A method for determining genotoxic impurities in urapidil hydrochloride raw material

Abstract

The invention discloses a method for detecting a genotoxic impurity 6-[[3-(2-methoxyphenylamino)propyl]amino]-1,3-dimethyluracil in an urapidil hydrochloride raw material medicine. The method has good separation, strong specificity, high sensitivity and good accuracy, and can control the genotoxic impurity 6-[[3-(2-methoxyphenylamino)propyl]amino]-1,3-dimethyluracil in the urapidil hydrochloride raw material medicine, thereby ensuring the quality of a final product and the safety of medication.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry and analysis, and particularly relates to a method for determining genotoxic impurities in urapidil hydrochloride raw material medicine. Background Art

[0002] Urapidil can block the postsynaptic α1 receptor and presynaptic α2 receptor, has a central hypotensive effect, can reduce peripheral resistance, lower blood pressure, and will not cause reflex tachycardia while lowering blood pressure. It is mainly used in the clinic for various types of hypertension and controlled blood pressure reduction before, during and after surgery. It can also be used to treat urination disorders associated with prostatic hypertrophy. Urapidil hydrochloride is the API of urapidil hydrochloride injection and urapidil sustained-release tablets used in the clinic, and its molecular structure is shown in Formula 1.

[0003]

[0004] 6-[[3-(2-methoxyphenylamino)propyl]amino]-1,3-dimethyluracil (hereinafter referred to as 6-PADP), the molecular structure of which is shown in Formula 2, is a potential impurity that may be produced in the synthesis process of urapidil. It is the product of a substitution reaction between the synthetic intermediate P2 of urapidil hydrochloride and the impurity SM3-4 in the starting material SM3. The specific production pathway is shown in Formula 3 and Formula 4. At present, almost all synthetic routes of urapidil hydrochloride contain the intermediate P2 and the starting material SM3, so it is very necessary to develop an analytical method for detecting the impurity 6-[[3-(2-methoxyphenylamino)propyl]amino]-1,3-dimethyluracil in the raw material of urapidil hydrochloride.

[0005]

[0006]

[0007] 6-PADP is a derivative of aniline and may have potential genotoxicity. The (Q)SAR method (expert rules and statistics, Derek and Sarah) was used to predict genotoxicity. The prediction results showed that 6-PADP is Class 3, that is, "a known mutagen with unknown carcinogenicity", so this impurity should be controlled as a genotoxic impurity in the raw material drug, and the acceptable intake should be calculated based on TTC (1.5μg / day).

[0008] According to the instructions for urapidil hydrochloride injection, the maximum daily dose of this product is 250 mg based on urapidil, and its limit is calculated as 1.5 (μg / day) × 1000 / 250 (mg / day) / 1.094 = 5.48 ppm. Therefore, the detection method for the impurity 6-PADP in urapidil hydrochloride should be developed according to the control limit of less than 5 ppm. This low-limit detection method cannot be effectively detected according to the related substance detection method under urapidil in the Chinese Pharmacopoeia. Therefore, a more sensitive and accurate method for detecting the genotoxic impurity 6-PADP is urgently needed to be established and used for the detection of this impurity. Summary of the invention

[0009] The purpose of the present invention is to establish an analytical method for the genotoxic impurity 6-PADP in the raw material of urapidil hydrochloride. The method has good separation, strong specificity, high sensitivity and good accuracy, and can control the impurity 6-PADP in the raw material of urapidil hydrochloride to ensure the quality of the final product.

[0010] In order to achieve the above object, the present invention adopts a method of combining high performance liquid chromatography with mass spectrometry to detect the genotoxic impurity 6-PADP in the raw material of urapidil hydrochloride, which is achieved by the following technical scheme:

[0011] Detection conditions: octadecylsilane bonded silica gel is used as filler (CORTECS C18 3.0mm×75mm, 2.7μm or chromatographic column with equivalent performance); 0.02mol / L ammonium formate solution is used as mobile phase A, acetonitrile is used as mobile phase B, and linear gradient elution is performed according to the table below; the flow rate is 0.5ml per minute; the column temperature is 30℃; the detector is a mass spectrometer detector; the ion source is an electrospray ion source (ESI); the acquisition mode is positive ion SIM mode acquisition; the monitoring ion is 319.2; the mass spectrometer acquisition time is 8min~13min; the injection volume is 5μl.

[0012]

[0013] Determination method: Accurately measure the test solution and the reference solution, inject them into the HPLC-MS instrument respectively, and record the mass spectrum. Calculate the impurity 6-PADP content by the peak area external standard method, and the calculation formula is as follows:

[0014]

[0015] Where:

[0016] M 对 : The weight of impurities in the reference solution, mg;

[0017] A 对 : The average value of the peak area of ​​impurities in the reference solution;

[0018] M 供 : The amount of the test solution, mg;

[0019] A 供 : Peak area of ​​impurities in the test solution.

[0020] The test solution: Take an appropriate amount of the product, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing about 4 mg per 1 mL.

