HPLC (High Performance Liquid Chromatography) characteristic chromatogram of andrographolide sulfonate injection and detection method of sulfonate content

Abstract

The invention discloses an HPLC (High Performance Liquid Chromatography) specific chromatogram of an andrographolide sulfonate injection and a detection method for the content of a sulfonate, wherein the HPLC specific chromatogram detection method adopts a high performance liquid chromatography method for detection. The HPLC characteristic chromatogram and sulfonated substance content detection of the andrographolide sulfonated substance injection is realized through specific chromatographic conditions, the method has the characteristics of strong specificity and high precision, the detection method is scientific and advanced, the result is accurate, the evaluation mode is reasonable, and the method has the characteristics of operability, high sensitivity and the like. The method can effectively replace an ultraviolet spectrophotometric method to measure the content of the total sulfonated substance, the result precision is higher, and the quality of the andrographolide sulfonated substance and the preparation thereof can be rapidly and effectively controlled.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a characteristic spectrum of andrographolide sulfonate injection and a method for detecting the sulfonate content. Background Art

[0002] Andrographolide is the main active ingredient of the natural plant Andrographis paniculata. It has the effects of removing heat and detoxification, anti-inflammatory and analgesic, and has special effects on bacterial and viral upper respiratory tract infections and dysentery. Andrographolide is insoluble in water. Through the sulfonation process, hydrophilic groups are introduced to prepare andrographolide sulfonate, which can be fused with water and has significant antibacterial and anti-inflammatory effects. Andrographolide sulfonate injection prepared by dissolving andrographolide sulfonate in water has been registered and sold by many companies in accordance with the "Regulations on Veterinary Drug Administration" and the "Veterinary Drug Registration Measures" for the auxiliary treatment of swine mycoplasma pneumonia.

[0003] At present, the content of andrographolide sulfonate in andrographolide sulfonate injection is determined by UV-visible spectrophotometry. UV-visible spectrometer is only applicable to microanalysis. For high concentration (generally refers to concentration> 0.01mol / L) substances, the relationship between the absorbance and concentration of the substance deviates, so Lambert-Beer's law is not applicable. There are many factors that affect the deviation of Beer's law, such as non-monochromatic light, stray light, noise, chemical factors, etc. And there are many external or internal factors that affect optical system parameters, and the error is difficult to correct well, which has a greater impact on the accuracy of the test results, and the test results cannot be used as a basis for certification. Therefore, it is necessary to provide a detection method for andrographolide sulfonate injection with higher accuracy so that its quality can be effectively controlled. Summary of the invention

[0004] In order to effectively control the quality of andrographolide sulfonate injection, the present invention provides a HPLC characteristic spectrum detection method of andrographolide sulfonate injection, which adopts high performance liquid chromatography detection, and the specific steps are as follows:

[0005] a. Preparation of test solution: Take andrographolide sulfonate injection, add water and mix well to obtain the solution;

[0006] b. Preparation of reference solution: Take the sulfonated reference substance and dissolve it in water to obtain the reference solution;

[0007] c. Take the test solution and the reference solution and inject them into the chromatograph respectively. The chromatographic conditions are as follows:

[0008] Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: acetonitrile as mobile phase A, potassium dihydrogen phosphate buffer as mobile phase B; gradient elution program:

[0009]

[0010]

[0011] Furthermore, the volume ratio of the andrographolide sulfonate injection in step a to water is 5:25-125.

[0012] Furthermore, the reference solution in step b contains 10 to 1000 μg of sulfonate per 1 ml.

[0013] Furthermore, the sulfonate includes 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and / or 17-hydro-9-dehydroandrographolide.

[0014] Furthermore, the chromatographic column in the chromatographic conditions of step c is: Diamonsil C 18 , column length 250 mm, inner diameter 4.6 mm, particle size 5 μm, column temperature 25-35 °C, injection volume 5-25 μL, flow rate 0.8-1.2 mL·min -1 , detection wavelength 220~230nm.

[0015] Furthermore, the chromatographic conditions include a column temperature of 25°C, an injection volume of 10 μL, and a flow rate of 0.1 mL·min -1 , detection wavelength 225nm.

