A method for determining the multi-component content of a four-ingredient Tibetan cat milk soup

Abstract

The present application belongs to the technical field of medicine detection, and particularly relates to a multi-component content determination method of four Tibet cat milk soup. The present application uses specific high performance liquid chromatography conditions to determine the four Tibet cat milk soup, realizes complete separation of 13 components of gallic acid, strychnine acid, gentiopicroside, magnoflorine, acacia acid, acerola acid, jatrorrhizine, berberine hydrochloride, quercetin, naringenin, apigenin, kaempferol and rhamnocitrin, can quickly, simply, accurately and reliably obtain the quality condition of the four Tibet cat milk soup, realizes the purpose of controlling the quality of the four Tibet cat milk soup by determining the content of the 13 chemical components, and provides a reference for development and quality standard establishment of the four Tibet cat milk soup which is a classic Tibetan medicine.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a method for determining the content of multiple components in a four-ingredient Tibetan cat milk soup. Background Technology

[0002] Tibetan cat milk is a shrub belonging to the genus *Corydalis* (green shrub) and the family Rhamnaceae. The Four-Ingredient Tibetan Cat Milk Soup comes from the Tibetan medical classic "Four Medical Tantras" and is a basic prescription for treating "yellow water disease" in Tibetan medicine. It consists of Tibetan cat milk, bark of *Berberis vulgaris*, *Gentiana macrophylla*, and *Terminalia chebula*. It has the effects of drying up hot yellow water and clearing heat and detoxifying. It can be used for "True Closing Disease", especially for the treatment of RA (rheumatoid arthritis).

[0003] The prior patent with application number 2022105268764 discloses a Tibetan medicine formula, preparation method and application for treating rheumatoid arthritis. It discloses a four-ingredient Tibetan cat milk soup composed of Tibetan cat milk, bark of berberis, gentian root and chebula. However, it does not disclose how to control the quality of the four-ingredient Tibetan cat milk soup, and cannot guarantee the effectiveness of clinical use.

[0004] Therefore, how to establish a method for determining the components of the four-ingredient Tibetan cat milk soup, ensure the effectiveness of clinical use, and provide standards for strictly controlling drug quality has become an urgent problem to be solved in this field. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for determining the content of multiple components in a four-ingredient Tibetan cat milk soup. By using specific liquid chromatography conditions, the quality of the four-ingredient Tibetan cat milk soup can be determined quickly and accurately.

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

[0007] A method for determining the content of multiple components in a four-ingredient Tibetan cat milk soup, the method comprising the following steps:

[0008] S1, Preparation of the test solution;

[0009] S2, a mixed reference solution was prepared using gallic acid, loganic acid, gentiopicrin, magnoflorine, chebulic acid, purslane, chebulic acid, berberine hydrochloride, quercetin, naringenin, apigenin, kaempferol and rhamnolimonin as reference standards.

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

[0011] The chromatographic conditions for the high-performance liquid chromatography method are as follows:

[0012] Column: Packed with octadecylsilane-bonded silica gel;

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

[0014] The elution method is gradient elution, and the gradient elution procedure is shown in Table 1.

[0015] Table 1 Gradient elution program

[0016]

[0017] S4, calculate the content of each component in the four-flavor Tibetan cat milk soup.

[0018] Preferably, in S1, the test solution is prepared by mixing four-flavor Tibetan cat milk soup with an alcohol solvent, mixing thoroughly, centrifuging or filtering, and taking the supernatant; the alcohol solvent is a methanol aqueous solution with a volume percentage of 75%; the volume ratio of the four-flavor Tibetan cat milk soup to the alcohol solvent is 1:9.

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

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

[0021] The concentration of magnoflorine was 0.00394 mg / mL to 0.06308 mg / mL.

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

[0023] 0.00238 mg / mL~0.03815 mg / mL; the concentration of chebulic acid is

[0024] 0.00480 mg / mL~0.07692 mg / mL; the concentration of phycocyanin is

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

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

[0027] The concentration of naringenin was 0.00480 mg / mL to 0.07692 mg / mL.