[0021] The reference substance solution: take about 2 mg of the impurity 6-PADP reference substance, accurately weigh it, put it in a 100 ml volumetric flask, add 2 ml of acetonitrile to dissolve it, and quantitatively dilute it with water to make a solution containing about 20 ng per 1 ml.

[0022] The present invention achieves the following beneficial technical effects:

[0023] Due to the adoption of the above analysis method, the present invention has good separation, strong specificity, high sensitivity and good accuracy, and can control the impurity 6-PADP in the urapidil hydrochloride raw material to ensure the quality of the final product. At the same time, it can also provide a useful reference and basis for the detection of other impurities with extremely low limits in urapidil hydrochloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0025] Figure 1 This is the chromatogram of the spiked mixed solution in Comparative Example 1;

[0026] Figure 2 This is the chromatogram of the spiked mixed solution in Comparative Example 2;

[0027] Figure 3 This is the chromatogram of the spiked mixed solution in Comparative Example 3;

[0028] Figure 4 This is the mass spectrum of the reference solution in Comparative Example 4. DETAILED DESCRIPTION

[0029] The present invention can be further described by the following examples, however, the present invention is not limited to the following examples, and these examples do not limit the scope of the present invention in any way. Certain changes and adjustments made by those skilled in the art within the scope of the claims should also be considered to belong to the scope of the present invention.

[0030] Reagents and Materials

[0031] Water: Laboratory-made Grade 1 water in accordance with GB / T6682-2008

[0032] Ammonium formate: analytical grade

[0033] Ammonium acetate: chromatographic grade

[0034] Acetonitrile: chromatographic grade

[0035] Formic acid: chromatographic grade

[0036] Instruments and Equipment

[0037] Agilent 1260+G6125c HPLC-MS

[0038] Mettler XS105DU Analytical Balance

[0039] Mettler MS304TS Analytical Balance

[0040] Sartorius MSA6.6S-OCE-DM Analytical Balance

[0041] Example:

[0042] 1. Solution preparation:

[0043] Stock solution of reference substance: Accurately weigh 2.029 mg of impurity 6-PADP reference substance, place it in a 100 ml volumetric flask, add 2.0 ml of acetonitrile to dissolve it, dilute to the scale with water, shake well, and obtain.

[0044] Reference substance mother solution: Accurately measure 1.0 ml of reference substance stock solution, place in a 100 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0045] (1) Specific solution:

[0046] Blank solution: water.

[0047] Reference substance solution: Accurately measure 5.0 ml of reference substance mother solution, place in a 50 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0048] Test solution: Accurately weigh 40.81 mg of the test sample (urapidil hydrochloride bulk drug), place it in a 10 ml volumetric flask, dissolve it in water and dilute it to the scale, shake well, and obtain.

[0049] Spiked test solution: Accurately weigh 40.92 mg of the test sample, place it in a 10 ml volumetric flask, dissolve and dilute to the scale with the reference solution, shake well, and obtain.

[0050] (2) Sensitivity solution:

[0051] Limit of quantitation solution (LOQ): Accurately measure 1.0 ml of the reference solution in the specific solution, place it in a 10 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0052] Detection limit solution (LOD): Accurately measure 3.0 ml of quantitation limit solution, place it in a 10 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0053] (3) Linear solution:

[0054] Linear solution #6: Accurately measure 10.0 ml of the mother solution of the reference substance, place it in a 50 ml volumetric flask, dilute to the mark with water, shake well, and the solution is ready.

[0055] Linear 1#~5# solutions: Accurately measure 1.0ml, 3.0ml, 5.0ml, 7.0ml, and 9.0ml of linear 6# solution respectively, place them in different 10ml volumetric bottles, dilute to the scale with water, shake well, and use them as linear 1#~5# solutions.

[0056] (4) Accuracy solution:

[0057] Reference substance solution: Accurately measure 10.0 ml of reference substance mother solution, place in a 100 ml volumetric flask, dilute to the scale with water, shake well, and obtain.

[0058] Test solution: Accurately weigh 40.28 mg of the test sample, place it in a 10 ml volumetric flask, dissolve it in water and dilute it to the scale, shake well, and obtain.

[0059] Spiked test solution: Take about 40 mg of the test sample, weigh accurately, place in a 10 ml volumetric flask, dissolve and dilute to the mark with the reference solution, shake well, and the solution is ready. Prepare 6 portions in parallel, weighing 40.13 mg, 40.57 mg, 40.85 mg, 40.25 mg, 40.59 mg, and 40.06 mg respectively.

[0060] (5) Solution stability Solution:

[0061] Reference solution: Take the reference solution from the specific solution, place it at room temperature, and test at 0h, 0.5h, 3h, 3.5h, and 6h.

[0062] Spiked test solution: Take the spiked test solution in the specific solution, place it at room temperature, and detect it at 0h, 0.5h, 3h, 3.5h, and 6h.

