As a 13 C Methacetin impurity reference substance p-aminoanisole and 13 C. Analytical method of methacetin

Abstract

The invention provides p-aminoanisole, an impurity of C13 methacetin, and use of the p-aminoanisole as an impurity reference substance, and establishes a method capable of detecting the p-aminoanisole impurity and other impurities in C13 methacetin. The method of the invention can detect the impurity content in C13 methacetin, has the advantages of short analysis time, high separation, good peak shape, simple operation, energy saving, etc., solves the problem of impurity detection in C13 methacetin, thereby ensuring that the quality of C13 methacetin products is controllable, and is of great significance to further improving the product quality of C13 methacetin.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a C13 methacetin impurity, use of the C13 methacetin impurity reference substance, and an analysis method of the C13 methacetin. Background Art

[0002] Stable isotope carbon-13 labeled breath reagents have gradually replaced radioactive reagents due to their high safety, simplicity, non-invasiveness, high sensitivity and specificity, and have played an increasingly important role in the diagnosis of clinical diseases and have been favored by the medical industry at home and abroad. Among them, the 13C methacetin breath test is a new method for detecting liver reserve function that has recently emerged and has high practical value. Through experiments, it was found that the 13C methacetin breath test value can distinguish between cirrhosis and non-cirrhosis.

[0003] 13C-methacetin, chemical name is acetaminophenoxy methyl ether (methoxy-13C), and its structural formula is as follows:

[0004]

[0005] According to current reports at home and abroad, the synthetic route of C13 methacetin is the reaction of 13C-labeled small molecules with acetaminophenol. The labeled small molecules mainly include methanol, methyl iodide and dimethyl sulfate. The main impurity reported so far is acetaminophenol. However, p-aminoanisole belongs to the list of Class 3 carcinogens published by the World Health Organization's International Agency for Research on Cancer. It is also an impurity that must be studied in C13 methacetin. There is no literature reporting on p-aminoanisole impurities in C13 methacetin, methods for detecting p-aminoanisole in C13 methacetin, and the use of p-aminoanisole as a reference substance for impurities in C13 methacetin. Summary of the invention

[0006] In order to strictly control the product quality, through research, p-aminoanisole is used as an impurity reference substance, and a method capable of detecting p-aminoanisole impurities and other impurities in C13 methacetin is established. The method of the present invention can detect the impurity content in C13 methacetin, has the advantages of short analysis time, high separation, good peak shape, simple operation, energy saving, etc., solves the problem of impurity detection in C13 methacetin, thereby ensuring the quality of C13 methacetin products to be controllable, and is of great significance to further improving the product quality of C13 methacetin.

[0007] On the one hand, the present invention relates to a C13 methacetin impurity, characterized in that the impurity is p-aminoanisole, and its structural formula is as follows:

[0008]

[0009] On the other hand, the present invention also relates to a use of p-aminoanisole as an impurity reference substance for C13 methacetin.

[0010] On the other hand, the present invention also relates to a method for analyzing C13 methacetin, characterized in that it comprises the following steps:

[0011] Step (1), preparation of mobile phase: preparing phosphate buffer by taking disodium hydrogen phosphate, sodium dihydrogen phosphate and water, and using a mixture of phosphate buffer and acetonitrile as the mobile phase;

[0012] Step (2), preparation of the test solution: take C13 methacetin, accurately weigh it, put it in a volumetric flask, add diluent to dissolve the C13 methacetin and dilute it to the scale, shake well, and use it as the test solution;

[0013] Step (3), preparation of reference solution: take the impurity p-aminoanisole reference substance, place it in a volumetric flask, add diluent to dissolve and dilute to the scale, shake well, accurately pipette a certain volume of the solution, place it in a volumetric flask, add diluent to dissolve and dilute to the scale, shake well, as the reference solution;

[0014] Step (4), accurately measuring the test solution and the reference solution, injecting them into a liquid chromatograph, recording the chromatogram, and calculating the peak area by the external standard method. The content of p-aminoanisole shall not exceed 0.10%, and other single impurities shall not exceed 0.10% by calculating the C13 methacetin reference area in the chromatogram of the reference solution. The sum of other impurities shall not exceed 0.5%.

