A method for simultaneously detecting the contents of six active ingredients in Lianhuang granules

Abstract

The present invention relates to the technical field of drug analysis and detection, and proposes a method for simultaneously detecting the contents of six active ingredients in Lianhuang granules, using acetonitrile and a 0.1% by volume phosphoric acid solution as mobile phases, and adopting high performance liquid chromatography to detect the six active ingredients in Lianhuang granules, wherein the six active ingredients are andrographolide, dehydroandrographolide, aesculetin, aesculetin, baicalin and gallic acid. Through the above technical scheme, the problem of no content detection method for Lianhuang granules in the prior art, inability to scientifically determine the quality and stability of the product, and resulting in no precise medication timing in the clinical application of Lianhuang granules is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis and detection, and in particular to a method for simultaneously detecting the contents of six active ingredients in Lianhuang granules. Background Art

[0002] Long-term and high-dose abuse of antibiotics leads to bacterial resistance, resulting in reduced or even ineffective clinical effects. At the same time, antibiotic residues also endanger food safety. In order to ensure the safety of animal-derived food and protect public health safety, my country has implemented measures to reduce antibiotics at the breeding end and ban antibiotics at the feed end. The implementation of measures to reduce and limit antibiotics has a certain impact on the prevention and treatment of livestock and poultry diseases, especially the higher incidence of intestinal diseases, which seriously affects the health and economic benefits of livestock and poultry. Lianhuang Granules are made of Andrographis paniculata, Scutellaria baicalensis, Pulsatilla striata, Fraxinus chinensis and Terminalia chebula, and have the effects of clearing heat and dampness, cooling blood and stopping diarrhea, and are widely used in veterinary clinics. Chinese veterinary preparations have the characteristics of multi-component, multi-pathway and multi-target action, with the advantages of small toxic and side effects, no drug resistance, no residue and no pollution. The quality standard of Lianhuang Granules is included in the 2017 edition of "Quality Standards for Veterinary Drugs" (Chinese Medicine Volume). The quality standard only has thin layer chromatography identification, no content detection items, and cannot scientifically determine the quality and stability of the product. At the same time, the application of Lianhuang Granules in clinical practice has no precise medication time, resulting in Lianhuang Granules failing to effectively solve clinical problems. Summary of the invention

[0003] The present invention proposes a method for simultaneously detecting the contents of six active ingredients in Lianhuang Granules, which solves the problem that there is no content detection method for Lianhuang Granules in the related art, and the quality and stability of the product cannot be scientifically determined, resulting in the problem of no precise medication timing in the clinical application of Lianhuang Granules.

[0004] The technical solution of the present invention is as follows:

[0005] A method for simultaneously detecting the contents of six active ingredients in Lianhuang granules, using acetonitrile and 0.1% by volume phosphoric acid solution as mobile phases, and adopting high performance liquid chromatography to detect the six active ingredients in Lianhuang granules, wherein the six active ingredients are andrographolide, dehydroandrographolide, aesculetin, aesculetin, baicalin and gallic acid.

[0006] As a further technical solution, it also includes the use of external standard method for quantitative determination of the contents of dehydroandrographolide, andrographolide, fraxinoside, fraxinoside, baicalin and gallic acid in Lianhuang Granules.

[0007] As a further technical solution, the Lianhuang granules are dissolved in a solvent to obtain a test solution, and the test solution is detected by high performance liquid chromatography. The solvent includes anhydrous methanol, anhydrous ethanol, 50% methanol by volume and 50% ethanol by volume.

[0008] As a further technical solution, the solvent is methanol with a volume fraction of 50%.

[0009] As a further technical solution, the mass volume ratio of the lotus yellow particles to the solvent is 1g:25~100mL.

[0010] As a further technical solution, the mass volume ratio of the lotus yellow particles to the solvent is 1g:50mL.

[0011] As a further technical solution, the dissolution is ultrasonic dissolution, and the ultrasonic time is 10 to 30 minutes.

[0012] As a further technical solution, the ultrasonic time is 20 minutes.