[0016] Furthermore, the potassium dihydrogen phosphate buffer in step c is a solution containing 1.361 g of potassium dihydrogen phosphate and 1.0 g of sodium heptane-1-sulfonate per 1000 ml of water.

[0017] Furthermore, there are 8 characteristic peaks in the chromatogram of the test solution, of which 3 peaks should correspond to the retention times of the corresponding reference chromatographic peaks, and the retention times of the remaining peaks are peak 2: 29.366±1, peak 4: 30.905±1, peak 5: 37.749±1, peak 6: 47.059±1, and peak 7: 52.957±1.

[0018] The present invention also provides a method for detecting the content of sulfonate in andrographolide sulfonate injection, comprising the following steps:

[0019] 1) taking andrographolide sulfonate injection and testing it according to the method described in any one of claims 1 to 7;

[0020] 2) Calculate the content of sulfonate in andrographolide sulfonate injection by peak area using the external standard method.

[0021] Furthermore, the sulfonate includes 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and / or 17-hydro-9-dehydroandrographolide; the total sulfonate content in the andrographolide sulfonate injection is 7.79 to 7.86 times the sum of the contents of 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and 17-hydro-9-dehydroandrographolide.

[0022] The HPLC characteristic spectrum of andrographolide sulfonate injection and the detection method of sulfonate content of the present invention realize the HPLC characteristic spectrum of andrographolide sulfonate injection and sulfonate content detection through specific chromatographic conditions, and have the characteristics of strong specificity and high precision, scientific and advanced detection method, accurate results, reasonable evaluation method, operability, can effectively replace ultraviolet spectrophotometry to carry out the content determination of total sulfonate and the result is more accurate. The present invention applies the characteristic spectrum technology to the quality control of Chinese veterinary drug injection, only needs to carry out one high-performance liquid phase detection, can simultaneously determine the content of total sulfonate and index components, embodies the advantage of one test and multiple evaluations, and enables andrographolide sulfonate and its preparation to obtain rapid and effective quality control.

[0023] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Characteristic spectrum of andrographolide sulfonate by HPLC

[0026] Figure 2 Results of HPLC of andrographolide sulfonate

[0027] Figure 3 Results of HPLC of Andrographolide Sulfonate Injection

[0028] Figure 4 Results of HPLC of Andrographolide Sulfonate Injection DETAILED DESCRIPTION

[0029] Example 1 Characteristic spectrum detection of andrographolide sulfonate injection of the present invention

[0030] (1) Preparation of reference solution: Take sodium 17-hydro-9-dehydroandrographolide-3,19-disulfate, sodium 17-hydro-9-dehydroandrographolide-19-sulfate and a reference substance of 17-hydro-9-dehydroandrographolide, accurately weigh them, and add water to prepare a mixed reference solution containing 100 μg of each of sodium 17-hydro-9-dehydroandrographolide-3,19-disulfate, sodium 17-hydro-9-dehydroandrographolide-19-sulfate and 17-hydro-9-dehydroandrographolide per ml.

[0031] (2) Preparation of test solution: Accurately measure 5 ml of andrographolide sulfonate injection and place it in a 50 ml volumetric flask. Add water to dilute to the mark and shake well.

[0032] (3) Accurately measure 10 μl of the reference solution and the test solution respectively and inject them into the liquid chromatograph. The chromatographic conditions are as follows:

[0033] Column: Diamonsil C 18 , column length is 250 mm, inner diameter is 4.6 mm, and particle size is 5 μm;

[0034] Mobile phase: acetonitrile as mobile phase A, potassium dihydrogen phosphate buffer (1.361 g potassium dihydrogen phosphate and 1.0 g sodium heptane-1-sulfonate per 1000 ml water) as mobile phase B;

[0035] The column temperature was 25°C; the detection wavelength was 225 nm, and the flow rate was 1.0 ml / min;

[0036] Gradient elution:

[0037]

[0038]

[0039] Theoretical plate number: calculated based on the peak of 17-hydro-9-dehydroandrographolide-19-sulfate sodium ester, should be no less than 10,000.