[0015] In some embodiments, the preparation of the mobile phase in step (1) is specifically as follows: 8000-9500 mg of disodium hydrogen phosphate and 3500-4500 mg of sodium dihydrogen phosphate are accurately weighed and placed in the same 1-liter mobile phase bottle, water is added to make up the volume to obtain phosphate buffer, and the volume ratio of phosphate buffer to acetonitrile is (50-90): (10-50) to prepare the mobile phase.

[0016] In some embodiments, the amount of disodium hydrogen phosphate is 8950 mg.

[0017] In some embodiments, the amount of sodium dihydrogen phosphate is 3900 mg.

[0018] In some embodiments, the volume ratio of phosphate buffer to acetonitrile is 80 / 20.

[0019] In some embodiments, the preparation of the test solution in step (2) is specifically as follows: 10-50 mg of C13 methacetin is taken, accurately weighed, placed in a 10 ml volumetric flask, diluted with diluent to dissolve and dilute to the scale, and shaken to obtain the test solution.

[0020] In some embodiments, in the step (2) of preparing the test solution, the amount of C13 methacetin weighed is 20 mg.

[0021] In some embodiments, the preparation of the reference solution in step (3) is specifically as follows: 10-50 mg of the impurity p-aminoanisole reference substance is placed in a 100 ml volumetric flask, diluted with diluent to dissolve and dilute to the scale, shake well, accurately pipette 1 ml of the solution, placed in a 100 ml volumetric flask, diluted with diluent to dissolve and dilute to the scale, shake well, and use as the reference solution.

[0022] In some embodiments, in the step (3) of preparing the reference solution, the amount of p-aminoanisole weighed is 20 mg.

[0023] In some embodiments, the analytical method of C13 methacetin further includes the step of preparing a system suitability test solution, specifically: taking an appropriate amount of the impurity p-aminoanisole reference substance, adding a diluent to dissolve and dilute to prepare a mixed solution containing 1-5 μg of p-aminoanisole and 0.1-0.5 mg of C13 methacetin per 1 ml, as the system suitability solution.

[0024] In some embodiments, the system suitability solution contains 2 μg of p-aminoanisole and 0.2 mg of C13 methacetin per 1 ml.

[0025] In some embodiments, the diluent is a mixed solution with a volume ratio of acetonitrile / water of (10-50):(50-90).

[0026] In some embodiments, the diluent is a mixed solution of acetonitrile / water in a volume ratio of 20:80.

[0027] In some embodiments, in step (4), the volume of the test solution and the reference solution is precisely measured to be 5-10 μl.

[0028] In some embodiments, in step (4), the volume of the test solution and the reference solution is precisely measured to be 5 μl.

[0029] In some embodiments, in step (4), the chromatographic conditions are: detection wavelength of 233 nm, column temperature of 30-40° C., flow rate of 0.5-1 mL·min -1 .

[0030] In some embodiments, in step (4), the chromatographic conditions are: detection wavelength of 233 nm, column temperature of 35° C., flow rate of 0.8 mL·min -1 .

[0031] In some embodiments, in step (4), the chromatographic column is a C18 bonded silica chromatographic column.

[0032] In some embodiments, in step (4), the chromatographic column is selected from the chromatographic column Kromasil 100-5-C18, 4.6*250 mm.

[0033] The beneficial effects of the present invention are:

[0034] (1) The present invention uses p-aminoanisole as an impurity reference substance of C13 methacetin, detects and quantifies p-aminoanisole in the C13 methacetin product, ensures that the quality of the C13 methacetin product is controllable, and further improves the product quality of C13 methacetin.

[0035] (2) The analysis method of the present invention can detect the impurity content in C13 methacetin, which has the advantages of short analysis time, high separation, good peak shape, simple operation, and energy saving. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Shown is the HPLC spectrum of the p-aminoanisole impurity reference substance of Example 1;

[0037] Figure 2 Shown is the HPLC spectrum of C13 methacetin of Example 1;

[0038] Figure 3 Shown is the long-term 0-month HPLC profile of the stability test of Example 2;

[0039] Figure 4 Shown is the HPLC profile of the stability test for 3 months in Example 3;

[0040] Figure 5 Shown is the long-term 6-month HPLC profile of the stability test of Example 4;

[0041] Figure 6 Shown is the HPLC profile of the long-term 9-month stability test of Example 5;

[0042] Figure 7 Shown is the long-term 12-month HPLC profile of the stability test of Example 6. DETAILED DESCRIPTION

[0043] The present invention is described in detail below in conjunction with embodiments and drawings, but the following embodiments should not be construed as limiting the scope of the present invention.