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

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

[0030] The concentrations of rhamnol range from 0.00480 mg / mL to 0.07692 mg / mL; the concentrations of rhamnol range from 0.00230 mg / mL to 0.03692 mg / mL.

[0031] The preferred standard curves for gallic acid, loganic acid, gentiopicrin, magnoflorine, chebulic acid, purslane, chebulic acid, berberine, quercetin, naringenin, apigenin, kaempferol, and rhamnolimonene 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: 274nm;

[0047] Flow rate: 1.0 mL / min;

[0048] Column temperature: 25℃;

[0049] Injection volume: 5 μL.

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

[0051] Preferably, in S4, based on the concentration of chemical components in the mixed reference solution in step S2, the peak area of ​​the mixed reference solution in the chromatogram, and the peak area of ​​the corresponding components in the four-flavor Tibetan cat milk soup in the reference solution in the chromatogram, the content of each component in the four-flavor Tibetan cat milk soup is calculated by external standard method.

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

[0053] (1) This invention uses specific high performance liquid chromatography conditions to determine four Tibetan cat milk soups, achieving complete separation of 13 components including gallic acid, loganic acid, gentiopicrin, magnoflorine, chebulic acid, chebulic acid, cyperine, berberine hydrochloride, quercetin, naringenin, apigenin, kaempferol, and rhamnolimonin. It has the advantages of high separation, strong specificity, and good peak shape.

[0054] (2) The method provided by this invention has high precision, accuracy and stability, and can quickly, easily, accurately and reliably determine the quality of the four-flavor Tibetan cat milk soup. It achieves the purpose of controlling the quality of the four-flavor Tibetan cat milk soup by measuring the content of 13 chemical components, provides a reference for the development and quality standard formulation of the classic Tibetan medicine formula four-flavor Tibetan cat milk soup, and has guiding significance for the quality detection and evaluation of the four-flavor Tibetan cat milk soup. Attached Figure Description

[0055] Figure 1 The HPLC chromatogram of Example 1;

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

[0057] Figure 3 HPLC chromatogram of the test solution of the four-ingredient Tibetan cat milk decoction;

[0058] Figure 4HPLC chromatogram of a negative sample solution of Siwei Tibetan Cat Milk Decoction (lacking Tibetan cat milk);

[0059] Figure 5 HPLC chromatogram of a negative sample solution of the Four-Ingredient Tibetan Cat Milk Decoction (lacking Terminalia chebula);

[0060] Figure 6 HPLC chromatogram of a negative sample solution of four-ingredient Tibetan cat milk decoction (lacking bark);

[0061] Figure 7 HPLC chromatogram of a negative sample solution of four-ingredient Tibetan cat milk decoction (lacking Gentiana macrophylla);

[0062] Figure 8 HPLC chromatogram of Tibetan cat milk;

[0063] Figure 9 HPLC chromatogram of Terminalia chebula;

[0064] Figure 10 HPLC chromatogram of bark;

[0065] Figure 11 HPLC chromatogram of Gentiana macrophylla;

[0066] Figure 12 The HPLC chromatogram is for Comparative Example 1;

[0067] Figure 13 The HPLC chromatogram is for Comparative Example 2;

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

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

[0070] Figure 16 The HPLC chromatogram is for Comparative Example 5;

[0071] Figure 17 The HPLC chromatograms for comparative examples are shown at column temperatures of 6–30 °C.

[0072] Figure 18 The HPLC chromatograms for comparative examples are shown at column temperatures of 6–35 °C.

[0073] Figure 19 The HPLC chromatogram for Comparative Example 7 is shown at a flow rate of 0.8 mL / min.

[0074] Figure 20 The HPLC chromatogram for Comparative Example 7 is shown at a flow rate of 1.2 mL / min.