[0013] As a further technical solution, in the high performance liquid chromatography method, the chromatographic column filler is octadecylsilane bonded silica gel, and the chromatographic column is a Symmetuy C18 chromatographic column.

[0014] As a further technical solution, the high performance liquid chromatography method adopts a gradient elution method, and the gradient elution conditions are specifically as follows:

[0015] .

[0016] As a further technical solution, the high performance liquid chromatography method adopts a segmented wavelength conversion method, and the segmented wavelength conversion conditions are specifically as follows:

[0017] .

[0018] Application of a method for simultaneously detecting the contents of six active ingredients in Lianhuang Granules in the quality inspection of Lianhuang Granules.

[0019] The working principle and beneficial effects of the present invention are:

[0020] The present invention adopts high performance liquid chromatography to simultaneously determine six effective components in Lianhuang granules, including andrographolide, dehydroandrographolide, aesculin, aesculetin, baicalin and gallic acid, and can quantitatively determine the contents of andrographolide, dehydroandrographolide, aesculin, aesculetin, baicalin and gallic acid in Lianhuang granules. The method is simple and economical, has good separation effect, strong specificity, good reproducibility, can scientifically determine the quality and stability of Lianhuang granules, and lays a foundation for the application opportunity of Lianhuang granules in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0022] Figure 1 The HPLC chromatogram of the mixed reference substance obtained in Example 1 of the present invention;

[0023] Figure 2 This is a high performance liquid chromatogram of Lianhuang granules obtained in Example 1 of the present invention;

[0024] Figure 3 This is a high performance liquid chromatogram of a negative sample of Lianhuang granules lacking Andrographis paniculata obtained in Example 1 of the present invention;

[0025] Figure 4 This is a high performance liquid chromatogram of a negative sample of Lianhuang granules lacking Qinpi obtained in Example 1 of the present invention;

[0026] Figure 5 This is a high performance liquid chromatogram of a negative sample of Lianhuang granules lacking Scutellaria baicalensis obtained in Example 1 of the present invention;

[0027] Figure 6 This is a high performance liquid chromatogram of a negative sample of Lianhuang granules of Sanguisorba officinalis obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

[0030] 1. Preparation of solution:

[0031] Test solution: weigh 1g of Lianhuang granule powder, add it to a 50mL volumetric flask, add 25mL of 50% methanol by volume, ultrasonically treat for 20min, cool, and then dilute to 50mL with 50% methanol by volume, shake well, and filter to obtain the test solution;

[0032] Mixed reference substance solution: weigh 1 g each of andrographolide, dehydroandrographolide, esculin, esculin, baicalin and gallic acid, and dilute to 25 mL with methanol to obtain andrographolide reference substance stock solution, dehydroandrographolide reference substance stock solution, esculin reference substance stock solution, esculin reference substance stock solution, baicalin reference substance stock solution and gallic acid reference substance stock solution. Measure 2 mL of andrographolide reference substance stock solution, 2 mL of dehydroandrographolide reference substance stock solution, 2 mL of esculin reference substance stock solution, 1 mL of esculin reference substance stock solution, 1 mL of baicalin reference substance stock solution and 2 mL of gallic acid reference substance stock solution, respectively, and mix them to obtain a mixed reference substance solution;

[0033] 2. Detection

[0034] 2.1. Test conditions

[0035] The mixed reference solution and the test solution were tested by high performance liquid chromatography under the following conditions:

[0036] The chromatographic column was Symmetuy C18 (250 mm × 4.60 mm, 5 μm), the filler was octadecylsilane bonded silica gel, the mobile phase was acetonitrile-0.1% phosphoric acid solution by volume, and the gradient elution method was adopted. The gradient elution conditions are shown in Table 1:

[0037] Table 1 Elution ratio

[0038]

[0039] The segmented wavelength conversion method is adopted, and the segmented wavelength conversion conditions are shown in Table 2:

[0040] Table 2 Segmented wavelength conversion conditions

[0041]

[0042] Carry out detection according to the above detection conditions and record the chromatogram.

