Method for detecting related substances in trimethylphloroglucinol

Abstract

The invention relates to the technical field of drug analysis and detection, and particularly discloses a method for detecting related substances in trimethylphloroglucinol. According to the method, high performance liquid chromatography is adopted for detection, and chromatographic conditions are as follows: a chromatographic column is an octadecylsilane chemically bonded silica column; the detection wavelength is 225-235 nm, a mobile phase A is a phosphoric acid aqueous solution with the pH value of 2.6-3.0, a mobile phase B is acetonitrile, and gradient elution is carried out. According to the present invention, the octadecylsilane chemically bonded silica gel chromatographic column is adopted, the phosphoric acid aqueous solution with the pH value of 2.6-3.0 and the acetonitrile are adopted as the mobile phase, and the effective separation of the trimethyl phloroglucinol and the six known impurities can be achieved through the high performance liquid chromatography in the gradient elution manner so as to accurately detect the impurity condition in the trimethyl phloroglucinol, the defect that related substances in the trimethylphloroglucinol cannot be quantitatively detected in the prior art is overcome, and reliable guarantee is provided for improving and better controlling the quality of the trimethylphloroglucinol product.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis and detection, and in particular to a method for detecting related substances in trimethylphloroglucinol. Background Art

[0002] Trimethyl phloroglucinol, chemical name 1,3,5-trimethoxybenzene, is an important organic synthesis intermediate, widely used in the synthesis of medicines, pesticides and fine chemical products, such as the synthesis of vasodilator buflomedil hydrochloride. At the same time, trimethyl phloroglucinol itself is an effective antispasmodic drug, which can directly act on the smooth muscles of the gastrointestinal tract and urogenital tract. Compared with other smooth muscle antispasmodics, the biggest feature of phloroglucinol is that it does not have anticholinergic effect. While relieving smooth muscle spasm, it will not produce a series of anticholinergic adverse reactions, nor will it cause hypotension, increased heart rate or arrhythmia and other symptoms. It has little effect on cardiovascular function. It only acts on spasmodic smooth muscle and has little effect on normal smooth muscle. It is clinically used to treat acute spasmodic pain caused by digestive system and biliary dysfunction, acute spasmodic pain of urethra, bladder and kidney, and gynecological spasmodic pain. It has fast spasmodic and analgesic effect, significant effect and good tolerance.

[0003] At present, trimethyl phloroglucinol is mainly prepared by methoxylation reaction using 1,3,5-tribromobenzene as raw material. Some impurity compounds may be produced in the process of preparing trimethyl phloroglucinol, which will affect the quality and safety of the product. Detection of these impurity compounds is an important part of ensuring product quality. However, the pharmacopoeias of various countries have not included the detection method of related substances in trimethyl phloroglucinol. Therefore, the development of a detection method that can detect multiple related substances in trimethyl phloroglucinol is of great significance for improving the quality control of trimethyl phloroglucinol and improving the safety of patients' medication. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a method for detecting related substances in trimethylphloroglucinol.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0006] A method for detecting related substances in trimethylphloroglucinol, wherein the related substances are 1-chloro-3,5-dimethoxybenzene, 1-bromo-3,5-dimethoxybenzene, 3-bromo-5-chloroanisole, 3,5-dibromoanisole, 1-chloro-3,5-dibromobenzene and 1,3,5-tribromobenzene, characterized in that high performance liquid chromatography is used for detection, and the chromatographic conditions are:

[0007] Chromatographic column: octadecylsilane bonded silica gel column;

[0008] UV detector, detection wavelength 225nm ~ 235nm;

[0009] Mobile phase A: phosphoric acid aqueous solution with a pH of 2.6 to 3.0, mobile phase B is acetonitrile;

[0010] The elution method was gradient elution.

[0011] Compared with the prior art, the method for detecting related substances in trimethyl phloroglucinol provided by the present invention adopts an octadecylsilane bonded silica gel chromatographic column, a phosphoric acid aqueous solution with a pH of 2.6 to 3.0 and acetonitrile as mobile phases, and uses high performance liquid chromatography in a gradient elution manner to achieve effective separation of trimethyl phloroglucinol and 6 known impurities (1-chloro-3,5-dimethoxybenzene, 1-bromo-3,5-dimethoxybenzene, 3-bromo-5-chloroanisole, 3,5-dibromoanisole, 1-chloro-3,5-dibromobenzene, and 1,3,5-tribromobenzene), and accurately detect the impurities in trimethyl phloroglucinol. Moreover, the method provided by the present invention has been studied and verified through methodological studies on specificity, sensitivity and the like, and it is found that the method provided by the present invention is sensitive, accurate and has good reproducibility, and can achieve accurate quantitative detection of related substances in trimethyl phloroglucinol in a simple and quick way, thus solving the existing problem that related substances in trimethyl phloroglucinol cannot be quantitatively detected, providing a reliable guarantee for improving and better controlling the quality of trimethyl phloroglucinol products, and also having a very important significance for improving drug safety.

