A method for determining related substances of paeoniflorin-6'-O-benzenesulfonate

Abstract

The invention relates to the technical field of drug analysis, and in particular to a method for determining related substances of paeoniflorin-6'-O-benzenesulfonate. The method adopts high performance liquid chromatography, a chromatographic column is YMC-Triart C18 (4.6 mm×250 mm, 3 μm), and gradient elution is performed according to the following mobile phases: mobile phase A is acetonitrile: tetrahydrofuran: 0.1% phosphoric acid aqueous solution (18:10:72); mobile phase B is acetonitrile; and a diluent is methanol. Related substances in paeoniflorin-6'-O-benzenesulfonate are separated and determined, and analyzed and calculated by a self-main component comparison method. The method provided by the invention has the characteristics of good separation, simple and fast operation, good durability, strong specificity, etc., and can be effectively used for quality control of related substances introduced into raw materials, production process, final product preparation process and storage process of paeoniflorin-6'-O-benzenesulfonate.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for determining paeoniflorin-6'-O-benzenesulfonate-related substances. Background Art

[0002] Paeoniflorin-6'-O-benzenesulfonate, CAS No. 1390658-79-3, chemical name is [(1aR, 2S, 3aR, 5R, 5aR, 5bS)-5b-[(Benzyloxy)methyl]-3a, 5, 5a, 5b-tetrahydro-5-hydroxy-2-methyl-2, 5-methylene-3, 4-dioxa-1H-cyclobutacyclopentadiene-1a(2H)]-6-oxy-benzenesulfonyl-β-D-pyranose, the structural formula is as follows:

[0003]

[0004] Sjögren's syndrome Spleen and stomach disease syndrome (SS) is a chronic inflammatory autoimmune disease that invades exocrine glands, especially salivary glands and lacrimal glands. It is coded as 4A43.2 in the International Classification of Diseases ICD-11 and M35.000 in my country's International Classification of Diseases GB / T 14396-2016. This disease is also the second most common autoimmune disease in my country after rheumatoid arthritis, with a prevalence of 0.33% to 0.77% and more than 8 million patients.

[0005] More than 80% of SS patients have symptoms such as dry mouth, dry eyes, fatigue and joint pain, which can be effectively controlled or relieved after appropriate treatment, but are prone to relapse. At the same time, symptoms of multi-system damage caused by involvement of other exocrine glands and other organs outside the glands may also appear. Long-term development can easily lead to pulmonary interstitial fibrosis combined with infection, pulmonary hypertension, renal failure, lymphoma, central nervous system disease and other diseases that may lead to death. The high-incidence age range of the disease coincides with the working age of patients, which may affect the employment of patients and bring huge burdens to families and society. The treatment of this disease is an area where clinical needs have not yet been met. Existing drugs have no fundamental therapeutic effect and can only control symptoms and slow the progression of the disease, accompanied by more serious adverse reactions.

[0006] The cause of SS has not been definitively determined. The current focus of treatment is to improve salivary and lacrimal gland dysfunction and use immunomodulatory or immunosuppressive drugs to treat certain extraglandular symptoms. The only botanical preparation recommended in the "Clinical Pathway for Sjögren's Syndrome" issued by the National Health and Family Planning Commission, total glucosides of paeony (TGP), can be used for the treatment of Sjögren's syndrome. TGP has a definite therapeutic effect and few adverse reactions with long-term use, but its slow onset limits its clinical application. The innovative drug paeoniflorin-6'-O-benzenesulfonate is a compound that has undergone structural modification of paeoniflorin (Pae), the most abundant active ingredient in TGP. Paeoniflorin-6'-O-benzenesulfonate works by regulating the immune system and conducts efficacy studies on the entire symptom of Sjögren's syndrome. It is different from the current drugs used in the clinical treatment of Sjögren's syndrome, which are mainly used to relieve symptoms, and therefore has good development prospects.

