Detection method for simultaneously determining main components and related substances of compound glycyrrhizin tablets by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a detection method for simultaneously determining main components and related substances of compound glycyrrhizin tablets by utilizing an HPLC (High Performance Liquid Chromatography) method. The HPLC-CAD method is adopted to measure the content of main components and related substances of the compound glycyrrhizin tablets, and high sensitivity and good reproducibility are achieved. Compared with a derivatization method, the detection method disclosed by the invention is simple and rapid to operate, and can realize the determination of the content of the main components and the detection of related impurities without carrying out derivatization reaction on the sample. Meanwhile, by optimizing chromatographic conditions, main components and impurities in the compound glycyrrhizin tablets can be well separated. Further, a plurality of unknown impurities can be identified by mass spectrometric detection. In conclusion, the accuracy and the sensitivity of impurity analysis in the compound glycyrrhizin tablet can be improved, and a targeted production process optimization scheme can be provided for related research.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a detection method for simultaneously determining the main component and related substances of compound glycyrrhizin tablets by utilizing an HPLC method. Background Art

[0002] Licorice is known as the king of traditional Chinese medicines, with excellent effects on tonifying the spleen and replenishing qi, clearing away heat and detoxifying, and relieving acute pain. Glycyrrhizic acid, an active ingredient extracted from licorice, has liver-protecting and detoxifying, anti-inflammatory, antiviral, anti-tumor, and immunomodulatory effects. It is used clinically to create oral or injectable preparations for the treatment of various acute and chronic hepatitis, bronchitis, and peptic ulcers. Compound Glycyrrhizic Acid Tablets, a compound preparation composed of glycyrrhizic acid, glycine, and methionine, are used as an adjuvant medication for liver disease, improving abnormal liver function and showing a certain therapeutic effect on experimental hepatocellular damage, alcoholic cirrhosis, and hepatitis cirrhosis. It also has clinical application value in treating chronic urticaria, chronic eczema, and postherpetic neuralgia.

[0003] In current determination methods, glycine and methionine, as highly polar amino acids, exhibit poor retention on conventional C18 columns and weak UV absorption, necessitating derivatization methods for content determination. Imported standards currently utilize HPLC and post-column derivatization HPLC methods for the determination of glycyrrhizin, glycine, and methionine, respectively. These methods are cumbersome, and the derivatization reagents significantly interfere with the detection of impurities, resulting in poor reproducibility. The included related substance inspection items are more suitable for testing for glycyrrhizic acid monoammonium impurities. However, since post-column derivatization methods for glycine and methionine target amino groups, they do not fully reflect the impurity profile. Therefore, the development of an effective and reliable method for the direct determination of the main components and related substances in compound glycyrrhizin tablets is urgently needed. Summary of the Invention

[0004] The object of the present invention is to provide a detection method for simultaneously determining the main components and related substances of compound glycyrrhizin tablets by HPLC. The HPLC method provided by the present invention can be directly used to determine the content of the main components and related substances of compound glycyrrhizin tablets, has few interfering factors and good reproducibility.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a method for simultaneously determining the main component and related substances of compound glycyrrhizin tablets by HPLC, comprising the following steps: The sample solution of compound glycyrrhizin tablets is subjected to high performance liquid chromatography to obtain a chromatogram of the main component and related substances; the main component includes glycine glycoside, methionine and glycine; According to the chromatogram of the main component, the content of the main component in compound glycyrrhizin tablets was obtained by external standard method; According to the chromatograms of the related substances, the content of the related substances in the compound glycyrrhizin tablets was obtained by self-control method; The conditions for high performance liquid chromatography detection include: the chromatographic column filler is octadecylsilane bonded silica gel; mobile phase A is trifluoroacetic acid-water system, and mobile phase B is acetonitrile; Elution was gradient elution; The gradient elution program is: 0 min~3 min: the volume percentage of the mobile phase A is 98%; 3 min~15 min: the volume percentage of the mobile phase A is reduced from 98% to 20%; 16 min to 20 min: the volume percentage of the mobile phase A is 98%; The detector is an electrospray detector.