[0040] (4) There are 8 characteristic peaks in the chromatogram of the test solution, of which 3 peaks should correspond to the retention times of the corresponding reference peaks, namely 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium (peak 1), 17-hydro-9-dehydroandrographolide-19-sulfate sodium (peak 3), and 17-hydro-9-dehydroandrographolide (peak 8). The retention times of the remaining peaks are 29.366±1 (peak 2), 30.905±1 (peak 4), 37.749±1 (peak 5), 47.059±1 (peak 6), and 52.957±1 (peak 7).

[0041] Example 2 Determination of the content of andrographolide sulfonate injection of the present invention

[0042] (1) Preparation of reference solution: Take sodium 17-hydro-9-dehydroandrographolide-3,19-disulfate, sodium 17-hydro-9-dehydroandrographolide-19-sulfate and a reference substance of 17-hydro-9-dehydroandrographolide, accurately weigh them, and add water to prepare a mixed reference solution containing 100 μg of each of sodium 17-hydro-9-dehydroandrographolide-3,19-disulfate, sodium 17-hydro-9-dehydroandrographolide-19-sulfate and 17-hydro-9-dehydroandrographolide per ml.

[0043] (2) Preparation of test solution: Accurately measure 5 ml of andrographolide sulfonate injection and place it in a 50 ml volumetric flask. Add water to dilute to the mark and shake well.

[0044] (3) Accurately measure 10 μl of the reference solution and the test solution respectively and inject them into the liquid chromatograph. The chromatographic conditions are as follows:

[0045] Column: Diamonsil C 18 , column length is 250 mm, inner diameter is 4.6 mm, and particle size is 5 μm;

[0046] Mobile phase: acetonitrile as mobile phase A, potassium dihydrogen phosphate buffer (1.361 g potassium dihydrogen phosphate and 1.0 g sodium heptane-1-sulfonate per 1000 ml water) as mobile phase B;

[0047] The column temperature was 25°C; the detection wavelength was 225 nm; the flow rate was 1.0 ml / min;

[0048] Gradient elution:

[0049]

[0050] Theoretical plate number: calculated based on the peak of 17-hydro-9-dehydroandrographolide-19-sulfate sodium ester, should be no less than 10,000.

[0051] (4) The contents of 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and 17-hydro-9-dehydroandrographolide in andrographolide sulfonate injection were calculated by the external standard method based on the peak area.

[0052] The content of total sulfonated andrographolide is 7.79 to 7.86 times the sum of the contents of 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and 17-hydro-9-dehydroandrographolide.

[0053] The beneficial effects of the present invention are further illustrated by test examples below.

[0054] Test Example 1 Study on the quality control method of andrographolide sulfonate injection of the present invention

[0055] 1. Detection of andrographolide sulfonate injection by traditional UV spectrophotometry

[0056] 1.1 Methods

[0057] Preparation of reference solution: Take about 0.1 g of andrographolide reference substance, weigh accurately, place in a 25 ml volumetric flask, accurately add 5 ml of water, add appropriate amount of anhydrous ethanol, seal, sonicate to dissolve, cool, add anhydrous ethanol to the scale, shake well, and the solution is obtained (each 1 ml contains 4 mg of andrographolide).

[0058] Preparation of reference solution: Accurately measure 1 ml, 2 ml, 3 ml, 4 ml and 5 ml of reference solution and place them in 25 ml volumetric bottles respectively. Accurately measure 3 ml of 1% m-dinitrobenzene ethanol solution and place it in a 25 ml volumetric bottle as reference solution. Add 80% ethanol to each bottle to make it 5.0 ml. Accurately add 3 ml of 1% m-dinitrobenzene ethanol solution and 10 ml of ethanol solution (take 240.0 g of anhydrous ethanol, place it in a 500 ml volumetric bottle, add water to the mark, and shake well) to each bottle, shake well, and place at 60°C. In a water bath, accurately add 5 ml of potassium hydroxide solution (take 1.37 g of potassium hydroxide and 5.9 g of sodium citrate, put them in the same 100 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well), shake well, heat for 40 minutes, cool, add the above ethanol solution to the scale, shake well, and irradiate with UV-visible spectrophotometry within 30 minutes (Appendix 0401 of Part II of the "Chinese Pharmacopoeia of Veterinary Medicine" 2020 edition), determine the absorbance at a wavelength of 407 nm, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0059] Accurately measure 5 ml of each andrographolide sulfonate injection sample, place in a 25 ml volumetric flask, add anhydrous ethanol to the mark, shake well, accurately measure 4 ml, place in a 25 ml volumetric flask, and follow the method under the standard curve preparation, starting from "add 80% ethanol to 5.0 ml each", determine the absorbance according to the law, read the amount of andrographolide in the test solution from the standard curve, calculate, and multiply the result by 1.291 to obtain the result.

