Method for determining impurity content in phloroglucinol trimethyl ether

Abstract

The invention belongs to the field of chemical medicine analysis, and particularly relates to a method for determining the content of impurities in phloroglucinol trimethyl ether. According to the method provided by the invention, the excessive methylation impurities (2, 4, 6-trimethoxytoluene, 2, 4, 6-trimethoxyxylene and 2, 4, 6-trimethoxytrimethylbenzene) generated in the methylation process can be detected, and the excessive methylation impurities are prevented from being further transferred and existing in a phloroglucinol trimethyl ether raw material medicine and a final product phloroglucinol injection preparation. Researches on specificity, accuracy, precision, limit of detection, limit of quantitation, linear range, durability and the like of the method prove that the method is suitable for measuring the content of the over-methylated impurities in the phloroglucinol trimethyl ether.

Description

Technical Field

[0001] The invention belongs to the field of chemical drug analysis, and particularly relates to a method for determining the content of over-methylated impurities in phloroglucinol trimethyl ether. Background Art

[0002] Phloroglucinol trimethyl ether is an effective antispasmodic drug that can directly act on smooth muscle. Its duration of action is about six times that of phloroglucinol. At the same time, it can make the sphincter and urethral smooth muscle more sensitive to phloroglucinol through the synergistic effect with phloroglucinol, thereby enhancing the antispasmodic and analgesic effects. Therefore, the phloroglucinol injection used clinically is a compound preparation made of phloroglucinol and phloroglucinol trimethyl ether.

[0003] Phloroglucinol is a new type of myotropic non-atropine non-pavoline pure smooth muscle antispasmodic drug used in clinical practice. It can directly act on the smooth muscles of the gastrointestinal tract and urogenital tract. Because phloroglucinol has the advantages of rapid antispasmodic and analgesic effects, significant effects, high safety numbers and no toxic side effects, it has extremely important clinical application value in the fields of urology, gastroenterology, obstetrics and gynecology. In France and European countries, phloroglucinol injection has been widely used in urology, gastroenterology, obstetrics and gynecology, etc., and has become the first choice for the treatment of spasmodic pain.

[0004] The preparation method of phloroglucinol trimethyl ether is generally to use phloroglucinol as a raw material, and under the action of a methylating agent, a methylation reaction occurs to obtain phloroglucinol trimethyl ether. During the methylation reaction, over-methylated impurities (2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene, 2,4,6-trimethoxytrimethylbenzene) are generated and further transferred to the phloroglucinol trimethyl ether raw material and the final product phloroglucinol injection preparation. Summary of the invention

[0005] The invention aims to provide a method for determining the impurity content in phloroglucinol trimethyl ether, which has strong specificity, simple operation, high sensitivity, and is accurate and reliable.

[0006] The method for detecting the content of impurities in phloroglucinol trimethyl ether of the present invention has the following specific implementation scheme: The present invention discloses a method for determining the impurity content in phloroglucinol trimethyl ether. The method uses acetonitrile-water as a solvent to prepare a reference solution and a test solution, wherein the impurity reference is one or more of 2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene and 2,4,6-trimethoxytrimethylbenzene.

[0007] The present invention discloses a method for determining the impurity content in phloroglucinol trimethyl ether, which is a high performance liquid chromatography method, comprising: Step 1) Prepare reference solution and test solution: dissolve 2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene and 2,4,6-trimethoxytrimethylbenzene reference substances in acetonitrile-water as solvent to prepare reference solution; dissolve phloroglucinol trimethyl ether in acetonitrile-water as solvent to prepare test solution; Step 2) Take samples of the test solution and the reference solution respectively, detect them by high performance liquid chromatography, and record the chromatograms.

[0008] The present invention discloses a method for determining the impurity content in phloroglucinol trimethyl ether. The high performance liquid chromatography detection conditions are as follows: using octadecylsilane bonded silica gel as a filler; the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is a phosphate buffer, the mobile phase B is a phosphate buffer-acetonitrile, and a gradient elution program is adopted.

[0009] The present invention discloses a method for determining the impurity content in phloroglucinol trimethyl ether, wherein the ratio of acetonitrile to water in the detection method is 20:80.

[0010] The present invention discloses a method for determining the impurity content in phloroglucinol trimethyl ether. The type of phosphate in the phosphate buffer described in the detection method is one or more of potassium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate and disodium phosphate.