[0006] Preferably, the column temperature of the HPLC is 30°C; and the atomization temperature is 35°C.

[0007] Preferably, the volume concentration of trifluoroacetic acid in the trifluoroacetic acid-water system is 0.5%.

[0008] Preferably, after obtaining the chromatograms of the main component and related substances, the method further comprises performing mass spectrometry on the separated sample to determine the structure of the related substances; The mass spectrometry detection conditions included: ESI ionization source, positive / negative ion scanning, positive ion mode spray voltage 4.5 kV; heater voltage 8 V; lens voltage 115 V; In negative ion mode, the spray voltage was -4.0 kV; the heater voltage was -25 V; and the tube lens voltage was -100 V.

[0009] Preferably, the sheath gas flow rate of the mass spectrometry detection is 30 arb, the purge gas flow rate is 10 arb, the ion transmission capillary temperature is 375°C, and the scanning range is m / z 50-1500.

[0010] Preferably, the secondary mass spectrometry of the mass spectrometry detection adopts data-dependent scanning, and the secondary mass spectrometry collision-induced dissociation normalized energy value is set to 35%.

[0011] Preferably, the related substances include one or more of diglycine, methionine sulfoxide, methionine sulfone, glycyrrhizin G2, and 18α-glycyrrhizic acid.

[0012] Preferably, the chromatographic column is a C18 AQ chromatographic column.

[0013] Preferably, the flow rate of mobile phase A and mobile phase B is 0.8 mL / min.

[0014] Preferably, the injection volume of the HPLC is 10 μL.

[0015] The present invention uses an HPLC-CAD method to determine the content of the main component and related substances in compound glycyrrhizin tablets, demonstrating high sensitivity and good reproducibility. Compared to derivatization methods, the present invention's detection method is simple and rapid to operate, eliminating the need for sample derivatization reactions and enabling determination of the main component content and detection of related impurities. Furthermore, by optimizing chromatographic conditions, the present invention achieves excellent separation of the main component and impurities in compound glycyrrhizin tablets.

[0016] Furthermore, multiple unknown impurities can be identified by mass spectrometry. In summary, the present invention not only helps to improve the accuracy and sensitivity of impurity analysis in compound glycyrrhizin tablets, but also provides a targeted production process optimization solution for related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 The chromatograms of the blank solution, reference solution, and test solution of Example 1 are shown, where 1 is glycine; 2 is methionine; and 3 is glycyrrhizin. Figure 2 The chromatograms of the specific experiment of Example 1 are shown in Figures a and b, respectively, where a is the reference solution, b is the test solution, and c is the blank solution. Figure 3 is the linear spectrum of glycine in Example 1; Figure 4 This is the linear spectrum of methionine in Example 1; Figure 5 This is the linear spectrum of glycine glycoside in Example 1; Figure 6 This is the linear spectrum of the control solution in Example 2. DETAILED DESCRIPTION

[0019] The present invention provides a method for simultaneously determining the main component and related substances of compound glycyrrhizin tablets by HPLC, comprising the following steps: The sample solution of compound glycyrrhizin tablets is subjected to high performance liquid chromatography to obtain a chromatogram of the main component and related substances; the main component includes glycine glycoside, methionine and glycine; According to the chromatogram of the main component, the content of the main component in compound glycyrrhizin tablets was obtained by external standard method; According to the chromatograms of the related substances, the content of the related substances in the compound glycyrrhizin tablets was obtained by self-control method; Elution was gradient elution; The gradient elution program is: 0 min~3 min: the volume percentage of the mobile phase A is 98%; 3 min~15 min: the volume percentage of the mobile phase A is reduced from 98% to 20%; 16 min~20 min: the volume percentage of the mobile phase A is 98%; The detector is an electrospray detector.

[0020] The invention conducts high performance liquid chromatography detection on a sample solution of the compound glycyrrhizin tablets to obtain a chromatogram of a main component and related substances.