[0007] Paeoniflorin-6'-O-benzenesulfonate is an innovative drug. At present, there is no report on the method of determining the impurities of paeoniflorin-6'-O-benzenesulfonate by high performance liquid chromatography in domestic and foreign patents and literature. Paeoniflorin-6'-O-benzenesulfonate is obtained by sulfonylation reaction and refining of paeoniflorin extracted from peony. The potential impurities of paeoniflorin-6'-O-benzenesulfonate mainly come from the introduction of materials and the generation during the production process. At the same time, this product is very unstable during storage, thus producing degradation products, which brings hidden dangers to the quality and safety of the raw materials. In order to monitor the related substances brought by the raw materials, production process, final product preparation process and storage process of paeoniflorin-6'-O-benzenesulfonate, it is necessary to effectively control the related substances of the raw materials. Therefore, it is particularly urgent for enterprises to develop and produce innovative drugs. Summary of the invention

[0008] The object of the present invention is to solve the problem that the degradation products are very easy to be produced in the process of synthesis, purification and drying of paeoniflorin-6'-O-benzenesulfonate, and the prior art lacks relevant detection means. After a large number of experiments, the related substance detection method of paeoniflorin-6'-O-benzenesulfonate raw material is explored, and the method is strictly verified to ensure the scientific rigor of the method. Therefore, the object of the present invention is to provide a method for determining the related substances of paeoniflorin-6'-O-benzenesulfonate, which has the characteristics of good separation, simple and fast operation, good durability, strong specificity, etc., and can be effectively used for the quality control of the related substances brought by the raw material, production process, final product preparation process and storage process of paeoniflorin-6'-O-benzenesulfonate.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] A method for determining related substances of paeoniflorin-6'-O-benzenesulfonate is provided, wherein a mixed solvent of acetonitrile-tetrahydrofuran-phosphoric acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and a reverse phase analytical column is used for gradient elution to determine related substances in a sample of paeoniflorin-6'-O-benzenesulfonate.

[0011] There are many commonly used mobile phases in liquid phase analysis, but how to mix and match mobile phases to achieve better separation effects is the research focus of this field. Since some impurities in the objects detected by the present invention are very similar in polarity and structure, and the number of impurities is large, this causes great difficulties for the selection and optimization of the mobile phase system. How to use the different polarities of the mobile phase system and the reasonable configuration of the composition ratio requires multiple groups of experiments to obtain various combinations of chromatographic information, and compare and summarize, and screen the optimal conditions to obtain innovative results. After long-term research, it was unexpectedly found that the use of a mixed solvent of acetonitrile-tetrahydrofuran-phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B can achieve excellent separation effects. The characteristics of tetrahydrofuran with low viscosity, medium polarity and strong relative elution power are cleverly utilized, which complements acetonitrile. While increasing the polarity range of the mobile phase system, the different elution abilities of the two on the target components are brought into play. Under the premise of different adsorption forces of YMC-TriartC18 (4.6mm×250mm, 3μm) on the target components, the purpose of effectively separating impurities and main components is achieved, and innovative results are obtained.

[0012] Preferably, in the mobile phase A, the volume ratio of acetonitrile, tetrahydrofuran and phosphoric acid aqueous solution is 18:10:72; the phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.1%; and the reverse phase analytical column is YMC-Triart C18.

[0013] Preferably, the specific operation of the gradient elution is: at 0 to 25 min, the initial volume ratio of the mobile phase A to the mobile phase B is 100:0; at 25 to 60 min, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 100:0 to 70:30; at 60 to 61 min, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 70:30 to 100:0; at 61 to 70 min, the volume ratio of the mobile phase A to the mobile phase B remains at 100:0.

[0014] Preferably, the mobile phase flow rate of the gradient elution is 0.8 ml / min, the injection volume is 20 μL, the column temperature is 20-30° C., the detector is an ultraviolet absorption detector, and the detection wavelength is 220-240 nm.

[0015] Preferably, the reverse phase analytical column is YMC-Triart C18, with a specification of 4.6 mm×250 mm and a filler particle size of 3 μm; the column temperature of the gradient elution is 25° C., and the detection wavelength of the ultraviolet absorption detector is 230 nm.

[0016] Preferably, with its own main component as a control, the related substances separated and identified include: paeoniflorin, paeoniflorin lactone glycosides, impurity A, impurity D, impurity E, impurity F, impurity H, and the rest are unknown impurities; the structure of the paeoniflorin is shown in Formula 1, the structure of the paeoniflorin lactone glycosides is shown in Formula 2, the structure of the impurity A is shown in Formula 3, the structure of the impurity D is shown in Formula 4, the structure of the impurity E is shown in Formula 5, the structure of the impurity F is shown in Formula 6, and the structure of the impurity H is shown in Formula 7:

[0017]

[0018] Preferably, the test solution is prepared before the gradient elution, specifically, the paeoniflorin-6'-O-benzenesulfonate raw material is dissolved and the volume is fixed to the scale, and the solution is shaken to obtain the test solution.

