Method for detecting nitrosamine impurity in diclofenac sodium preparation

Abstract

According to the method for detecting the nitrosamine impurity in the diclofenac sodium preparation, disclosed by the invention, the N-nitroso-diclofenac impurity in the diclofenac sodium preparation can be quantitatively and efficiently detected, and auxiliary materials in the diclofenac sodium preparation can be effectively separated from the N-nitroso-diclofenac impurity through limited chromatographic conditions; the separation degree between the N-nitroso-diclofenac and adjacent impurities reaches 1.5 or above, and effective monitoring on the N-nitroso-diclofenac impurities is realized. The sensitivity is high, and N-nitroso-diclofenac impurities with the concentration equivalent to 0.96 ppm of a test sample can be detected.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of drug detection, and particularly relates to a detection method of nitrosamine impurities in diclofenac sodium preparation. BACKGROUND

[0002] Diclofenac sodium is a non-steroidal anti-inflammatory analgesic. It is used in clinic for treating various rheumatism, rheumatoid arthritis, ankylosing spondylitis, postoperative pain caused by cancer, and fever caused by various reasons. Diclofenac sodium has the characteristics of good curative effect and low side effect, but it is rapidly absorbed and has a short biological half-life. Therefore, it is developed into an enteric preparation with a double-release mechanism, so as to maintain a long-time blood drug concentration, reduce the frequency of drug administration, reduce the adverse reactions of the drug, and make the in-vivo drug-time curve show a double-peak shape.

[0003] Diclofenac sodium and its impurities contain secondary amine structures, and secondary amine is a chemical precursor of nitrosamine. It is speculated that nitrosamine impurities may be produced. Nitrosamine impurities are known high-risk carcinogens, and therefore N-nitroso-diclofenac needs to be researched. According to the latest FDA regulations, the recommended acceptable intake of N-nitroso-diclofenac impurities is 1500 ng / day, and the maximum daily dose of diclofenac sodium double-release enteric capsules is 150 mg. It is calculated that the limit of N-nitroso-diclofenac impurities should not exceed 10 ppm.

[0004] The detection methods of compounds usually include gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, etc. The chromatography-mass spectrometry technology needs to ionize the target impurities during detection, and the ion source is easily affected by matrix effect and ion suppression. Therefore, the sample matrix has a high requirement, and complex pretreatment is needed to remove the above influences. In addition, the sample matrix is easy to contaminate the ion source, and the operation cost of maintenance and repair is high. For example, patent CN119198986A discloses a method for determining N-nitroso-diclofenac in traditional Chinese medicine by liquid chromatography-mass spectrometry. The test sample is dissolved with a solvent (50% acetonitrile aqueous solution), and then extracted with an extracting agent dichloromethane to remove the matrix effect. The sample pretreatment is complex, and the solvent cannot disperse and dissolve the particles in the diclofenac sodium double-release enteric capsules. Therefore, it is of great significance to develop a detection method with the advantages of easy access to instruments and equipment, low maintenance and operation cost, simple pretreatment, simple operation, and meeting the requirements of specificity and sensitivity for the quality control and safety of diclofenac sodium preparation. At present, no patent related to the detection method of nitrosamine impurities N-nitroso-diclofenac in diclofenac sodium preparation has been searched. SUMMARY

[0005] Invention purposes: The purpose of the present application is to provide a method for detecting N-nitroso-diclofenac, a nitrosamine impurity in diclofenac sodium preparation and raw material, using high performance liquid chromatography, so as to detect N-nitroso-diclofenac in diclofenac sodium preparation and facilitate monitoring of its content during production and storage. The method has good specificity, high sensitivity, and can quickly and accurately monitor the content of N-nitroso-diclofenac in diclofenac sodium preparation.

