Urapidil sustained-release capsule oxide impurity IV and preparation method and detection method thereof

Abstract

The invention relates to an urapidil sustained-release capsule oxide impurity IV as well as a preparation method and a detection method thereof. And the oxidation impurity IV of the urapidil sustained-release capsule is 6-[[3-[4-(2-methoxyphenyl)-4-oxy-1-piperazinyl]-propyl] amino]-1, 3-dimethyluracil. According to the invention, the oxidation impurity IV with relatively high purity is obtained through a proper preparation way, and the structure of the oxidation impurity IV is confirmed to confirm the structure of the compound; meanwhile, the invention further provides a detection method of the oxidation impurity IV, the detection method is good in separation degree, good in reproducibility and high in accuracy, the accurate content of the oxidation impurity IV in the urapidil product can be measured, and the change condition of impurities in urapidil can be effectively monitored. The implementation of the invention is helpful for improving the quality standard of urapidil, so that the product quality of urapidil sustained-release capsules is better controlled.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceuticals, and in particular to an oxidized impurity IV of urapidil sustained-release capsules and a preparation method and a detection method thereof. Background Art

[0002] Urapidil, English name Urapidil, chemical formula is C20H29N5O3, chemical name is 6-[[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]amino]-1,3-dimethyluracil, its structural formula is as follows:

[0003]

[0004] Urapidil is a highly selective α-receptor blocker with both peripheral and central hypotensive effects. Peripheral effects: It mainly blocks postsynaptic α1 receptors, significantly reducing peripheral resistance, thereby dilating blood vessels and lowering blood pressure. It also moderately blocks the vasoconstriction effect of catecholamines. Central effects: It lowers blood pressure by activating 5-hydroxytryptamine-1A receptors and reducing the sympathetic feedback regulation of the medullary cardiovascular center. It also has no side effect of reflex heart rate acceleration and can improve various hemodynamic indicators. Urapidil can be used to treat hypertensive crisis (such as a sharp increase in blood pressure), severe and extremely severe hypertension, refractory hypertension, essential hypertension, renal hypertension, and hypertension caused by pheochromocytoma, and can also be used to control perioperative hypertension. Since urapidil entered clinical use in the 1970s, it has been increasingly recognized and trusted by doctors and patients for its significant blood pressure lowering effect and unique blood pressure lowering mechanism. In particular, it has no obvious effect on blood sugar, blood lipids, uric acid and liver function, blood urea nitrogen and creatinine, heart rate, etc. It can be taken by patients with renal insufficiency, coronary heart disease, diabetes, and gout. It is a blood pressure lowering drug that is rapid, reliable, relatively safe and promising.

[0005] Urapidil is available in a variety of dosage forms, including capsules, sustained-release tablets, and injections. Urapidil sustained-release capsules were tested under strong oxidative and high-temperature conditions (30% H2O2, temperature 60°C), and the results showed that there was a significant increase in oxidative degradation products. These oxide impurities are present in urapidil raw materials or finished products and may be toxic or otherwise harmful to patients. For these reasons, their presence in the substance should be minimal; it is very important to accurately detect the level of these impurities, so their correct structure should be determined and their content should be accurately measured by appropriate methods.

[0006] Chinese patent CN201410713922.7 discloses an urapidil oxidation product and a preparation method thereof. The invention obtains an urapidil oxidation product and performs structural confirmation to confirm the structure of the compound, which is used as an impurity reference substance for the inspection of urapidil-related substances, thereby directly and effectively monitoring the changes in impurities in urapidil. The urapidil oxidation product obtained in the patent is 6-[[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]nitrogenoxy]-1,3-dimethyl-2,4(1H,3H)-pyrimidinedione, and the structural formula is:

[0007]

[0008] However, the analysis of impurity content shows that the oxidative degradation impurities of urapidil are not limited to the structures disclosed in the above-mentioned prior patents, but also contain oxide impurities with other structures. In order to further clarify the oxidative impurity spectrum of urapidil, it is necessary to separate and structurally confirm other oxide impurities, provide reference materials for the analysis of urapidil-related substances, and thus improve the quality standard of urapidil sustained-release capsules. Summary of the invention

[0009] The purpose of the present invention is to provide an oxidized impurity IV of urapidil sustained-release capsules and a preparation method and a detection method thereof in view of the drawbacks of the prior art.