[0019] Preferably, the paeoniflorin-6'-O-benzenesulfonate sample is dissolved in methanol; the content of paeoniflorin-6'-O-benzenesulfonate in the test solution is 5 mg / ml; the operation of using the main component itself as a control includes: measuring 1 ml of the test solution, placing it in a 100 ml volumetric flask, diluting it to the scale with methanol, mixing it, and using it as the self-reference solution.

[0020] An application of the above-mentioned method for determining related substances of paeoniflorin-6'-O-benzenesulfonate is used for quality control of one or more related substances introduced during the raw materials, production process, final product preparation process and storage process of paeoniflorin-6'-O-benzenesulfonate.

[0021] The content of related substances was analyzed and calculated by the self-principal component comparison method. Except for the solvent peak and the main peak of paeoniflorin-6'-O-benzenesulfonate, the quality standard stipulates that the sum of the impurity peak areas shall not be greater than 5 times (5.0%) the peak area of ​​paeoniflorin-6'-O-benzenesulfonate reference substance.

[0022] Preferably, any peak in the test solution that is less than 0.05 times the area of ​​the main peak of paeoniflorin-6'-O-benzenesulfonate is ignored.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. Paeoniflorin-6'-O-benzenesulfonate is an innovative drug. Currently, there is no report on the determination of impurities of paeoniflorin-6'-O-benzenesulfonate by high-performance liquid chromatography in domestic and foreign patents and literature. This method is extremely advanced. The present invention establishes a method for determining related substances of paeoniflorin-6'-O-benzenesulfonate, which can quickly and accurately detect the impurities and degradation products of paeoniflorin-6'-O-benzenesulfonate, meeting the urgent needs of drug research and development and production of enterprises.

[0025] 2. After research, it was found that the functional groups of some impurities are basically the same as those of the main product, such as the carbon chain length is basically the same, which makes the polarity of the impurity very close to that of the main component. Therefore, when using the traditional C18 column for detection, even if the mobile phase and gradient elution program are repeatedly optimized, it is difficult to effectively separate the main product and the impurity. Therefore, the separation between the main peak of the product and the impurity peak is difficult to meet the detection requirements, and even the main peak of the product overlaps with the impurity peak, and the presence of the impurity cannot be detected. Experiments have found that the separation of impurity A and the main component of paeoniflorin-6'-O-benzenesulfonate, as well as the poor separation effect between impurities with similar structures, result in the inability to accurately detect impurities. After many research experiments, it was confirmed that the YMC-Triart C18 (4.6mm×250mm, 3μm) was used, the mobile phase A was acetonitrile:tetrahydrofuran:0.1% phosphoric acid aqueous solution (18:10:72), and the mobile phase B was acetonitrile. Gradient elution was performed, and unexpectedly a good separation effect between impurities and main components was obtained. Impurity A peaked before the main component. The chromatographic information of other mobile phase systems such as acetonitrile-phosphoric acid or methanol-phosphoric acid was that impurity A peaked at the tail of the main component. The impurities could not achieve baseline separation, resulting in poor measurement accuracy and unreliable results.

[0026] 3. The detection method of the present invention has the characteristics of good separation, simple and fast operation, good durability, strong specificity, etc. It can fully meet the requirements of relevant substance inspection and decomposition product determination, better control special impurities in samples, ensure product quality, and has strong practicality in work; this method has also been strictly verified to ensure the scientific rigor of the method. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The chromatogram of the test solution of paeoniflorin-6'-O-benzenesulfonate under the chromatographic conditions of the present invention;

[0028] Figure 2 The chromatogram of the elution of paeoniflorin-6'-O-benzenesulfonate and its impurities under the chromatographic conditions of the present invention;

[0029] Figure 3 The chromatogram of paeoniflorin-6'-O-benzenesulfonate and its impurities eluted by the conventional mobile phase acetonitrile-phosphoric acid system in Example 1;

[0030] Figure 4 The chromatogram of paeoniflorin-6'-O-benzenesulfonate and its impurities eluted by the same type of conventional chromatographic column in Example 1;