[0006] Technical solutions: The technical solutions of the present application are as follows:

[0007] A method for detecting nitrosamine impurities in diclofenac sodium preparation, which is detected by high performance liquid chromatography, and the nitrosamine impurity is N-nitroso-diclofenac. The method comprises the following steps:

[0008] 1) Preparation of solution, preparation of blank excipient solution, reference substance solution, test sample solution and test sample standard addition solution;

[0009] 2) High performance liquid chromatography is used, and the chromatographic conditions include:

[0010] C18 chromatographic column with octadecylsilane bonded silica gel as filler;

[0011] Mobile phase: 0.02 mol / L potassium dihydrogen phosphate-methanol;

[0012] Flow rate: 0.8-1.2 ml / min;

[0013] Column temperature: 25℃;

[0014] Injection volume: 100ul;

[0015] Detection wavelength: 242nm.

[0016] Preferably, the chromatographic column specifications are YMC-Pack ODS-A, 4.6mmx250mm, 5um.

[0017] Preferably, the volume ratio of 0.02 mol / L potassium dihydrogen phosphate to methanol in the mobile phase is 50:50-30:70; the mobile phase is adjusted to a pH value range of 2.0-4.0 by phosphoric acid.

[0018] Preferably, the flow rate is 1.0ml / min.

[0019] The present application also provides a detection method for diclofenac sodium double-release enteric-coated capsules, comprising the following steps:

[0020] 1) Preparation of blank excipient solution: take a certain amount of blank excipient, accurately weigh and determine, add a certain amount of solvent A, ultrasonic and dilute to the mark, shake well, and filter; accurately take a certain amount, dilute to the mark with solvent B, and shake well;

[0021] 2) Control solution: accurately weigh N-nitroso-diclofenac control sample, add methanol to dissolve, as control stock solution; accurately take control stock solution, dilute to the mark with solvent B, shake well;

[0022] 3) Test solution: accurately weigh diclofenac sodium double-release enteric-coated capsules, add solvent A to ultrasonic to micro-pellet disintegration and dilute to the mark, shake well, filter, as test stock solution; accurately take test stock solution, dilute to the mark with solvent B, shake well;

[0023] 4) Test sample with standard solution: accurately take 5ml of test stock solution of step (3) in a 10ml volumetric flask, add 10ul of control stock solution of step (2), dilute to the mark with solvent B, shake well;

[0024] 5) take blank auxiliary material solution, control solution, test solution, test sample with standard solution, inject into liquid chromatograph, record chromatogram.

[0025] Preferably, the solvent A is a mixture of 0.1mol / L sodium hydroxide aqueous solution and methanol, the volume ratio of 0.1mol / L sodium hydroxide aqueous solution and methanol is 1:9, the selection of the solvent can dissolve part of the auxiliary materials in diclofenac sodium double-release enteric-coated capsules, so that diclofenac sodium is fully released.

[0026] Preferably, the solvent B is a mixture of 0.02mol / L potassium dihydrogen phosphate aqueous solution and methanol.

[0027] Preferably, the detection limit concentration of N-nitroso-diclofenac is 0.29ppm, and the quantification limit concentration is 0.96ppm.

[0028] Preferably, the concentration of the control solution is 0.10ug / ml; the concentration of the test solution is 5-15mg / ml solution. The concentration of the test sample refers to the concentration of the main component of the drug, diclofenac sodium.

[0029] Beneficial effects: the high performance liquid chromatography provided by the application can quantitatively and efficiently detect N-nitroso-diclofenac impurities in diclofenac sodium preparation, the chromatographic conditions of the application limit can effectively separate the auxiliary materials and N-nitroso-diclofenac impurities in diclofenac sodium preparation, the separation degree between N-nitroso-diclofenac and adjacent impurities reaches 1.5 or more, realizing effective monitoring of N-nitroso-diclofenac impurities. High sensitivity, can detect N-nitroso-diclofenac impurities equivalent to 0.96ppm of test sample concentration.

[0030] The method provided by the application can avoid the interference of impurities and adjuvants possibly existing in the preparation on the N-nitroso-diclofenac impurity, and through the research on the specificity, sensitivity and precision of the method, it is proved that the method provided by the application is suitable for the detection of the N-nitroso-diclofenac impurity in the diclofenac sodium preparation. The high performance liquid chromatography method defined in the application has the advantages of simple sample pretreatment, simple operation, high detection efficiency, easy-to-obtain instruments and equipment, and wide applicability. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The chromatogram of the blank adjuvant solution is shown in Figure 1;

[0032] Figure 2 The chromatogram of the reference substance solution is shown in Figure 2;

[0033] Figure 3 The chromatogram of the test sample solution is shown in Figure 3;

[0034] Figure 4 The chromatogram of the test sample solution of the diclofenac sodium double-release enteric-coated capsule is shown in Figure 4; DETAILED DESCRIPTION

[0035] In order to deepen the understanding of the application, the application will be further described in combination with the embodiments and the drawings, and the embodiments are only used to explain the application and do not constitute a limitation on the protection scope of the application.