[0044] Example 1

[0045] 1. Test drugs and reagents: acetonitrile, water, disodium hydrogen phosphate, sodium dihydrogen phosphate

[0046] 2. Instruments and equipment: 1 / 100,000 balance, chromatographic column (Kromasil (100-5-C18, 4.6*250mm or chromatographic column with equivalent performance)), high performance liquid chromatograph, volumetric flask, beaker, pipette

[0047] 3. Solvent: Phosphate buffer (take 8950 mg of disodium hydrogen phosphate and 3900 mg of sodium dihydrogen phosphate, weigh accurately, put them in the same 1-liter mobile phase bottle, and add water to make up to volume) / acetonitrile = 80 / 20 as the mobile phase.

[0048] 4. Preparation of system suitability test solution: Take an appropriate amount of the impurity p-aminoanisole reference substance, add diluent [acetonitrile-water (20:80)] to dissolve and dilute to make a mixed solution containing 2 μg of impurity and 0.2 mg of test sample per 1 ml, which is used as the system suitability solution.

[0049] 5. Preparation of test solution: Take about 20 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use as the test solution.

[0050] 6. Preparation of reference solution: Take about 20 mg of the impurity p-aminoanisole reference substance, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, accurately pipette 1 ml of the solution, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use it as the reference solution.

[0051] 7. Chromatographic conditions: Use Kromasil (100-5-C18, 4.6*250mm or a chromatographic column with equivalent performance) as the chromatographic column, phosphate buffer (take 8950 mg of disodium hydrogen phosphate and 3900 mg of sodium dihydrogen phosphate, accurately weigh, put into the same volumetric flask, add water to make up to volume) / acetonitrile = 80 / 20 as the mobile phase, the detection wavelength is 233 nm, the column temperature is 35°C, and the flow rate is 0.8 mL·min-1.

[0052] 8. Accurately measure 5 μl of the test solution and the reference solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the peak area using the external standard method. The content of p-aminoanisole shall not exceed 0.10%. Other individual impurities shall be calculated using the methacetin reference area in the chromatogram of the reference solution and shall not exceed 0.10%. The sum of other impurities shall not exceed 0.5%.

[0053] The obtained spectrum is as Figure 1 and Figure 2 shown.

[0054] The acceptable standards for validation of the impurity analysis methodology of the present invention are shown in Table 1.

[0055] Table 1

[0056]

[0057] Example 2

[0058] According to the analysis method of Example 1, the C13 methacetin that was placed for 0 months was analyzed and tested. The HPLC spectrum is as follows Figure 3 shown.

[0059] Example 3

[0060] According to the analysis method of Example 1, the C13 methacetin that had been placed for 3 months was analyzed and tested. The HPLC spectrum is as follows: Figure 4 shown.

[0061] Example 4

[0062] According to the analysis method of Example 1, the C13 methacetin that had been placed for 6 months was analyzed and tested. The HPLC spectrum is as follows Figure 5 shown.

[0063] Example 5

[0064] According to the analysis method of Example 1, the C13 methacetin that had been placed for 9 months was analyzed and tested. The HPLC spectrum is as follows: Figure 6 shown.

[0065] Example 6

[0066] According to the analysis method of Example 1, C13 methacetin that had been placed for 12 months was analyzed and tested. The HPLC spectrum is as follows: Figure 7 shown.

[0067] By the attached Figure 3-7 It can be seen that the impurity contents in C13 methacetin after long-term storage are in compliance with regulations. The specific results are shown in Table 2.

[0068] Table 2

[0069]

[0070] By the attached Figure 1-2 It can be seen that the analysis method of the present invention can effectively separate p-aminoanisole and C13 methacetin, and has a short analysis time, high separation degree, and good peak shape. The analysis method of the present invention has the advantages of simple operation and energy saving.

