Multi-component content determination method for four-ingredient Tibet cat milk soup

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a multi-component content determination method of four-ingredient Tibetan cat milk soup. According to the method disclosed by the invention, the four-ingredient Tibet cat milk soup is determined by using specific high performance liquid chromatography conditions, so that complete separation of 13 components including gallic acid, loganic acid, gentiopicroside, magnoflorine, chebulagic acid, chebulagic acid, jateorhizine, berberine hydrochloride, quercetin, naringenin, apigenin, kaempferol and rhamnocitrin is realized; the quality condition of the four-ingredient Tibetan cat milk soup can be rapidly, simply, accurately and reliably known, the purpose of controlling the quality of the four-ingredient Tibetan cat milk soup by measuring the content of the 13 chemical components is achieved, and reference is provided for development and quality standard formulation of the four-ingredient Tibetan cat milk soup of the classic famous formula of Tibetan medicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug detection, and particularly relates to a method for determining the contents of multiple components in a Tibetan cat's milk decoction of four herbs. Background Art

[0002] Tibetan cat's milk belongs to the genus Rhamnella (green firewood), and is a shrub of the Rhamnaceae family; the Tibetan cat's milk decoction of four herbs is derived from the Tibetan medical classic "Four Medical Tantras", and is a basic prescription for Tibetan medicine to treat "yellow water disease". It is composed of Tibetan cat's milk, berberis bark, gentian root, and terminalia chebula, and has the effects of drying up hot and humid yellow water, clearing heat and detoxifying, and can be used for the treatment of "zhenbu" disease, especially RA (rheumatoid arthritis).

[0003] The prior patent with the application number 2022105268764 discloses a Tibetan medicine formula, preparation method and application with the effect of treating rheumatoid arthritis, and discloses the Tibetan cat's milk decoction of four herbs composed of Tibetan cat's milk, berberis bark, gentian root, and terminalia chebula. However, it does not disclose how to control the quality of the Tibetan cat's milk decoction of four herbs, and it is impossible to ensure the effectiveness of clinical medication.

[0004] Therefore, how to establish a method for determining the components of the Tibetan cat's milk decoction of four herbs, ensure the effectiveness of clinical medication, and provide a standard for strictly controlling the quality of drugs has become an urgent problem to be solved in this field. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a method for determining the contents of multiple components in a Tibetan cat's milk decoction of four herbs. By using specific liquid chromatography conditions, the quality of the Tibetan cat's milk decoction of four herbs can be quickly and accurately known.

[0006] The technical solution of the present invention is as follows:

[0007] A method for determining the contents of multiple components in a Tibetan cat's milk decoction of four herbs, the content determination method comprising the following steps:

[0008] S1, preparation of a test solution;

[0009] S2, preparation of a mixed reference solution using gallic acid, loganic acid, gentiopicroside, magnoflorine, chebulinic acid, jatrorrhizine, chebulic acid, berberine hydrochloride, quercetin, naringenin, apigenin, kaempferol, and rhamnocitrin as reference substances;

[0010] S3, respectively determining the chromatograms of the test solution and the mixed reference solution by high performance liquid chromatography;

[0011] The chromatographic conditions of the high performance liquid chromatography are as follows:

[0012] Chromatographic column: packed with octadecylsilane chemically bonded silica gel;

[0013] Mobile phase: acetonitrile (B) and aqueous solution containing phosphoric acid (A);

[0014] The elution method is gradient elution, and the gradient elution program is shown in Table 1;

[0015] Table 1 Gradient elution program

[0016]

[0017] S4. Calculate the content of each component in the Tibetan cat's milk soup of four flavors.

[0018] Preferably, in S1, the preparation method of the test solution is: mix the Tibetan cat's milk soup of four flavors with an alcohol solvent, mix well, centrifuge or filter, and take the supernatant to obtain it; the alcohol solvent is a methanol aqueous solution with a volume percentage of 75%; the volume ratio of the Tibetan cat's milk soup of four flavors to the alcohol solvent is 1:9.