[0021] As an embodiment of the present invention, the conditions for the high performance liquid chromatography detection include: the filler of the chromatographic column is octadecylsilane bonded silica gel, and the chromatographic column can specifically be a C18 AQ chromatographic column. In the embodiment of the present invention, a SHIMADZU Shim-pack C18-AQ chromatographic column with a specification of 150 mm × 4.6 mm and 3 μm is used for illustration; the column temperature of the chromatographic column can be 30 ° C; As an embodiment of the present invention, the mobile phase A is a trifluoroacetic acid-water system; the volume concentration of trifluoroacetic acid in the trifluoroacetic acid-water system can be 0.5%; Mobile phase B is acetonitrile; the flow rate of mobile phase A and mobile phase B can be 0.8 mL / min; Elution is gradient elution; the program of gradient elution is: 0 min~3 min: the volume percentage of the mobile phase A is 98%; 3 min~15 min: the volume percentage of the mobile phase A is reduced from 98% to 20%; 16 min~20 min: the volume percentage of the mobile phase A is 98%; The injection volume can be 10 μL; the detector is an electrospray detector; and the nebulization temperature can be 35 °C.

[0022] As an embodiment of the present invention, after obtaining the chromatogram of the main component, the content of the main component in the compound glycyrrhizin tablets is obtained according to the chromatogram of the main component using an external standard method.

[0023] The external standard method is to obtain the content of the main component in the compound glycyrrhizin tablets based on the peak area of ​​the main component and the corresponding predetermined standard curve of the main component.

[0024] As an embodiment of the present invention, obtaining the predetermined standard curve of the corresponding principal component includes the following steps: A series of concentrations of glycine, methionine and glycyrrhizin mixed reference solutions were prepared and subjected to high performance liquid chromatography to obtain the peak area of ​​each main component; A linear fit is performed with the concentration of each main component as the abscissa and the peak area as the ordinate to obtain a predetermined standard curve for each main component.

[0025] As an embodiment of the present invention, the predetermined standard curve of glycine can be specifically Y = 9.1203X + 1.3847; the predetermined standard curve of methionine can be specifically Y = 13.517X + 1.7408; the predetermined standard curve of glycyrrhizin can be specifically Y = 16.659X + 1.4282.

[0026] As an embodiment of the present invention, after obtaining the chromatogram of the related substances, the present invention uses a self-control method based on the chromatogram of the related substances to obtain the content of the related substances in the compound glycyrrhizin tablets.

[0027] By combining high-performance liquid chromatography with a CAD detector, this method overcomes the difficulties in the retention and detection of amino acids (methionine and glycine), improving detection sensitivity and accuracy. This method enables the determination of glycine and methionine while simultaneously ensuring the retention and effective separation of glycyrrhizin and its impurities. This method offers the advantages of accuracy, rapidity, high sensitivity, and good reproducibility, enabling accurate detection of the content of compound glycyrrhizin tablets and related substances, thereby ensuring their safety and effectiveness.

[0028] As an embodiment of the present invention, after obtaining the chromatograms of the main component and related substances, the method further includes performing mass spectrometry on the separated samples to determine the structures of the related substances.

[0029] Mass spectrometry detection conditions included an ESI ionization source with positive / negative ion scanning. In positive ion mode, the spray voltage was 4.5 kV, the heater voltage was 8 V, and the lens voltage was 115 V. In negative ion mode, the spray voltage was -4.0 kV, the heater voltage was -25 V, and the lens voltage was -100 V. A sheath gas flow rate of 30 arb and a purge gas flow rate of 10 arb were used. The ion transfer capillary temperature was 375°C, and the scan range was m / z 50–1500. Data-dependent scanning was used for secondary mass spectrometry, and the normalized energy of collision-induced dissociation (CID) was set to 35%.

[0030] As an embodiment of the present invention, the secondary mass spectrometer adopts data-dependent MS / MS to monitor the secondary mass spectra of ions with determined mass numbers in the full scan of the primary mass spectrometer.