[0011] The present invention discloses a method for determining the impurity content in phloroglucinol trimethyl ether. The volume ratio of the phosphate buffer solution to acetonitrile in the mobile phase B is 0.5:100-5:95.

[0012] The present invention discloses a method for determining the impurity content in phloroglucinol trimethyl ether. The phosphate concentration in the phosphate buffer described in the detection method is 5-20 mmol / L, and phosphoric acid is used to adjust the pH value to 2-3.

[0013] The present invention discloses a method for determining the impurity content in phloroglucinol trimethyl ether. The detection column temperature of the detection method is 20-35° C., the detection wavelength is 220 nm, and the flow rate is 0.8-1.2 ml per minute.

[0014] The present invention discloses a method for determining the impurity content in phloroglucinol trimethyl ether, wherein the gradient elution procedure of the detection method is: .

[0015] The method for determining the impurity content in phloroglucinol trimethyl ether of the present invention has an injection volume of 20 μl. Beneficial effects of the present invention: The method for analyzing impurities in phloroglucinol trimethyl ether provided by the present invention is the first method developed in the art, and fills the technical gap in the detection method of phloroglucinol and similar drug impurities.

[0016] The present invention provides a method for determining the impurity content in phloroglucinol trimethyl ether, which has the characteristics of strong specificity, simple operation, high sensitivity, accuracy and reliability. The method has a short detection time, improves the sample analysis efficiency, and reduces the experimental cost. At the same time, by optimizing the solvent, the influence of the solvent on the degradation of impurities in the analysis method is effectively reduced, and the accuracy and reliability of the analysis result are improved. Combined with comparative examples 1-2, the beneficial effect description of the solvent selection is supplemented. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the blank solvent chromatogram; Figure 2 The chromatogram is of the test sample spiked solution. DETAILED DESCRIPTION

[0018] The following examples are only for further illustrating the present invention and are not intended to limit the scope of the present invention in any form.

[0019] Example 1: Specificity (1) Chromatographic conditions: Testing instrument: Shimadzu LC2030 high performance liquid chromatograph Chromatographic column: Thermo Hypersil GOLD 4.6mm×250mm, 5μm Detection wavelength: 220nm Column temperature: 30°C Flow rate: 1.0ml / min Injection volume: 20 μl Mobile phase A: 10mmol / L potassium dihydrogen phosphate aqueous solution (adjust pH to 3.0 with phosphoric acid) Mobile phase B: acetonitrile Gradient elution program:

[0020] (2) Test methods Take 20 µl of each of the blank solution (solvent), each impurity location solution, test sample solution, and test sample spiked solution and inject them into the liquid chromatograph.

[0021] (3) Test results As shown in Table 1, the blank solvent has no interference with the components to be tested; phloroglucinol trimethyl ether, 2,4,6-trimethoxytoluene, 2,4,6-trimethoxytrimethylbenzene, unknown impurities, and 2,4,6-trimethoxyxylene peak in sequence, and the separation between the peaks of each component is greater than 1.5, which meets the detection requirements. Figure 1-2 .

[0022] Table 1: Example 1 specificity test results .

[0023] Example 2: Repeatability (1) Test method The chromatographic conditions were the same as those in Example 1. Six mixed solutions of the test sample containing 100% of 2,4,6-trimethoxyxylene, 2,4,6-trimethoxyxylene, and 2,4,6-trimethoxytrimethylbenzene within the specified limits were prepared in parallel, and the contents of 2,4,6-trimethoxyxylene, 2,4,6-trimethoxyxylene, and 2,4,6-trimethoxytrimethylbenzene were determined.

[0024] (2) Test results As shown in Table 2, the RSDs of the six test results of the spiked solution were all 0. This method has good repeatability.

[0025] Table 2: Repeatability test results of Example 2 .

[0026] Example 3: Intermediate Precision (1) Test method The chromatographic conditions were the same as those in Example 1. Different analysts used different equipment on different dates to repeatedly measure the contents of 2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene, and 2,4,6-trimethoxytrimethylbenzene in six spiked solutions of the test sample.

[0027] (2) Test results As shown in Table 3, different personnel used different instruments on different days to parallel measure 6 spiked test solutions, and the RSD of each impurity content was less than 5.0%; a total of 12 spiked test solutions were tested for repeatability, and the RSD of each impurity content was less than 10.0%, indicating that the method has good precision.

[0028] Table 3: Example 3 Intermediate precision test results .