[0075] Among them, 1. gallic acid; 2. loganic acid; 3. gentiopicrin; 4. magnoflorine; 5. chebulic acid; 6. chebulic acid; 7. purslane; 8. berberine; 9. quercetin; 10. naringenin; 11. apigenin; 12. kaempferol; 13. rhamnolimonin. Detailed Implementation

[0076] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0077] The preparation method of the four-ingredient Tibetan cat milk soup involved in this invention embodiment is as follows:

[0078] Weigh the medicinal materials according to the mass ratio of Tibetan cat milk: Gentiana macrophylla: Berberis bark: Terminalia chebula = 12:10:6.5:3, mix them, pulverize them, and pass them through a 20-mesh sieve to obtain powder raw materials;

[0079] Add the powdered raw materials to water at a weight-to-volume ratio of 1:20, reflux and extract twice, each extraction time being 1.5 hours. Filter, combine the filtrates, and you will get the four-flavor Tibetan cat milk soup.

[0080] Example 1

[0081] A method for determining the content of multiple components in a four-ingredient Tibetan cat milk soup, the method comprising the following steps:

[0082] S1, Preparation of the test solution:

[0083] Accurately measure 1 mL of the Four-Flavor Tibetan Cat Milk Decoction and place it in a 10 mL volumetric flask. Dilute to the mark with 75% methanol (the volume ratio of the Four-Flavor Tibetan Cat Milk Decoction to the volume of the alcohol solvent is 1:9). Mix thoroughly and centrifuge at 12000 rpm for 10 min. Take the supernatant to obtain the test solution.

[0084] S2, take appropriate amounts of 13 reference standards, and place the reference standards of gallic acid, loganic acid, gentiopicrin, magnoflorine, chebulic acid, purslane, chebulic acid, berberine, quercetin, naringenin, apigenin, kaempferol, and rhamnolic acid in a 25 mL volumetric flask. Dissolve in methanol and dilute to the mark to obtain a mixed reference solution. Ensure that the mixed reference solution contains gallic acid, loganic acid, gentiopicrin, magnoflorine, chebulic acid, purslane, chebulic acid, berberine, quercetin, naringenin, and apigenin. The mass concentrations of kaempferol, kaempferol, and rhamnolimonene were 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, and 36.92 μg / mL, respectively.

[0085] S3, the chromatograms of the test solution and the reference solution were determined by high performance liquid chromatography, respectively;

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

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

[0088] Mobile phase: acetonitrile (B) and 0.10% (v / v) aqueous solution of phosphoric acid (A);

[0089] Detection wavelength: 274nm;

[0090] Flow rate: 1.0 mL / min;

[0091] Column temperature: 25℃;

[0092] Injection volume: 5 μL;

[0093] The elution method is gradient elution, and the gradient elution procedure is shown in Table 1 of the invention.

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

[0095] Test results as follows Figure 1 As shown, by Figure 1 As can be seen, using the chromatographic conditions of this embodiment, the components to be measured can be detected well in a single chromatogram, interference can be removed, and the content of each component can be accurately calculated.

[0096] Example 2: Preparation of negative and single herbal sample solutions

[0097] Following the preparation method of the Four-Ingredient Tibetan Cat Milk Decoction, negative samples without Tibetan cat milk, Gentiana macrophylla, Berberis vulgaris, and Terminalia chebula, as well as single-herb samples containing Tibetan cat milk, Gentiana macrophylla, Berberis vulgaris, and Terminalia chebula, were prepared.

[0098] Taking the preparation of a negative sample without Tibetan cat milk as an example: according to the formula ratio of the four-ingredient Tibetan cat milk decoction, weigh out Gentiana macrophylla, Berberis vulgaris, and Terminalia chebula respectively, and prepare a negative sample without Tibetan cat milk according to the preparation method of the four-ingredient Tibetan cat milk decoction.

[0099] The method for preparing a negative sample solution that does not contain Tibetan cat milk is as follows:

[0100] Accurately measure 1 mL of the extract of the negative sample containing no Tibetan cat milk and place it in a 10 mL volumetric flask. Dilute to the mark with 75% methanol, mix thoroughly, and centrifuge at 12000 rpm for 10 min. Take the supernatant to obtain the negative sample solution containing no Tibetan cat milk.