[0031] In order to further illustrate the present invention, the scheme of the present invention is described in detail below with reference to the embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0032] In the examples of the present invention, the reagents, drugs and equipment used are as follows: 1) Reagents and drugs: Glycine (batch number: 140689-202308, content: 100%, China Food and Drug Inspection Institute); methionine (batch number: 140684-202404, content: 99.9%, China Food and Drug Inspection Institute); and ammonium glycyrrhizate (batch number: 110731-202122, content: 94.4%, China Food and Drug Inspection Institute); trifluoroacetic acid (batch number: L9A0U0A, Beijing Bailingwei Technology Co., Ltd.); acetonitrile (batch number: L9A0U0A, Beijing Bailingwei Technology Co., Ltd.); ultrapure water (Milli-Q, laboratory-made, resistivity >18.2); Compound Glycyrrhizin Tablets (batch numbers: 20210901, 20210902, 20210904, Xinjiang Tefeng Pharmaceutical Co., Ltd.), each tablet containing 25 mg of glycyrrhizin, 25 mg of glycine, and 25 mg of DL-methionine.

[0033] Table 1 Prescription ingredients of compound glycyrrhizin tablets

[0034] 2) Instruments and equipment: Ultimate 3000 high-performance liquid chromatograph, Corona Veo RS electrospray detector, LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, USA); XP205 1 / 100,000 electronic balance (METTLER TOLEDO, Switzerland), IQ7000 ultrapure water machine (Millipore, USA).

[0035] Example 1 1. Chromatographic conditions A SHIMADZU Shim-pack C18-AQ column (150 mm×4.6 mm, 3 μm) was used; mobile phase A was 0.5% trifluoroacetic acid-water, mobile phase B was acetonitrile, and gradient elution was performed. The gradient elution program was as follows: 0 min~3 min: the volume percentage of the mobile phase A was 98%; 3 min~15 min: the volume percentage of the mobile phase A decreased from 98% to 20%; 16 min~20 min: the volume percentage of the mobile phase A was 98%, the flow rate was 0.8 mL / min, the column temperature was 30 °C, the nebulization temperature of the electrospray detector was 35 °C, and the injection volume was 10 μL.

[0036] 2. Solution Preparation Blank solution: 50 vol.% ethanol solution.

[0037] Reference solution: Take appropriate amounts of glycine reference, methionine reference, and ammonium glycyrrhizate reference, accurately weigh them, dissolve them in 50 vol.% ethanol solution, and quantitatively dilute them to make a solution containing approximately 250 μg of each per 1 mL.

[0038] Test solution: Take 20 tablets of this product (Compound Glycyrrhizin Tablets), accurately weigh, grind, accurately weigh an appropriate amount (equivalent to 25 mg of glycine, 25 mg of DL-methionine, and 25 mg of glycyrrhizin in each tablet of Compound Glycyrrhizin), place in a 100 mL volumetric flask, add 30 mL of 50 vol.% ethanol solution, shake thoroughly for more than 5 minutes, dilute to the scale with 50 vol.% ethanol solution, shake well, centrifuge, filter the supernatant, and take the filtrate, which is the test solution.

[0039] 3. Methodological Investigation (1) Specificity test Inject the reference solution and the test solution into the HPLC instrument and detect according to "1. Chromatographic conditions" and record the corresponding chromatograms. Under the chromatographic conditions, the retention times of glycine, methionine and glycyrrhizin are 2.476 min, 8.918 min and 13.796 min, respectively. The test results are shown in Figures 1 and 2 .

[0040] (2) Degradation experiment Compound glycyrrhizin tablets were accurately weighed and ground into a fine powder. An appropriate amount was then accurately weighed and subjected to strong acid degradation (1 mL of 1 mol / L hydrochloric acid solution for 4 hours), strong alkaline degradation (1 mL of 1 mol / L sodium hydroxide solution for 4 hours), oxidative degradation (1 mL of 3% hydrogen peroxide solution for 4 hours), high temperature degradation (heating at 80°C for 48 hours), and photodegradation (irradiation at 365 nm UV light for 7 days). After acid-base degradation, the tablets were neutralized, diluted to the mark with 50 vol.% ethanol, shaken, and filtered. The test solutions after each forced degradation were assayed. The results showed that the blank solution did not interfere with the determination of the analyte. However, the test solution was unstable under oxidative conditions, resulting in the formation of significant impurities. The impurity peaks in the chromatogram were well resolved from the main component peaks.