[0060] 2. Results

[0061] In the actual operation of the above method, as the concentration of the reference substance changes, the absorbance varies. At the same time, the maximum absorption wavelength changes significantly, does not meet the deviation of ±2nm, and decreases with increasing concentration. The actual test results are shown in Table 1.

[0062] Table 1 UV spectrophotometric content determination

[0063]

[0064] The above results indicate that the current traditional method for determining the total sulfonate content has certain systematic defects, and the test results cannot effectively represent the changes in the content of total sulfonate of andrographolide.

[0065] 2. Detection of andrographolide sulfonate injection by high performance liquid chromatography of the present invention

[0066] According to Example 1, the sample of andrographolide sulfonate injection was tested to obtain the characteristic spectrum of andrographolide sulfonate ( Figure 1 ), there are 8 main common characteristic peaks, and the relative retention times are 4.125min (peak 1), 29.366 (peak 2), 30.250 (peak 3) (S), 30.905 (peak 4), 37.749 (peak 5), 47.059 (peak 6), 52.957 (peak 7), 53.904 (peak 8)

[0067] The retention time of Peak 1 (17-hydrogen-9-dehydroandrographolide-3,19-sodium disulfate) is 4.125 min. In the sample chromatogram, the peak area of ​​Peak 1 is 664.72766-673.81287, and the peak area of ​​the reference substance is 533.08983-537.81921. After conversion, it can be known that the amount of 17-hydrogen-9-dehydroandrographolide-3,19-sodium disulfate contained in each 1 ml of andrographolide sulfonate injection is 0.8 mg.

[0068] The retention time of Peak 3 (17-hydrogen-9-dehydroandrographolide-19-sodium sulfate) is 30.250 min. In the sample chromatogram, the peak area of ​​Peak 3 is 1751.29819-1779.28973, and the peak area of ​​the reference substance is 1348.89192-1357.28191. After conversion, it can be known that the amount of 17-hydrogen-9-dehydroandrographolide-19-sodium sulfate contained in each 1 ml of andrographolide sulfonate injection is 1.3 mg.

[0069] The retention time of Peak 8 (17-hydrogen-9-dehydroandrographolide) is 53.904 min. In the sample chromatogram, the peak area of ​​Peak 8 is 1238.81927-1246.29917, and the peak area of ​​the reference substance is 1121.28937-1125.29808. After conversion, it can be known that the amount of 17-hydrogen-9-dehydroandrographolide contained in each 1 ml of andrographolide sulfonate injection is 1.1 mg.

[0070] From the above research results, it can be seen that peaks 1, 3, and 8 can be quantitatively analyzed, and the content determination results are stable, with good precision and accuracy. The sum of the mass of peaks 1, 3, and 8 is 3.2 mg / ml. Combined with the content determination results of andrographolide total sulfonate (25±0.07 mg / ml) and the dosage of andrographolide sulfonate injection (25 mg / ml), it can be calculated that the sum of the mass of peaks 1, 3, and 8 has a fixed multiple relationship with the content determination results of andrographolide total sulfonate, that is, the content determination results of andrographolide total sulfonate are 7.79 to 7.86 times the sum of the mass of peaks 1, 3, and 8. Combined with the stability test data, the sum of the mass of peaks 1, 3, and 8 is compared with the content determination results of andrographolide total sulfonate, and the results are shown in Tables 2 and 3:

[0071] Table 2 Accelerated stability test results

[0072]

[0073] Table 3 Long-term stability test results

[0074]

[0075] From the test results in the table above, it can be seen that the content change of total sulfonated andrographolide has a fixed multiple relationship with the sum of the mass of peaks 1, 3, and 8, and the coefficient is 7.79-7.86, which is stable and durable. The experimental results have proved that the sum of the mass of peaks 1, 3, and 8 can effectively represent the total sulfonated content in andrographolide sulfonates and their preparations. The total sulfonated andrographolide in each 1 ml of andrographolide sulfonate preparations should be 3.0-3.3 mg, calculated as the sum of the mass of peaks 1, 3, and 8.

