Method for detecting nitrosamine impurities in medicine

Abstract

The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting nitrosamine impurities in drugs. According to the detection method, an HPLC method is adopted to detect the content of the nitrosamine impurities in the medicine, the nitrosamine impurities are desloratadine nitrosamine, and the structural formula of the nitrosamine impurities is shown in the formula I. The method provided by the invention not only can be used for effectively determining the content of the desloratadine nitrosamine in the medicine such as a desloratadine oral solution, but also is high in sensitivity and strong in specificity; the detection result is reliable and accurate, and a technical means is provided for detecting impurities in the medicine such as the desloratadine oral solution, so that the quality of the medicine can be controlled. Moreover, the quality detection method provided by the invention can provide a reference standard for quality control of drugs such as desloratadine oral solutions and safety detection of clinical safe medication, so that the safety and reliability of clinical medication are ensured. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for detecting nitrosamine impurities in drugs. Background Art

[0002] Nitrosamine compounds are classified as Class 2A carcinogens by the International Agency for Research on Cancer. Nitrosamine impurities belong to the "concern queue" substances mentioned in the ICHM7 (R1) (Evaluation and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk) guideline. N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitroso-N-ethylisopropylamine (NEIPA), N-nitrosodiisopropylamine (NDIPA), N-nitrosodi-n-propylamine (NDPA), N-nitroso-N-methylaniline (NMPA), N-nitrosodibutylamine (NDBA) and N-nitroso-N-methyl-4-aminobutyric acid (NMBA) are more common nitrosamine impurities in drugs, which will affect the efficacy and safety of drugs to a certain extent. Since nitrosamine impurities were detected in valsartan raw materials in July 2018, many domestic and foreign pharmaceutical companies have initiated recalls due to the detection of excessive nitrosamine impurities in other sartan drugs and non-sartan drugs (such as ranitidine, metformin, etc.).

[0003] On May 8, 2020, the State Food and Drug Administration issued the "Technical Guidelines for the Study of Nitrosamine Impurities in Chemical Drugs (Trial)", requiring that appropriate analytical methods should be established for products with a clear risk of residual nitrosamine impurities to ensure that nitrosamine impurities in the finished product are below the limit requirements. There are many reasons for the generation of nitrosamine impurities, such as process generation, degradation pathways, and pollution introduction. Since the acceptable limit of nitrosamine impurities in the human body is relatively small, the detection and control of trace impurities is difficult. Therefore, in order to better detect the quality of nitrosamine impurities in drugs, it is necessary to provide a detection method to accurately detect the content of nitrosamine impurities, thereby ensuring the safety of medication.

[0004] CN113899834A discloses a method for detecting nitrosamine impurities in drugs, which uses a high performance liquid chromatography-triple quadrupole tandem mass spectrometer for detection, and uses an external standard method to calculate the content of nitrosamine impurities in the drug test sample. The method can simultaneously detect 8 nitrosamine impurities, the signal-to-noise ratio of the 8 nitrosamine impurities is greater than 10, and the separation degree of N-nitrosodiisopropylamine and its isomer N-nitrosodi-n-propylamine is greater than 1.5. The method has the advantages of high sensitivity, strong specificity, rapid analysis, and strong anti-interference, and is particularly suitable for the detection of nitrosamine impurities in metformin hydrochloride.

[0005] The FDA database found that desloratadine also contains a new nitrosamine impurity, nitrosamine desloratadine impurity, the limit of which is 400ng / day. Nitrosamine desloratadine, CAS: 1246819-22-6, molecular formula: C 19 H 18 C1N 3 O, the structural formula is as follows:

[0006]

[0007] Nitrosamine desloratadine impurities belong to the nitrosamine impurities, which belong to the "concern queue" substances mentioned in the ICH M7 (R1) (Evaluation and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk) guideline. Relevant guidelines have been issued both at home and abroad, requiring the evaluation and research of nitrosamine impurities in products. However, for the above-mentioned nitrosamine desloratadine impurities, no relevant detection methods have been found.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The object of the present invention is to provide a method for detecting nitrosamine impurities in a drug. The method can not only effectively determine the content of nitrosamine desloratadine in a drug such as desloratadine oral solution, but also has high sensitivity and strong specificity, and the test results are reliable and accurate, which is conducive to providing technical means for detecting impurities in drugs such as desloratadine oral solution, thereby performing quality control on the impurities. Moreover, the detection method of the present invention can provide a reference standard for the quality control of drugs such as desloratadine oral solution and the safety detection of clinical safe drug use, thereby ensuring the safety and reliability of clinical drug use.