[0010] The technical solution adopted by the present invention to solve its technical problem is:

[0011] An oxidized impurity IV of urapidil sustained-release capsules, wherein the chemical name of the oxidized impurity IV is 6-[[3-[4-(2-methoxyphenyl)-4-oxy-1-piperazinyl]-propyl]amino]-1,3-dimethyluracil;

[0012] Its chemical formula is:

[0013]

[0014] The preparation method of urapidil sustained-release capsule oxidized impurity IV comprises four steps: oxidation reaction, separation and purification, two-step separation and drying;

[0015] S1. Oxidation reaction

[0016] Dissolve urapidil hydrochloride in water, add 30% hydrogen peroxide, carry out oxidation reaction at 60°C, and obtain oxidation reaction liquid after post-treatment;

[0017] S2. Separation and purification

[0018] The oxidation reaction solution is subjected to gradient elution by HPLC preparative liquid chromatography, and the first component before the main peak is collected to obtain a one-step extract with a purity greater than 80%;

[0019] S3, two-step separation

[0020] The first-step extract is subjected to gradient elution by HPLC preparative liquid chromatography to further separate the oxidation product, and the main peak component is collected to obtain a second-step extract with a purity greater than 90%;

[0021] S4. Drying

[0022] The second step extract is freeze-dried to obtain a solid oxidized impurity IV product.

[0023] In step S1, the mass volume ratio of urapidil hydrochloride and 30% hydrogen peroxide is 100 mg:2 ml, and the oxidation reaction temperature is 60° C.;

[0024] The specific process of step S1 is as follows: weigh 100 mg of urapidil hydrochloride, put it into a 100 ml volumetric flask, add 5 ml of water to dissolve it, add 2 ml of 30% hydrogen peroxide, place it in a 60° C. water bath and stir to react for 5 minutes. After the reaction is completed, it naturally cools to room temperature and is diluted to 100 ml with a methanol aqueous solution with a methanol:water ratio of 1:1 to obtain an oxidation reaction solution.

[0025] The chromatographic conditions of the gradient elution in step S2 are:

[0026] Injection solution: The oxidation reaction solution in step S1 is mixed with methanol aqueous solution (methanol: water = 1:1) in a volume ratio of 1:3;

[0027] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0028] Mobile phase A: 10 mmol / L ammonium formate solution at pH 3.5;

[0029] Mobile phase B: chromatographic methanol;

[0030] Flow rate: 2ml / min;

[0031] Injection volume: 5ml;

[0032] Detection wavelength: 236nm and 270nm;

[0033] The specific conditions of gradient elution are:

[0034] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 5 90 10 23 10 90 27 10 90 35 90 10 40 90 10

[0035] The first component before the main peak was collected to obtain a one-step extract.

[0036] The two-step separation chromatographic conditions of step S3 are:

[0037] Injection solution: Mix the one-step extraction solution and water in a volume ratio of 1:3;

[0038] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0039] Mobile phase A: ultrapure water;

[0040] Mobile phase B: chromatographic methanol;

[0041] Flow rate: 3ml / min;

[0042] Injection volume: 5ml;

[0043] Detection wavelength: 236nm and 270nm;

[0044] The specific conditions of gradient elution are:

[0045] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 20 10 90 24 10 90 25 90 10 30 90 10

[0046] The specific process of step S4 is: the two-step extract is first pre-frozen at -45°C, then vacuumed, gradually heated and sublimated, and finally heated to 20°C for 3 hours to obtain a solid oxidized impurity IV product. During the freeze-drying and sublimation process, the vacuum degree is controlled at 0-30Pa.

[0047] The method for detecting the urapidil oxidation product IV is to use high performance liquid chromatography for gradient elution to achieve separation between multiple oxidation impurities.

[0048] The specific chromatographic conditions are:

[0049] Chromatographic column: hexadecylamide bonded silica gel chromatographic column;

[0050] Mobile phase: 0.02 mol / L sodium perchlorate solution as mobile phase A, methanol as mobile phase B;

[0051] Injection solution: Dissolve the sample in a 1:1 volume ratio of mobile phase A-methanol solution to prepare a solution containing about 1 mg / ml of urapidil;

[0052] Flow rate: 1.0ml / min;

[0053] Column temperature: 40°C;

[0054] Detection wavelength: 270nm;

[0055] Injection volume: 10 μl;

[0056] Sample chamber temperature: 4°C;

[0057] The specific conditions of gradient elution are:

[0058] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 10 80 20 35 62 38 38 90 10 43 90 10

[0059] The relative retention time of the oxidized impurity IV is 0.60.

[0060] Through the above technical solution, the beneficial effects obtained by the present invention are:

[0061] The invention provides an oxidized impurity IV of urapidil sustained-release capsule and a preparation method thereof, which can prepare an oxidized impurity IV product with a purity higher than 80%. The structure of the compound is confirmed to be oxidized impurity IV. The oxidized impurity IV product can be used as a liquid phase reference.