[0019] Preferably, in S2, in the mixed reference solution, the concentration of gallic acid is 0.00480 mg / mL to 0.07692 mg / mL; the concentration of loganic acid is

[0020] 0.00480 mg / mL to 0.07692 mg / mL; the concentration of gentiopicroside is

[0021] 0.00394 mg / mL to 0.06308 mg / mL; the concentration of magnoflorine is

[0022] 0.00236 mg / mL to 0.03785 mg / mL; the concentration of chebulic acid is

[0023] 0.00238 mg / mL to 0.03815 mg / mL; the concentration of chebulinic acid is

[0024] 0.00480 mg / mL to 0.07692 mg / mL; the concentration of jatrorrhizine is

[0025] 0.00241 mg / mL to 0.03854 mg / mL; the concentration of berberine hydrochloride is

[0026] 0.00480 mg / mL to 0.07692 mg / mL; the concentration of quercetin is

[0027] 0.00480 mg / mL to 0.07692 mg / mL; the concentration of naringenin is

[0028] 0.00480 mg / mL to 0.07692 mg / mL; the concentration of apigenin is

[0029] 0.00480 mg / mL to 0.07692 mg / mL; the concentration of kaempferol is

[0030] 0.00480 mg / mL to 0.07692 mg / mL; the concentration of rhamnocitrin is 0.00230 mg / mL to 0.03692 mg / mL.

[0031] Preferably, the standard curves of gallic acid, loganin acid, gentiopicroside, magnoflorine, chebulinic acid, jatrorrhizine, chebulic acid, berberine, quercetin, naringenin, apigenin, kaempferol, and rhamnocitrin are as follows:

[0032] Y = 5.0608X + 5.5723, R 2 = 0.9997;

[0033] Y = 6.7316X + 7.8526, R 2 = 0.9991;

[0034] Y = 6.1605X + 0.8729, R 2 = 0.9998;

[0035] Y = 4.6313X + 4.7529, R 2 = 0.9994;

[0036] Y = 6.4391X + 2.3765, R 2 = 0.9994;

[0037] Y = 5.6931X + 5.3979, R 2 = 0.9996;

[0038] Y = 2.5262X + 0.3208, R 2 = 0.9995;

[0039] Y = 15.3152X + 19.0663, R 2 = 0.9991;

[0040] Y = 14.1467X + 1.8294, R 2 = 0.9992;

[0041] Y = 2.9034X + 1.4126, R 2 = 0.9993;

[0042] Y = 10.0042X + 19.0123, R 2 = 0.9998;

[0043] Y = 16.2083X + 21.4323, R 2 = 0.9998;

[0044] Y = 30.6154X + 32.7763, R 2 = 0.9999.

[0045] Preferably, in S3, the chromatographic conditions further include:

[0046] Detection wavelength: 274 nm;

[0047] Flow rate: 1.0 mL / min;

[0048] Column temperature: 25 °C;

[0049] Injection volume: 5 μL.

[0050] Preferably, in S3, the volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.10%.

[0051] Preferably, in S4, according to the concentrations of the chemical components in the mixed reference solution in step S2, the peak areas of the mixed reference solution in the chromatogram, and the peak areas of the corresponding components in the four-flavor Schisandra tamnoides soup in the chromatogram, the contents of each component in the four-flavor Schisandra tamnoides soup are calculated by the external standard method.

[0052] The beneficial effects of the present invention are as follows:

[0053] (1) The present invention uses specific high-performance liquid chromatography conditions to determine the four-flavor Schisandra tamnoides soup, achieving complete separation of 13 components including gallic acid, loganic acid, gentiopicroside, magnoflorine, chebulagic acid, chebulinic acid, jatrorrhizine, berberine hydrochloride, quercetin, naringenin, apigenin, kaempferol, and rhamnocitrin, with the advantages of high resolution, strong specificity, and good peak shape;

[0054] (2) The method provided by the present invention has high precision, accuracy, and stability, can quickly, simply, accurately, and reliably obtain the quality of the four-flavor Schisandra tamnoides soup, realizes the purpose of controlling the quality of the four-flavor Schisandra tamnoides soup by measuring the contents of 13 chemical components, provides a reference for the development and quality standard formulation of the classic Tibetan medicine prescription four-flavor Schisandra tamnoides soup, and has guiding significance for the quality detection and evaluation of the four-flavor Schisandra tamnoides soup. Description of the Drawings

[0055] Figure 1 Is the HPLC chromatogram of Example 1;

[0056] Figure 2 Is the HPLC chromatogram of the mixed reference solution;

[0057] Figure 3 Is the HPLC chromatogram of the test solution of the four-flavor Schisandra tamnoides soup;