[0043] 2.2 Test results

[0044] The HPLC chromatogram of the mixed reference substance is as follows Figure 1 As shown;

[0045] The HPLC chromatogram of Lianhuang granules is shown in Figure 2 As shown, it can be seen from the figure that the peaks of andrographolide, dehydroandrographolide, esculin, esculetin, baicalin and gallic acid in Lianhuang granules, indicating that the detection method of the present invention can simultaneously detect the six effective ingredients of andrographolide, dehydroandrographolide, esculin, esculetin, baicalin and gallic acid in Lianhuang granules;

[0046] Quantitative analysis: The contents of andrographolide, dehydroandrographolide, aesculetin, aesculetin, baicalin and gallic acid in Lianhuang granules were calculated by peak area using the external standard method. The calculation formula is as follows:

[0047] X=(A 供 ×W 对 ×V 供 ) / (W 供 ×A 对 ×V 对 )×100%;

[0048] Where: X is the content of the component to be tested in Lianhuang granules, %;

[0049] A 供is the peak area of ​​the component to be tested in the chromatogram of the test solution;

[0050] A 对 It is the average peak area of ​​the component to be tested in the chromatogram of the mixed reference solution;

[0051] W 供 is the sample weight of the test product (mg);

[0052] W 对 is the weight of the component to be tested in the mixed reference solution (mg);

[0053] V 供 is the dilution factor of the test solution;

[0054] V 对 is the dilution factor of the mixed reference solution.

[0055] After calculation, the contents of gallic acid, fraxinoside, fraxinoside, baicalin, andrographolide and dehydroandrographolide in Lianhuang Granules are 1.63 mg / g, 1.42 mg / g, 0.13 mg / g, 19.54 mg / g, 1.53 mg / g and 0.21 mg / g, respectively.

[0056] Example 2

[0057] The only difference from Example 1 is that the test sample Lianhuang Granules in this example is prepared according to the method of the Chinese medicine volume of the 2017 edition of the "Quality Standards for Veterinary Drugs".

[0058] After calculation, the contents of gallic acid, fraxinusin, baicalin and andrographolide in Lianhuang Granules are 1.55 mg / g, 1.44 mg / g, 18.99 mg / g and 1.62 mg / g, respectively.

[0059] Since the contents of dehydroandrographolide and aconitine in the test sample Lianhuang granules of this embodiment are relatively low, quantitative analysis is difficult.

[0060] 3. Method Validation

[0061] 3.1. Specificity test

[0062] (1) Test method:

[0063] With reference to the 2017 edition of the Chinese medicine volume of "Quality Standards for Veterinary Drugs", negative samples of Lianhuang Granules lacking Andrographis paniculata, Fraxinus fraseri, Scutellaria baicalensis and Sanguisorba officinalis were prepared respectively.

[0064] Test solution: Weigh 1 g of the negative sample prepared above, add it into a 50 mL volumetric flask, add 25 mL of 50% methanol by volume, treat it with ultrasound for 20 min, cool it, and then make the volume to 50 mL with 50% methanol by volume. Shake well and filter to obtain 4 test solutions, namely, the negative test solution of Lianhuang Granules lacking Andrographis paniculata, the negative test solution of Lianhuang Granules lacking Fragrant Cortex, the negative test solution of Lianhuang Granules lacking Scutellaria baicalensis, and the negative test solution of Lianhuang Granules lacking Sanguisorba officinalis. Inject them in sequence for determination.

[0065] Mixed reference substance solution: weigh 1 g each of andrographolide, dehydroandrographolide, esculin, esculin, baicalin and gallic acid, and dilute to 25 mL with methanol to obtain andrographolide reference substance stock solution, dehydroandrographolide reference substance stock solution, esculin reference substance stock solution, esculin reference substance stock solution, baicalin reference substance stock solution and gallic acid reference substance stock solution. Measure 2 mL of andrographolide reference substance stock solution, 2 mL of dehydroandrographolide reference substance stock solution, 2 mL of esculin reference substance stock solution, 1 mL of esculin reference substance stock solution, 1 mL of baicalin reference substance stock solution and 2 mL of gallic acid reference substance stock solution, respectively, and mix them to obtain a mixed reference substance solution;