[0101] Negative sample solutions containing no Gentiana macrophylla, Berberis thunbergii, or Terminalia chebula, as well as sample solutions containing Tibetan cat milk, Gentiana macrophylla, Berberis thunbergii, and Terminalia chebula, were prepared using the same method.

[0102] Example 3

[0103] Exclusivity Examination

[0104] Accurately pipette 5 μl each of the test solution and mixed reference solution from Example 1, and each negative sample solution from Example 2, and inject them into the liquid chromatograph. Perform chromatographic analysis according to the chromatographic conditions in Example 1. Compare and analyze the chromatograms of the above solutions. The results are as follows: Figures 2-11 As shown.

[0105] Depend on Figure 2-11 As shown, the chromatographic peaks of the four Tibetan cat milk decoction test solution and reference solution all had the same peaks at corresponding positions, indicating good peak separation. The resolution of each peak from adjacent peaks was greater than 1.5. The peaks of the individual herbs were accurately assigned, and there was no interference from negative samples. This demonstrates that the chromatographic method has good specificity.

[0106] Example 2

[0107] Examining linear relationships

[0108] Take 5 μl of the mixed reference solution from Example 1 and inject it into the liquid chromatograph. Perform the determination according to the chromatographic conditions of Example 1.

[0109] A standard curve was plotted with the concentration X (μg / mL) of the reference solution as the abscissa and the peak area Y of each reference compound as the ordinate. The corresponding regression equations, correlation coefficients, and linear ranges of the 13 references were calculated. Standard curves were plotted with the peak area (Y) of gallic acid, loganic acid, gentiopicrin, magnoflorine, chebulic acid, chebulic acid, purslane, berberine, quercetin, naringenin, apigenin, kaempferol, and rhamnolimonene as the ordinate and the mass concentration (X) as the abscissa. The limit of detection was the concentration of the compound with a signal-to-noise ratio S / N = 3, and the limit of quantitation was the concentration of the compound with a signal-to-noise ratio S / N = 10. The linear regression equations, limits of detection, and limits of quantitation are shown in Table 2.

[0110] Table 2. Linear regression equations, limits of detection, and limits of quantitation for the 13 reference standards.

[0111]

[0112] Table 2 shows that the 13 reference standards in the four Tibetan cat milk samples exhibited good linearity within their respective concentration ranges, with correlation coefficients R0. 2 All are greater than 0.999.

[0113] Example 3

[0114] Precision test

[0115] Take the mixed reference solution from Example 1, inject it 6 times consecutively under the chromatographic conditions of Example 1, record the peak area of ​​each component and calculate its RSD value. The results are shown in Table 3.

[0116] Table 3. Precision test results (n=6)

[0117]

[0118]

[0119] As shown in Table 3, the RSD values ​​of the peak areas of each reference standard were all less than 1.82%, indicating that the method has good precision.

[0120] Example 4

[0121] Stability test

[0122] Take the test solution from Example 1, place it at room temperature, and inject it at 0, 2, 4, 6, 8, and 10 h according to the chromatographic conditions of Example 1. Record the peak area of ​​each component and calculate its RSD value. The results are shown in Table 4.

[0123] Table 4. Stability test results (n=6)

[0124]

[0125]

[0126] As shown in Table 4, the RSD values ​​of each active ingredient in the stability test were all less than 1.98%, indicating that the four Tibetan cat milk soup test solutions had good stability within 10 hours under room temperature conditions.

[0127] Example 5

[0128] Repeatability test

[0129] Six parallel preparations of the four-ingredient Tibetan cat milk soup test solution from Example 1 were performed and analyzed under the chromatographic conditions of Example 1. The peak areas of each compound were measured, and the content and RSD values ​​of each compound were calculated. The results are shown in Table 5.

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

[0131]

[0132]

[0133] Table 5 shows that the average percentage contents of gallic acid, loganic acid, gentiopicrin, magnoflorine, chebulic acid, baicalein, berberine, quercetin, naringenin, kaempferol, apigenin, and rhamnolimonin were 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, with RSD% less than 1.62%, indicating good reproducibility of the method.