[0031] Figure 5 This is a chromatogram of paeoniflorin-6'-O-benzenesulfonate and its impurities eluted with mobile phase A containing 5% tetrahydrofuran in Example 1;

[0032] Figure 6 This is a chromatogram of paeoniflorin-6'-O-benzenesulfonate and its impurities eluted with mobile phase A containing 20% ​​tetrahydrofuran in Example 1. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0034] Example 1

[0035] The present invention provides a method for determining related substances of paeoniflorin-6'-O-benzenesulfonate, and the specific operation process is as follows:

[0036] (1) Preparation of test solution: Weigh the raw material of paeoniflorin-6'-O-benzenesulfonate to be tested, dilute it with methanol to a solution concentration of 5 mg / ml, mix well, and filter to use as the test solution; then measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with methanol, mix well, and use as the self-reference solution.

[0037] (2) Setting the HPLC detection conditions: the chromatographic column was YMC-Triart C18 (4.6 mm × 250 mm, 3 μm), acetonitrile:tetrahydrofuran:0.1% phosphoric acid aqueous solution (18:10:72) was used as the mobile phase A, acetonitrile was used as the mobile phase B, the flow rate was 0.8 ml / min for gradient elution, the detection wavelength was 230 nm, and the column temperature was 25 °C;

[0038] (3) Take 20 μl of the test solution and self-reference solution respectively and inject them into the high performance liquid chromatograph for analysis.

[0039] Each test parameter of the liquid chromatography detection method is obtained through screening, and the screening process is as follows:

[0040] 1. Comparative screening of mobile phase systems,

[0041] The gradient elution program for mobile phase system screening is shown in Table 1.

[0042] Table 1 Gradient elution program

[0043]

[0044] The results showed that when mobile phase system I (conventional mobile phase reported in the literature) was used for gradient elution, the order of elution of all components was as follows: peony lactone glycosides → peony lactone glycosides → impurity H → peony lactone glycoside-6'-O-benzenesulfonate → impurity A → impurity F → impurity D → impurity E; when mobile phase system II was used for gradient elution, the order of elution of all components was as follows: peony lactone glycosides → peony lactone glycosides → impurity H → impurity A → peony lactone glycoside-6'-O-benzenesulfonate → impurity F → impurity D → impurity E; when mobile phase system I was used for gradient elution, the main component peak of peony lactone glycoside-6'-O-benzenesulfonate had tailing because its concentration was required to be high in order to achieve the purpose of measuring sample impurities. If the sample concentration was low, the chance of missing related impurities would increase, and the main component peak and impurity A could not achieve baseline separation. After adding an appropriate amount of tetrahydrofuran to the mobile phase system II, it was unexpectedly found that the order of elution of impurity A and the main component peak changed, avoiding the situation where impurities could not be separated due to tailing of the main component peak, proving that the mobile phase system has achieved significant improvement.

[0045] 2. Screening of tetrahydrofuran ratio

[0046] The chromatographic conditions for the tetrahydrofuran ratio screening study of mobile phase A are shown in Table 2, and the comparative study results are shown in Table 3.

[0047] Table 2 Chromatographic conditions

[0048]

[0049] Table 3 Comparative study results of different proportions of tetrahydrofuran mobile phase A

[0050] Acetonitrile:Tetrahydrofuran:0.1% phosphoric acid aqueous solution Number of components detected Minimum resolution between peaks (25：5：70) 6 1.8 (18：10：72) 8 1.7 (10：20：70) 7 1.8

[0051] The results show that the appropriate ratio of tetrahydrofuran in mobile phase A is very important. Too much or too little tetrahydrofuran cannot effectively separate all impurities. When mobile phase A contains 5% tetrahydrofuran (the volume ratio of acetonitrile-tetrahydrofuran-0.1% phosphoric acid water is 25:5:70), impurity A cannot be detected. When mobile phase A contains 20% tetrahydrofuran (the volume ratio of acetonitrile-tetrahydrofuran-0.1% phosphoric acid water is 10:20:70), impurity E cannot be detected, and the peak of paeoniflorin overlaps with the solvent peak. When mobile phase A contains 10% tetrahydrofuran (the volume ratio of acetonitrile-tetrahydrofuran-0.1% phosphoric acid water is 18:10:72), the number of related substances detected and the separation between peaks are better. Therefore, we set the solvent ratio of acetonitrile:tetrahydrofuran:0.1% phosphoric acid aqueous solution in mobile phase A to 18:10:72 (volume ratio). It was demonstrated that the determination of the proportion of mobile phase A achieved a significant improvement effect.