[0012] It should be noted that the structural formula of the relevant substances in trimethylphloroglucinol is shown in the following table:

[0013]

[0014] Preferably, the elution procedure of the gradient elution is as follows:

[0015] 0min, 70%-80% mobile phase A, 30%-20% mobile phase B;

[0016] 10 min, 20% mobile phase A, 80% mobile phase B;

[0017] 25 min, 20% mobile phase A, 80% mobile phase B;

[0018] 25.1 min, 70%-80% mobile phase A, 30%-20% mobile phase B;

[0019] 35min, 70%-80% mobile phase A, 30%-20% mobile phase B.

[0020] Since impurities A and B, and impurities C and D have similar structures and physical and chemical properties, it is difficult to separate them during the detection process. Through creative thinking, the inventors used a phosphoric acid aqueous solution with a pH of 2.6 to 3.0 and acetonitrile as mobile phases, and C18 as a chromatographic column, and achieved effective separation of impurities A and B, and impurities C and D through a specific gradient elution, which is conducive to accurate qualitative and quantitative detection of impurities in trimethylphloroglucinol, and facilitates more accurate monitoring of the quality of trimethylphloroglucinol.

[0021] More preferably, the elution procedure of the gradient elution is as follows:

[0022] 0 min, 75% mobile phase A, 25% mobile phase B;

[0023] 10 min, 20% mobile phase A, 80% mobile phase B;

[0024] 25 min, 20% mobile phase A, 80% mobile phase B;

[0025] 25.1 min, 75% mobile phase A, 25% mobile phase B;

[0026] 35 min, 75% mobile phase A, 25% mobile phase B.

[0027] The preferred gradient elution sequence can improve the separation degree and detection sensitivity between trimethylphloroglucinol and six known impurities, making the detection results quantitatively accurate and highly precise.

[0028] Preferably, the specification of the chromatographic column is 250 mm*4.6 mm, and the filler diameter is 5 μm.

[0029] More preferably, the chromatographic column is ZORBAX SB-C18, 250 mm*4.6 mm, 5 μm.

[0030] The preferred chromatographic column specifications can make the peak shape, separation and detection sensitivity of each component excellent, and the baseline interference is small, which is conducive to the effective separation of the raw materials and various impurities in trimethylphloroglucinol, and the results are accurate, reliable and reproducible.

[0031] Preferably, the flow rate is 0.8 mL / min to 1.2 mL / min, and the column temperature is 25°C to 35°C.

[0032] More preferably, the flow rate is 1.0 mL / min and the column temperature is 30°C.

[0033] Preferably, the detection wavelength is 230 nm.

[0034] Preferably, the injection volume is 20 μL.

[0035] The preferred detection conditions can achieve a higher separation degree between trimethyl phloroglucinol and various impurities to ensure effective detection of impurities, thereby achieving the purpose of effectively and accurately controlling the content of related substances in trimethyl phloroglucinol.

[0036] Preferably, mobile phase A is a phosphoric acid aqueous solution at pH 2.8.

[0037] The preferred pH value of mobile phase A can reduce spectral tailing and improve peak shape, thereby facilitating improved separation between components and making the detection results more accurate and precise.

[0038] Furthermore, the preparation process of the test solution is as follows: take the trimethylphloroglucinol test sample, accurately weigh it, add acetonitrile to dissolve it, and dilute it to make a 1 mg / mL test solution.

[0039] Furthermore, the preparation process of the reference solution is as follows: accurately measure an appropriate amount of the test solution, add acetonitrile and quantitatively dilute to prepare a 1 μg / mL reference solution.