[0010] To achieve the above object, the present invention adopts the following technical solution:

[0011] A method for detecting nitrosamine impurities in a drug, wherein the quality detection method is to use HPLC to detect the content of nitrosamine impurities in the drug, and the nitrosamine impurity is nitrosamine desloratadine, whose structural formula is shown in Formula I:

[0012]

[0013] Furthermore, the content of nitrosamine desloratadine in the medicine is less than 0.008%.

[0014] Furthermore, the detection method comprises the following steps:

[0015] 1) Prepare the drug test solution and the nitrosamine desloratadine impurity reference solution respectively;

[0016] 2) injecting the drug test solution and the nitrosamine desloratadine impurity reference solution obtained in step 1) into a high performance liquid chromatograph respectively, recording the chromatogram, and calculating the content of the nitrosamine desloratadine.

[0017] Furthermore, in step 2), the high performance liquid chromatography test conditions are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel, the detector is an ultraviolet detector, the mobile phase is a solution of 0.003 mol / L sodium dodecyl sulfate solution and acetonitrile mixed in a volume ratio of (58-60): (42-40), the detection wavelength is 239-243 nm, the flow rate is 0.8-1.2 ml / min, the column temperature is 30-40° C., and the injection volume is 60 μl-80 μl.

[0018] Furthermore, in step 1), the concentration of desloratadine in the prepared drug test solution is 0.2-0.3 mg / mL; the concentration of nitrosamine desloratadine in the prepared nitrosamine desloratadine impurity reference solution is 0.01-0.03 μg / mL.

[0019] Further, in step 1), the concentration of desloratadine in the prepared drug test solution is 0.25 mg / mL; the concentration of nitrosamine desloratadine in the prepared nitrosamine desloratadine impurity reference solution is 0.02 μg / mL.

[0020] Furthermore, the last diluent used in the preparation of the drug test solution and the nitrosamine desloratadine impurity reference solution is the mobile phase.

[0021] Furthermore, the chromatographic column is YMC J'Sphere ODS-M80, 250×4.6 mm, 4 μm or YMC PackPro C18, 4.6 mm×250 mm, 5 μm.

[0022] Furthermore, in step 2), the content of nitrosamine desloratadine is calculated by external standard method.

[0023] Furthermore, the drug is desloratadine and its intermediates or preparations.

[0024] In the present invention, the 0.003 mol / L sodium dodecyl sulfate solution is prepared by the following method: 0.865 g of sodium dodecyl sulfate is taken, weighed, added with 1000 ml of water to dissolve, and 0.5 ml of trifluoroacetic acid is added and mixed to obtain the solution.

[0025] Furthermore, the drug is desloratadine and its intermediates or preparations.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] (1) The HPLC method provided by the present invention can effectively determine the content of nitrosamine desloratadine in drugs such as desloratadine oral solution, and has high sensitivity and strong specificity. The test results are reliable and accurate, which is conducive to providing technical means for the detection of impurities in drugs such as desloratadine oral solution, thereby performing quality control thereof.

[0028] (2) The quality detection method provided by the present invention can provide a reference standard for the quality control of drugs such as desloratadine oral solution and the safety detection of clinical safe drug use, thereby ensuring the safety and reliability of clinical drug use. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 YMC J'Sphere ODS-M80, 250×4.6mm, 4μm, 0.25mg / ml desloratadine test solution spectrum;

[0030] Figure 2 This is the spectrum of the nitrosamine desloratadine;

[0031] Figure 3 It is an overlay diagram of 0.5 mg / ml desloratadine test solution and nitrosamine desloratadine quantitative limit concentration solution;

[0032] Figure 4 YMC J'Sphere ODS-M80, 250×4.6mm, 4μm spiked sample spectrum;

[0033] Figure 5 YMC Pack Pro C18, 4.6mm×250mm, 5μm spiked sample spectrum; DETAILED DESCRIPTION

[0034] The following are specific embodiments of the present invention, and the embodiments are intended to further describe the present invention rather than to limit the present invention.