[0058] Figure 4It is the HPLC chromatogram of the negative sample solution of the Four-Ingredient Gouania wardiana Soup (lacking Gouania wardiana);

[0059] Figure 5 It is the HPLC chromatogram of the negative sample solution of the Four-Ingredient Gouania wardiana Soup (lacking Terminalia chebula);

[0060] Figure 6 It is the HPLC chromatogram of the negative sample solution of the Four-Ingredient Gouania wardiana Soup (lacking Berberis dasystachya);

[0061] Figure 7 It is the HPLC chromatogram of the negative sample solution of the Four-Ingredient Gouania wardiana Soup (lacking Gentiana macrophylla);

[0062] Figure 8 It is the HPLC chromatogram of Gouania wardiana;

[0063] Figure 9 It is the HPLC chromatogram of Terminalia chebula;

[0064] Figure 10 It is the HPLC chromatogram of Berberis dasystachya;

[0065] Figure 11 It is the HPLC chromatogram of Gentiana macrophylla;

[0066] Figure 12 It is the HPLC chromatogram of Comparative Example 1;

[0067] Figure 13 It is the HPLC chromatogram of Comparative Example 2;

[0068] Figure 14 It is the HPLC chromatogram of Comparative Example 3;

[0069] Figure 15 It is the HPLC chromatogram of Comparative Example 4;

[0070] Figure 16 It is the HPLC chromatogram of Comparative Example 5;

[0071] Figure 17 It is the HPLC chromatogram of Comparative Example 6 at a column temperature of -30°C;

[0072] Figure 18 It is the HPLC chromatogram of Comparative Example 6 at a column temperature of -35°C;

[0073] Figure 19 It is the HPLC chromatogram of Comparative Example 7 at a flow rate of 0.8 mL / min;

[0074] Figure 20 It is the HPLC chromatogram of Comparative Example 7 at a flow rate of 1.2 mL / min;

[0075] Among them, 1. gallic acid; 2. loganic acid; 3. gentiopicroside; 4. magnoflorine; 5. chebulagic acid; 6. chebulinic acid; 7. jatrorrhizine; 8. berberine; 9. quercetin; 10. naringenin; 11. apigenin; 12. kaempferol; 13. rhamnocitrin. Detailed implementation manners

[0076] In order to enable those skilled in the art to better understand the present invention, the present invention will be further elaborated below in conjunction with the specific implementation manners.

[0077] The preparation method of the four-flavor Tibet cat milk soup involved in the embodiment of the present invention is as follows:

[0078] Weigh the medicinal materials according to the mass ratio of Tibet cat milk: gentian: berberis bark: chebula fruit = 12:10:6.5:3, mix and crush them, and pass through a 20-mesh sieve to obtain powder raw materials;

[0079] Add water to the powder raw materials according to the weight-to-volume ratio of 1:20, reflux and extract twice, with the extraction time of 1.5 hours each time, filter, and combine the filtrates to obtain the four-flavor Tibet cat milk soup.

[0080] Example 1

[0081] A method for determining the contents of multiple components in a four-flavor Tibet cat milk soup, the content determination method includes the following steps:

[0082] S1, preparation of the test solution:

[0083] Precisely measure 1 mL of the four-flavor Tibet cat milk soup, place it in a 10 mL volumetric flask, and make up the volume to the scale with 75% methanol (the volume ratio of the four-flavor Tibet cat milk soup to the alcohol solvent is 1:9), mix well, centrifuge at 12000 rpm for 10 min, and take the supernatant to obtain the test solution;

[0084] S2. Weigh appropriate amounts of 13 reference substances. Place the reference substances of gallic acid, loganic acid, gentiopicroside, magnoflorine, chebulinic acid, jatrorrhizine, chebulin, berberine, quercetin, naringenin, apigenin, kaempferol, and rhamnocitrin in a 25-mL volumetric flask. Dissolve them with methanol and make up to the mark to obtain a mixed reference substance solution. Ensure that the mass concentrations of gallic acid, loganic acid, gentiopicroside, magnoflorine, chebulinic acid, jatrorrhizine, chebulin, berberine, quercetin, naringenin, apigenin, kaempferol, and rhamnocitrin in the mixed reference substance solution are 76.92 μg / mL, 76.92 μg / mL, 63.08 μg / mL, 37.85 μg / mL, 76.92 μg / mL, 38.54 μg / mL, 38.15 μg / mL, 76.92 μg / mL, 76.92 μg / mL, 76.92 μg / mL, 76.92 μg / mL, 76.92 μg / mL, and 36.92 μg / mL, respectively.