[0040] The present invention has determined, through a large number of studies, that 1-chloro-3,5-dimethoxybenzene, 1-bromo-3,5-dimethoxybenzene, 3-bromo-5-chloroanisole, 3,5-dibromoanisole, 1-chloro-3,5-dibromobenzene, and 1,3,5-tribromobenzene are six impurities as indicator impurities in trimethyl phloroglucinol, and the quality of trimethyl phloroglucinol can be more effectively controlled by detecting these six impurities. The detection method provided by the present invention can simultaneously detect many types of impurities, is simple and fast to operate, has high sensitivity, provides a reliable method for quality control of trimethyl phloroglucinol and its preparation products, and is beneficial to improving the medication safety of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the chromatogram of the blank solvent under the specificity item in Example 2;

[0042] Figure 2 This is the chromatogram of the system suitability solution under the specificity item in Example 2. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] Example 1

[0045] Materials and Methods

[0046] Instruments: High performance liquid chromatograph, UV detector, volumetric flask, electronic balance.

[0047] Reagents: acetonitrile, phosphoric acid, water.

[0048] Blank solvent: acetonitrile.

[0049] 1.1 Solution preparation

[0050] Preparation of test solution: Take an appropriate amount of trimethylphloroglucinol test sample, accurately weigh it, add acetonitrile to dissolve it and quantitatively dilute it to make a solution containing about 1 mg per 1 mL to obtain the test solution.

[0051] Preparation of reference solution: Accurately measure an appropriate amount of the test solution, add acetonitrile to quantitatively dilute it to make a solution containing approximately 1 μg per 1 mL to obtain the reference solution.

[0052] System suitability solution: Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and trimethylphloroglucinol reference substance, accurately weigh, dissolve in acetonitrile and dilute to make a solution containing approximately 1 μg of each impurity and approximately 1 mg of trimethylphloroglucinol per 1 mL to obtain the system suitability solution.

[0053] HPLC conditions:

[0054] Chromatographic column: ZORBAX SB-C18 (250 mm*4.6 mm, 5 μm);

[0055] Mobile phase A: pH 2.8 phosphoric acid aqueous solution;

[0056] Mobile phase B: acetonitrile;

[0057] Flow rate: 1.0 mL / min;

[0058] Detection wavelength: 230nm;

[0059] Column temperature, 30 °C;

[0060] Injection volume: 20 μL.

[0061] Elution was performed according to the following gradient program:

[0062] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 10 20 80 25 20 80 25.1 75 25 35 75 25

[0063] Example 2

[0064] Methodological validation:

[0065] 2.1 Exclusivity

[0066] Take blank solvent and system suitability solution and perform injection test according to the above HPLC conditions, and record the chromatogram. Figure 1 As shown, the chromatographic peaks of system suitability are as follows Figure 2As shown, peaks 1 to 7 in the chromatogram of the test solution are, from left to right, the chromatographic peaks of trimethylphloroglucinol, impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F. The experimental results of the system suitability are shown in Table 1.

[0067] Table 1 System suitability test results

[0068]

[0069] The test results show that the baseline is stable and free of interference, and the blank solvent has no interference with the detection of impurities and main components; the separation between each component and each impurity is greater than 1.5, and there is no interference between each impurity or between each impurity and trimethylphloroglucinol, which is suitable for the detection of related substances, indicating that this method has good specificity.

[0070] 2.2 Limit of detection and limit of quantification

[0071] Trimethyl phloroglucinol stock solution: Take an appropriate amount of trimethyl phloroglucinol reference substance, dissolve it in acetonitrile and dilute it to make a solution containing about 10 μg per 1 mL of solution to obtain trimethyl phloroglucinol stock solution.

[0072] Stock solutions of each reference substance: Take appropriate amount of each reference substance of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F, dissolve them in acetonitrile and dilute them to make solutions containing about 10 μg per 1 mL of solution, to obtain the corresponding stock solutions of each reference substance.

[0073] Quantitation limit solution: Pipette 0.25 mL of the above-mentioned trimethylphloroglucinol stock solution, 0.5 mL of impurity A stock solution, 0.5 mL of impurity B stock solution, 1 mL of impurity C stock solution, 1 mL of impurity D stock solution, 2 mL of impurity E stock solution, and 2 mL of impurity F stock solution into the same 100 mL volumetric flask, dilute to the scale with acetonitrile, shake well, and obtain.

[0074] The above quantitative limit solution was diluted step by step, and the concentration when the signal-to-noise ratio was 3:1 was taken as the detection limit, and the concentration when the signal-to-noise ratio was 10:1 was taken as the quantitative limit. The results are shown in Table 2. The quantitative limit solution was injected 6 times continuously, and the RSD of the peak area was calculated. The results are shown in Table 3.