[0041] (3) Precision test The reference solution was injected into the HPLC six times for testing according to "1. Chromatographic Conditions." The peak area RSD of the six test results was required to be ≤2.0% to confirm the good precision of the test method. The results are shown in Table 2.

[0042] Table 2 Precision test results

[0043] Table 2 shows that the RSDs of glycine, methionine, and glycyrrhizin were 1.95%, 0.86%, and 1.30%, respectively, indicating good precision of the method.

[0044] (4) Repeatability test The reference solution and six test sample solutions were injected into the HPLC according to "1. Chromatographic Conditions". The RSD of the glycine, methionine, and glycyrrhizin content test results for six times was required to be ≤2.0% to confirm that the method had good repeatability. The results are shown in Table 3.

[0045] Table 3 Repeatability test results

[0046] Table 3 shows that the RSDs of glycine, methionine, and glycyrrhizin were 0.90%, 1.11%, and 1.14%, respectively, indicating good repeatability of the method.

[0047] (5) Solution stability test The reference and test solutions were left at room temperature for 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, and 24 h, respectively. Afterwards, they were injected into a high-performance liquid chromatograph and tested and calculated according to "1. Chromatographic Conditions." The RSD values ​​for the peak areas of glycine, methionine, and glycyrrhizin in the reference and test solutions were required to be no more than 2.0% within 24 h to confirm good solution stability. The results are shown in Table 4.

[0048] Table 4 Solution stability test results

[0049] As shown in Table 4, after being placed at room temperature for 24 hours, the RSD values ​​of the peak areas of glycine, methionine and glycyrrhizin in the reference solution were 1.77%, 1.95% and 1.28%, respectively; the RSD values ​​of the peak areas of glycine, methionine and glycyrrhizin in the test solution were 0.82%, 1.39% and 1.74%, respectively, indicating good solution stability.

[0050] (6) Linear test Appropriate amounts of the stock solutions of the precise glycine, methionine, and glycyrrhizin mixed reference substances were diluted with 50 vol.% ethanol solution to obtain a series of mixed reference substance solutions. The glycine concentrations were 0.1325, 0.2120, 0.2650, 0.3180, and 0.5300 mg mL -1 ; Methionine concentrations were 0.1249, 0.1998, 0.2498, 0.2997, and 0.4996 mg·mL -1 ; Glycyrrhizin concentrations were 0.1151, 0.1842, 0.2302, 0.2762, and 0.4604 mg·mL -1 10 μL of each concentration reference solution was injected into a high performance liquid chromatograph for detection, and the peak area was recorded. A linear regression equation was performed with the concentration X as the abscissa and the chromatographic peak area Y as the ordinate to obtain the linear regression equation of glycine, methionine, and glycyrrhizin in the compound glycyrrhizin tablets, see Figures 3-5 The results of linearity and range tests met the acceptance criteria.

[0051] Table 5 Linear range and regression equation of glycine, methionine and glycyrrhizin