[0076] In the process of quality monitoring of andrographolide sulfonates and their preparations, the content of total andrographolide sulfonates can be directly calculated through quantitative analysis of peaks 1, 3, and 8, avoiding the systematic errors of the ultraviolet method. The total sulfonates, index component contents, and characteristic spectrum similarity can be evaluated simultaneously through a single measurement.

[0077] Test Example 2 Quality Control Verification of Andrographolide Sulfonate Injection of the Present Invention

[0078] 1. Verification ①

[0079] 1. Detection of active ingredients by the method of the present invention

[0080] Preparation of reference solution: Take appropriate amounts of 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and 17-hydro-9-dehydroandrographolide reference substance, accurately weigh, and add water to prepare a mixed reference solution containing 100 μg of each of 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and 17-hydro-9-dehydroandrographolide per 1 ml.

[0081] Preparation of the test solution: Accurately measure 0.125 g of andrographolide sulfonate injection from three batches respectively, place in a 50 ml volumetric flask, add water to dilute to the scale, shake well, and obtain the solution.

[0082] Chromatographic conditions and system suitability tests were performed using octadecylsilane bonded silica gel as filler (Diamonsil C 18 , column length 250mm, inner diameter 4.6mm, particle size 5μm); acetonitrile as mobile phase A, potassium dihydrogen phosphate buffer (1.361g potassium dihydrogen phosphate and 1.0g sodium heptane-1-sulfonate per 1000ml water) as mobile phase B, gradient elution as specified in the table below; column temperature 25℃; detection wavelength 225nm, flow rate 1.0ml / min. The theoretical plate number calculated based on the 17-hydrogen-9-dehydroandrographolide-19-sulfate sodium peak should not be less than 10000.

[0083]

[0084] Determination method: Accurately measure 10μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and calculate by peak area according to external standard method.

[0085] Typical chromatograms of sample detection are shown in Figure 2 :

[0086] It can be seen from the test results that the content determination results of andrographolide sulfonate are clear and the retention time of each main chromatographic peak is stable.

[0087] 2. UV spectrophotometry to detect total sulfonates

[0088] The contents of total andrographolide sulfonates in three batches of andrographolide sulfonate injections were detected according to the method under “Detection of andrographolide sulfonate injection by traditional ultraviolet spectrophotometry” in Experimental Example 1.

[0089] 3. Results

[0090] The results of UV content determination and HPLC content determination were statistically analyzed, and the results are shown in Table 4:

[0091] Table 4 Content determination results

[0092]

[0093] As can be seen from the table above, the content change of total andrographolide sulfonates has a fixed multiple relationship with the sum of the mass of peaks 1, 3, and 8. The experimental results show that the coefficient is in the range of 7.79-7.86. The total andrographolide sulfonates contained in each 1 ml of andrographolide sulfonate preparation are calculated as the sum of the mass of peaks 1, 3, and 8, which is 3.18-3.21 mg.

[0094] 2. Verification ②

[0095] 1. The method of the present invention detects active ingredients

[0096] Preparation of reference solution: Take appropriate amounts of 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and 17-hydro-9-dehydroandrographolide reference substance, accurately weigh, and add water to prepare a mixed reference solution containing 100 μg of each of 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and 17-hydro-9-dehydroandrographolide per 1 ml.