[0062] The present invention also provides a method for detecting urapidil oxidized impurities, by which the oxidized impurity IV can be effectively separated from urapidil and oxidized impurities I to III, with good separation, good reproducibility and high accuracy, thereby effectively obtaining the precise content of oxidized impurity IV in the urapidil product, and directly and effectively monitoring the changes of impurities in urapidil raw materials and related preparations. The implementation of the detection method of the present invention is conducive to improving the quality standards of urapidil-related preparations, thereby better controlling the product quality of urapidil-related preparations, which is of great significance to the safe use of drugs by the people. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is the HPLC spectrum of the oxidative degradation experiment of urapidil sustained-release capsules;

[0064] Figure 2 It is the fragmentation mass spectrum of RRT0.40 impurity of the oxidative degradation test solution;

[0065] Figure 3 This is the fragmentation mass spectrum of the homemade RRT0.40 oxidation impurity IV;

[0066] Figure 4 It is the specificity spectrum of the oxidation impurity IV detection method;

[0067] Figure 5 This is the purity detection spectrum of the oxidized impurity IV in Example 1. DETAILED DESCRIPTION

[0068] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0069] Embodiment 1 to Embodiment 3

[0070] Examples 1 to 3 are preparation examples of urapidil sustained-release capsules with oxidized impurity IV, and three batches were prepared in parallel using the same method.

[0071] An oxidized impurity IV of urapidil sustained-release capsules, whose chemical name is 6-[[3-[4-(2-methoxyphenyl)-4-oxy-1-piperazinyl]-propyl]amino]-1,3-dimethyluracil;

[0072] Its chemical formula is:

[0073]

[0074] The method for preparing the oxidized impurity IV of urapidil sustained-release capsules comprises four steps: oxidation reaction, separation and purification, two-step separation and drying.

[0075] S1. Oxidation reaction

[0076] Weigh 100 mg of urapidil hydrochloride and place it in a 100 ml volumetric flask. First, add 5 ml of water to dissolve it, then add 2 ml of 30% hydrogen peroxide, place it in a 60°C water bath for heating and reaction for 5 min, cool it to room temperature, and dilute it to 100 ml with a methanol aqueous solution with a methanol: water ratio of 1:1 (volume ratio) to obtain an oxidation reaction solution;

[0077] S2. Separation and purification

[0078] Using HPLC preparative liquid chromatography to perform gradient elution on the oxidation reaction solution to separate the oxidation product and obtain a one-step extract with a purity greater than 80%;

[0079] The specific chromatographic conditions are:

[0080] Injection solution: The oxidation reaction solution in step S1 is mixed with methanol aqueous solution (methanol: water = 1:1) in a volume ratio of 1:3;

[0081] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0082] Mobile phase A: 10 mmol / L ammonium formate solution at pH 3.5;

[0083] Mobile phase B: chromatographic methanol;

[0084] Flow rate: 2ml / min;

[0085] Injection volume: 5ml;

[0086] Detection wavelength: 236nm and 270nm;

[0087] The specific conditions of gradient elution are:

[0088] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 5 90 10 23 10 90 27 10 90 35 90 10 40 90 10

[0089] The first eluting component before the main peak (its ultraviolet absorption spectrum shows that the peak height ratio of 270 nm to 236 nm is about 10) is collected to obtain a one-step extract with a chromatographic purity greater than 80%.

[0090] S3, two-step separation

[0091] The first-step extract is subjected to gradient elution by HPLC preparative liquid chromatography, the oxidation product is further separated, and the main peak component is collected to obtain a second-step extract with a purity greater than 90%.

[0092] The specific chromatographic conditions are:

[0093] Injection solution: Mix the one-step extraction solution with water in a volume ratio of 1:3 as the injection solution;

[0094] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0095] Mobile phase A: ultrapure water;

[0096] Mobile phase B: chromatographic methanol;

[0097] Flow rate: 3ml / min;

[0098] Injection volume: 5ml;

[0099] Detection wavelength: 236nm and 270nm;

[0100] The specific conditions of gradient elution are:

[0101] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 20 10 90 24 10 90 25 90 10 30 90 10

[0102] In this elution process, only the main peak component is collected to obtain a two-step extract with a chromatographic purity greater than 90%.

[0103] S4. Drying

[0104] The second-step extract was freeze-dried, first pre-frozen at -45°C, then evacuated (vacuum degree 0-30 Pa), gradually heated to sublime, and finally heated to 20°C and maintained for 3 hours to obtain the oxidized impurity IV solid.