[0029] Example 4: Accuracy (1) Test method The chromatographic conditions were the same as those in Example 1. 2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene, 2,4,6-trimethoxytrimethylbenzene and the test sample were taken, and three test sample mixed solutions at three concentration levels of 50%, 100% and 150% were prepared according to the limit concentration. The mixed solutions were injected into the chromatograph, and the recoveries of 2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene and 2,4,6-trimethoxytrimethylbenzene were calculated.

[0030] (2) Test results As shown in Tables 4 to 6, the impurity recoveries were all between 80% and 120%, and the RSD values ​​were all less than 5.0%, indicating that the method had good accuracy.

[0031] Table 4-6: Accuracy results of Example 4 Table 4 2,4,6-trimethoxytoluene recovery experimental results

[0032] Table 5 2,4,6-trimethoxyxylene recovery experimental results

[0033] Table 6 2,4,6-Trimethoxytrimethylbenzene recovery experimental results .

[0034] Example 5: Linear (1) Experimental methods The chromatographic conditions were the same as those in Example 1. 2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene, and 2,4,6-trimethoxytrimethylbenzene were used as reference substances to prepare 7 series of solutions at different concentration levels, which were injected into the liquid chromatograph respectively. The function curves of the peak area of ​​each impurity at different concentration levels and its concentration were plotted, and the intercept and correlation coefficient r were calculated.

[0035] (2) Test results As shown in Tables 7-9, within the concentration range from the limit of quantification to 200% limit, the concentration and peak area of ​​each component showed a good linear relationship, the correlation coefficient r was greater than 0.995, and the ratio of the absolute value of the Y-axis intercept to the 100% response value was less than 20.0%, indicating a good linear relationship.

[0036] Table 7 2,4,6-Trimethoxytoluene linearity and range

[0037]

[0038] Table 8 2,4,6-Trimethoxyxylene linearity and range

[0039] Table 9 2,4,6-Trimethoxytrimethylbenzene linearity and range .

[0040] Example 6: Limit of Quantitation and Limit of Detection (1) Experimental methods The chromatographic conditions were the same as those in Example 1. The reference solutions of 2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene and 2,4,6-trimethoxytrimethylbenzene were diluted for determination. When the S / N (signal-to-noise ratio) was 3-10, this concentration was the detection limit; when the S / N (signal-to-noise ratio) was ≥10, this concentration was the quantification limit. The quantification limit solution was injected 6 times continuously, the peak area was recorded, and the relative standard deviation of the peak area was calculated.

[0041] (2) Test results As shown in Table 10, the quantitative limit concentrations of the components to be tested were all no greater than 20% of the limit concentration, and the signal-to-noise ratios were all greater than 10; the signal-to-noise ratios of the detection limit concentration solutions were all greater than 3, indicating that the detection method met the sensitivity requirements for impurity detection.

[0042] Table 10 Results of quantitative limit and detection limit tests in Example 6 .

[0043] Example 7: Solution Stability (1) Chromatographic conditions The chromatographic conditions were the same as those in Example 1. Acetonitrile-water (20:80) was used as the solvent to dissolve 2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene, 2,4,6-trimethoxytrimethylbenzene reference substance and phloroglucinol trimethyl ether to prepare a spiked test solution.

[0044] Experimental methods Take the mixed reference solution and the spiked test solution at room temperature and measure them according to the law at intervals. Accurately measure 20µl of the mixed reference solution and the spiked test solution at different time periods and inject them into the liquid chromatograph respectively.

[0045] Test results The spiked test solution was placed at room temperature for 35 hours, and the RSD of the peak areas of phloroglucinol trimethyl ether, 2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene, and 2,4,6-trimethoxytrimethylbenzene at each time point was less than 2.0%, indicating that the spiked test solution was relatively stable after being placed at room temperature for 35 hours.

[0046] Table 11 Solution stability test results .

[0047] Comparative Example 1 (1) Chromatographic conditions The chromatographic conditions were the same as those in Example 1. 10 mmol / L potassium dihydrogen phosphate aqueous solution (pH adjusted to 3.0 with phosphoric acid)-acetonitrile (90:10) was used as the solvent to dissolve 2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene, 2,4,6-trimethoxytrimethylbenzene reference substance and phloroglucinol trimethyl ether to prepare a spiked test solution.

[0048] (2) Experimental methods: Take the mixed reference solution and the spiked test solution at room temperature and measure them according to the law at intervals. Accurately measure 20µl of the mixed reference solution and the spiked test solution at different time periods and inject them into the liquid chromatograph respectively.