[0052] (7) Accuracy test Accurately weigh approximately 12.5 mg of each of glycine, methionine, and glycyrrhizin reference substances into a 10 mL volumetric flask, dissolve in 50 vol.% ethanol solution, and dilute to the mark to serve as reference substance stock solutions. Accurately measure 2 mL of the reference substance stock solution and place it in a 10 mL volumetric flask. Dilute to the mark with 50 vol.% ethanol solution and shake well to prepare the reference substance solution. Take 20 tablets of this product, accurately weigh them, grind them into powder, accurately weigh an appropriate amount (equivalent to 25 mg of glycine, 25 mg of methionine, and 25 mg of glycyrrhizin in each tablet of compound glycyrrhizin), place them in a 100 mL volumetric flask, add 30 mL of 50 vol.% ethanol solution, shake thoroughly for more than 5 minutes, and dilute to the scale with 50 vol.% ethanol solution to prepare the test solution; Accurately measure 5 mL of each test solution and place it in a 10 mL volumetric flask. Accurately add 1.0 mL of the reference substance stock solution to each flask. Dilute to the mark with 50 vol.% ethanol solution, shake well, and prepare 6 replicates as spike recovery solutions. According to the test regulations, the recovery rate of the spiked recovery solution should be between 95% and 102%, and the RSD value of the recovery rate of the six samples should be no greater than 5.0% to confirm good accuracy. The results are shown in Table 6.

[0053] Table 6 Accuracy test results

[0054] The recovery rate is calculated as follows:

[0055] Where m 测 : measured masses of glycine, methionine and glycyrrhizin; m 原有 : The mass of glycine, methionine and glycyrrhizin contained in the original test solution; m 加入 : The actual mass of glycine, methionine and glycyrrhizin added.

[0056] As shown in Table 6, the average recoveries of glycine, methionine, and glycyrrhizin were 100.6%, 99.70%, and 97.62%, respectively, and the RSD values ​​were 2.25%, 2.09%, and 2.01%, respectively. The method had good accuracy.

[0057] (8) Durability test The reference solution and the test solution were separately injected into a high-performance liquid chromatograph for detection and calculation. Using the chromatographic conditions described in Example 1 as standard, minor changes in column temperature, flow rate, and CAD atomization temperature did not affect the determination of glycine, methionine, and glycyrrhizin in Compound Glycyrrhizin Tablets (RSD ≤ 5.0%). The conditions investigated are shown in Table 7, and the test results are shown in Tables 8-10.

[0058] Table 7 Durability consideration factors

[0059] Table 8 Glycine durability test results

[0060] Table 9 Methionine durability test results

[0061] Table 10 Glycyrrhizin durability test results

[0062] As can be seen from Tables 8 to 10, slight changes in column temperature, flow rate, and CAD atomization temperature do not affect the determination of glycine, methionine, and glycyrrhizin in compound glycyrrhizin tablets, indicating that the detection method has good durability.

[0063] 4. Sample Measurement Prepare reference and test solutions of Compound Glycyrrhizin Tablets from different batches according to the solution preparation method. Analyze according to "1. Chromatographic Conditions" and determine the peak areas. Calculate the contents of glycine, methionine, and glycyrrhizin using the external standard method based on the peak areas and the pre-determined standard curve. The results are shown in Table 11.

[0064] Table 11 Content determination results of compound glycyrrhizin tablets

[0065] Example 2 The method for detecting related substances in compound glycyrrhizin tablets comprises the following steps: Chromatographic conditions: Same as Example 1. Solution Preparation: Blank solution: 50 vol.% ethanol solution.

[0066] Test solution: Take 20 tablets of this product, accurately weigh, grind, accurately weigh an appropriate amount (equivalent to 25 mg of compound glycyrrhizin glycine, 25 mg of DL-methionine, and 25 mg of glycyrrhizin per tablet), place in a 100 mL volumetric flask, add 30 mL of 50 vol.% ethanol solution, shake thoroughly for more than 5 minutes, dilute to the scale with 50 vol.% ethanol solution, shake well, centrifuge, filter the supernatant, and take the filtrate.

[0067] Reference solution: Accurately measure 1 mL of the test solution into a 50 mL volumetric flask and dilute to the mark with 50% ethanol solution.

[0068] Sensitivity solution: Accurately measure 1 mL of the control solution into a 20 mL volumetric flask and dilute to the mark with solvent.

[0069] 3. Methodological Review (1) System suitability test: Inject the control solution and the sensitivity solution into the HPLC for detection and record the corresponding chromatograms. The control solution should be injected six times continuously, and the peak area RSD should not exceed 2.0%. The signal-to-noise ratio of the glycyrrhizin peak height should be greater than 10.