[0105] The three batches of oxidized impurity IV products of Examples 1 to 3 were tested by HPLC. The chromatograms are shown in Figure 5 , the chromatographic purity and the maximum impurity (area normalization method) are:

[0106] Example Oxidation impurity IV content (%) Maximum impurity (%) Example 1 88.387 3.249 Example 2 87.580 3.382 Example 3 88.029 3.443

[0107] It can be seen from the data in the above table that the content of the oxidized impurity IV solid obtained by the three batches of parallel preparation examples using the method of the present invention is higher than 85%, the content of the maximum impurity is no more than 3.5%, the purity control is stable, and it can be used as a qualitative standard.

[0108] The following is a description of the process for confirming the structure of the oxidized impurity IV.

[0109] During the structural confirmation process, the chromatographic conditions for the detection of related substances in urapidil sustained-release capsules are as follows:

[0110] Injection solution: Dissolve urapidil in solvent (mobile phase A: methanol mixed in a volume ratio of 1:1) to a concentration of 10 mg / 10 ml;

[0111] Chromatographic column: hexadecylamide bonded silica gel as filler (Agilent Zorbax Bonus-RP 4.6×150mm, 3.5μm or chromatographic column with equivalent performance);

[0112] Mobile phase A: 0.02 mol / L sodium perchlorate solution (take 2.81 g of sodium perchlorate monohydrate, add 900 ml of water to dissolve, adjust the pH value to 2.0 with 70% perchloric acid solution, and add water to 1000 ml);

[0113] Mobile phase B: methanol;

[0114] Flow rate: 1.0ml / min;

[0115] Column temperature: 40°C;

[0116] Detection wavelength: 236nm and 270nm;

[0117] Injection volume: 10 μl;

[0118] Sample chamber temperature: 4°C.

[0119] Time (min) Mobile phase A (%) Mobile phase B (%) 0 85 15 40 55 45 45 85 15 50 85 15

[0120] During the research and development process, the applicant investigated the degradation of urapidil sustained-release capsules under various damage conditions, such as using different concentrations of oxidants and different oxidation reaction times, and then tested the content and impurities of urapidil to determine its oxidation impurity situation. It was determined experimentally that when the oxidation test conditions were adding 30% H2O2 and placing at 60°C for 5 minutes, urapidil would experience strong degradation.

[0121] The above chromatographic conditions were used to detect the degradation experimental samples of urapidil sustained-release capsules. The HPLC degradation spectrum was as follows: Figure 1The chromatographic results showed that under the oxidative conditions, the content of urapidil decreased significantly and the unknown impurities increased. The main oxidative degradation impurities were unknown impurities with relative peak retention times (hereinafter referred to as RRT) of 0.40 and 1.44, with contents of about 2% and 4%, respectively. Figure 1 .

[0122] 1. Inference of oxidative degradation impurities

[0123] According to the theoretical analysis of the structure of urapidil, the positions prone to oxidation reactions are mainly the two N atoms in the piperazine ring and the amino (NH) part in the uracil fragment in the molecular structure. Based on this theory, it is inferred that there are four potential oxidation impurities, namely oxidation impurity I, oxidation impurity II, oxidation impurity III, and oxidation impurity IV. The specific structural formulas of the four oxidation impurities are as follows:

[0124]

[0125] 2. Oxidative degradation impurity reference substances

[0126] At present, the three substances of oxidized impurity I, oxidized impurity II, and oxidized impurity III can all be purchased as reference substances. The HPLC method was used to locate the oxidized impurities I, II, and III standards, and their relative retention times were determined to be 0.45, 1.44, and 1.44, respectively. Therefore, it is speculated that the RRT1.44 impurity may be oxidized impurity II, oxidized impurity III, or a mixture of the two, while the structure of the RRT0.40 impurity has not been directly confirmed, and it is suspected to be oxidized impurity IV.

[0127] Since it was impossible to purchase an oxidized impurity IV reference substance for direct comparison with the RRT0.40 impurity, an attempt was made to prepare and purify the RRT0.40 impurity to determine its structure. The applicant used the preparation solution to treat urapidil hydrochloride according to the oxidation conditions of the degradation experiment and conducted multiple rounds of impurity preparation. The optimal preparation method for the RRT0.40 unknown impurity was finally determined as follows:

[0128] S1. Oxidation reaction

[0129] Weigh 100 mg of urapidil hydrochloride and place it in a 100 ml volumetric flask. First add 5 ml of water to dissolve it, then add 2 ml of 30% hydrogen peroxide. Place it in a 60°C water bath and stir to react for 5 minutes. After the reaction is completed, naturally cool to room temperature and dilute to the scale with methanol: water (1:1) to obtain the oxidation reaction solution.

