Method for detecting related substances in imaric acid dihydrochloride by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention discloses a method for detecting related substances in imaric acid dihydrochloride by HPLC (High Performance Liquid Chromatography), and belongs to the technical field of chemical engineering and pharmacy, the related substances in the imaric acid dihydrochloride are detected by high performance liquid chromatography, a phosphate buffer solution (0.05 mol / L of monopotassium phosphate solution, and pH adjusted to 2.5 by phosphoric acid) is used as a mobile phase A, acetonitrile is used as a mobile phase B, gradient elution is carried out, the flow rate is 1.0 ml / min, the detection wavelength is 250nm, and the detection wavelength is 250nm. The column temperature is 40 DEG C, and the detection wavelength is 235nm; the method can be used for effectively separating and quantitatively detecting imatamide, demethylated imaric acid dihydrochloride, ortho-imaric acid, meta-imaric acid, p-hydroxymethyl benzamide, an intermediate B, p-hydroxymethyl benzoic acid, an imaric acid dimer, p-cyanobenzyl alcohol, p-chloromethyl benzamide, p-toluic acid and p-chloromethyl benzoic acid in the imaric acid dihydrochloride. The method disclosed by the invention is simple and convenient to operate, strong in specificity, high in sensitivity and accuracy, good in linearity and capable of effectively controlling the quality of a p-imaric acid dihydrochloride product.

Description

Technical Field

[0001] The invention relates to the technical field of chemical industry and pharmaceutical industry, and in particular to a method for detecting related substances in imamic acid dihydrochloride by HPLC. Background Art

[0002] Imatinib mesylate dihydrochloride is a key starting material for the preparation of imatinib mesylate. Imatinib mesylate is a protein tyrosine kinase inhibitor that effectively inhibits the activity of BCR-ABL tyrosine kinase and can be used to treat chronic myeloid leukemia (Ph+CML), malignant gastrointestinal stromal tumors (GIST), and acute lymphoblastic leukemia. Numerous clinical trials have demonstrated that imatinib mesylate can significantly prolong patient survival and reduce the risk of tumor progression, with relatively mild adverse reactions, simple use, and significant clinical efficacy. As a key structural unit of imatinib mesylate, imatinib dihydrochloride is an organic synthesis intermediate and pharmaceutical intermediate that can be used in laboratory research and development processes and chemical pharmaceutical synthesis. It has broad application prospects in pharmaceutical therapy and chemical synthesis.

[0003] The impurity content of imatinib dihydrochloride is an important factor affecting the quality of imatinib mesylate products. Currently, there is no public method for detecting related substances in imatinib dihydrochloride in the existing technology. Therefore, it is very necessary to develop an HPLC method that can effectively detect related substances in imatinib dihydrochloride. Summary of the Invention

[0004] In order to solve the technical problems mentioned in the above background technology, the present invention provides a method for detecting related substances in imamic acid dihydrochloride by HPLC, comprising the following steps:

[0005] 1) Instrument selection: High-performance liquid chromatography (HPLC); ACE Excel 5 Super C18 column, 4.6 mm × 250 mm, 5 μm, or equivalent; mobile phase A: 0.05 mol / L potassium dihydrogen phosphate solution, pH adjusted to 2.5 with phosphoric acid; mobile phase B: acetonitrile; ultraviolet detection at a wavelength of 200–300 nm; mobile phase flow rate: 0.5–1.5 ml / min; column temperature: 25–45°C; column length: 100–250 mm;

[0006] 2) Prepare the following solutions:

[0007] a. Diluent: mobile phase A-acetonitrile (95:5);

[0008] b. Blank solution: take an appropriate amount of diluent;

[0009] c. System suitability solution: Weigh appropriate amounts of impurity imamamide, demethylimalic acid dihydrochloride, o-imalic acid, m-imalic acid, p-hydroxymethylbenzamide, intermediate B, p-hydroxymethylbenzoic acid, imalic acid dimer, p-cyanobenzyl alcohol, p-chloromethylbenzamide, p-toluic acid, p-chloromethylbenzoic acid, p-cyanobenzyl chloride reference substance, and imalic acid dihydrochloride reference substance, accurately weigh, dissolve in diluent, and quantitatively dilute to prepare an imalic acid dihydrochloride solution containing 0.4 μg each of impurity imamamide, demethylimalic acid dihydrochloride, o-imalic acid, m-imalic acid, p-hydroxymethylbenzamide, intermediate B, p-hydroxymethylbenzoic acid, imalic acid dimer, p-cyanobenzyl alcohol, p-chloromethylbenzamide, p-toluic acid, p-chloromethylbenzoic acid, and p-cyanobenzyl chloride per 1 ml, and the content of imalic acid dihydrochloride in this solution is 0.4 mg;

[0010] d. Test solution: Take an appropriate amount of imamic acid dihydrochloride test sample, accurately weigh it, dissolve it in diluent and quantitatively dilute it to make a solution containing approximately 0.4 mg per 1 ml;

[0011] e. Control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with diluent to make a solution containing 0.8 μg per 1 ml;

[0012] Inject samples according to the following sequence:

[0013] name Number of injection needles blank solution ≥1 injection System suitability solution 1 needle Test solution 1 needle Control solution 1 needle

[0014] The related substances in the test solution of imamic acid dihydrochloride are calculated according to the self-control method.