[0066] (2) Test results:

[0067] The test was carried out according to the above test conditions, and the chromatogram was recorded. The HPLC chromatogram of the negative sample of Lianhuang Granules lacking Andrographis paniculata is as follows Figure 3 As shown in the figure, the HPLC chromatogram of the negative sample of Lianhuang granules lacking Qinpi is as follows Figure 4 As shown in the figure, the HPLC chromatogram of the negative sample of Lianhuang Granule lacking Scutellaria baicalensis is as follows Figure 5 As shown, the HPLC chromatogram of the negative sample of Lianhuang granules lacking Sanguisorba officinalis is as follows Figure 6 shown.

[0068] from Figures 3 to 6 It can be seen that the retention time of the HPLC chromatograms of the negative samples of each Lianhuang granule is the same as that of the mixed reference solution HPLC chromatogram, and there is no interference between the peaks. Therefore, the method has good specificity.

[0069] 3.2 Linear Relationship

[0070] (1) Test method:

[0071] Gallic acid reference solution: Prepare gallic acid reference solutions with concentrations of 10, 25, 30, 40, and 50 μg / mL in methanol;

[0072] Aesculin reference solution: Prepare aesculin reference solution with concentrations of 4.8, 9.6, 14.4, 19.2 and 24 μg / mL in methanol;

[0073] Aesculetin reference solution: Prepare baicalin reference solutions with aesculetin concentrations of 0.1, 0.2, 0.4, 0.8 and 1.0 g / mL in methanol;

[0074] Baicalin reference solution: Prepare baicalin reference solutions with baicalin concentrations of 80, 160, 240, 320 and 400 μg / mL in methanol;

[0075] Andrographolide reference solution: Prepare andrographolide reference solutions with andrographolide concentrations of 4.8, 9.6, 14.4, 19.2 and 24 μg / mL in methanol;

[0076] Dehydroandrographolide reference solution: Prepare baicalin reference solutions with dehydroandrographolide concentrations of 0.2, 0.8, 1.2, 1.6 and 2.0 g / mL in methanol;

[0077] Inject samples and measure them sequentially.

[0078] (2) Test results:

[0079] The concentration of the reference solution was used as the abscissa and the peak area as the ordinate to establish a linear regression curve. The test results are shown in Table 3:

[0080] Table 3 Linear detection results

[0081]

[0082] 3.3 Precision test

[0083] (1) Test method:

[0084] Mixed reference solution: weigh 1 g each of andrographolide, dehydroandrographolide, fraxinotin, fraxinotin, baicalin and gallic acid, and dilute to 25 mL with methanol to obtain andrographolide reference stock solution, dehydroandrographolide reference stock solution, fraxinotin reference stock solution, fraxinotin reference stock solution, baicalin reference stock solution and gallic acid reference stock solution. Measure 2 mL of andrographolide reference stock solution, 2 mL of dehydroandrographolide reference stock solution, 2 mL of fraxinotin reference stock solution, 1 mL of fraxinotin reference stock solution, 1 mL of baicalin reference stock solution and 2 mL of gallic acid reference stock solution, respectively, mix them to obtain a mixed reference solution; prepare 6 portions in parallel and inject them continuously.

[0085] (2) Test results:

[0086] The peak area RSD values ​​of gallic acid, aesculin, aesculetin, baicalin, andrographolide and dehydroandrographolide were calculated, and the results are shown in Table 4:

[0087] Table 4 Precision test results

[0088]

[0089] 3.4 Repeatability test

[0090] (1) Test method:

[0091] Test solution: Weigh 1 g of Lianhuang granule powder, add it to a 50 mL volumetric flask, add 25 mL of 50% methanol by volume, ultrasonically treat for 20 min, cool, and then dilute to 50 mL with 50% methanol by volume, shake well, filter, and obtain the test solution; prepare 6 copies in parallel and inject continuously.