[0130] S2, one-step separation

[0131] The oxidation reaction solution prepared in step S1 was mixed with methanol aqueous solution (methanol: water = 1:1) at a volume ratio of 1:3 as an injection solution, and HPLC preparative liquid chromatography was used for gradient elution to separate the oxidation product in one step. The specific chromatographic conditions were:

[0132] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0133] Mobile phase A: 10 mmol / L ammonium formate solution at pH 3.5;

[0134] Mobile phase B: chromatographic methanol;

[0135] Flow rate: 2ml / min;

[0136] Injection volume: 5ml;

[0137] Detection wavelength: 236nm and 270nm;

[0138] The specific conditions of gradient elution are:

[0139] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 5 90 10 23 10 90 27 10 90 35 90 10 40 90 10

[0140] The first eluting component before the main peak (the peak height ratio of 270 nm to 236 nm in the UV absorption spectrum is about 10) was collected to obtain a one-step extract.

[0141] S3, two-step separation

[0142] The first step extract was mixed with water in a volume ratio of 1:3, and gradient eluted by HPLC preparative liquid chromatography to separate the oxidation product in one step, and only the main peak was collected, and further purified to obtain the second step extract. The second step extract was measured by HPLC, and the spectrum showed that the RRT of the main peak in the extract was consistent with the RRT of the oxidative degradation impurity IV, and the purity was >90%.

[0143] The specific chromatographic conditions are:

[0144] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0145] Mobile phase A: ultrapure water;

[0146] Mobile phase B: chromatographic methanol;

[0147] Flow rate: 3ml / min;

[0148] Injection volume: 5ml;

[0149] Detection wavelength: 236nm and 270nm;

[0150] The specific conditions of gradient elution are:

[0151] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 20 10 90 24 10 90 25 90 10 30 90 10

[0152] S4. Drying

[0153] The second step extract was dried on a rotary evaporator at 60°C for 4 hours. The chromatographic purity of the substance after drying was 2.8%, which proved that the impurity was extremely unstable when heated, and the main impurity peak was basically decomposed at 60°C.

[0154] The second step extract was treated with low temperature rotary evaporation and dried by rotary evaporation instrument at 40℃ for 6 hours. The dried material was tested again by HPLC, and its chromatographic purity was found to be 13.6%. The impurity peaks increased and the main impurity peak was degraded, which proved that the impurity was also very easy to decompose at 40℃.

[0155] The second step extract was freeze-dried, first pre-frozen at -45°C, then evacuated (vacuum degree 0-30Pa), gradually heated to sublime, and finally heated to 20°C for 3 hours to obtain oxidized impurity IV; HPLC detection showed that its chromatographic purity reached 88.0%.

[0156] Therefore, it was decided to use freeze-drying method to minimize the decomposition of the impurity.

[0157] 3. Determination of the structure of oxidative degradation impurities

[0158] In order to determine the accuracy of the inference of oxidative impurities, relevant literature was consulted.

[0159] Chinese patent CN201410713922.7 discloses an oxidation product of urapidil. After structural comparison, the oxidation product is the oxidation impurity III.

[0160] Ernst Sturm et al. isolated and identified the metabolites of Urapidil in animals and humans (Reference 1: BIOMEDICAL MASS SPECTROMETRY, VOL. 11, NO. 5, 1984 Biotransformation of Urapidil: Isolation and Identification of Metabolites in Mouse, Rat, Dog and Man). The study found that there are five main metabolites, among which the in vivo metabolite 4 (whose structure could not be confirmed by mass spectrometry in the literature) has the same chromatographic behavior as urapidil after PCl3 reduction, and it is speculated that it should be the dioxide of urapidil, that is, oxidized impurity I. The literature inferred from the mass spectrometry data that the oxygen in the structural formula of the in vivo metabolite 5 is located at the center of piperazine-N, and compared the mass spectra with the metabolite 5 reference substance to determine its structure, which is the same as the above-mentioned oxidized impurity II. The structural formula of metabolite 5 in the literature is as follows:

[0161]

[0162] Laximan Velip et al. identified and characterized the forced degradation products of urapidil hydrochloride (Reference 2: Identification and characterization of urapidil stress degradation products by LC-Q-TOF-MS and NMR: Toxicity prediction of degradation products Journal of Pharmaceutical and Biomedical Analysis 211 (2022) 114612). In this document, five degradation impurities of urapidil hydrochloride (denoted as DP1, DP2, DP3, DP4, and DP5) were identified and characterized, and the main oxidative degradation impurities of urapidil hydrochloride were described as DP3 and DP5. The peak times of the impurities were before and after the main peak, respectively, and the cleavage pathway and mass spectrometry data were provided. In order to further determine the oxidation position of DP5, the document conducted nuclear magnetic resonance detection on it and determined its structure. According to the impurity structure information provided in this document, DP3 and DP5 correspond to oxidized impurity IV and oxidized impurity II, respectively. The applicant used the impurity detection method in the document to test the raw materials under oxidation conditions (the raw materials were added with 0.3% hydrogen peroxide and placed in the dark at room temperature for 3 hours, the reaction was terminated with sodium thiosulfate, and then diluted with 50% acetonitrile). The peak positions of the two main degradation impurities were consistent with the peak positions of DP3 and DP5, further confirming the structures of the oxidized impurities IV and II.