[0134] Example 6

[0135] Recovery test

[0136] Six samples of four-ingredient Tibetan cat milk decoction were prepared. Each sample was prepared by adding corresponding mass of the control substance at 80%, 100%, and 120% of the known content (known content in Example 5) of each chemical component (gallic acid, loganic acid, gentiopicrin, magnoflorine, chebulic acid, baicalein, berberine, quercetin, naringenin, kaempferol, apigenin, and rhamnolimonene) according to the method described in Example 1. The samples were then analyzed under the chromatographic conditions of Example 1. The peak areas of each component were recorded, and the recoveries and RSDs were calculated. The results are shown in Table 6.

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

[0138]

[0139]

[0140]

[0141]

[0142] As shown in Table 6, the average recovery rates of the 13 control compounds in the six samples of Tibetan cat milk soup ranged from 98.54% to 100.68%, and the RSD values ​​were all less than 2%, indicating that the content determination method had good recovery rate and was accurate and feasible.

[0143] Example 7

[0144] Twenty batches of four-flavor Tibetan cat milk soup were prepared, and corresponding 20 batches of test solutions were prepared according to the method in Example 1. The content of each active ingredient in each batch was determined according to the method in Example 1. The results are shown in Table 7.

[0145] Table 7. Results of determination of the content (%) of each effective component in 20 batches of four-flavor Tibetan cat milk soup.

[0146]

[0147] Table 7 shows that the gallic acid content in the 20 batches of four-flavor Tibetan cat milk soup samples was [missing information].

[0148] 0.132–0.294%, loganic acid content 0.415–0.574%, gentiopicroside content 0.132–0.294%.

[0149] The content of magnoflorine was 1.095–1.359%, the content of magnoflorine was 0.686–0.805%, and the content of chebulic acid was...

[0150] 0.575–0.788%, chebulic acid content was 0.394–0.489%, and herba alkaloid content was...

[0151] The content of berberine was 0.399–0.489%, the content of quercetin was 0.376–0.599%, and the content of quercetin was...

[0152] The content of naringenin was 0.253–0.311%, and the content of kaempferol was 0.332–0.423%.

[0153] The content of apigenin is 0.298–0.388%, the content of rhamnolimonin is 0.173–0.356%, and the content of rhamnolimonin is 0.128–0.267%. The method of the present invention can determine the content of components in the four-ingredient Tibetan cat milk soup.

[0154] Comparative Example 1

[0155] The difference from Example 1 is that the detection wavelength is 245 nm; all other steps are the same as in Example 1, and the results are as follows. Figure 12 As shown.

[0156] Depend on Figure 12 It can be seen that when using a wavelength of 245nm for detection, the separation of each detection peak is poor between 0 and 40 minutes, and it is impossible to achieve good separation of the 13 components.

[0157] Comparative Example 2

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

[0159] Depend on Figure 13 It is known that when using a wavelength of 360nm for detection, some components (such as gallic acid, loganic acid, gentiopicrin, etc.) have low absorption and are difficult to detect, making it impossible to accurately detect all 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) column was used; all other steps were the same as in Example 1, and the results are as follows. Figure 14 As shown.

[0162] Depend on Figure 14 It is known that when using the Elite Hypersil BDS C18 (4.6×250mm, 5μm) column for separation and detection, some components, such as gallic acid, were not separated and difficult to detect within 0–5 minutes, and gentiopicrin and magnoflorine were not separated and difficult to detect within 10–20 minutes. Therefore, this type of column is not suitable for the separation and detection of samples in this invention.

[0163] Comparative Example 4

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

[0165] Depend on Figure 15 It was found that using an Agilent SB-C18 (4.6×250mm, 5μm) column for detection and separation resulted in the failure to separate gentiopicrin and magnoflorine within 10–20 minutes, making them difficult to detect. Therefore, this type of column is not suitable for the sample separation and detection in this invention.

[0166] Comparative Example 5

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

[0168] Depend on Figure 16 It is known that the methanol-0.1% phosphoric acid aqueous solution system has slow elution of peaks, slow elution of compounds, and poor separation of peaks, making it impossible to effectively detect the sample of this invention.