[0052] 3. Chromatographic column screening

[0053] The chromatographic column comparison screening conditions are shown in Table 4, and the chromatographic column comparison study results are shown in Table 5.

[0054] Table 4 Column screening conditions

[0055]

[0056] Table 5 Column comparison study results

[0057] Chromatographic columns Number of components detected Minimum resolution between peaks DIKMA 7 1.11 YMC 8 1.70

[0058] The results show that the number of impurities detected by column 2 (YMC-Tiart C18 4.6mm×250mm, 3μm) is higher than that of column 1 (conventional column, DIKMA C18 (2), 4.6mm×250mm, 3μm), when the chromatographic column 1 was used to measure the test sample, the gradient was changed and the unknown impurity peak (RRT1.18) between the main component peak and the impurity F could not be detected. After research, the inventor found that the polarity of some impurities is very close to that of the main component. Therefore, when using the traditional C18 chromatographic column for detection, even if the mobile phase and gradient elution program are repeatedly optimized, it is difficult to effectively separate the main component and its related impurities. Therefore, the separation between the main peak of the product and the impurity peak is difficult to meet the detection requirements, and even the main peak of the product and the impurity peak overlap, and the presence of impurities cannot be detected. When this special chromatographic column and mobile phase are used for detection, the impurity peak and the main peak of the product can be well separated, and the impurity content can be accurately detected. Therefore, the chromatographic column finally selected is chromatographic column 2 (YMC-Triart C18 4.6mm×250mm, 3μm).

[0059] Example 2

[0060] Taking the solution containing this product as an example, the following methodological validation was performed using established chromatographic conditions:

[0061] Investigation of the specificity of the detection method of the present invention

[0062] (1) Solution preparation

[0063] Preparation of test solution: Take 125 mg of paeoniflorin-6'-O-benzenesulfonate raw material, put it in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well to obtain the test solution;

[0064] Preparation of single impurity localization solution: Take appropriate amount of paeoniflorin, paeoniflorin lactone glycosides, impurity A, impurity D, impurity E, impurity F, and impurity H separated from the structure confirmation of paeoniflorin-6'-O-benzenesulfonate, place them in a 25 ml volumetric flask, add methanol to dissolve and dilute, as a single impurity localization solution;

[0065] Specific solution for related substances of paeoniflorin-6'-O-benzenesulfonate: Take a single impurity location solution and mix it with the test sample solution respectively, shake well, and use it as the specific solution for related substances of paeoniflorin-6'-O-benzenesulfonate.

[0066] (2) Determination: The chromatographic column was YMC-Triart C18 (4.6 mm × 250 mm, 3 μm), acetonitrile:tetrahydrofuran:0.1% phosphoric acid aqueous solution (18:10:72) was used as mobile phase A, acetonitrile was used as mobile phase B, the column temperature was 25°C, the detection wavelength was 230 nm, 20 μl of methanol solution, test sample solution, single impurity location solution, and paeoniflorin-6'-O-benzenesulfonate related substance specific solution were injected respectively, and eluted according to the gradient in Table 6.

[0067] Table 6 Elution gradient

[0068] time Mobile phase A (%) Mobile phase B (%) Flow rate (ml / min) 0 100 0 0.8 25 100 0 0.8 60 70 30 0.8 61 100 0 0.8 70 100 0 0.8

[0069] The results of methanol solution, test sample solution, single impurity localization solution, and paeoniflorin-6'-O-benzenesulfonate related substance specificity solution are shown in Table 7.

[0070] Table 7 Specific separation results

[0071]

[0072] As shown in Table 5, the diluent methanol has no interference with the determination, the retention time of the impurities separated from each structure confirmation is consistent with the retention time of the impurities in the test sample, and the resolution of the related substances and the main peak of paeoniflorin-6'-O-benzenesulfonate is greater than 1.5, indicating that the method has good specificity and can be used for the determination of related substances of paeoniflorin-6'-O-benzenesulfonate.