[0070] The results showed that the RSD of the peak area of ​​the control solution after six consecutive injections was 0.99%, and the signal-to-noise ratio of the glycyrrhizin peak height in the sensitivity solution was 14.5, which met the requirements.

[0071] (2) Repeatability test: The control solution and 6 test sample solutions were injected into the HPLC instrument respectively. The impurity content test results were required to have an RSD of ≤3.0% to verify that the method has good repeatability. The results are shown in Table 12 (the impurity numbers in Table 12 correspond to Figure 2 Serial number in ).

[0072] Table 12 Repeatability test results

[0073] The results of 6 repeatability tests showed that the RSDs of impurities 4 and 5 in the selected compound glycyrrhizin tablets were 2.15% and 1.52%, respectively, indicating that the method had good repeatability.

[0074] (3) Linear test: An appropriate amount of the test solution was measured and diluted with 50 vol.% ethanol solution to obtain a series of glycyrrhizin concentration control solutions of 10.0 wt.%, 5.0 wt.%, 2.0 wt.%, 1.0 wt.%, and 0.5 wt.%. Each concentration control solution was injected into a high-performance liquid chromatograph, and the peak area was recorded. A linear regression equation was performed with the concentration X as the abscissa and the chromatographic peak area Y as the ordinate to obtain the linear regression equation for the glycyrrhizin series control solutions in compound glycyrrhizin tablets. The correlation coefficient met the requirements within the range of 0.2% to 10.0% of the main component glycyrrhizin; y = 0.1534x + 0.0483 ( r =0.9994, n=5). Linear spectrum see Figure 6 .

[0075] (4) Limit of quantification: The control solution was diluted stepwise with 50 vol.% ethanol solution to the required concentrations, and the sample concentration with a signal-to-noise ratio (S / N) of 10:1 was set as the limit of quantification. The results showed that when the test solution was diluted to 0.1%, the average S / N of the glycyrrhizin peak for 6 injections was 17.2, and the average value (Average) was >10SD (0.016>0.012), which met the method limit of quantification requirements.

[0076] (5) Detection limit: The detection limit was set at a sample concentration with a signal-to-noise ratio (S / N) of 3:1. Results showed that when the test solution was diluted to 0.05%, the average S / N of the three injections of glycyrrhizin peak was 7.9, with the average value (Average) > 3SD (0.0085 > 0.0036), meeting the method's detection limit.

[0077] (6) Durability: The test solution and the control solution were injected into the high performance liquid chromatograph for detection and calculation. The chromatographic conditions in Example 1 were used as standard conditions, and impurities 4 and 5 in the test sample were used as the investigation targets. The results showed that slight changes in column temperature, flow rate, CAD atomization temperature, etc. did not affect the determination of the main impurities in the compound glycyrrhizin tablets (the impurity numbers in Table 13 correspond to Figure 2 Serial number in ).

[0078] Table 13 Durability evaluation factors

[0079] 4. Sample Measurement Take compound glycyrrhizin tablets of different batches, prepare test solution and control solution according to the solution preparation method, analyze according to the chromatographic conditions, and determine the impurity peak area. Select the impurities with higher content and calculate them by the glycyrrhizin main component self-reference method. The results are shown in Table 14 (the impurity numbers in Table 14 correspond to Figure 2 Serial number in ).

[0080] Table 14 Detection results of major impurities in compound glycyrrhizin tablets

[0081] Example 3 Analysis and identification of related substances in compound glycyrrhizin tablets: Compound glycyrrhizin tablets are a combination preparation consisting of glycyrrhizin, glycine, and methionine. Different stereochemical configurations of the hydrogen at position 18 in the glycyrrhizic acid molecule result in the existence of two diastereomers: 18α-glycyrrhizin and 18β-glycyrrhizin. Currently, compound glycyrrhizin preparations mostly use the β-glycyrrhizic acid monoammonium salt as the primary ingredient. Glycine and methionine, as amino acid components, regulate renal function and enhance liver detoxification capacity. Methionine, however, is unstable under oxidative conditions.