[0035] Example 1

[0036] This embodiment provides a method for detecting desloratadine oral solution, which specifically comprises the following steps:

[0037] 1) Prepare the drug test solution and the nitrosamine desloratadine impurity reference solution separately; the details are as follows:

[0038] Impurity reference solution: Take the nitrosamine desloratadine reference substance, first dissolve it in methanol as solvent, and then dilute it with the mobile phase as diluent to prepare a 0.02μg / ml solution.

[0039] Test solution: Take an appropriate amount of desloratadine oral solution, add mobile phase to quantitatively dilute to a solution containing 0.25 mg of desloratadine per 1 mL, filter, and take the filtrate;

[0040] 2) Inject the test solution and impurity reference solution obtained in step 1) into a high performance liquid chromatograph and record the chromatogram (e.g. Figure 1 As shown), the content of nitrosamine desloratadine was calculated according to the external standard method. Wherein, the chromatographic conditions are as follows:

[0041] Chromatographic column: YMC J'Sphere ODS-M80, 250×4.6mm, 4μm;

[0042] Detector: UV detector;

[0043] Mobile phase: a solution of 0.003 mol / L sodium dodecyl sulfate solution (take about 0.865 g of sodium dodecyl sulfate, weigh it, add 1000 ml of water to dissolve it, add 0.5 ml of trifluoroacetic acid, mix well, and obtain it) and acetonitrile in a volume ratio of 58:42;

[0044] Detection wavelength: 241nm;

[0045] Flow rate: 1.0ml / min;

[0046] Column temperature: 35°C;

[0047] Injection volume: 80 μl;

[0048] Running time: 35min;

[0049] Example 2

[0050] This embodiment is the same as embodiment 1, except that the chromatographic column used in the chromatographic conditions is YMCPack Pro C18, 4.6 mm×250 mm, 5 μm.

[0051] Example 3

[0052] This embodiment is the same as embodiment 1, except that the chromatographic conditions are different, as follows:

[0053] Mobile phase: a mixture of 0.003 mol / L sodium dodecyl sulfate solution and acetonitrile in a volume ratio of 59:41;

[0054] Detection wavelength: 239nm;

[0055] Flow rate: 0.8ml / min;

[0056] Column temperature: 30℃.

[0057] Example 4

[0058] This embodiment is the same as embodiment 1, except that the chromatographic conditions are different, as follows:

[0059] Mobile phase: a solution of 0.003 mol / L sodium dodecyl sulfate solution and acetonitrile in a volume ratio of 60:40;

[0060] Detection wavelength: 243nm;

[0061] Flow rate: 1.2 ml / min;

[0062] Column temperature: 40℃.

[0063] Example 5

[0064] The same as Example 1, except that desloratadine raw material was used in the preparation of the test solution.

[0065] Example 6

[0066] The same as Example 1, except that desloratadine tablet powder was used in the preparation of the test solution.

[0067] Example 7

[0068] The same as Example 1, except that loratadine raw material was used in the preparation of the test solution.

[0069] Example 8

[0070] The same as Example 1, except that loratadine tablet powder was used in the preparation of the test solution.

[0071] Example 9

[0072] The same as Example 1, except that loratadine syrup was used in the preparation of the test solution.

[0073] Example 10: Quality analysis of desloratadine oral solution using the method of the present invention

[0074] Desloratadine oral solution comes from Beijing Xiuzhen Innovative Pharmaceutical Research Institute Co., Ltd., with a total of 3 batches, batch numbers are 231201, 231202, and 231203 respectively.

[0075] The above three batches of desloratadine oral solution were tested for the content of the nitrosamine desloratadine represented by formula I in the desloratadine oral solution according to the method of Example 1. The results are shown in Table 1:

[0076] Table 1. Determination results of nitrosamine impurities shown in formula I in desloratadine oral solution

[0077] Sample batch number Content of nitrosamine desloratadine shown in formula I 231201 Not detected 231202 Not detected 231203 Not detected

[0078] The results showed that in the three batches of samples, no nitrosamine desloratadine represented by formula I was detected in the desloratadine oral solution.