[0015] ① The conditions of the high performance liquid chromatography method include: using an ultraviolet detector, the detection wavelength is preferably 235 nm; the optimal flow rate of the mobile phase is 1.0 ml / min; the column temperature is 40°C; the chromatographic column is preferably ACE Excel 5Super C18, 4.6 mm × 250 mm, 5 μm; and the column length is 250 mm.

[0016] ② The impurities are imamide, demethylimalic acid dihydrochloride, o-imalic acid, m-imalic acid, p-hydroxymethylbenzamide, intermediate B, p-hydroxymethylbenzoic acid, imalic acid dimer, p-cyanobenzyl alcohol, p-chloromethylbenzamide, p-methylbenzoic acid, p-chloromethylbenzoic acid, and p-cyanobenzyl chloride.

[0017] The present invention has the following advantages: the present invention can provide a method for simultaneously detecting the contents of impurities such as imamamide, demethylimamic acid dihydrochloride, o-imamic acid, m-imamic acid, p-hydroxymethylbenzamide, intermediate B, p-hydroxymethylbenzoic acid, imamic acid dimer, p-cyanobenzyl alcohol, p-chloromethylbenzamide, p-methylbenzoic acid, p-chloromethylbenzoic acid, and p-cyanobenzyl chloride in imamic acid dihydrochloride; the method is simple to operate, has strong specificity, and is highly sensitive and accurate, thereby ensuring that the quality of imamic acid dihydrochloride can be controlled. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the blank solution spectrum of the present invention;

[0019] Figure 2 is a solution map of the system suitability of the present invention;

[0020] Figure 3 It is the sample solution atlas of the present invention;

[0021] Figure 4 This is the reference solution spectrum of the present invention. DETAILED DESCRIPTION

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0023] Example 1, specificity investigation:

[0024] A method for detecting related substances in imamic acid dihydrochloride comprises the following steps: instrument and chromatographic conditions: instrument: high performance liquid chromatograph; chromatographic column: ACE Excel 5Super C18, 4.6 mm×250 mm, 5 μm; mobile phase A: 0.05 mol / L potassium dihydrogen phosphate solution, with the pH value adjusted to 2.5 by phosphoric acid; mobile phase B: acetonitrile; wavelength: 235 nm, column temperature: 40° C., flow rate: 1.0 ml / min, injection volume: 10 μL, and run time: 45 min.

[0025] The gradient elution table is as follows:

[0026]

[0027] Stock solutions of each impurity: Take 20 mg each of the impurities imamide, demethylimalic acid dihydrochloride, o-imalic acid, m-imalic acid, p-hydroxymethylbenzamide, intermediate B, p-hydroxymethylbenzoic acid, imalic acid dimer, p-cyanobenzyl alcohol, p-chloromethylbenzamide, p-methylbenzoic acid, p-chloromethylbenzoic acid, and p-cyanobenzyl chloride, accurately weigh, place in 50 ml volumetric flasks respectively, add diluent to dissolve and dilute to the scale, and shake well.

[0028] Each impurity location solution: Accurately measure 1 ml of each of the above impurity stock solutions, place them in different 50 ml volumetric flasks, dilute to the scale with diluent, and shake well; accurately measure 1 ml, place them in different 20 ml volumetric flasks, dilute to the scale with diluent, and shake well.

[0029] Mixed solution: Weigh appropriate amounts of impurity imamamide, demethylimalic acid dihydrochloride, ortho-imalic acid, meta-imalic acid, p-hydroxymethylbenzamide, intermediate B, p-hydroxymethylbenzoic acid, imalic acid dimer, p-cyanobenzyl alcohol, p-chloromethylbenzamide, p-toluic acid, p-chloromethylbenzoic acid, p-cyanobenzyl chloride reference substance and imalic acid dihydrochloride reference substance, accurately weigh, dissolve in diluent and quantitatively dilute to prepare an imalic acid dihydrochloride solution containing 0.4 μg each of impurity imamamide, demethylimalic acid dihydrochloride, ortho-imalic acid, meta-imalic acid, p-hydroxymethylbenzamide, intermediate B, p-hydroxymethylbenzoic acid, imalic acid dimer, p-cyanobenzyl alcohol, p-chloromethylbenzamide, p-toluic acid, p-chloromethylbenzoic acid and p-cyanobenzyl chloride per 1 ml, and the content of imalic acid dihydrochloride in the solution is 0.4 mg.

