linearity in the linear range of 15.015 to 65.065 μg / mL.

[0090] Example 6

[0091] Accurately pipette 100.1 μg / mL of the bufotoxin reference stock solution and dilute it to 42.58 μg / mL with 95% acetonitrile aqueous solution. Measure the absorbance six times consecutively at 295 nm using a UV spectrophotometer, record the absorbance, and calculate the RSD%. The RSD% result is 0.05, indicating good instrument precision.

[0092] Example 7

[0093] Six 100 mg samples of fresh toad venom extract (batch number 202206-4) were accurately weighed and added to 12.5 mL of ultrapure water. The mixture was vortexed until a uniform milky white color was obtained. Then, 12.5 mL of a reference solution containing 35.02 μg / mL bufotoxin was added to each sample, and the mixture was sonicated for 30 min and cooled to obtain the extract. The test solution was then prepared according to the pretreatment method in Example 1, and the absorbance was measured at a wavelength of 295 nm according to Example 4. The specific results are shown in Table 2. Table 2 shows that the recoveries ranged from 96.34% to 104.76% over six tests, with an RSD of 3.93, indicating that the method is relatively accurate.

[0094] Table 2

[0095]

[0096] Example 8

[0097] Six samples of fresh toad venom slurry (batch number 202206-4) were taken and prepared into test solutions according to the pretreatment method in Example 1. The contents were detected at a wavelength of 295 nm and were 4.32%, 4.41%, 4.32%, 4.23%, 4.34%, and 4.31%, respectively, with an RSD% of 1.39, indicating that the method has high repeatability.

[0098] Example 9

[0099] Fresh toad venom extract (batch number 202206-4) was extracted using different methods, and the resulting test solutions were analyzed using the method (HPLC method) for the determination of toad venom content in the 2020 edition of the Chinese Pharmacopoeia.

[0100] (1) Vortexing: Take 200mg of fresh toad venom juice (batch number 202206-4), weigh it accurately, put it in a 50mL centrifuge tube, add 12.5mL of ultrapure water accurately, vortex until the fresh juice is dissolved and there are no lumps, add 12.5mL of methanol, vortex for 30min, mix well, cool, and filter through a 0.45μm microporous membrane.

[0101] (2) Ultrasound: Take 200mg of fresh toad venom juice (batch number 202206-4), accurately weigh it, put it in a 50mL centrifuge tube, add 25mL of 50% methanol, sonicate for 30 minutes, mix well, cool, and filter through a 0.45μm microporous membrane.

[0102] (3) Prepared using the method described in Example 1. Specifically, vortexing + sonication: Take 200 mg of fresh toad venom (batch number 202206-4), accurately weigh it, place it in a 50 mL centrifuge tube, accurately add 12.5 mL of ultrapure water, vortex until the fresh venom dissolves and there are no lumps, add 12.5 mL of methanol, sonicate for 30 minutes, mix well, cool, and filter through a 0.45 μm microporous membrane.

[0103] The results of the three extraction methods are shown in Table 3. As can be seen from Table 3, the vortex + ultrasound extraction method used in this application has the best extraction efficiency for the three components in toad venom juice.

[0104] Table 3 Results of the Extraction Method Examination

[0105] Extraction method Bufotoxin + Bufotoxin Base + Bufotoxin Acid Base % Deviation between parallel samples % vortex 4.59 1.76 ultrasound 3.56 1.34 Vortex + Ultrasound 4.80 1.70

[0106] Example 10

[0107] Fresh toad venom samples from different origins were collected, with two replicates per batch. Test solutions were prepared according to Example 1, and absorbance values ​​were measured according to Example 4. The total content of bufotoxin, bufotoxin base, and bufotoxin ligand in the test solutions was calculated. Simultaneously, HPLC was used for detection, and the results are shown in Table 4.

[0108] Table 4 Content of Fresh Toad Venom Extract

[0109]

[0110]

[0111] As shown in Table 4, for toad venom extract samples within the linear range (absorbance greater than 0.2), the content detected by the UV method and the HPLC method showed small deviations (deviation < 4%), indicating accurate results. However, the toad venom extract samples with lower content (batch numbers 202205-1, 202206-3, and 202206-8) had lower absorbances, exceeding the accurate quantitative range of this method. Their detected values ​​were higher than those detected by the HPLC method, showing larger deviations (deviation > 5%).

[0112] Example 11

[0113] Fresh toad venom sample (batch number 202206-5) was taken, and a test solution was prepared according to Example 1. A control solution was prepared according to Example 3. The total content of bufotoxin, bufotoxin base, and bufotoxin ligand in the test solution was determined by UV detection according to Example 4.

[0114] Then, 1g of fresh toad venom sample (batch number 202206-5) was taken and the moisture content was measured at 120℃ using a HE83 rapid moisture analyzer in standard drying mode B. The measurement was stopped after the displayed value stabilized, and the value was read. The total dry weight content of bufotoxin, bufotoxin base, and bufotoxin ligand in the fresh toad venom sample was calculated according to formula (1). The test results showed that the fresh toad venom sample contained a total of 15.89% bufotoxin, bufotoxin base, and bufotoxin ligand on a dry weight basis.

[0115] Meanwhile, samples of fresh toad venom pulp from this batch were processed and dried using traditional methods to produce toad venom. The content was then tested according to the content determination items in the 2020 edition of the Chinese Pharmacopoeia. After deducting moisture, the dry content was: the toad venom material, calculated on a dried basis, contained a total of 15.36% bufotoxin, bufotoxin base, and bufotoxin ligand.