[0085] S3. Use high-performance liquid chromatography to separately determine the chromatograms of the test solution and the reference solution.

[0086] The chromatographic conditions of the high-performance liquid chromatography are as follows:

[0087] Chromatographic column: Thermo Hypersil GOLD TM C18 chromatographic column (4.6×250 mm, 5 μm);

[0088] Mobile phase: acetonitrile (B) and phosphoric acid aqueous solution with a volume percentage of 0.10% (A);

[0089] Detection wavelength: 274 nm;

[0090] Flow rate: 1.0 mL / min;

[0091] Column temperature: 25 °C;

[0092] Injection volume: 5 μL;

[0093] The elution method is gradient elution, and the gradient elution program is shown in Table 1 in the detailed description of the invention;

[0094] The detector is a variable wavelength ultraviolet / visible detector.

[0095] The detection results are as Figure 1 shown. It can be seen from Figure 1 that by using the chromatographic conditions of this example, the determined components can be better detected in a single chromatogram, interference can be removed, which is conducive to the accurate calculation of the content of each component.

[0096] Preparation of each negative and single-herb sample solution in Example 2

[0097] According to the preparation method of the Four-Ingredient Schizophragma integrifolium (Franch.) Oliv. Decoction, negative samples without Schizophragma integrifolium (Franch.) Oliv., Gentiana macrophylla Pall., Berberis dasystachya Maxim., and Terminalia chebula Retz. and single-herb samples of Schizophragma integrifolium (Franch.) Oliv., Gentiana macrophylla Pall., Berberis dasystachya Maxim., and Terminalia chebula Retz. were prepared respectively.

[0098] Taking the preparation of the negative sample without Schizophragma integrifolium (Franch.) Oliv. as an example: Weigh Gentiana macrophylla Pall., Berberis dasystachya Maxim., and Terminalia chebula Retz. respectively according to the formula ratio of the Four-Ingredient Schizophragma integrifolium (Franch.) Oliv. Decoction, and prepare the negative sample without Schizophragma integrifolium (Franch.) Oliv. according to the preparation method of the Four-Ingredient Schizophragma integrifolium (Franch.) Oliv. Decoction.

[0099] The method for preparing the solution of the negative sample without Schizophragma integrifolium (Franch.) Oliv. is as follows:

[0100] Precisely measure 1 mL of the extract of the negative sample without Schizophragma integrifolium (Franch.) Oliv., place it in a 10 mL volumetric flask, dilute it to the mark with 75% methanol, mix well, centrifuge at 12000 rpm for 10 min, and take the supernatant to obtain the solution of the negative sample without Schizophragma integrifolium (Franch.) Oliv.

[0101] Prepare the solution of the negative samples without Gentiana macrophylla Pall., Berberis dasystachya Maxim., and Terminalia chebula Retz. and the solution of the single-herb samples of Schizophragma integrifolium (Franch.) Oliv., Gentiana macrophylla Pall., Berberis dasystachya Maxim., and Terminalia chebula Retz. in the same way.

[0102] Example 3

[0103] Specificity investigation

[0104] Precisely pipette 5 μL of the test solution and the mixed reference solution in Example 1 and each negative sample solution in Example 2, inject them into the liquid chromatograph, and perform the determination respectively according to the chromatographic conditions in Example 1. Compare and investigate the chromatograms of the above solutions. The results are as Figures 2 - 11 shown.

[0105] As Figure 2 - 11 shown, there are the same chromatographic peaks at the corresponding positions of the chromatograms of the test solution and the reference solution of the Four-Ingredient Schizophragma integrifolium (Franch.) Oliv. Decoction. The chromatographic peaks are well separated, and the resolution from the adjacent peaks is greater than 1.5. The attribution of the chromatographic peaks of the single herbs is accurate, and there is no interference from the negative samples. It shows that this chromatographic method has good specificity.

[0106] Example 2

[0107] Linearity relationship investigation

[0108] Pipette 5 μL of the mixed reference solution in Example 1, inject it into the liquid chromatograph, and perform the determination according to the chromatographic conditions in Example 1.