[0073] Detection limit test of the detection method of the present invention: Take the test solution in Example 2, add methanol to dilute it to 0.05%, use it as the detection limit test solution, and measure it according to the determination method in Example 2. The response value of the main component is 1.1684.

[0074] Precision test of the detection method of the present invention - repeatability test, intermediate precision test

[0075] Weigh 6 portions of the same batch of paeoniflorin-6'-O-benzenesulfonate raw materials, measure them according to the detection method of this patent, and calculate the relative standard deviation of the total impurity content, which should be RSD ≤ 3%. Take the same batch of paeoniflorin-6'-O-benzenesulfonate raw materials in the same way, and have different personnel measure them according to the law on different dates, record the chromatogram, and calculate the relative standard deviation of the total impurity content. Compare the results of this experiment with the results of the repeatability experiment, and the RSD of the 12 data obtained should be ≤ 3.

[0076] (1) Solution preparation

[0077] Preparation of test solution: Take an appropriate amount of the product, weigh accurately, dissolve it in methanol and quantitatively dilute it to make a solution containing about 5 mg per 1 ml, shake well, filter, and use it as the test solution. Prepare 6 copies in parallel;

[0078] Preparation of self-reference solution: Take an appropriate amount of the test sample solution and use methanol to make a solution containing about 50ug per 1ml, shake well, filter, and use it as the self-reference solution.

[0079] (2) Determination: The chromatographic column was YMC-Triart C18 (4.6 mm × 250 mm, 3 μm), acetonitrile:tetrahydrofuran:0.1% phosphoric acid aqueous solution (18:10:72) was used as mobile phase A, acetonitrile was used as mobile phase B, the column temperature was 25°C, the detection wavelength was 230 nm, 20 μl of the test solution and the self-reference solution were injected, and eluted according to the gradient in Table 8.

[0080] Table 8 Elution gradient

[0081] time Mobile phase A (%) Mobile phase B (%) Flow rate (ml / min) 0 100 0 0.8 25 100 0 0.8 60 70 30 0.8 61 100 0 0.8 70 100 0 0.8

[0082] If there are impurity peaks in the chromatogram other than the solvent peak and the paeoniflorin-6'-O-benzenesulfonate peak, calculate the amount of total impurities using the principal component control method. Any peak in the test solution that is less than 0.05 times the area of ​​the main peak of paeoniflorin-6'-O-benzenesulfonate can be ignored.

[0083] The formula for calculating the proportion of the paeoniflorin-6'-O-benzenesulfonate-related substances, i.e., impurities, in the paeoniflorin-6'-O-benzenesulfonate in the embodiment is:

[0084] Total impurities (%) = At / As

[0085] At: the sum of the peak areas of impurities in the test solution;

[0086] As: Peak area of ​​the main peak in the self-reference solution.

[0087] The results of the precision test are shown in Table 9.

[0088] Table 9 Precision results of related substances of paeoniflorin-6'-O-benzenesulfonate

[0089]

[0090] As shown in Table 9, in the repeatability test, the RSD of the repeatability results of the related substances of the 6 samples was 2.8%, indicating that the repeatability of this method is good. In the intermediate precision test, the RSD of the total impurity content of the 12 samples was 1.8%, indicating that the intermediate precision of this method is good.

[0091] Durability test of the detection method of the present invention

[0092] (1) Solution preparation

[0093] Preparation of test solution: Take an appropriate amount of the product, weigh accurately, dissolve it with methanol and quantitatively dilute it to make a solution containing about 5 mg per 1 ml, shake well, filter, and use it as the test solution;

[0094] Preparation of self-reference solution: Take an appropriate amount of the test solution and use methanol to make a solution containing about 50ug (1%) per 1ml, shake well, filter, and use as the self-reference solution.

[0095] (2) Determination: Using a chromatographic column [YMC-Triart C18 (4.6 mm × 250 mm, 3 μm)], acetonitrile:tetrahydrofuran:0.1% phosphoric acid aqueous solution (18:10:72) as mobile phase A and acetonitrile as mobile phase B, inject 20 μl of the test solution and self-reference solution at different column temperatures (20°C, 25°C and 30°C), different detection wavelengths (228 nm, 230 nm and 240 nm) and different times (within 24 hours), and elute according to the gradient shown in Table 10.