[0097] Preparation of the test solution: Accurately measure 5 ml of 3 batches respectively, place in a 50 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0098] Chromatographic conditions and system suitability tests were performed using octadecylsilane bonded silica gel as filler (Diamonsil C 18 , column length 250mm, inner diameter 4.6mm, particle size 5μm); acetonitrile as mobile phase A, potassium dihydrogen phosphate buffer (1.361g potassium dihydrogen phosphate and 1.0g sodium heptane-1-sulfonate per 1000ml water) as mobile phase B, gradient elution as specified in the table below; column temperature 25℃; detection wavelength 225nm, flow rate 1.0ml / min. The theoretical plate number calculated based on the 17-hydrogen-9-dehydroandrographolide-19-sulfate sodium peak should not be less than 10000.

[0099]

[0100] Determination method: Accurately measure 10μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and calculate by peak area according to external standard method.

[0101] Typical chromatograms of sample detection are shown in Figure 3 :

[0102] It can be seen from the test results that the content determination results of andrographolide sulfonate are clear and the retention time of each main chromatographic peak is stable.

[0103] 2. UV spectrophotometry to detect total sulfonates

[0104] The contents of total andrographolide sulfonates in three batches of andrographolide sulfonate injections were detected according to the method under “Detection of andrographolide sulfonate injection by traditional ultraviolet spectrophotometry” in Experimental Example 1.

[0105] 3. Results

[0106] The results of UV content determination and HPLC content determination were statistically analyzed, and the results are shown in Table 5:

[0107] Table 5 Content determination results

[0108]

[0109] As can be seen from the table above, the content change of total andrographolide sulfonates has a fixed multiple relationship with the sum of the mass of peaks 1, 3, and 8. The experimental results show that the coefficient is in the range of 7.79-7.86. The total andrographolide sulfonates contained in each 1 ml of andrographolide sulfonate preparation is 3.17-3.21 mg, calculated as the sum of the mass of peaks 1, 3, and 8.

[0110] 3. Verification ③

[0111] 1. Methods

[0112] Preparation of reference solution: Take appropriate amounts of 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and 17-hydro-9-dehydroandrographolide reference substance, accurately weigh, and add water to prepare a mixed reference solution containing 200 μg of each of 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and 17-hydro-9-dehydroandrographolide per 1 ml.

[0113] Preparation of the test solution: Accurately measure 5 ml of 3 batches of andrographolide sulfonate injection respectively, place in a 50 ml volumetric flask, add water to dilute to the scale, shake well, and obtain the solution.

[0114] Chromatographic conditions and system suitability tests were performed using octadecylsilane bonded silica gel as filler (Diamonsil C 18, column length 250mm, inner diameter 4.6mm, particle size 5μm); acetonitrile as mobile phase A, potassium dihydrogen phosphate buffer (1.361g potassium dihydrogen phosphate and 1.0g sodium heptane-1-sulfonate per 1000ml water) as mobile phase B, gradient elution as specified in the table below; column temperature 25℃; detection wavelength 225nm, flow rate 1.0ml / min. The theoretical plate number calculated based on the 17-hydrogen-9-dehydroandrographolide-19-sulfate sodium peak should not be less than 10000.

[0115]

[0116] Determination method: Accurately measure 10μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and calculate by peak area according to external standard method.

[0117] Typical chromatograms of sample detection are shown in Figure 4 :

[0118] It can be seen from the test results that the content determination results of andrographolide sulfonate are clear and the retention time of each main chromatographic peak is stable.

[0119] 2. UV spectrophotometry to detect total sulfonates

[0120] 3. Results

[0121] The results of UV content determination and HPLC content determination were statistically analyzed, and the results are shown in Table 6:

[0122] Table 6 Content determination results

[0123]

[0124] As can be seen from the table above, the content change of total andrographolide sulfonates has a fixed multiple relationship with the sum of the mass of peaks 1, 3, and 8. The experimental results show that the coefficient is in the range of 7.79-7.86. The total andrographolide sulfonates contained in each 1 ml of andrographolide sulfonate preparation are calculated as the sum of the mass of peaks 1, 3, and 8, which is 3.19-3.33 mg.