[0049] (3) Test results The spiked test solution was placed at room temperature for 35 h. The RSDs of the peak areas of phloroglucinol trimethyl ether, 2,4,6-trimethoxytoluene, and 2,4,6-trimethoxytrimethylbenzene at each time point were all less than 2.0%. The peak area of ​​4,6-trimethoxyxylene showed a decreasing trend, and the RSDs of the peak areas were all greater than 2.0%, indicating that impurity F was degraded when 10 mmol / L potassium dihydrogen phosphate aqueous solution (pH adjusted to 3.0 with phosphoric acid)-acetonitrile (90:10) was used as the solvent.

[0050] Table 12 Experimental results of comparative example 1 .

[0051] Comparative Example 2 (1) Chromatographic conditions Testing instrument: Shimadzu LC2030 high performance liquid chromatograph Chromatographic column: Thermo Hypersil GOLD 4.6mm×250mm, 5μm Detection wavelength: 220nm Column temperature: 30°C Flow rate: 1.0ml / min Injection volume: 20 μl Mobile phase A: 10mmol / L potassium dihydrogen phosphate aqueous solution (adjust pH to 3.0 with phosphoric acid) Mobile phase B: 10mmol / L potassium dihydrogen phosphate aqueous solution (adjust pH to 3.0 with phosphoric acid)-acetonitrile (90:10) Gradient elution program: .

[0052] (2) Test methods Take 20 µl of each impurity location solution and inject it into the liquid chromatograph.

[0053] (3) Test results As shown in Table 13, the peak times of phloroglucinol trimethyl ether, 2,4,6-trimethoxytoluene, 2,4,6-trimethoxytrimethylbenzene, and 2,4,6-trimethoxyxylene are relatively late, and the analysis time is relatively long, which cannot meet the detection requirements.

[0054] Table 13: Comparative Example 2 Test Results .

Claims

1. A method for determining the impurity content in phloroglucinol trimethyl ether, characterized in that: The detection method uses acetonitrile-water as a solvent to prepare a reference solution and a test solution, wherein the impurity reference is one or more of 2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene, and 2,4,6-trimethoxytrimethylbenzene.

2. A method for determining the impurity content in phloroglucinol trimethyl ether according to claim 1, characterized in that: The test method is high performance liquid chromatography, including: Step 1) Prepare reference solution and test solution: dissolve 2,4,6-trimethoxytoluene, 2,4,6-trimethoxyxylene and 2,4,6-trimethoxytrimethylbenzene reference substances in acetonitrile-water as solvent to prepare reference solution; dissolve phloroglucinol trimethyl ether in acetonitrile-water as solvent to prepare test solution; Step 2) Take samples of the test solution and the reference solution respectively, detect them by high performance liquid chromatography, and record the chromatograms.

3. A method for determining the impurity content in phloroglucinol trimethyl ether according to any one of claims 1 to 2, characterized in that: The high performance liquid chromatography detection conditions are: using octadecylsilane bonded silica gel as a filler; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is phosphate buffer, the mobile phase B is phosphate buffer-acetonitrile, and a gradient elution program is used.

4. A method for determining the impurity content in phloroglucinol trimethyl ether according to any one of claims 1 to 2, characterized in that: The ratio of acetonitrile to water in the detection method is 20:

80.

5. A method for determining the impurity content in phloroglucinol trimethyl ether according to claim 3, characterized in that: The type of phosphate in the phosphate buffer described in the detection method is one or more of potassium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, and disodium phosphate.

6. A method for determining the impurity content in phloroglucinol trimethyl ether according to claim 3, characterized in that: The volume ratio of phosphate buffer solution to acetonitrile in the mobile phase B of the detection method is 0.5:100-5:

95.

7. A method for determining the impurity content in phloroglucinol trimethyl ether according to claim 3, characterized in that: The phosphate concentration in the phosphate buffer described in the detection method is 5-20 mmol / L, and the pH value is adjusted to 2-3 with phosphoric acid.

8. A method for determining the impurity content in phloroglucinol trimethyl ether according to claim 3, characterized in that: The detection column temperature of the detection method is 20-35°C, the detection wavelength is 220nm, and the flow rate is 0.8-1.2ml per minute.

9. A method for determining the impurity content in phloroglucinol trimethyl ether according to claim 3, characterized in that: The gradient elution procedure described in this detection method is: 。

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