[0096] Table 10 Elution gradient

[0097] time Mobile phase A (%) Mobile phase B (%) Flow rate (ml / min) 0 100 0 0.8 25 100 0 0.8 60 70 30 0.8 61 100 0 0.8 70 100 0 0.8

[0098] ①Comparison of different column temperatures

[0099] Take the same batch of paeoniflorin-6'-O-benzenesulfonate raw material, prepare 2 test solutions according to (1), and follow the determination method (2). Other chromatographic conditions remain unchanged. Set the column temperature to 20℃, 25℃ and 30℃ respectively for injection and record the chromatogram. The solvent peak in the chromatogram is ignored; except for the solvent peak and the paeoniflorin-6'-O-benzenesulfonate peak, if there is an impurity peak, calculate the amount of total impurities by the self-main component comparison method. Any peak in the test solution that is less than 0.05 times the area of ​​the main peak of paeoniflorin-6'-O-benzenesulfonate can be ignored. The test results are shown in Table 11.

[0100] Table 11 Experimental results of the effects of different column temperatures on the related substances of paeoniflorin-6'-O-benzenesulfonate

[0101]

[0102] ②Comparison of different detection wavelengths

[0103] Take the same batch of paeoniflorin-6'-O-benzenesulfonate raw material, prepare 2 test solutions according to (1), and follow the determination method (2). Other chromatographic conditions remain unchanged. Set the wavelengths to 228nm, 230nm and 240nm for injection, and record the chromatogram. In the chromatogram, except for the solvent peak and the paeoniflorin-6'-O-benzenesulfonate peak, if there are impurity peaks, calculate the amount of total impurities by the main component comparison method. Any peak in the test solution that is less than 0.05 times the main peak area of ​​paeoniflorin-6'-O-benzenesulfonate can be ignored. The test results are shown in Table 12.

[0104] Table 12 Experimental results of the effects of different wavelengths on related substances of paeoniflorin-6'-O-benzenesulfonate

[0105]

[0106] ③Solution stability test

[0107] Take the same batch of paeoniflorin-6'-O-benzenesulfonate raw material and prepare one test solution according to (1). According to the determination method (2), the solution was determined once at 0, 2, 4, 6, 8, 12, and 24 hours respectively. The relative standard deviation of the impurity peak area was calculated. The results are shown in Table 12 below.

[0108] Table 14 Paeoniflorin-6'-O-benzenesulfonate related substance stability test results

[0109]

[0110] From the durability results, it can be seen that the determination of paeoniflorin-6'-O-benzenesulfonate-related substances has no effect on the determination under changes in column temperature, wavelength, and the test solution has good stability within 24 hours.

[0111] Example 3

[0112] Determination of three batches of paeoniflorin-6'-O-benzenesulfonate related substances by the method of the present invention

[0113] Take 3 batches of paeoniflorin-6'-O-benzenesulfonate and measure it according to the following detection method:

[0114] (1) Solution preparation

[0115] Preparation of test solution: Take an appropriate amount of the product, weigh accurately, dissolve it with methanol and quantitatively dilute it to make a solution containing about 5 mg per 1 ml, shake well, filter, and use it as the test solution;

[0116] Preparation of self-reference solution: Take an appropriate amount of the test solution and use methanol to make a solution containing about 50ug (1%) per 1ml, shake well, filter, and use as the self-reference solution.

[0117] (2) Determination: The chromatographic column was YMC-Triart C18 (4.6 mm × 250 mm, 3 μm), acetonitrile:tetrahydrofuran:0.1% phosphoric acid aqueous solution (18:10:72) was used as mobile phase A, acetonitrile was used as mobile phase B, the column temperature was 25°C, the detection wavelength was 230 nm, 20 μl of the test solution and the self-reference solution were injected, and eluted according to the gradient shown in Table 15.

[0118] Table 15 Elution gradient

[0119] In the chromatogram, except for the solvent peak and the peak of paeoniflorin-6'-O-benzenesulfonate, if there are impurity peaks, use the main

[0120] time Mobile phase A (%) Mobile phase B (%) Flow rate (ml / min) 0 100 0 0.8 25 100 0 0.8 60 70 30 0.8 61 100 0 0.8 70 100 0 0.8

[0121] The amount of total impurities is calculated by the component control method. Any peak in the test solution that is less than 0.05 times the area of ​​the main peak can be ignored.