[0163] The structures of the oxidative degradation impurities DP3 and DP5 in Reference 2 are:

[0164]

[0165] To further confirm the structure of RRT0.40 oxidation impurities, the diluted S1 oxidation reaction solution (diluted with methanol and water in a volume ratio of 1:1) was subjected to mass spectrometry scanning, and the homemade RRT0.40 impurities and the degradation solution of the test sample were pyrolyzed using different voltages. The pyrolysis mass spectra are shown in Figure 2 , Figure 3 .

[0166] The data after cleavage were compared with the oxidative degradation impurity DP3 in Reference 2. The results showed that the homemade RRT0.40 impurity and the RRT0.40 impurity in the degradation solution of the test sample were consistent with the literature data of the oxidative degradation impurity DP3 (oxidative impurity IV), and the RRT0.40 impurity was determined to be DP3. The comparison of mass spectrometry data is shown in Table 1-3.

[0167] Table 1-3 Comparison of mass spectrometry data of RRT0.40 impurity and impurity DP3

[0168]

[0169] The above data clarified the structure of the main oxidation degradation product of urapidil. The RRT0.40 impurity is the impurity DP3 (oxidation impurity IV) in Reference 2, and its chemical name is: 6-[[3-[4-(2-methoxyphenyl)-4-oxy-1-piperazinyl]-propyl]amino]-1,3-dimethyluracil;

[0170] The chemical formula is:

[0171]

[0172] 4. Research on detection methods of oxidative impurities

[0173] The detection of the oxidized impurity IV was performed by high performance liquid chromatography with gradient elution. The specific chromatographic conditions are as follows:

[0174] Chromatographic column: hexadecylamide bonded silica gel as filler (Agilent Zorbax Bonus-RP 4.6×150mm, 3.5μm or chromatographic column with equivalent performance);

[0175] Mobile phase A: 0.02 mol / L sodium perchlorate solution (take 2.81 g of sodium perchlorate monohydrate, add 900 ml of water to dissolve, adjust the pH value to 3.6 with 70% perchloric acid solution, and add water to 1000 ml);

[0176] Mobile phase B: methanol;

[0177] Injection solution: Dissolve the sample in a mobile phase A-methanol solution with a volume ratio of 1:1, with a concentration of 10 mg / 10 ml;

[0178] Flow rate: 1.0ml / min;

[0179] Column temperature: 40°C;

[0180] Detection wavelength: 270nm;

[0181] Injection volume: 10 μl;

[0182] Sample chamber temperature: 4°C.

[0183] The specific conditions of gradient elution are:

[0184] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 10 80 20 35 62 38 38 90 10 43 90 10

[0185] The gradient elution conditions used in the impurity structure study were derived from the import registration standard of urapidil hydrochloride injection. The specific conditions for gradient elution were:

[0186] Time (min) Mobile phase A (%) Mobile phase B (%) 0 85 15 40 55 45 45 85 15 50 85 15

[0187] Compared with the gradient elution procedure used in the impurity structure research process, the revised gradient elution method reduces the proportion of the initial organic phase and improves the separation degree of each degradation impurity. After the degradation impurities are completely eluted, the initial organic phase proportion is restored, which greatly shortens the elution time and improves the separation efficiency.

[0188] Since the oxidized impurity IV (RRT0.40 impurity) is unstable at room temperature, no higher purity reference substance was obtained after many attempts, and only the specificity, detection limit and quantification limit of its detection were studied.

[0189] 1. Specificity research

[0190] Specific detection method:

[0191] Blank solvent: Mobile phase A-methanol (1:1)

[0192] Peak identification solution: Take about 5 mg each of oxidized impurity I, oxidized impurity II, oxidized impurity III, and oxidized impurity IV, place them in different 50 ml volumetric flasks, add methanol to dissolve and dilute to the scale, shake well, and use them as their respective stock solutions.

[0193] Specificity test solution: weigh about 11 mg of urapidil hydrochloride reference substance, put it into a precisely weighed 10 ml volumetric flask, add 1 ml of each peak identification stock solution, dilute to the scale with solvent, shake well, and obtain.

[0194] Determination method: Accurately measure 10 μl of the blank solvent and specific test solution mentioned above, inject them into the liquid chromatograph respectively, determine according to the method, and record the chromatogram.