[0109] Using the concentration X (μg / mL) of the reference solution as the abscissa and the peak areas Y of the respective reference compounds as the ordinate, a standard curve was plotted; the regression equations, correlation coefficients, and linear ranges for the 13 reference standards were calculated. Using the peak areas (Y) of gallic acid, loganin acid, gentiopicroside, magnoflorine, chebulinic acid, chebulin, jatrorrhizine, berberine, quercetin, naringenin, apigenin, kaempferol, and rhamnocitrin as the ordinate and the mass concentration (X) as the abscissa, a standard curve was plotted; the detection limit was the compound concentration at a signal-to-noise ratio S / N = 3, and the quantification limit was the compound concentration at a signal-to-noise ratio S / N = 10. The linear regression equations, detection limits, and quantification limits are shown in Table 2.

[0110] Table 2 Linear regression equations, detection limits, and quantification limits of 13 reference standards

[0111]

[0112] As can be seen from Table 2, the 13 reference standards in the four Tibetan Rhamnella franguloides samples had good linear relationships within the corresponding concentration ranges, and their correlation coefficients R 2 were all greater than 0.999.

[0113] Example 3

[0114] Precision test

[0115] Taking the mixed reference solution in Example 1, injecting samples continuously 6 times under the chromatographic conditions of Example 1 for detection, recording the peak areas of each component and calculating their RSD values. The results are shown in Table 3.

[0116] Table 3 Results of precision test (n = 6)

[0117]

[0118]

[0119] As can be seen from Table 3, the RSD values of the peak areas of each reference standard were all less than 1.82%, indicating that the precision of this method was good.

[0120] Example 4

[0121] Stability test

[0122] Taking the test solution in Example 1, placing it at room temperature, injecting samples for determination at 0, 2, 4, 6, 8, and 10 h under the chromatographic conditions of Example 1, recording the peak areas of each component and calculating their RSD values. The results are shown in Table 4.

[0123] Table 4 Results of stability test (n = 6)

[0124]

[0125]

[0126] As can be seen from Table 4, the RSD values of the stability tests for each active ingredient are all less than 1.98%, indicating that under room temperature conditions, the test solution of the Four-Ingredient Tibet Cat's Milk Decoction has good stability within 10 h.

[0127] Example 5

[0128] Repeatability test

[0129] Six test solutions of the Four-Ingredient Tibet Cat's Milk Decoction in Example 1 were prepared in parallel, and were determined according to the chromatographic conditions in Example 1. The peak areas of the chromatographic peaks of each compound were measured, and the contents and RSD values of each compound were calculated. The results are shown in Table 5.

[0130] Table 5 Results of repeatability test (n = 6)

[0131]

[0132]

[0133] As can be seen from Table 5, the average percentage contents of gallic acid, loganic acid, gentiopicroside, magnoflorine, chebulic acid, chebulinic acid, jatrorrhizine, berberine, quercetin, naringenin, kaempferol, apigenin, and rhamnocitrin are 0.13%, 0.45%, 1.10%, 0.73%, 0.61%, 0.46%, 0.36%, 0.44%, 0.22%, 0.43%, 0.27%, 0.24%, and 0.15% respectively, and the RSD% are all less than 1.62%, indicating that the method has good repeatability.

[0134] Example 6

[0135] Spiked recovery test

[0136] Six samples of the Four-Ingredient Tibet Cat's Milk Decoction were prepared. Appropriate masses of each reference single product were added to the six samples at 80%, 100%, and 120% of the known contents of each chemical component (gallic acid, loganic acid, gentiopicroside, magnoflorine, chebulic acid, chebulinic acid, jatrorrhizine, berberine, quercetin, naringenin, kaempferol, apigenin, and rhamnocitrin) in the sample solution (the known contents in Example 5). The six test solutions were prepared according to the preparation method of the test solution in Example 1. The test was carried out according to the chromatographic conditions in Example 1. The peak areas of each component were recorded, and the spiked recoveries and RSD values were calculated. The results are shown in Table 6.

[0137] Table 6 Results of spiked recovery test (n = 6)

[0138]

[0139]

[0140]

[0141]

[0142] As can be seen from Table 6, the average spike recovery rates of 13 control compounds in 6 samples of the Four-Ingredient Tibet Raspberry-like Buckthorn Decoction were between 98.54% and 100.68%, and the RSD values were all less than 2%, indicating that the spike recovery rate of this content determination method was good and the method was accurate and feasible.