[0092] (2) Test results:

[0093] The peak area RSD values ​​of gallic acid, aesculin, aesculetin, baicalin, andrographolide and dehydroandrographolide were calculated, and the results are shown in Table 5:

[0094] Table 5 Repeatability test results

[0095]

[0096] 3.5 Stability test

[0097] (1) Test method:

[0098] Test solution: Weigh 1g of Lianhuang granule powder, add it to a 50mL volumetric flask, add 25mL of 50% methanol by volume, ultrasonically treat for 20min, cool, and then dilute to 50mL with 50% methanol by volume, shake well, filter, and obtain the test solution; prepare 6 copies in parallel, and sample and measure at 0, 2, 4, 8, 16, and 24h respectively.

[0099] (2) Test results:

[0100] The peak area RSD values ​​of gallic acid, aesculin, aesculetin, baicalin, andrographolide and dehydroandrographolide were calculated, and the results are shown in Table 6:

[0101] Table 6 Stability test results

[0102]

[0103] 3.6. Sample recovery test

[0104] (1) Test method:

[0105] Gallic acid reference substance sample solution: weigh 5.43 mg of gallic acid, dilute to 20 mL with methanol, measure 3 mL of the dilute solution and place it in a 50 mL volumetric flask, add 0.5 g of Lianhuang granules with known content and 20 mL of 50% methanol by volume, sonicate for 20 min, cool, and dilute to 50 mL with 50% methanol by volume;

[0106] Fraxinus reference substance sample solution: weigh 7.1 mg of fraxinus, dilute to 20 mL with methanol, measure 2 mL of the dilute solution and place it in a 50 mL volumetric flask, add 0.5 g of Lianhuang granules with known component content and 20 mL of 50% methanol by volume, ultrasonicate for 20 min, cool, and dilute to 50 mL with 50% methanol by volume;

[0107] Fraxinus acetogenin reference substance sample solution: weigh 4.0 mg of fraxinus acetogenin, dilute to 50 mL with methanol, measure 1 mL of the dilute solution and place it in a 50 mL volumetric flask, add 0.5 g of Lianhuang granules with known component content and 20 mL of 50% methanol by volume, ultrasonicate for 20 min, cool, and dilute to 50 mL with 50% methanol by volume;

[0108] Baicalin reference substance sample solution: weigh 9.77 mg of baicalin, dilute to 20 mL with methanol, measure 20 mL of the dilute solution and place it in a 50 mL volumetric flask, add 0.5 g of Lianhuang granules with known component content and 10 mL of 50% methanol by volume, ultrasonicate for 20 min, cool, and dilute to 50 mL with 50% methanol by volume;

[0109] Andrographolide reference substance sample solution: weigh 5.1 mg of andrographolide, dilute to 20 mL with methanol, measure 3 mL of the dilute solution and place it in a 50 mL volumetric flask, add 0.5 g of Lianhuang granules with known component content and 20 mL of 50% methanol by volume, ultrasonicate for 20 min, cool, and dilute to 50 mL with 50% methanol by volume;

[0110] Dehydroandrographolide reference substance sample solution: weigh 6.1 mg of dehydroandrographolide, dilute to 50 mL with methanol, measure 1 mL of the dilute solution and place it in a 50 mL volumetric flask, add 0.5 g of Lianhuang granules with known component content and 20 mL of 50% methanol by volume, ultrasonicate for 20 min, cool, and dilute to 50 mL with 50% methanol by volume;

[0111] (2) Test results:

[0112] The average sample recovery rates and peak area RSD values ​​of gallic acid, aesculin, aesculetin, baicalin, andrographolide and dehydroandrographolide were calculated. The results are shown in Table 7:

[0113] Table 7 Results of sample recovery test

[0114]

[0115] Experiment 1: Effect of mobile phase

[0116] (1) Test method

[0117] Experimental group 1: same as Example 1, except that the mobile phase was acetonitrile-0.1% phosphoric acid solution;

[0118] Experimental group 2: The only difference from Example 1 is that the mobile phase is methanol-water solution;

[0119] Experimental group 3: The only difference from Example 1 is that the mobile phase is acetonitrile-water solution;

[0120] Experimental group 4: The only difference from Example 1 is that the mobile phase is methanol-0.1% by volume phosphoric acid solution;

[0121] The detection was carried out at a constant volume ratio of 50:50 for the mobile phase, and the other detection conditions were the same as those in Example 1.