[0082] Mass spectrometry conditions employed an ESI ionization source with positive / negative ion scanning. In positive ion mode, the spray voltage was 4.5 kV, the heater voltage was 8 V, and the tube lens voltage was 115 V. In negative ion mode, the spray voltage was -4.0 kV, the heater voltage was -25 V, and the tube lens voltage was -100 V. Sheath gas flow rate was 30 arb, purge gas flow rate was 10 arb, the ion transfer capillary temperature was 375°C, and the scan range was m / z 50–1500. Data-dependent MS / MS was used to monitor the mass of ions with confirmed masses during the full mass spectrometry scan. The normalized energy for collision-induced dissociation (CID) in the MS was set to 35%.

[0083] Impurity component analysis was combined with forced degradation test. The test solution was injected and analyzed by LC-CAD-MS. The possible compound structures were speculated by positioning some reference substances (bis-glycine, methionine sulfoxide, methionine sulfone) and mass spectrometry analysis. The results are shown in Table 15. (The impurity numbers in Table 15 correspond to Figure 2 Serial number in ).

[0084] Table 15 Information on main related substances in compound glycyrrhizin tablets

[0085] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for simultaneously determining the main components and related substances of compound glycyrrhizin tablets by HPLC, characterized in that: The following steps are involved: The sample solution of compound glycyrrhizin tablets is subjected to high performance liquid chromatography to obtain a chromatogram of the main component and related substances; the main component includes glycine glycoside, methionine and glycine; According to the chromatogram of the main component, the content of the main component in compound glycyrrhizin tablets was obtained by external standard method; According to the chromatograms of the related substances, the content of the related substances in the compound glycyrrhizin tablets was obtained by self-control method; The conditions for high performance liquid chromatography detection include: the chromatographic column filler is octadecylsilane bonded silica gel; mobile phase A is trifluoroacetic acid-water system, and mobile phase B is acetonitrile; Elution was gradient elution; The gradient elution program is: 0 min~3 min: the volume percentage of the mobile phase A is 98%; 3 min~15 min: the volume percentage of the mobile phase A is reduced from 98% to 20%; 16 min to 20 min: the volume percentage of the mobile phase A is 98%; The detector is an electrospray detector.

2. The detection method according to claim 1, wherein The column temperature of the HPLC was 30°C and the atomization temperature was 35°C.

3. The detection method according to claim 1, wherein The volume concentration of trifluoroacetic acid in the trifluoroacetic acid-water system is 0.5%.

4. The detection method according to claim 1, wherein After obtaining the chromatograms of the main components and related substances, the method further includes performing mass spectrometry on the separated samples to determine the structures of the related substances; The mass spectrometry detection conditions included: ESI ionization source, positive / negative ion scanning, positive ion mode spray voltage 4.5 kV; heater voltage 8 V; lens voltage 115 V; In negative ion mode, the spray voltage was -4.0 kV; the heater voltage was -25 V; and the tube lens voltage was -100 V.

5. The detection method according to claim 4, wherein The sheath gas flow rate of the mass spectrometer detection was 30 arb, the purge gas flow rate was 10 arb, the ion transmission capillary temperature was 375°C, and the scanning range was m / z 50-1500.

6. The detection method according to claim 4, wherein The secondary mass spectrometry of the mass spectrometry detection adopts data-dependent scanning, and the secondary mass spectrometry collision-induced dissociation normalized energy value is set to 35%.

7. The detection method according to claim 1, wherein The related substances include one or more of diglycine, methionine sulfoxide, methionine sulfone, glycyrrhizin G2 and 18α-glycyrrhizic acid.

8. The detection method according to claim 1, wherein The chromatographic column is a C18 AQ chromatographic column.

9. The detection method according to claim 1, wherein The flow rate of the mobile phase A and the mobile phase B was 0.8 mL / min.

10. The detection method according to claim 1, wherein The injection volume of the HPLC was 10 μL.

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