[0143] Example 7

[0144] Prepare 20 batches of the Four-Ingredient Tibet Raspberry-like Buckthorn Decoction, and prepare the corresponding 20 batches of test solutions according to the method of Example 1. Determine the contents of the active ingredients in each batch according to the method of Example 1. The results are shown in Table 7.

[0145] Table 7 Determination results of the contents of the active ingredients in 20 batches of the Four-Ingredient Tibet Raspberry-like Buckthorn Decoction (%)

[0146]

[0147] As can be seen from Table 7, the gallic acid content in 20 samples of the Four-Ingredient Tibet Raspberry-like Buckthorn Decoction was

[0148] 0.132 - 0.294%, the loganic acid content was 0.415 - 0.574%, the gentiopicroside content was

[0149] 1.095 - 1.359%, the magnoflorine content was 0.686 - 0.805%, the chebulagic acid content was

[0150] 0.575 - 0.788%, the chebulinic acid content was 0.394 - 0.489%, the jatrorrhizine content was

[0151] 0.399 - 0.489%, the berberine content was 0.376 - 0.599%, the quercetin content was

[0152] 0.253 - 0.311%, the naringenin content was 0.332 - 0.423%, the kaempferol content was

[0153] 0.298 - 0.388%, the apigenin content was 0.173 - 0.356%, the rhamnocitrin content was 0.128 - 0.267%. The method of the present invention can determine the component contents in the Four-Ingredient Tibet Raspberry-like Buckthorn Decoction.

[0154] Comparative Example 1

[0155] The difference from Example 1 was that the detection wavelength was 245 nm, and other steps were the same as those in Example 1. The results were as Figure 12 shown.

[0156] As can be seen from Figure 12 the figure, when detecting at a wavelength of 245 nm, within 0 - 40 min, the resolution of each detection peak is poor, and it is impossible to achieve good separation of 13 components.

[0157] Comparative Example 2

[0158] The difference from Example 1 is that the detection wavelength is 360 nm, and other steps are the same as those in Example 1. The results are as Figure 13 shown.

[0159] As can be seen from Figure 13 the figure, when detecting at a wavelength of 360 nm, the absorption of some components (such as gallic acid, loganic acid, gentiopicroside, etc.) is small, making it difficult to detect, and it is impossible to complete the accurate detection of 13 components.

[0160] Comparative Example 3

[0161] The difference from Example 1 is that an Elite Hypersil BDS C18 (4.6×250 mm, 5 μm) chromatographic column is used, and other steps are the same as those in Example 1. The results are as Figure 14 shown.

[0162] As can be seen from Figure 14 the figure, when using an Elite Hypersil BDS C18 (4.6×250 mm, 5 μm) chromatographic column for separation and detection, for some components, such as gallic acid could not be separated and was difficult to detect within 0 - 5 min, and gentiopicroside and magnoflorine could not be separated and were difficult to detect within 10 - 20 min. Therefore, this type of chromatographic column is not suitable for the separation and detection of the samples of the present invention.

[0163] Comparative Example 4

[0164] The difference from Example 1 is that an Agilent SB-C18 (4.6×250 mm, 5 μm) chromatographic column is used, and other steps are the same as those in Example 1. The results are as Figure 15 shown.

[0165] As can be seen from Figure 15 the figure, when using an Agilent SB-C18 (4.6×250 mm, 5 μm) chromatographic column for detection and separation, the results show that gentiopicroside and magnoflorine could not be separated and were difficult to detect within 10 - 20 min. Therefore, this type of chromatographic column is not suitable for the separation and detection of the samples of the present invention.

[0166] Comparative Example 5

[0167] The difference from Example 1 is that the mobile phase is methanol - 0.1% phosphoric acid aqueous solution, and other steps are the same as those in Example 1. The results are as Figure 16as shown

[0168] It can be seen from Figure 16 that in the methanol - 0.1% phosphoric acid aqueous solution system, the peak emergence is slow, the compound elution is slow, the resolution of each peak is poor, and the samples of the present invention cannot be effectively detected.

[0169] Comparative Example 6

[0170] The difference from Example 1 is that the column temperature was investigated at 30°C and 35°C respectively, and other steps were the same as those in Example 1. The results are as shown in Figure 17 and Figure 18 as shown

[0171] Figure 17 The results show that in the time period of 0 - 20 minutes, the peaks piled up and were not effectively separated. Therefore, a column temperature of 30°C is not suitable for the high - performance liquid chromatography detection of the samples of the present invention.