[0075] Table 2 Results of detection limit and quantification limit tests

[0076]

[0077] Table 3 Results of repeatability test of limit of quantitation

[0078]

[0079] The test results show that the detection limit of trimethyl phloroglucinol is 0.0077μg / mL (0.0008%), and the quantitative limit is 0.0256μg / mL (0.0026%). The detection limit of impurity A is 0.0156μg / mL (0.0016%), and the quantitative limit is 0.0519μg / mL (0.0052%). The detection limit of impurity B is 0.0153μg / mL (0.0015%), and the quantitative limit is 0.0510μg / mL (0.0051%). The detection limit of impurity C is 0.0306μg / mL (0.0031%), and the quantitative limit is 0.1018μg / mL (0.0102%). The detection limit of impurity D is 0.0308μg / mL (0.0031%), and the quantification limit is 0.1027μg / mL (0.0103%). The detection limit of impurity E is 0.0597μg / mL (0.0060%), and the quantification limit is 0.1991μg / mL (0.0199%). The detection limit of impurity F is 0.0598μg / mL (0.0060%), and the quantification limit is 0.1994μg / mL (0.0199%). This shows that this method has high sensitivity.

[0080] 2.3 Linear range

[0081] Linear stock solution: Take appropriate amount of trimethylphloroglucinol, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F reference substances, dissolve and dilute with acetonitrile to make a solution containing about 10 μg per 1 mL of solution, shake well to obtain linear stock solution.

[0082] A series of reference substance solutions: Take the above linear stock solution and dilute it in a gradient manner with acetonitrile to prepare a series of reference substance mixed solutions with the main component and each impurity concentration being approximately 0.2%, 0.15%, 0.1%, 0.08%, 0.05%, and 0.02% of the test sample concentration (1 mg / mL).

[0083] The detection was carried out according to the chromatographic conditions of Example 1: 20 μL of each reference solution was accurately measured and injected into the liquid chromatograph, the chromatogram was recorded, the peak area was determined, and a linear regression was performed with the peak area A as the ordinate and the concentration C as the abscissa. The results are shown in Tables 4 to 10.

[0084] Table 4 Trimethylphloroglucinol linear results

[0085]

[0086] Table 5 Impurity A linear results

[0087]

[0088]

[0089] Table 6 Impurity B linear results

[0090]

[0091] Table 7 Impurity C linearity results

[0092]

[0093] Table 8 Impurity D linear results

[0094]

[0095] Table 9 Impurity E linearity results

[0096]

[0097] Table 10 Impurity F linearity results

[0098]

[0099]

[0100] The above results show that trimethyl phloroglucinol has good linearity in the range of 0.206μg / mL to 2.062μg / mL, r=0.9997; impurity A has good linearity in the range of 0.208μg / mL to 2.076μg / mL, r=0.9998; impurity B has good linearity in the range of 0.204μg / mL to 2.041μg / mL, r=0.9998; impurity C has good linearity in the range of 0.204μg / mL to 2.037μg / mL The linearity was good within the range of 0.205 μg / mL to 2.055 μg / mL, r=0.9998; the linearity was good within the range of 0.199 μg / mL to 1.991 μg / mL, r=0.9997; the linearity was good within the range of 0.199 μg / mL to 1.994 μg / mL, r=0.9997; the linearity was good between the concentration and peak area of ​​each impurity.

[0101] 2.4 Accuracy

[0102] Reference substance stock solution: Take appropriate amount of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F reference substances, dissolve and dilute with acetonitrile to make a solution containing about 10 μg per 1 ml of solution, and shake well.

[0103] Accurately weigh about 10 mg of trimethylphloroglucinol reference substance and place it in a 10 mL volumetric flask. Weigh 9 portions in parallel and add appropriate amount of acetonitrile to dissolve them respectively. Then add 0.2 mL, 1 mL, and 1.5 mL of the above-mentioned reference substance stock solution and dilute to the scale with acetonitrile respectively. Prepare 3 portions in parallel for each concentration as the recovery test solution.

[0104] Accurately measure 20 μL of each of the above solutions, inject into the liquid chromatograph, record the chromatogram, calculate the recovery rate of each impurity and the RSD value of the recovery rate. The results are shown in Tables 11 to 16 below.

[0105] Table 11 Impurity A recovery results

[0106]

[0107]

[0108] Table 12 Impurity B recovery results

[0109]

[0110] Table 13 Impurity C recovery results

[0111]

[0112]

[0113] Table 14 Impurity D recovery results

[0114]

[0115] Table 15 Impurity E recovery results

[0116]

[0117] Table 16 Impurity F recovery results

[0118]

[0119] The above results show that the recoveries of each impurity at each concentration are all in the range of 90% to 110%, and the RSD values ​​are all less than 5%, indicating that the accuracy of this method is good.