[0036] The various raw materials used in the application are all from commercially available industrial products, unless otherwise specified.

[0037] In the following examples, the source of the N-nitroso-diclofenac impurity is QCSRM, with a purity of 95.8%; the diclofenac sodium double-release enteric-coated capsule is self-prepared according to the prescription of the Russian listed product, and the specific prescription is shown in Table 1

[0038] Table 1 Prescription and process of diclofenac sodium double-release enteric-coated capsule

[0039]

[0040]

[0041] Example 1 Detection of impurities in diclofenac sodium double-release enteric-coated capsule

[0042] The specific steps are as follows:

[0043] (1) Solvent A: 100 ml of 0.1 mol / L sodium hydroxide aqueous solution (0.4 g→100 ml) and 900 ml of methanol are mixed uniformly;

[0044] (2) Solvent B: 0.02 mol / L potassium dihydrogen phosphate aqueous solution (2.72 g→1000 ml)-methanol (40:60), adjust pH to 3.0 with phosphoric acid.

[0045] (3) Preparation of blank excipient: weigh microcrystalline cellulose about 19.124 g, povidone K25 about 3.00 g, colloidal silicon dioxide about 0.376 g, methyl methacrylate-ethyl acrylate copolymer about 2.00 g, propylene glycol about 0.20 g, quaternary ammonium methacrylate copolymer type B about 0.498 g, quaternary ammonium methacrylate copolymer type A about 0.10 g, triethyl citrate about 0.06 g, talc about 0.452 g, mix well, pass through 40 mesh sieve, and obtain.

[0046] (4) Blank excipient solution: weigh blank excipient about 180 mg, put into 5 ml volumetric flask, add solvent A, ultrasonic for 20 min, cool, dilute to the mark with solvent A, shake well, filter, accurately take 2.5 ml of the filtrate, put into 5 ml volumetric flask, dilute to the mark with solvent B, shake well, the concentration of blank excipient is 18 mg / ml.

[0047] (5) Control solution: weigh N-nitroso-diclofenac control substance, accurately weigh and determine, dissolve and dilute to prepare a solution containing about 100 μg per 1 ml as control stock solution; accurately take the control stock solution, quantitatively dilute with solvent B to prepare a solution containing about 0.1 μg per 1 ml.

[0048] (6) Test solution: weigh diclofenac sodium double-release enteric-coated capsules about 280 mg, accurately weigh and determine, put into 5 ml volumetric flask, add solvent A, ultrasonic for 20 min until the pellets are disintegrated, cool, dilute to the mark with solvent B, shake well, filter, take the filtrate as test stock solution; accurately take 2.5 ml of the test stock solution, put into 5 ml volumetric flask, dilute to the mark with solvent B, shake well. The concentration of test solution is 10 mg. / ml, and the concentration of test solution is the concentration of main component diclofenac sodium.

[0049] (7) Test sample addition solution: accurately take 5 ml of test stock solution of step (6), put into 10 ml volumetric flask, add 10 μl of control stock solution of step (5), dilute to the mark with solvent B, shake well, the concentration of diclofenac sodium in the prepared test sample addition solution is 10 mg / ml, and the concentration of N-nitroso-diclofenac is 0.1 μg / ml.

[0050] (8) Take blank excipient solution, control solution, test solution, test sample addition solution, inject into liquid chromatograph, record chromatogram as chromatogram Figure 1 , Figure 2 , Figure 4 and Figure 3 , respectively.

[0051] Wherein the chromatographic conditions are:

[0052] Chromatographic column: YMC-Pack ODS-A, 4.6 mm x 250 mm, 5 μm;

[0053] Mobile phase: 0.02 mol / L potassium dihydrogen phosphate-methanol (40:60, adjust pH value to 3.0 with phosphoric acid).