[0030] Accurately measure 10 μL of each blank solution, impurity-targeting solution, and mixed solution, inject them into the liquid chromatograph, and record the chromatogram. The specificity test results are shown in the table below. The minimum resolution between each impurity and the peaks of imamic acid dihydrochloride in the table is 1.96, which is greater than 1.0, indicating good specificity of the method.

[0031] The following table is the specificity test results table:

[0032] name Retention time (min) Separation Imamid 3.947 / Demethylimamic acid dihydrochloride 5.443 11.30 o-Imalic acid 6.507 6.88 Imalic acid dihydrochloride 7.563 6.74 Meta-imamic acid 8.673 7.23 p-Hydroxybenzamide 8.970 1.96 Intermediate B 12.903 22.93 Parabens 15.280 11.97 Imalic acid dimer 18.160 14.65 p-Cyanobenzyl alcohol 22.217 16.73 p-Chloromethylbenzamide 25.313 14.83 p-Toluic acid 27.957 22.81 p-Chloromethylbenzoic acid 28.390 3.82 p-Cyanobenzyl chloride 31.850 20.59

[0033] Example 2, detection limit investigation:

[0034] Accurately pipette an appropriate amount of each impurity stock solution, add diluent to dilute to prepare a detection limit solution, take 10 μL of the detection limit solution, inject it into the liquid chromatograph, and record the chromatogram. The specific results are shown in the table below. The detection limit determination results show that the detection limits of impurities imamamide, demethylimalic acid dihydrochloride, o-imalic acid, m-imalic acid, p-hydroxymethylbenzamide, intermediate B, p-hydroxymethylbenzoic acid, imalic acid dimer, p-cyanobenzyl alcohol, p-chloromethylbenzamide, p-methylbenzoic acid, p-chloromethylbenzoic acid and p-cyanobenzyl chloride are all less than 0.005% of the test sample concentration, indicating that the method has good sensitivity.

[0035] The following table is the detection limit test results:

[0036] name Concentration / μg / ml Ratio to limit Concentration ratio of the test sample Signal-to-noise ratio Imamid 0.012 3.0％ 0.003％ 4.1 Demethylimamic acid dihydrochloride 0.013 3.3％ 0.003％ 2.4 o-Imalic acid 0.011 2.7％ 0.003％ 2.3 Meta-imamic acid 0.010 2.6％ 0.003％ 3.1 p-Hydroxymethylbenzamide 0.006 1.5％ 0.001％ 2.6 Intermediate B 0.009 2.4％ 0.002％ 2.7 p-Hydroxymethylbenzoic acid 0.007 1.7％ 0.002％ 2.7 Imalic acid dimer 0.013 3.3％ 0.003％ 2.4 p-Cyanobenzyl alcohol 0.010 2.4％ 0.002％ 2.2 p-Chloromethylbenzamide 0.006 1.5％ 0.002％ 4.4 p-Toluic acid 0.004 1.1％ 0.001％ 2.8 p-Chloromethylbenzoic acid 0.004 1.0％ 0.001％ 2.4 p-Cyanobenzyl chloride 0.007 1.8％ 0.002％ 2.9

[0037] Example 3, accuracy inspection:

[0038] Accurately pipette 1 ml each of the impurity imamamide, demethylimalic acid dihydrochloride, o-imalic acid, m-imalic acid, p-hydroxymethylbenzamide, intermediate B, p-hydroxymethylbenzoic acid, imalic acid dimer, p-cyanobenzyl alcohol, p-chloromethylbenzamide, p-methylbenzoic acid, p-chloromethylbenzoic acid, and p-cyanobenzyl chloride stock solutions into a 20 ml volumetric flask, dilute to the mark with diluent, shake well, and use as the impurity mixed stock solution. Accurately weigh 20 mg of imalic acid dihydrochloride sample, a total of 9 portions, and place them in 50 ml volumetric flasks, 3 portions as a group. Add 0.8 ml, 1.0 ml, and 1.2 ml of the impurity mixed stock solution according to low, medium, and high concentrations, respectively. Add diluent solvent and dilute to the mark, shake well, inject and analyze separately, record the chromatogram, and calculate the recovery rate. The results are shown in the table below:

[0039] The following table is the accuracy test results:

[0040] Limit concentration 80％ 100％ 120％ RSD Imamid 99.0％ 99.5％ 99.0％ 0.6％ Demethylimamic acid dihydrochloride 101.8％ 101.0％ 100.6％ 0.7％ o-Imalic acid 100.1％ 100.4％ 99.5％ 0.7％ Meta-imamic acid 99.4％ 99.0％ 98.2％ 0.7％ p-Hydroxybenzamide 100.0％ 100.3％ 99.9％ 0.4％ Intermediate B 100.6％ 100.4％ 99.7％ 0.8％ Parabens 98.8％ 99.1％ 99.4％ 0.4％ Imalic acid dimer 103.8％ 102.5％ 101.6％ 1.0％ p-Cyanobenzyl alcohol 99.1％ 99.8％ 99.1％ 0.4％ p-Chloromethylbenzamide 94.5％ 94.7％ 93.6％ 0.7％ p-Toluic acid 100.8％ 100.3％ 99.8％ 0.7％ p-Chloromethylbenzoic acid 100.6％ 98.8％ 97.4％ 1.9％ p-Cyanobenzyl chloride 102.7％ 102.3％ 101.3％ 0.7％

[0041] The present invention is simple to operate, easy to use, and suitable for comprehensive promotion and application. Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting related substances in imamic acid dihydrochloride by HPLC, characterized in that: The following steps are involved: 1) Equipment selection: High performance liquid chromatography; UV detector; chromatographic column using octadecylsilane bonded silica gel as filler; 2) Preparation of diluent: Mobile phase A - acetonitrile (95:5); 3) Preparation of blank solution: Take an appropriate amount of diluent; 4) Preparation of system suitability solution: Weigh appropriate amounts of the impurities imamamide, demethylimalic acid dihydrochloride, ortho-imalic acid, meta-imalic acid, p-hydroxymethylbenzamide, intermediate B, p-hydroxymethylbenzoic acid, imalic acid dimer, p-cyanobenzyl alcohol, p-chloromethylbenzamide, p-toluic acid, p-chloromethylbenzoic acid, p-cyanobenzyl chloride reference substance, and imalic acid dihydrochloride reference substance, accurately weigh them, dissolve them in a diluent, and quantitatively dilute them to produce an imalic acid dihydrochloride solution containing 0.4 μg each of the impurities imamamide, demethylimalic acid dihydrochloride, ortho-imalic acid, meta-imalic acid, p-hydroxymethylbenzamide, intermediate B, p-hydroxymethylbenzoic acid, imalic acid dimer, p-cyanobenzyl alcohol, p-chloromethylbenzamide, p-toluic acid, p-chloromethylbenzoic acid, and p-cyanobenzyl chloride per 1 ml, resulting in an imalic acid dihydrochloride content of 0.4 mg. 5) Preparation of test solution: Take an appropriate amount of imamic acid dihydrochloride test sample, accurately weigh it, dissolve it in diluent, and quantitatively dilute it to make a solution containing approximately 0.4 mg per 1 ml; 6) Preparation of control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with diluent to produce a solution containing 0.8 μg per 1 ml; 7) Determination of substances: Accurately measure blank solution, system suitability solution, control solution, and test solution and inject them into the liquid chromatograph respectively. Record the liquid chromatogram and calculate the peak area by the self-control method.

2. a method for detecting related substances in imamic acid dihydrochloride by HPLC according to claim 1, characterized in that, The chromatographic column selected in step 1) is ACE Excel 5Super C18 or a chromatographic column of equivalent performance, and the column length is 100-250 mm.

3. a method for detecting related substances in imamic acid dihydrochloride by HPLC according to claim 1, characterized in that, An ultraviolet detector was used in the high performance liquid chromatography detection, and the detection wavelength was 200 to 300 nm.

4. a method for detecting related substances in imamic acid dihydrochloride by HPLC according to claim 1, characterized in that, Mobile phase A was 0.05 mol / L potassium dihydrogen phosphate solution, and the pH value was adjusted to 2.5 with phosphoric acid; mobile phase B was acetonitrile.

5. a method for detecting related substances in imamic acid dihydrochloride by HPLC method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.5-1.5 ml / min.

6. a method for detecting related substances in imamic acid dihydrochloride by HPLC according to claim 1, characterized in that, Gradient elution was used, and the gradient elution table is as follows:

7. a method for detecting related substances in imamic acid dihydrochloride by HPLC according to claim 1, characterized in that, The column temperature is 25-45°C.

8. The method for detecting related substances in imamic acid dihydrochloride by a HPLC method according to claim 1, wherein The injection volume was 10 μL.

9. a method for detecting related substances in imamic acid dihydrochloride by HPLC according to claim 1, characterized in that, The substances measured in the substance measuring step include at least one or a combination of two or more of imamamide, demethylimalic acid dihydrochloride, o-imalic acid, m-imalic acid, p-hydroxymethylbenzamide, intermediate B, p-hydroxymethylbenzoic acid, imalic acid dimer, p-cyanobenzyl alcohol, p-chloromethylbenzamide, p-methylbenzoic acid, p-chloromethylbenzoic acid, and p-cyanobenzyl chloride.

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