[0120] 2.5 Repeatability

[0121] The test solution was prepared according to the method of Example 1 and the test was repeated 6 times. The results are shown in Table 17.

[0122] Table 17 Repeatability test results

[0123] Element Impurity A(%) Maximum single impurity (%) Total impurities (%) Repeatability -1 0.017 0.009 0.026 Repeatability -2 0.017 0.009 0.026 Repeatability - 3 0.017 0.010 0.027 Repeatability - 4 0.017 0.009 0.026 Repeatability -5 0.016 0.009 0.025 Repeatability - 6 0.017 0.009 0.026 Mean 0.017 0.009 0.026 Very bad 0.0006 0.0011 0.0015

[0124] The results showed that impurities B, C, D, E and F were not detected in the test solution after repeated testing for 6 times, and the maximum single impurity and total detection amount were consistent, indicating that the method had good repeatability.

[0125] 2.6 Durability

[0126] The system suitability solution was prepared in the same manner as in Example 1, and the durability test was carried out according to the test conditions shown in Table 18.

[0127] Table 18 Durability test conditions

[0128] Inspection conditions Standard conditions Range of variation Mobile phase pH pH 2.8 pH3.0, pH2.6 Initial ratio of mobile phase Water (pH 2.8)-acetonitrile = 75:25 80:20、70:30 Column temperature 30℃ 35℃、25℃ Flow rate 1mL / min 1.2mL / min、0.8mL / min wavelength 230nm 235nm, 225nm

[0129] The separation results are shown in Table 19.

[0130] Table 19 System suitability solution separation results under different chromatographic conditions

[0131]

[0132]

[0133] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting related substances in trimethylphloroglucinol, wherein the related substances are 1-chloro-3,5-dimethoxybenzene, 1-bromo-3,5-dimethoxybenzene, 3-bromo-5-chloroanisole, 3,5-dibromoanisole, 1-chloro-3,5-dibromobenzene and 1,3,5-tribromobenzene, characterized in that: High performance liquid chromatography was used for detection, and the chromatographic conditions were: Chromatographic column: octadecylsilane bonded silica gel column; UV detector, detection wavelength 225nm ~ 235nm; Mobile phase A: phosphoric acid aqueous solution with a pH of 2.6 to 3.0, mobile phase B is acetonitrile; The elution method was gradient elution.

2. The method for detecting related substances in trimethyl phloroglucinol as claimed in claim 1, characterized in that: The elution procedure of the gradient elution is as follows: 0min, 70%-80% mobile phase A, 30%-20% mobile phase B; 10 min, 20% mobile phase A, 80% mobile phase B; 25 min, 20% mobile phase A, 80% mobile phase B; 25.1 min, 70%-80% mobile phase A, 30%-20% mobile phase B; 35min, 70%-80% mobile phase A, 30%-20% mobile phase B.

3. The method for detecting related substances in trimethyl phloroglucinol as claimed in claim 2, characterized in that: The elution procedure of the gradient elution is as follows: 0 min, 75% mobile phase A, 25% mobile phase B; 10 min, 20% mobile phase A, 80% mobile phase B; 25 min, 20% mobile phase A, 80% mobile phase B; 25.1 min, 75% mobile phase A, 25% mobile phase B; 35 min, 75% mobile phase A, 25% mobile phase B.

4. The method for detecting related substances in trimethyl phloroglucinol as claimed in claim 1, characterized in that: The specification of the chromatographic column is 250mm*4.6mm, and the filler diameter is 5μm.

5. The method for detecting related substances in trimethyl phloroglucinol as claimed in claim 4, characterized in that: The chromatographic column is ZORBAX SB-C18, 250 mm*4.6 mm, 5 μm.

6. The method for detecting related substances in trimethyl phloroglucinol as claimed in claim 1, characterized in that: The flow rate is 0.8 mL / min to 1.2 mL / min, and the column temperature is 25°C to 35°C.

7. The method for detecting related substances in trimethyl phloroglucinol as claimed in claim 6, characterized in that: The flow rate was 1.0 mL / min and the column temperature was 30 °C.

8. The method for detecting related substances in trimethyl phloroglucinol as claimed in claim 1, characterized in that: The detection wavelength is 230 nm.

9. The method for detecting related substances in trimethyl phloroglucinol as claimed in claim 1, characterized in that: The injection volume was 20 μL.

10. The method for detecting related substances in trimethyl phloroglucinol as claimed in claim 1, characterized in that: Mobile phase A was a phosphoric acid solution at pH 2.8.

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