[0125] From the above-mentioned multiple batches of sample testing and verification, it was determined that the content of total sulfonates in andrographolide sulfonate injection fluctuates in the range of 90%-110% of the labeled amount. Statistical analysis found that the change in the content of total sulfonates of andrographolide was correlated with the sum of the masses of peaks 1, 3, and 8. As the content of total sulfonates increased, the sum of the masses of peaks 1, 3, and 8 also increased. Through stability research, the above two indicators have a strong correlation, and a fixed conversion coefficient of 7.79 to 7.86 can be converted, that is, 7.79 to 7.86 times the sum of the masses of peaks 1, 3, and 8, which is the mass of the total sulfonates of andrographolide in this product.

[0126] In summary, the present invention has the characteristics of strong specificity and high precision, the detection method is scientific and advanced, the result is accurate, the evaluation method is reasonable, and it is operable, and it can effectively replace the ultraviolet spectrophotometry to determine the content of total sulfonates with higher result accuracy. The present invention applies the characteristic spectrum technology to the quality control of Chinese veterinary drug injections, and only needs to perform one high-performance liquid phase detection to simultaneously determine the content of total sulfonates and index components, reflecting the advantage of one test and multiple evaluations, so that andrographolide sulfonates and preparations thereof can be quickly and effectively controlled in quality.

Claims

1. A HPLC characteristic spectrum detection method for andrographolide sulfonate injection, characterized in that: It is detected by high performance liquid chromatography, and the specific steps are as follows: a. Preparation of test solution: Take andrographolide sulfonate injection, add water and mix well to obtain the solution; b. Preparation of reference solution: Take the sulfonated reference substance and dissolve it in water to obtain the reference solution; c. Take the test solution and the reference solution and inject them into the chromatograph respectively. The chromatographic conditions are as follows: Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: acetonitrile as mobile phase A, potassium dihydrogen phosphate buffer as mobile phase B; gradient elution program:

2. The HPLC characteristic spectrum detection method according to claim 1, wherein: The volume ratio of the andrographolide sulfonate injection in step a to water is 5:25-125.

3. The HPLC characteristic spectrum detection method according to claim 1, wherein: The reference solution in step b contains 10 to 1000 μg of sulfonate per 1 ml.

4. The HPLC characteristic spectrum detection method according to claim 1 or 3, characterized in that: The sulfonated product includes 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and / or 17-hydro-9-dehydroandrographolide.

5. The HPLC characteristic spectrum detection method according to claim 1, characterized in that: The chromatographic column in the chromatographic conditions of step c is: Diamonsil C 18 , column length 250 mm, inner diameter 4.6 mm, particle size 5 μm, column temperature 25-35 °C, injection volume 5-25 μL, flow rate 0.8-1.2 mL·min -1 , detection wavelength 220~230nm.

6. The HPLC characteristic spectrum detection method according to claim 5, characterized in that: The chromatographic conditions were as follows: column temperature 25°C, injection volume 10 μL, flow rate 1 mL·min -1 , detection wavelength 225nm.

7. The HPLC characteristic spectrum detection method according to claim 1, characterized in that: The potassium dihydrogen phosphate buffer in step c is a solution containing 1.361 g of potassium dihydrogen phosphate and 1.0 g of sodium heptane-1-sulfonate per 1000 ml of water.

8. The HPLC characteristic spectrum detection method according to claim 1, characterized in that: There are 8 characteristic peaks in the chromatogram of the test solution, of which 3 peaks should correspond to the retention times of the corresponding reference chromatographic peaks, and the retention times of the remaining peaks are peak 2: 29.366±1, peak 4: 30.905±1, peak 5: 37.749±1, peak 6: 47.059±1, and peak 7: 52.957±1.

9. A method for detecting the content of sulfonate in andrographolide sulfonate injection, characterized in that: The steps include: 1) taking andrographolide sulfonate injection and testing it according to the method described in any one of claims 1 to 7; 2) Calculate the content of sulfonate in andrographolide sulfonate injection by peak area using the external standard method.

10. The content detection method according to claim 9, characterized in that: The sulfonated product includes 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and / or 17-hydro-9-dehydroandrographolide; the total sulfonated product content in the andrographolide sulfonate injection is 7.79 to 7.86 times the sum of the contents of 17-hydro-9-dehydroandrographolide-3,19-disulfate sodium, 17-hydro-9-dehydroandrographolide-19-sulfate sodium and 17-hydro-9-dehydroandrographolide.

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