[0071] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A 13 C. The analytical method of methacetin, characterized in that The following steps are involved: Step (1), preparation of mobile phase: prepare phosphate buffer by taking disodium hydrogen phosphate, sodium dihydrogen phosphate and water, and use a mixture of phosphate buffer and acetonitrile as the mobile phase, wherein the volume ratio of the phosphate buffer to acetonitrile is 80 / 20; Step (2), preparation of test solution: 13 C. Methacetin, weigh accurately, place in a volumetric flask, add diluent to dissolve 13 C methacetin and dilute to the mark, shake well, and use as the test solution; Step (3), preparation of reference solution: take 10-50 mg of the impurity p-aminoanisole reference substance, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to scale, shake well, accurately pipette 1 ml of the solution, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to scale, shake well, as a reference solution, the structural formula of the p-aminoanisole is as follows: ; Step (4), accurately measure the test solution and the reference solution, inject them into the liquid chromatograph, record the chromatogram, calculate the peak area according to the external standard method, and the content of p-aminoanisole shall not exceed 0.10%. Other single impurities are calculated by the peak area in the chromatogram of the reference solution. 13 C. Methacetin control area shall not exceed 0.10%, and the sum of other impurities shall not exceed 0.5%; The 13 The analytical method of C methacetin also includes the steps of preparing a system suitability test solution, specifically: taking an appropriate amount of the impurity p-aminoanisole reference substance, adding a diluent to dissolve and dilute it to make each 1 ml contain 1-5 μg of p-aminoanisole, and 13 C. A mixed solution of 0.1-0.5 mg of methacetin as a system suitability solution; The diluent is a mixed solution of acetonitrile and water in a volume ratio of (10-50): (50-90); In step (4), the chromatographic conditions are: detection wavelength 233 nm, column temperature 35 ° C, flow rate 0.8 mL • min -1 ; In the step (4), the chromatographic column is a C18 bonded silica gel chromatographic column, and the C18 bonded silica gel chromatographic column is a chromatographic column Kromasil100-5-C18.

2. An analysis method according to claim 1, characterized in that The preparation of the mobile phase in step (1) is as follows: 8000-9500 mg of disodium hydrogen phosphate and 3500-4500 mg of sodium dihydrogen phosphate are accurately weighed and placed in a 1-liter mobile phase bottle, water is added to make up the volume to obtain a phosphate buffer, and phosphate buffer and acetonitrile are taken to prepare a mobile phase, wherein the volume ratio of the phosphate buffer to acetonitrile is 80 / 20.

3. An analysis method according to claim 2, characterized in that: In the step (1), 8950 mg of disodium hydrogen phosphate and 3900 mg of sodium dihydrogen phosphate are used; and the volume ratio of the phosphate buffer and acetonitrile is 80 / 20.

4. An analysis method according to claim 1, characterized in that: The preparation of the test solution in step (2) is specifically as follows: 13 C. Weigh 10-50 mg of methacetin accurately and place it in a 10 ml volumetric flask. Add diluent to dissolve and dilute to the mark. Shake well and use this as the test solution.

5. An analytical method according to claim 4, wherein in the step (2) of preparing the test solution, weigh 13 The amount of C methacetin is 20 mg.

6. An analysis method according to claim 1, characterized in that: In the step (3) of preparing the reference solution, 20 mg of p-aminoanisole is weighed.

7. An analysis method according to claim 1, characterized in that: The system suitability solution contains 2 μg of p-aminoanisole per 1 ml, and 13 C Methacetin 0.2mg.

8. An analysis method according to claim 1, characterized in that: The diluent is a mixed solution of acetonitrile and water in a volume ratio of 20:

80.

9. An analysis method according to claim 1, characterized in that: In the step (4), the volume of the test solution and the reference solution is accurately measured to be 5-10 μl.

10. An analysis method according to claim 9, characterized in that: In the step (4), the volume of the test solution and the reference solution is precisely measured to be 5 μl.

11. An analysis method according to claim 1, characterized in that: In the step (4), the specification of the chromatographic column is 4.6*250mm.

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