[0054] Flow rate: 1.0 ml / min;

[0055] Column temperature: 25°C;

[0056] Detection wavelength: 242 nm;

[0057] Injection volume: 100 μl.

[0058] The content of N-nitroso-diclofenac impurity in the diclofenac sodium double-release enteric-coated capsule is calculated by external standard method, and the calculation method is as follows:

[0059] A portion of N-nitroso-diclofenac reference substance is weighed and prepared into a limit concentration of 0.10 μg / ml, and parallel injection is performed, and the average value of f value is calculated, and the content of N-nitroso-diclofenac is calculated by external standard method.

[0060] f = (Ms x Cs) / (Vs x As)

[0061] N-nitroso-diclofenac content (%) = (f x Ai x Vi x average loading amount) / (Mi x I) x 100%

[0062] Ms is the sample weight of the reference substance;

[0063] CS is the content of the reference substance;

[0064] Vs is the dilution multiple of the reference substance solution;

[0065] As is the peak area of N-nitroso-diclofenac in the reference substance solution;

[0066] Ai is the peak area of N-nitroso-diclofenac in the test sample solution;

[0067] Vi is the dilution multiple of the test sample solution;

[0068] Mi is the sample weight of the test sample;

[0069] I is the labeled amount of 75 mg.

[0070] Example 1 detection result analysis: the detection amount of N-nitroso-diclofenac in the diclofenac sodium double-release enteric-coated capsule is not detected (below the limit of quantification).

[0071] Example 2: Specificity

[0072] The chromatographic conditions were the same as in Example 1, and the blank solvent, blank excipient, test sample spiked solution and reference substance solution were injected separately, and the chromatograms were recorded. The interference of the blank solvent and blank excipient on the detection of N-nitroso-diclofenac was investigated. According to the results of the chromatograms, the results are shown in Table 2. The separation degree of N-nitroso-diclofenac impurity in the test sample spiked solution from the adjacent peak before it was 1.60, and the separation degree from the adjacent peak after it was 1.82. The blank solvent and blank excipient had no interference on the detection of N-nitroso-diclofenac, and the specificity was good.

[0073] Table 2: Results of specificity experiment

[0074]

[0075] Example 3: Injection precision

[0076] The chromatographic conditions were the same as in Example 1, and the reference substance solution was continuously injected for 6 times, and the RSD of the peak area was calculated to investigate the injection precision. The test results are shown in Table 3. The reference substance solution was continuously injected for 6 times, and the RSD value of the retention time was 0.04%, which was less than 1.0%; the RSD value of the peak area was 3.66%, which was less than 5.0%, and the precision of the method was good.

[0077] Table 3: Test results of injection precision

[0078] Name jmd1 jmd2 jmd3 jmd4 jmd5 jmd6 RSD (%) Peak area 13.135 13.685 12.753 12.741 12.485 12.433 3.66 Retention time 7.944 7.944 7.944 7.941 7.939 7.938 0.04

[0079] Example 4: Detection limit and quantification limit

[0080] The chromatographic conditions were the same as in Example 1, and the N-nitroso-diclofenac reference substance solution was diluted for determination, and the chromatogram was recorded. When the S / N of N-nitroso-diclofenac was ≥3, this concentration was the detection limit concentration; when the S / N of N-nitroso-diclofenac was ≥10, this concentration was the quantification limit concentration. The quantification limit solution was continuously injected for 6 times, and the chromatogram was recorded, and the relative standard deviation of the peak area was calculated. The detection results are shown in Table 4.

[0081] Table 4: Test detection results of detection limit and quantification limit

[0082]

[0083] As can be seen from Table 4, the detection limit concentration of N-nitroso-diclofenac was 0.29 ppm; the peak area RSD value of the quantification limit solution continuously injected for 6 times was 3.93%, which was less than 15%, and the quantification limit concentration was 0.96 ppm, which was lower than 10% of the nitrosamine impurity limit (10 ppm) of the product. The present application meets the detection requirements of N-nitroso-diclofenac in the test sample, and the method has good sensitivity.