[0063] 2. Detection conditions: chromatographic column (CORTECS C18 3.0mm×75mm, 2.7μm or chromatographic column with equivalent performance); 0.02mol / L ammonium formate solution as mobile phase A, acetonitrile as mobile phase B, linear gradient elution according to the table below; flow rate of 0.5ml per minute; column temperature of 30℃; detector: mass spectrometer; ion source: electrospray ion source (ESI); acquisition mode: positive ion SIM mode; monitoring ion is 319.2; mass spectrometer acquisition time is 8min~13min; injection volume is 5μl.

[0064]

[0065]

[0066] 3. Determination method: Accurately measure each test solution, inject it into the high performance liquid chromatography-mass spectrometer respectively, and record the mass spectrum.

[0067] This example verifies the specificity, accuracy, precision (repeatability, injection precision), sensitivity (detection limit, quantitation limit), linearity and range, and durability (solution stability) of the method. The verification results are summarized as follows:

[0068]

[0069] According to the results in the above table, this method has strong specificity, good accuracy and high sensitivity, so it can achieve quantitative monitoring of the genotoxic impurity 6-[[3-(2-methoxyphenylamino)propyl]amino]-1,3-dimethyluracil in the raw material of urapidil hydrochloride, and also provide a useful reference and basis for the detection of other impurities with low limits in urapidil hydrochloride.

[0070] Comparative Example 1: The spiked mixed solution was determined using the following chromatographic conditions: Figure 1 It can be seen that the main peak of the test sample and the impurity 6-PADP cannot be baseline separated.

[0071]

[0072] Comparative Example 2: The spiked mixed solution was determined using the following chromatographic conditions: Figure 2 It can be seen that the retention times of the main peak of the test sample and the impurity 6-PADP are 11.3min and 11.8min respectively, and the separation degree is 2.8. Under this condition, 6-PADP is at the tail of the main peak of the test sample, and the main component will interfere with the detection of impurity 6-PADP during mass spectrometry detection.

[0073]

[0074] Comparative Example 3: The spiked mixed solution was determined using the following chromatographic conditions: Figure 3 It can be seen that under this condition, the retention times of the main peak of the test sample and the impurity 6-PADP are 5.0min and 9.0min respectively, and the separation degree is 7.8. Under this condition, 6-PADP is still at the tail of the main peak of the test sample, and the main component will still interfere with the impurity detection during mass spectrometry detection.

[0075]

[0076] Comparative Example 4: The target impurities in the reference solution were determined using the following chromatographic and mass spectrometric conditions: Figure 4 It can be seen that under this condition, when the acquisition mode is scan mode, the sensitivity of the impurity 6-PADP is relatively low and does not meet the detection requirements.

[0077]

[0078]

Claims

1. A method for determining genotoxic impurities in urapidil hydrochloride bulk drug, characterized in that: The genotoxic impurity is 6-[[3-(2-methoxyphenylamino)propyl]amino]-1,3-dimethyluracil, and the determination method comprises the following steps: (1) Take an appropriate amount of urapidil hydrochloride bulk drug, accurately weigh it, dissolve it in water and quantitatively dilute it to prepare a test solution; (2) Take the impurity 6-[[3-(2-methoxyphenylamino)propyl]amino]-1,3-dimethyluracil reference substance, accurately weigh it, add acetonitrile to dissolve it, and quantitatively dilute it with water to prepare a reference substance solution; (3) Accurately measure the test solution and the reference solution, inject them into the high performance liquid chromatography-mass spectrometer respectively, record the mass spectrum, and calculate the content of the impurity 6-[[3-(2-methoxyphenylamino)propyl]amino]-1,3-dimethyluracil by the peak area external standard method; the detection conditions of the high performance liquid chromatography-mass spectrometer are as follows: ammonium formate solution is used as mobile phase A, acetonitrile is used as mobile phase B, and linear gradient elution is performed according to the table below; the flow rate is 0.5 ml per minute; the column temperature is 30° C.; the detector is a mass spectrometer detector; The high performance liquid chromatography-mass spectrometry instrument uses octadecylsilane bonded silica gel as a chromatographic column filler; the chromatographic column is CORTECS C18 3.0mm×75mm, 2.7μm or a chromatographic column with equivalent performance.

2. The measuring method according to claim 1, characterized in that The concentration of the test solution in step (1) is 4 mg / mL, and the concentration of the reference solution in step (2) is 20 ng / mL.

3. The measuring method according to claim 1, characterized in that The calculation method of the impurity content in step (3) is as follows: Where: M 对 : The weight of impurities in the reference solution, mg; A 对 : The average value of the peak area of ​​impurities in the reference solution; M 供 : The amount of the test solution, mg; A 供 : Peak area of ​​impurities in the test solution.

4. The measuring method according to claim 1, characterized in that The concentration of the ammonium formate solution is 0.02 mol / L.

5. The measuring method according to claim 1, characterized in that The ion source of the mass spectrometer detector is an electrospray ion source; the acquisition mode is a positive ion SIM mode; the monitoring ion is 319.2; and the mass spectrometer acquisition time is 8 minutes to 13 minutes.

6. The measuring method according to claim 1, characterized in that The injection volume was 5 μL.

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