[0122] (2) Test results

[0123] Under constant ratio conditions, experimental groups 1 to 4 could not achieve the separation of gallic acid, aesculin, aesculetin, baicalin, andrographolide and dehydroandrographolide. However, the HPLC chromatograms of Lianhuang Granules obtained from experimental groups 2 to 4 showed low peak separation and many impurity peaks. Compared with experimental groups 2 to 4, the HPLC chromatogram of Lianhuang Granules obtained from experimental group 1 showed high peak separation and few impurity peaks. Therefore, acetonitrile-0.1% phosphoric acid solution was selected as the mobile phase.

[0124] Experiment 2: Effect of solvent

[0125] (1) Test method

[0126] Experimental group 1: Same as Example 1, test solution: weigh 1 g of Lianhuang granule powder, add it to a 50 mL volumetric flask, add 25 mL of 50% methanol by volume, ultrasonically treat for 20 min, cool, and then dilute to 50 mL with 50% methanol by volume, shake well, and filter to obtain the test solution;

[0127] Experimental group 2: The only difference from Example 1 is the test solution: weigh 1g of Lianhuang granule powder, add it to a 50mL volumetric flask, add 25mL of anhydrous methanol, ultrasonically treat for 20min, cool, and then dilute to 50mL with anhydrous methanol, shake well, and filter to obtain the test solution;

[0128] Experimental group 3: The only difference from Example 1 is the test solution: weigh 1g of Lianhuang granule powder, add it to a 50mL volumetric flask, add 25mL of anhydrous ethanol, ultrasonically treat for 20min, cool, and then dilute to 50mL with anhydrous ethanol, shake well, filter, and obtain the test solution;

[0129] Experimental group 4: The only difference from Example 1 is the test solution: weigh 1 g of Lianhuang granule powder, add it to a 50 mL volumetric flask, add 25 mL of 50% ethanol by volume, ultrasonically treat for 20 min, cool, and then dilute to 50 mL with 50% ethanol by volume, shake well, and filter to obtain the test solution;

[0130] (2) Test results

[0131] Experimental groups 1 to 4 were able to achieve qualitative analysis of andrographolide, dehydroandrographolide, aesculin, aesculetin, baicalin and gallic acid in Lianhuang Granules. However, the HPLC chromatograms of Lianhuang Granules obtained from experimental groups 2 to 4 had low separation and many impurity peaks, while the extraction rate of experimental group 1 was high, and the HPLC chromatograms of Lianhuang Granules obtained from experimental group 1 had high separation and few impurity peaks.

[0132] Experiment 3: Effect of test solution concentration

[0133] (1) Test method

[0134] Experimental group 1: The only difference from Example 1 is the test solution: weigh 1 g of Lianhuang granule powder, add it to a 50 mL volumetric flask, add 20 mL of 50% methanol by volume, ultrasonically treat for 30 min, cool, and then dilute to 50 mL with 50% methanol by volume, shake well, and filter to obtain the test solution;

[0135] Experimental group 2: The only difference from Example 1 is the test solution: weigh 1 g of Lianhuang granule powder, add it to a 25 mL volumetric flask, add 20 mL of 50% methanol by volume, ultrasonically treat for 30 min, cool, and then dilute to 25 mL with 50% methanol by volume, shake well, and filter to obtain the test solution;

[0136] Experimental group 3: The only difference from Example 1 is the test solution: weigh 1 g of Lianhuang granule powder, add it to a 100 mL volumetric flask, add 20 mL of 50% methanol by volume, ultrasonically treat for 30 min, cool, and then dilute to 100 mL with 50% methanol by volume, shake well, and filter to obtain the test solution;

[0137] (2) Test results

[0138] Experimental groups 1 to 3 were able to achieve qualitative analysis of andrographolide, dehydroandrographolide, aesculin, aesculetin, baicalin and gallic acid in Lianhuang Granules. However, the HPLC chromatograms of Lianhuang Granules obtained from experimental groups 2 to 3 had low separation and many impurity peaks, while the HPLC chromatogram of Lianhuang Granules obtained from experimental group 1 had high separation and few impurity peaks.