[0195] The test data of specific test solution and blank solvent are as follows:

[0196] name Retention time (min) Relative retention time (RRT) Solvents Oxidation impurities Ⅰ 12.114 0.58 No interfering peaks Oxidation impurities Ⅳ 12.491 0.60 No interfering peaks Urapidil 20.843 1.0 No interfering peaks Oxidation Impurities Ⅱ 28.410 1.36 No interfering peaks Oxidation impurities Ⅲ 28.410 1.36 No interfering peaks

[0197] See the specific map Figure 4 .

[0198] The experimental results are shown in the spectrum Figure 4 As shown in the figure, it can be seen that the chromatographic peak of the oxidized impurity IV is clearly separated from the chromatographic peaks of the oxidized impurity I, oxidized impurity II, oxidized impurity III and urapidil, and there is no interfering peak of the solvent. This method has good specificity.

[0199] 2. Limit of Detection and Limit of Quantitation

[0200] The detection method of the detection limit and quantification limit is:

[0201] The stock solution of the oxidized impurity IV under the specificity item is diluted step by step with solvent. When S / N≥10, it is the quantitative limit solution. When 3≤S / N<10, it is the detection limit solution.

[0202] Determination method: Accurately measure 10 μl of the quantitative limit solution and the detection limit solution mentioned above, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0203] The test data are as follows:

[0204]

[0205] It can be seen from the test data that the quantitative limit of the oxidized impurity IV is 0.14894 μg / ml, equivalent to 0.015% of the content in the preparation; the detection limit is 0.0298 μg / ml, equivalent to 0.003% of the content in the preparation, which fully demonstrates that for the oxidized impurity IV, the detection method of the present invention has high sensitivity and meets the requirements of drug-related analysis.

[0206] Embodiment 4 to Embodiment 6

[0207] Examples 4 to 6 are examples of detecting the content of oxidized impurity IV in urapidil sustained-release capsules. The test samples in Examples 4 to 6 are urapidil sustained-release capsules produced by Hebei Yipin Biopharmaceutical Co., Ltd., and the three batches have batch numbers: 230106, 230903, and 240408. In addition, two batches of urapidil sustained-release capsules (batch numbers C18580 and B13890) produced by the original manufacturer Japan Science and Technology Pharmaceutical Co., Ltd. were taken as comparative examples 1 and 2 to compare the accuracy of the detection method of the present invention.

[0208] In the following examples, the method for preparing the sample of urapidil sustained-release capsules is as follows:

[0209] Take the content (equivalent to 50 mg of urapidil) and place it in a 50 ml volumetric flask. Add solvent and sonicate for 30 minutes to dissolve the urapidil. Dilute to the scale with solvent (0.02 mol / L sodium perchlorate solution: methanol mixed in a volume ratio of 1:1), shake well, filter with a nylon filter membrane, discard 2 ml of the initial filtrate, and the test injection solution is obtained.

[0210] The content detection method is:

[0211] Column: Agilent Zorbax Bonus-RP 4.6×150 mm, 3.5 μm;

[0212] Mobile phase A: 0.02 mol / L sodium perchlorate solution;

[0213] Mobile phase B: methanol;

[0214] Flow rate: 1.0ml / min;

[0215] Column temperature: 40°C;

[0216] Detection wavelength: 270nm;

[0217] Injection volume: 10 μl;

[0218] Sample chamber temperature: 4°C;

[0219] Calculation method of impurity content: normalization method;

[0220] The specific conditions of gradient elution are:

[0221]

[0222]

[0223] Through the above test, the results of the content of each oxidized impurity in five batches of urapidil sustained-release capsules are as follows:

[0224]

[0225] It can be seen from the above data that the detection method of the present invention can accurately detect the content of oxidized impurity IV in urapidil sustained-release capsules with high precision; at the same time, it can detect the mixed content of oxidized impurity II + oxidized impurity III, which has guiding significance for the quality control of urapidil sustained-release capsules.

[0226] The urapidil sustained-release capsules of Example 4 (Batch No. 230106) and Comparative Example 2 (Batch No. B13890) were used for accelerated test, and the accelerated conditions (40°C ± 2°C, 75% RH ± 5% RH) and long-term conditions (25°C ± 2°C, 60% RH ± 5% RH) were set. The above method was used to detect the changes in the content of oxidized impurities in the urapidil sustained-release capsules after 3 months of acceleration, 6 months of acceleration, 6 months of long-term, and 12 months of long-term, respectively. The test data are shown in the following table:

[0227]

[0228] It can be seen from the accelerated and long-term test data that, whether it is the company's homemade urapidil sustained-release capsules or the original urapidil sustained-release capsules, the oxidized impurity IV will increase significantly over time under accelerated conditions, proving that the detection accuracy of the oxidized impurity IV is of great significance for improving the efficacy and safety of drugs.