[0079] Comparative Example 1

[0080] Referring to the detection method of Example 1, the only difference is that the concentration of the test solution is changed to 0.1 mg / ml. The results show that when the concentration of the test solution is 0.1 mg / ml, the sensitivity does not meet the requirements.

[0081] Comparative Example 2

[0082] Referring to the detection method of Example 1, the only difference is that the concentration of the test solution is changed to 0.5 mg / ml. The results show that when the concentration of the test solution is 0.5 mg / ml, the separation does not meet the requirements.

[0083] Comparative Example 3

[0084] Referring to the detection method of Example 1, the only difference is that the volume ratio of 0.003 mol / L sodium dodecyl sulfate solution and acetonitrile in the mobile phase is 57:43. The results show that when the mobile phase ratio is 57:43, the recovery rate of the spiked test sample is significantly high, and the content of nitrosamine desloratadine cannot be accurately determined.

[0085] Experimental examples, method development and analytical methodology validation

[0086] 1. Method development

[0087] The nitrosamine desloratadine impurity detection was carried out by high performance liquid chromatography, and the development process was as follows:

[0088] (1) Detection wavelength selection

[0089] Prepare a mixed solution containing 0.02 μg / ml nitrosamine desloratadine and desloratadine. During the preparation process, it may involve multiple dilution preparations. The sample is dissolved in any of methanol, ethanol or acetonitrile, and the mobile phase is used as a diluent in the last dilution step. Inject the mixed solution into a liquid chromatograph, record the chromatogram, and screen the detection wavelength. The results show that the maximum absorption wavelength of nitrosamine desloratadine is 241nm. The spectrum is shown in Figure 2 shown.

[0090] (2) Selection of test sample concentration

[0091] Prepare 0.1mg / ml, 0.25mg / ml, 0.5mg / ml test sample solutions and 0.005μg / ml nitrosamine desloratadine solution (quantitative limit solution, the signal-to-noise ratio (S / N) of this solution is about 12.5) respectively. When the test sample concentration is 0.1mg / ml, the quantitative limit concentration is about 0.005% of the test sample concentration, the impurity limit is 0.008%, and the quantitative limit concentration is not below 50% of the limit, so the sensitivity does not meet the requirements. When the test sample concentration is 0.5mg / ml, the separation degree of nitrosamine desloratadine and desloratadine peaks does not meet the requirements, the two peaks basically overlap, and the specificity does not meet the requirements. See the spectrum Figure 3 shown. Figure 3 In the figure, data 1: 0.5 mg / ml test solution; data 2: quantitation limit solution.

[0092] Conclusion: When the test sample concentration is 0.1mg / ml, the sensitivity does not meet the requirements, and when the test sample concentration is 0.5mg / ml, the specificity does not meet the requirements, so the test sample concentration is temporarily set at 0.25m / ml.

[0093] (3) Selection of mobile phase ratio

[0094] The ratio of mobile phase was adjusted to 57:43, 58:42 and 59:41 for aqueous phase: organic phase respectively. The spiked test solutions containing limit concentration impurities were tested respectively and the recovery rate of impurities was calculated. The results are shown in the table below.

[0095] Spiking results table

[0096]

[0097] When the mobile phase ratio was 57:43, the recovery rate of the spiked sample was significantly higher, and the content of nitrosamine desloratadine could not be accurately determined. When the mobile phase ratio was 58:42 and 59:41, the recovery rate of the spiked sample met the requirements, and the content of nitrosamine desloratadine could be accurately determined.

[0098] The results showed that the mobile phase ratio was tentatively set at 58:42.

[0099] 2. Validation of analytical methodology

[0100] The nitrosamine desloratadine was detected by high performance liquid chromatography, and the method was validated by analytical methodology. All indicators were in compliance with regulations and were applicable to the detection of nitrosamine desloratadine impurities in this product.

[0101] (1) Specificity and sensitivity

[0102] Neither blank excipients nor blank solvents interfere with impurity detection and have good specificity.