[0116] The content measured by the two methods showed a small deviation, with an RD of 1.70%. Specific data are shown in Table 5. Table 5 demonstrates that the rapid detection method for toad venom extract samples is accurate and reliable, and can quickly characterize the quality of toad venom extract.

[0117] Table 5 Comparison of UV detection results and pharmacopoeia methods

[0118]

[0119] Example 12

[0120] The total dry matter content of bufotalin, bufotoxin, and bufotalin in the test solution obtained in Example 11 was compared with the specified total dry matter content value, which was 7.0%. This is in accordance with the 2020 edition of the Chinese Pharmacopoeia, which stipulates that the total content of bufotalin, bufotoxin, and bufotalin, calculated on a dried basis, shall not be less than 7.0%.

[0121] When the total dry content is limited to 7.0%, the absorbance values ​​corresponding to fresh toad venom pulp with different moisture contents are different. This shows that the quality of fresh toad venom pulp depends not only on absorbance but also on moisture content. For specific calculations, the absorbance value at the 7.0% content limit can be used: A = 0.0127 + 0.0035 × m × (1 - moisture).

[0122] Therefore, the pass / fail status of toad venom extract samples is related not only to absorbance but also to moisture content. The relationship between the pass / fail threshold, moisture content, and absorbance is shown in Table 6. For example, if the moisture content of the toad venom extract is 70% and the absorbance is below 0.22, the sample does not meet the pharmacopoeia requirements. In this method, if the absorbance is at the lower limit of the quantifiable range (0.2), and the moisture content is above 73%, the batch of toad venom extract does not meet the pharmacopoeia requirements. This method can quickly determine the pass / fail status of toad venom extract, providing a fast and convenient method and basis for the procurement of medicinal materials.

[0123] Table 6. Absorbance limits for different moisture contents of fresh toad venom pulp samples (m = 200 mg)

[0124] Moisture% 7.0% content limit absorbance 40 0.4327 50 0.3627 60 0.2927 70 0.2227 73 0.2000 80 0.1527

[0125] In summary, this invention provides a rapid method for detecting the content of active ingredients in fresh toad venom using HLB solid-phase extraction-UV method. This method purifies and separates the fresh toad venom using an HLB solid-phase extraction column, obtaining a mixture of three components: bufotoxin, bufotoxin base, and bufotoxin ligand. The content of these components can be directly detected using a UV spectrophotometer, enabling rapid characterization of the quality of toad venom and providing a basis for quality control of fresh toad venom. Therefore, this invention overcomes the various shortcomings of existing technologies and has high industrial application value.

[0126] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for rapid detection of the content of active ingredients in fresh venin by HLB solid phase extraction-UV method, comprising the following steps: 1) dispersing the fresh venin sample with a first solvent, then adding a second solvent to ultrasonically extract, taking the supernatant, mixing, and cooling to obtain an extract; 2) loading the extract obtained in step 1) into a HLB solid phase extraction column, then washing with a third solvent, eluting with a fourth solvent, and constant volume to obtain a test solution; 3) dissolving the control sample of cinobufagin in the second solvent and constant volume to obtain a control solution; 4) determining the test solution and the control solution by UV spectrophotometry, and quantifying by standard curve method to determine the total content of bufadienolides, bufalin and cinobufagin in the test solution; In step 1), the first solvent is water; the second solvent is selected from one of methanol, ethanol or acetone; In step 2), the third solvent is a 28-30% (v / v) acetonitrile aqueous solution; the fourth solvent is a 90-98% (v / v) acetonitrile aqueous solution. 2.The method for rapid detection of the content of effective components in the fresh extract of toad venom according to claim 1, characterized in that, In step 1), any one or more of the following conditions are included: A1) the ratio of the mass of the fresh venin sample to the volume of the first solvent is 0.2:5-50, g / mL; A2) the dispersing is performed by vortexing until the fresh venin sample is free of lumps; A3) the second solvent is methanol; A4) the ratio of the mass of the fresh venin sample to the volume of the second solvent is 0.2:5-50, g / mL; A5) the ultrasonic extraction time is 10-50 min.

3. The method for rapid detection of the content of effective components in fresh cantharides extract according to claim 1, characterized in that, In step 2), any one or more of the following conditions are included: B1) the HLB solid phase extraction column is an Oasis PRiME HLB solid phase extraction cartridge; B2) the loading amount of the extract on the HLB solid phase extraction column is 0.5-2 mL; B3) the ratio of the volume of the extract to the volume of the third solvent is 0.5-2:4.5-5.5; B4) the ratio of the volume of the extract to the volume of the fourth solvent is 0.5-2:5.5-6.5; B5) the elution speed is 5-30 s per mL flow rate.

4. The method for rapid detection of the content of effective components in fresh cantharides extract according to claim 1, characterized in that, In step 3), the concentration of the control solution is 15-66 μg / mL.

5. The method for rapid detection of the content of effective components in fresh cantharides extract according to claim 1, characterized in that, In step 4), the detection wavelength of the UV spectrophotometry is 294-296 nm.

6. Use of the method for rapid detection of the content of active ingredients in fresh venin by HLB solid phase extraction-UV method according to any one of claims 1-5 in analyzing the quality of fresh venin.

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