[0122] The formula for calculating the proportion of the paeoniflorin-6'-O-benzenesulfonate-related substances, i.e., impurities, in the paeoniflorin-6'-O-benzenesulfonate in the embodiment is:

[0123] Total impurities (%) = At / As

[0124] At: the sum of the peak areas of impurities in the test solution;

[0125] As: Peak area of ​​the main peak in the self-reference solution.

[0126] The test results are shown in Table 16.

[0127] batch number M210301 M210801 M210802 Related substances (%) 0.96 0.57 0.92

[0128] The individual impurity components during the detection process were identified and the results are shown in Table 17.

[0129] Table 17 Impurity information of paeoniflorin-6'-O-benzenesulfonate

[0130]

[0131] In summary, the detection method of the present invention has the characteristics of good separation, simple and quick operation, good durability, strong specificity, etc.

[0132] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for determining related substances of paeoniflorin-6'-O-benzenesulfonate, characterized in that: A mixed solvent of acetonitrile-tetrahydrofuran-phosphoric acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and a reverse phase analytical column is used for gradient elution to determine related substances in a paeoniflorin-6'-O-benzenesulfonate sample; using its own main component as a control, the separated and identified related substances include: paeoniflorin, paeoniflorin lactone glycosides, impurity A, impurity D, impurity E, impurity F, impurity H, and the rest are unknown impurities; the structure of the paeoniflorin is shown in Formula 1, the structure of the paeoniflorin lactone glycosides is shown in Formula 2, the structure of the impurity A is shown in Formula 3, the structure of the impurity D is shown in Formula 4, the structure of the impurity E is shown in Formula 5, the structure of the impurity F is shown in Formula 6, and the structure of the impurity H is shown in Formula 7: In the mobile phase A, the volume ratio of acetonitrile, tetrahydrofuran and phosphoric acid aqueous solution is 18:10:72; the phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.1%; the reverse phase analytical column is YMC-Triart C18; The specific operation of the gradient elution is: at 0 to 25 minutes, the initial volume ratio of the mobile phase A to the mobile phase B is 100:0; at 25 to 60 minutes, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 100:0 to 70:30; at 60 to 61 minutes, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 70:30 to 100:0; at 61 to 70 minutes, the volume ratio of the mobile phase A to the mobile phase B is maintained at 100:

0.

2. The method for determining the related substances of paeoniflorin-6'-O-benzenesulfonate according to claim 1, characterized in that: The mobile phase flow rate of the gradient elution is 0.8 ml / min, the injection volume is 20 μL, the column temperature is 20-30° C., the detector is an ultraviolet absorption detector, and the detection wavelength is 220-240 nm.

3. The method for determining the related substances of paeoniflorin-6'-O-benzenesulfonate according to claim 2, characterized in that: The reverse phase analytical column is YMC-Ttiart C18, with a specification of 4.6 mm×250 mm and a filler particle size of 3 μm; the column temperature of the gradient elution is 25° C., and the detection wavelength of the ultraviolet absorption detector is 230 nm.

4. The method for determining the related substances of paeoniflorin-6'-O-benzenesulfonate according to claim 1, characterized in that: The test solution is prepared before the gradient elution, specifically: dissolving the paeoniflorin-6'-O-benzenesulfonate raw material and diluting to the scale, and shaking well to obtain the test solution.

5. The method for determining the related substances of paeoniflorin-6'-O-benzenesulfonate according to claim 4, characterized in that: The paeoniflorin-6'-O-benzenesulfonate sample is dissolved in methanol; the solution concentration in the test solution is 5 mg / ml; the operation of using the main component itself as a control includes: measuring 1 ml of the test solution, placing it in a 100 ml volumetric flask, diluting it to the scale with methanol, and mixing it to use as the self-control solution.

6. An application of the method for determining related substances of paeoniflorin-6'-O-benzenesulfonate as claimed in claim 1, characterized in that: Used for quality control of one or more related substances introduced into the raw materials, production process, final product preparation process and storage process of paeoniflorin-6'-O-benzenesulfonate.

7. The use of the method for determining related substances of paeoniflorin-6'-O-benzenesulfonate as claimed in claim 6, characterized in that: Ignore any peak in the test solution that is less than 0.05 times the area of ​​the main peak of paeoniflorin-6'-O-benzenesulfonate.

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