[0103] (2) Sensitivity

[0104] 0.005μg / ml and 0.001μg / ml nitrosamine desloratadine solutions were prepared and injected into the liquid chromatograph to record the chromatogram. The results showed that the signal-to-noise ratio of the quantitative limit was greater than 10, and the signal-to-noise ratio of the detection limit was greater than 3. The sensitivity of the detection method met the requirements and could achieve the expected effect.

[0105] Detection limit results table

[0106]

[0107] (3) Linearity and range

[0108] A nitrosamine desloratadine solution with a concentration range of 0.005 μg / ml to 0.048 μg / ml (0.002% to 0.02%) was prepared, and the concentration of the analyte and the peak area showed a good linear relationship, R2 was not less than 0.998, the Y-axis intercept was within 25% of the 100% response value, and the linear equation was y=267,212.3970x-262.2000.

[0109] (4) Accuracy

[0110] This method measured three concentrations of impurities in this product (solutions of 50%, 100%, and 150% of the limit concentration), and the average recovery rate of 9 spiked samples was 89.9%, between 70% and 130%, and the RSD value was 11.34%, not exceeding 30%. This shows that the test results meet the verification requirements and have good accuracy.

[0111] (5) Repeatability

[0112] The same batch of spiked test samples were measured six times, and the RSD value of the impurity content was 1.16%, no more than 10%, and there was no significant difference in the test results, indicating that the method had good repeatability.

[0113] (6) Intermediate precision

[0114] The same batch of spiked test samples were measured 12 times by different operators on different instruments on different dates. The RSD value of the impurity content was 4.41%, no more than 15%. There was no significant difference in the test results, indicating that the method has good precision.

[0115] (7) Durability

[0116] After changing the column temperature, flow rate, organic phase ratio, detection wavelength, and different chromatographic columns, the detection amount of impurities in this method did not change significantly, indicating that the method has good durability.

Claims

1. A method for detecting nitrosamine impurities in a drug, characterized in that: The detection method is to use HPLC to detect the content of nitrosamine impurities in the drug. The nitrosamine impurities are nitrosamine desloratadine, and its structural formula is shown in Formula I:

2. The detection method according to claim 1, characterized in that: The content of nitrosamine desloratadine in the medicine is less than 0.008%.

3. The detection method according to claim 2, characterized in that: The detection method comprises the following steps: 1) Prepare the drug test solution and the nitrosamine desloratadine impurity reference solution respectively; 2) injecting the drug test solution and the nitrosamine desloratadine impurity reference solution obtained in step 1) into a high performance liquid chromatograph respectively, recording the chromatogram, and calculating the content of the nitrosamine desloratadine.

4. The detection method according to claim 3, characterized in that: In step 2), the high performance liquid chromatography test conditions are: the chromatographic column is filled with octadecylsilane bonded silica gel, the detector is an ultraviolet detector, the mobile phase is a solution of 0.003 mol / L sodium dodecyl sulfate solution and acetonitrile mixed in a volume ratio of (58-60): (42-40), the detection wavelength is 239-243 nm, the flow rate is 0.8-1.2 ml / min, the column temperature is 30-40° C., and the injection volume is 60 μl-80 μl.

5. The detection method according to claim 3, characterized in that: In step 1), the concentration of desloratadine in the prepared drug test solution is 0.2-0.3 mg / mL; the concentration of nitrosamine desloratadine in the prepared nitrosamine desloratadine impurity reference solution is 0.01-0.03 μg / mL.

6. The detection method according to claim 5, characterized in that: In step 1), the concentration of desloratadine in the prepared drug test solution is 0.25 mg / mL; the concentration of nitrosamine desloratadine in the prepared nitrosamine desloratadine impurity reference solution is 0.02 μg / mL.

7. The detection method according to claim 6, characterized in that: The last diluent used in the preparation of the drug test solution and the nitrosamine desloratadine impurity reference solution is the mobile phase.

8. The detection method according to claim 4, characterized in that: The chromatographic column is YMC J'Sphere ODS-M80, 250×4.6 mm, 4 μm or YMC Pack Pro C18, 4.6 mm×250 mm, 5 μm.

9. The detection method according to claim 3, characterized in that: In step 2), the content of nitrosamine desloratadine is calculated by external standard method.

10. The detection method according to any one of claims 1 to 9, characterized in that: The drug is desloratadine and its intermediates or preparations.

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