[0169] Comparative Example 6

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

[0171] Figure 17 The results showed that during the 0-20 minute time period, the peaks piled up and were not effectively separated. Therefore, a column temperature of 30℃ is not suitable for the high-performance liquid chromatography detection of the samples of this invention.

[0172] Figure 18 It was observed that the component peaks were difficult to separate during the 0-20 minute timeframe. Berberine separation was unsatisfactory during the 55-60 minute timeframe. Naringenin, apigenin, and kaempferol could not be effectively separated during the 75-85 minute timeframe. Therefore, a column temperature of 35°C is unsuitable for the detection of samples in this invention.

[0173] Comparative Example 7

[0174] The difference from Example 1 is that the investigation was conducted at flow rates of 0.8 mL / min and 1.2 mL / min, respectively. All other steps were the same as in Example 1, and the results are as follows: Figure 19 and Figure 20 As shown.

[0175] Depend on Figure 19 The results showed that the peaks in the spectrum were poorly separated and difficult to detect within 0–20 minutes. Therefore, a flow rate of 0.8 mL / min is not suitable for the detection of samples in this invention.

[0176] Depend on Figure 20 It is evident that gallic acid could not be effectively separated within 0–10 minutes, making detection difficult. Within 55–60 minutes, the peak shape difference of berberine could not be effectively calculated, resulting in poor resolution. Therefore, a flow rate of 1.2 mL / min is not suitable for the detection of samples in this invention.

Claims

1. A method for determining the content of multiple components in a four-ingredient Tibetan cat milk soup, characterized in that, Includes the following steps: S1, Preparation of the test solution; S2, a mixed reference solution was prepared using gallic acid, loganic acid, gentiopicrin, magnoflorine, chebulic acid, purslane, chebulic acid, berberine hydrochloride, quercetin, naringenin, apigenin, kaempferol and rhamnolimonin as reference standards. S3, the chromatograms of the test solution and the mixed reference solution were determined by high performance liquid chromatography, respectively; The chromatographic conditions for the high-performance liquid chromatography method are as follows: Column: Packed with octadecylsilane-bonded silica gel; Mobile phase: Mobile phase B is acetonitrile, and mobile phase A is an aqueous solution containing 0.10% phosphoric acid by volume; Detection wavelength: 274 nm; Flow rate: 1.0 mL / min; Column temperature: 25 ℃; Injection volume: 5 μL; The elution method is gradient elution, and the gradient elution procedure is shown in Table 1. Table 1 Gradient elution program ； S4, calculate the content of each component in the four flavors of Tibetan cat milk soup.

2. The method as described in claim 1, characterized in that, In S1, the test solution is prepared by mixing four kinds of Tibetan cat milk soup with an alcohol solvent, mixing thoroughly, centrifuging or filtering, and taking the supernatant to obtain the test solution.

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

9.

4. The method as described in claim 1, characterized in that, In S2, the concentrations of gallic acid, strychnine, gentiopicroside, magnoflorine, chebulic acid, and gentiopicroside in the mixed reference solution are as follows: gallic acid: 0.00480 mg / mL to 0.07692 mg / mL; loganic acid: 0.00480 mg / mL to 0.07692 mg / mL; gentiopicroside: 0.00394 mg / mL to 0.06308 mg / mL; magnoflorine: 0.00236 mg / mL to 0.03785 mg / mL; chebulic acid: 0.00238 mg / mL to 0.03815 mg / mL; chebulic acid: 0.00480 mg / mL to 0.07692 mg / mL; magnoflorine:

0. The concentrations of berberine hydrochloride, quercetin, naringenin, apigenin, kaempferol, and rhamnocitric acid were 0.00241 mg / mL to 0.03854 mg / mL; berberine hydrochloride, quercetin, naringenin, apigenin, kaempferol, and rhamnocitric acid were 0.00230 mg / mL to 0.03692 mg / mL.

5. The method as described in claim 1, characterized in that, In S4, the content of each component in the four Tibetan cat milk soups was calculated using the external standard method.

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