[0172] Figure 18 It can be seen that in the time period of 0 - 20 minutes, it is difficult to separate the component peaks. In the time period of 55 - 60 minutes, the separation of berberine is not ideal. In the time period of 75 - 85 minutes, the three components of naringenin, apigenin and kaempferol cannot be effectively separated. Therefore, a column temperature of 35°C is not suitable for the detection of the samples of the present invention.

[0173] Comparative Example 7

[0174] The difference from Example 1 is that the flow rates were investigated at 0.8 mL / min and 1.2 mL / min respectively, and other steps were the same as those in Example 1. The results are as shown in Figure 19 and Figure 20 as shown

[0175] It can be seen from Figure 19 the results that in the time period of 0 - 20 minutes, the separation of each peak in the chromatogram is poor and it is difficult to detect. Therefore, a flow rate of 0.8 mL / min is not applicable to the detection of the samples of the present invention.

[0176] It can be seen from Figure 20 that in the time period of 0 - 10 minutes, gallic acid cannot be effectively separated and the detection is difficult. In the time period of 55 - 60 minutes, the peak shape of berberine is poor, it cannot be effectively calculated, and the resolution is poor. Therefore, a flow rate of 1.2 mL / min is not applicable to the detection of the samples of the present invention.

Claims

1. A method for determining the content of multiple components in four-flavor Tibetan cat milk soup, characterized in that: The following steps are involved: S1, preparation of test solution; S2, preparing a mixed reference solution using gallic acid, loganinic acid, gentiopicroside, magnolamine, chebulic acid, jatrorrhizine, chebulic acid, berberine hydrochloride, quercetin, naringenin, apigenin, kaempferol and rhamnocitin as reference substances; S3, using high performance liquid chromatography to measure the chromatograms of the test solution and the mixed reference solution respectively; The chromatographic conditions of the high performance liquid chromatography are as follows: Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Mobile phase: Mobile phase B is acetonitrile, mobile phase A is an aqueous solution containing phosphoric acid; The elution method is gradient elution, and the gradient elution program is shown in Table 1; Table 1 Gradient elution program S4, calculate the content of each component in the four-flavor Tibetan cat milk soup.

2. The method according to claim 1, characterized in that In S1, the preparation method of the test solution is: mix the four-flavor Tibetan cat milk soup with an alcohol solvent, mix thoroughly, centrifuge or filter, and take the supernatant to obtain the test solution.

3. The method according to claim 2, characterized in that The alcohol solvent is a methanol aqueous solution with a volume percentage of 75%; the ratio of the volume of the four-flavor Tibetan cat milk soup to the volume of the alcohol solvent is 1:

9.

4. The method according to claim 1, characterized in that In S2, in the mixed reference solution, the concentration of gallic acid is 0.00480mg / mL to 0.07692mg / mL; the concentration of loganinic acid is 0.00480mg / mL to 0.07692mg / mL; the concentration of gentiopicroside is 0.00394mg / mL to 0.06308mg / mL; the concentration of magnolamine is 0.00236mg / mL to 0.03785mg / mL; the concentration of chebulic acid is 0.00238mg / mL to 0.03815mg / mL; the concentration of chebulic acid is 0.00480mg / mL to 0.07692mg / mL; the concentration of jatrorrhizine is 0 .00241mg / mL~0.03854mg / mL; the concentration of berberine hydrochloride is 0.00480mg / mL~0.07692mg / mL; the concentration of quercetin is 0.00480mg / mL~0.07692mg / mL; the concentration of naringenin is 0.00480mg / mL~0.07692mg / mL; the concentration of apigenin is 0.00480mg / mL~0.07692mg / mL; the concentration of kaempferol is 0.00480mg / mL~0.07692mg / mL; the concentration of rhamnolimonin is 0.00230mg / mL~0.03692mg / mL.

5. The method according to claim 1, characterized in that In S3, the chromatographic conditions further include: Detection wavelength: 274nm; Flow rate: 1.0 mL / min; Column temperature: 25°C; Injection volume: 5 μL.

6. The method according to claim 1, characterized in that In S3, the volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.10%.

7. The method according to claim 1, characterized in that In S4, the content of each component in the four-flavor Tibetan cat milk soup was calculated by the external standard method.

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