[0084] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting nitrosamine impurities in diclofenac sodium preparations, characterized in that, The nitrosamine impurity was determined to be N-nitroso-diclofenac by high performance liquid chromatography.

2. The method for detecting nitrosamine impurities in diclofenac sodium preparations according to claim 1, characterized in that, Includes the following steps: 1) Prepare solutions, including blank excipient solution, reference solution, test solution, and spiked solution for the test sample; 2) High-performance liquid chromatography (HPLC) is used, and the chromatographic conditions include: A C18 chromatographic column packed with octadecylsilane-bonded silica gel; Mobile phase: 0.02 mol / L potassium dihydrogen phosphate - methanol; Flow rate: 0.8–1.2 ml / min; Column temperature: 25℃; Injection volume: 100 μl; Detection wavelength: 242nm.

3. The method for detecting nitrosamine impurities in diclofenac sodium preparations according to claim 2, characterized in that, The chromatographic column is a YMC-Pack ODS-A, 4.6mm × 250mm, 5μm.

4. The method for detecting nitrosamine impurities in diclofenac sodium preparations according to claim 2, characterized in that, The volume ratio of 0.02 mol / L potassium dihydrogen phosphate to methanol in the mobile phase is 50:50 to 30:70; the pH of the mobile phase is adjusted to 2.0 to 4.0 using phosphoric acid.

5. The method for detecting nitrosamine impurities in diclofenac sodium preparations according to claim 2, characterized in that, The flow rate is 1.0 ml / min.

6. The method for detecting the nitrosamine impurity N-nitroso-diclofenac in diclofenac sodium preparations according to claim 1, characterized in that, The diclofenac sodium preparation is a diclofenac sodium dual-release enteric-coated capsule, and the specific detection method includes the following steps: 1) Preparation of blank excipient solution: Take an appropriate amount of blank excipient, weigh it accurately, add an appropriate amount of solvent A, sonicate and dilute to the mark, shake well, and filter; accurately measure an appropriate amount and dilute to the mark with solvent B. 2) Reference solution: Weigh an appropriate amount of N-nitroso-diclofenac reference standard accurately, dissolve it in an appropriate amount of methanol to prepare the reference standard stock solution; then accurately measure an appropriate amount of the reference standard stock solution and dilute it to the mark with solvent B. 3) Test solution: Take an appropriate amount of diclofenac sodium dual-release enteric-coated capsules, accurately weigh them, add an appropriate amount of solvent A, sonicate until the microcapsules disintegrate and dilute to the mark, shake well, filter, and use as the test solution; accurately measure an appropriate amount and dilute to the mark with solvent B. 4) Spiked solution for test sample: Accurately measure 5 ml of the test sample stock solution in step (3), place it in a 10 ml volumetric flask, add 10 μl of the reference stock solution in step (2), and dilute to the mark with solvent B; 5) Take blank excipient solution, reference solution, test solution, and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of N-nitroso-diclofenac in the test sample using the external standard method.

7. The method for detecting the nitrosamine impurity N-nitroso-diclofenac in diclofenac sodium preparations according to claim 6, characterized in that, Solvent A is a mixture of 0.1 mol / L sodium hydroxide aqueous solution and methanol, wherein the volume ratio of the 0.1 mol / L sodium hydroxide aqueous solution to methanol is 1:

9.

8. The method for detecting the nitrosamine impurity N-nitroso-diclofenac in diclofenac sodium preparations according to claim 6, characterized in that, Solvent B is a mixture of 0.02 mol / L potassium dihydrogen phosphate aqueous solution and methanol.

9. The method for detecting the nitrosamine impurity N-nitroso-diclofenac in diclofenac sodium preparations according to claim 6, characterized in that, The limit of detection for N-nitroso-diclofenac is 0.29 ppm, and the limit of quantitation is 0.96 ppm.

10. The method for detecting the nitrosamine impurity N-nitroso-diclofenac in diclofenac sodium preparations according to claim 6, characterized in that, The concentration of the reference solution is 0.10 μg / ml; the concentration of the test solution is 5–15 mg / ml.

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