[0229] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Urapidil sustained-release capsules oxidized impurity IV, characterized in that: The chemical name of the oxidized impurity IV is 6-[[3-[4-(2-methoxyphenyl)-4-oxy-1-piperazinyl]-propyl]amino]-1,3-dimethyluracil; Its chemical formula is:

2. A method for preparing the oxidized impurity IV of urapidil sustained-release capsules according to claim 1, characterized in that: It includes four steps: oxidation reaction, separation and purification, two-step separation and drying; S1. Oxidation reaction Dissolve urapidil hydrochloride in water, add 30% hydrogen peroxide, carry out oxidation reaction at 60°C, and obtain oxidation reaction liquid after post-treatment; S2. Separation and purification The oxidation reaction solution is subjected to gradient elution by HPLC preparative liquid chromatography, and the first component before the main peak is collected to obtain a one-step extract with a purity greater than 80%; S3, two-step separation The first-step extract is subjected to gradient elution by HPLC preparative liquid chromatography to further separate the oxidation product, and the main peak component is collected to obtain a second-step extract with a purity greater than 90%; S4. Drying The second step extract is freeze-dried to obtain a solid oxidized impurity IV product.

3. The method for preparing urapidil sustained-release capsules containing oxidized impurities IV according to claim 2, characterized in that: In step S1, the mass volume ratio of urapidil hydrochloride and 30% hydrogen peroxide is 100 mg:2 ml, and the oxidation reaction temperature is 60° C.; The specific process of step S1 is as follows: weigh 100 mg of urapidil hydrochloride, add 5 ml of water to dissolve it, then add 2 ml of 30% hydrogen peroxide to the clear liquid, place it in a 60° C. water bath and stir to react for 5 minutes, and after the reaction is completed, naturally cool to room temperature, and dilute it to 100 ml with a methanol aqueous solution with a methanol:water ratio of 1:1 to obtain an oxidation reaction liquid.

4. The method for preparing urapidil sustained-release capsules containing oxidized impurities IV according to claim 2, characterized in that: The chromatographic conditions of the gradient elution in step S2 are: Injection solution: The oxidation reaction solution of step S1 is mixed with a methanol aqueous solution with a methanol:water ratio of 1:1 in a volume ratio of 1:3; Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Mobile phase A: 10 mmol / L ammonium formate solution at pH 3.5; Mobile phase B: chromatographic methanol; Flow rate: 2ml / min; Injection volume: 5ml; Detection wavelength: 236nm and 270nm; The specific conditions of gradient elution are: The first component before the main peak was collected to obtain a one-step extract.

5. The method for preparing urapidil sustained-release capsules containing oxidized impurities IV according to claim 2, characterized in that: The two-step separation chromatographic conditions of step S3 are: Injection solution: Mix the one-step extraction solution and water in a volume ratio of 1:3; Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Mobile phase A: ultrapure water; Mobile phase B: chromatographic methanol; Flow rate: 3ml / min; Injection volume: 5ml; Detection wavelength: 236nm and 270nm; The specific conditions of gradient elution are:

6. The method for preparing urapidil sustained-release capsules containing oxidized impurities IV according to claim 2, characterized in that: The specific process of step S4 is as follows: the second-step extract is first pre-frozen at -45°C, then vacuumed, gradually heated and sublimated, and finally heated to 20°C and maintained for 3 hours to obtain a solid oxidized impurity IV product.

7. The method for preparing urapidil sustained-release capsules containing oxidized impurities IV according to claim 6, characterized in that: During the freeze-drying sublimation process in step S4, the vacuum degree is controlled at 0-30 Pa.

8. The method for detecting oxidative impurity IV in urapidil sustained-release capsules according to claim 1, characterized in that: High performance liquid chromatography (HPLC) with gradient elution was used to separate various oxidative impurities.

9. The method for detecting oxidative impurity IV in urapidil sustained-release capsules according to claim 8, characterized in that The specific chromatographic conditions of HPLC are: Chromatographic column: hexadecylamide bonded silica gel chromatographic column; Mobile phase: 0.02 mol / L sodium perchlorate solution as mobile phase A, methanol as mobile phase B; Injection solution: Dissolve the sample in a mobile phase A-methanol solution with a volume ratio of 1:1, and the concentration of urapidil in the injection solution is 1 mg / ml; Flow rate: 1.0ml / min; Column temperature: 40°C; Detection wavelength: 270nm; Injection volume: 10 μl; Sample chamber temperature: 4°C; The specific conditions of gradient elution are:

10. The method for detecting oxidation impurity IV in urapidil sustained-release capsules according to claim 9, characterized in that: The relative retention time of the oxidized impurity IV is 0.60.

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