[0139] Experiment 4: Effect of ultrasound time

[0140] (1) Test method

[0141] Experimental group 1: Same as Example 1, test solution: weigh 1 g of Lianhuang granule powder, add it to a 50 mL volumetric flask, add 25 mL of 50% methanol by volume, ultrasonically treat for 20 min, cool, and then dilute to 50 mL with 50% methanol by volume, shake well, and filter to obtain the test solution;

[0142] Experimental group 2: The only difference from Example 1 is the test solution: weigh 1 g of Lianhuang granule powder, add it to a 50 mL volumetric flask, add 25 mL of 50% methanol by volume, ultrasonically treat for 10 min, cool, and then dilute to 50 mL with 50% methanol by volume, shake well, and filter to obtain the test solution;

[0143] Experimental group 3: The only difference from Example 1 is the test solution: weigh 1g of Lianhuang granule powder, add it to a 50mL volumetric flask, add 25mL of 50% methanol by volume, ultrasonically treat for 30min, cool, and then dilute to 50mL with 50% methanol by volume, shake well, and filter to obtain the test solution;

[0144] (2) Test results

[0145] Experimental groups 1 to 3 were able to achieve qualitative analysis of andrographolide, dehydroandrographolide, aesculin, aesculetin, baicalin and gallic acid in Lianhuang Granules. However, the HPLC chromatograms of Lianhuang Granules obtained from experimental groups 2 to 3 had low separation and many impurity peaks, while the extraction rate of experimental group 1 was high, and the HPLC chromatograms of Lianhuang Granules obtained had high separation and few impurity peaks.

[0146] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting the contents of six active ingredients in Lianhuang granules, characterized in that: The six active ingredients in Lianhuang granules were detected by high performance liquid chromatography using acetonitrile and 0.1% phosphoric acid solution as mobile phases. The six active ingredients were andrographolide, dehydroandrographolide, aesculetin, aesculetin, baicalin and gallic acid. After dissolving the Lianhuang granules with a solvent, a test solution is obtained, and the test solution is tested, wherein the solvent includes one of anhydrous methanol, anhydrous ethanol, methanol with a volume fraction of 50%, and ethanol with a volume fraction of 50%; In the high performance liquid chromatography method, the chromatographic column filler is octadecylsilane bonded silica gel, and the chromatographic column is a Symmetuy C18 chromatographic column; The high performance liquid chromatography method adopts a gradient elution method, and the gradient elution conditions are specifically as follows: ； The high performance liquid chromatography method adopts a segmented wavelength conversion method, and the segmented wavelength conversion conditions are specifically as follows: 。 2. The method for simultaneously detecting the contents of six active ingredients in Lianhuang granules according to claim 1, characterized in that: It also includes the use of external standard method for quantitative determination of the contents of dehydroandrographolide, andrographolide, fraxinoside, fraxinoside, baicalin and gallic acid in Lianhuang Granules.

3. The method for simultaneously detecting the contents of six active ingredients in Lianhuang granules according to claim 1, characterized in that: The solvent is methanol with a volume fraction of 50%.

4. The method for simultaneously detecting the contents of six active ingredients in Lianhuang granules according to claim 1, characterized in that: The mass volume ratio of the lotus yellow granules to the solvent is 1g:25~100mL.

5. The method for simultaneously detecting the contents of six active ingredients in Lianhuang granules according to claim 1, characterized in that: The dissolution is ultrasonic dissolution, and the ultrasonic time is 10 to 30 minutes.

6. Application of the method for simultaneously detecting the contents of six active ingredients in Lianhuang Granules as described in any one of claims 1 to 5 in the quality inspection of Lianhuang Granules.

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