Method for detecting content of N-nitroso-demethylated olopatadine in olopatadine hydrochloride bulk drug

Abstract

The invention relates to the technical field of medicine analysis, in particular to a method for detecting the content of N-nitroso-demethylated olopatadine in an olopatadine hydrochloride raw material medicine. The analysis method comprises the following steps: preparing a blank solution, a system applicability solution and an olopatadine hydrochloride bulk drug test solution; sequentially injecting the blank solution, the system applicability solution and the olopatadine hydrochloride raw material medicine test solution, injecting into a high performance liquid chromatography-tandem mass spectrometer, and detecting the content of N-nitroso-demethylated olopatadine according to an external standard method; the invention discloses a method for detecting the content of N-nitroso-demethylated olopatadine in an olopatadine hydrochloride bulk drug, which has the advantages of high sensitivity, simplicity, rapidness, strong specificity, good accuracy, high precision, good stability and good linear relationship, and can be used for detecting the content of N-nitroso-demethylated olopatadine in the olopatadine hydrochloride bulk drug.

Description

Technical Field

[0001] The present application relates to the technical field of drug analysis, and in particular to a method for detecting the content of N-nitrosodemethylolopatadine in olopatadine hydrochloride raw material. Background Art

[0002] The original drug of Olopatadine Hydrochloride was developed by Novartis Pharmaceuticals of Switzerland. It is a second-generation histamine H1 receptor blocker with anti-histamine, anti-inflammatory, anti-neurotransmitter and mast cell membrane stabilizing effects. Olopatadine can be used for skin allergic diseases such as urticaria, skin itching, eczema, psoriasis, etc. It can also be used for the treatment of allergic rhinitis, allergic conjunctivitis and allergic asthma.

[0003] Nitrosamine impurities are a class of compounds containing N-nitroso structures in their molecules. Unlike most organic impurities in drugs, nitrosamine impurities are highly carcinogenic at extremely low exposure levels. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) M7(R1) guideline "Evaluation and Control of DNA-Reactive (Mutagenic) Impurities in Drugs to Limit Potential Carcinogenic Risk" clearly defines nitrosamine impurities as "concern queue" substances.

[0004] The generation mechanism of N-nitrosamine impurities is as follows: There are potential risks when using sodium nitrite (NaNO2) or other nitrosating agents in the presence of secondary amines, tertiary amines, quaternary ammonium salts or amides in the same or different process steps.

[0005] According to the above production mechanism, N-nitrosodemethyl olopatadine first undergoes degradation and demethylation of the dimethylamine functional group of olopatadine hydrochloride under acidic conditions, removing a methyl molecule, and then possible nitrosating agents attack the secondary amine functional group to produce N-nitrosodemethyl olopatadine impurities.

[0006] Nitrosamine compounds are strong carcinogens. Due to their strong genotoxicity, they can cause cancer at extremely low exposure levels and damage human genetic material at extremely low concentrations, leading to gene mutations and possibly promoting the occurrence of tumors. Therefore, it is particularly important to accurately measure their content during the drug development process. However, since genotoxic impurities are controlled at trace levels in drugs, and the detection process needs to reduce the interference of various impurities that far exceed the limit of nitrosamine compounds in the raw materials, detection and control are relatively difficult.

[0007] Since the prior art does not provide an effective detection method, developing an effective detection method with good accuracy, high sensitivity, strong specificity, and for detecting N-nitrosodemethylolopatadine in olopatadine hydrochloride raw materials is a technical problem to be solved urgently in this field. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present application provides a method for detecting the content of N-nitrosodesmethylolopatadine in olopatadine hydrochloride raw material.

[0009] In the first aspect, the present application provides a method for detecting the content of N-nitrosodesmethylolopatadine in olopatadine hydrochloride raw material, using the following technical solution: A method for detecting the content of N-nitrosodesmethyl olopatadine in an olopatadine hydrochloride raw material, wherein the structure of N-nitrosodesmethyl olopatadine is as shown in formula (1), The detection method comprises the following steps: Prepare blank solution, system suitability solution, and olopatadine hydrochloride API test solution; Take blank solution, system suitability solution, and olopatadine hydrochloride bulk drug test solution and inject them into high performance liquid chromatography-tandem mass spectrometry in sequence, and detect the content of N-nitrosodesmethylolopatadine according to the external standard method; Preferably, the HPLC conditions include: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: methanol; Elution was performed using gradient elution; The gradient elution program is: 0-2 min, 30% B; 2-6 min, 30%-70% B; 6-8 min, 70% B; 8-8.1 min, 70%-30% B; 8.1-10 min, 30% B.

[0010] Preferably, the HPLC conditions further include: the chromatographic column is Infinitylab Porshell I20EC-C18, 2.1×50 mm, 1.9 μm.

[0011] Preferably, the HPLC conditions also include a flow rate of 0.2-0.4 mL / min.

[0012] Further preferably, the flow rate is 0.2 mL / min, 0.3 mL / min or 0.4 mL / min.

[0013] More preferably, the flow rate is 0.3 mL / min.

[0014] Preferably, the HPLC conditions also include an injection volume of 1-3 μL.

[0015] Further preferably, the injection volume is 1 μL, 2 μL or 3 μL.

[0016] More preferably, the injection volume is 2 μL.

[0017] Preferably, the HPLC conditions also include a column temperature of 30-40°C.

[0018] Further preferably, the column temperature is 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

[0019] More preferably, the column temperature is 35°C.

[0020] Preferably, the mass spectrometry conditions include: Detector: tandem mass spectrometry; Scan mode: mass spectrometry multiple reaction detection; Ion source: electrospray ion source; Polarity: positive mode; Valve switching: 4.5-10min To MS, the rest of the time To waste.

[0021] Preferably, the ion pair parameters for detecting N-nitrosodesmethylolopatadine are as follows:

[0022] Preferably, the method for preparing the olopatadine hydrochloride bulk drug test solution comprises the following steps: Accurately weigh the test sample, add the diluent to dilute it, and prepare a test solution of olopatadine hydrochloride raw material with a mass concentration of 0.1 mg / mL.

[0023] Wherein, the diluent is methanol.

[0024] Preferably, the method for preparing the system suitability solution comprises the following steps: Accurately weigh N-nitrosodesmethyl olopatadine, add diluent to dissolve, and prepare system suitability solutions with concentrations of L-10 solution (quantification limit solution, 0.75 ng / mL), L-50 solution (3.75 ng / mL), L-100 solution (7.5 ng / mL), L-150 solution (11.25 ng / mL), and L-200 solution (15 ng / mL).

[0025] Wherein, the diluent is methanol.

[0026] Preferably, the calculation formula of N-nitrosodesmethylolopatadine content is m=(Ai / Ar)×(Cr / Ci); Wherein, m is the detection result of N-nitrosodesmethylolopatadine, ppm, Ai is the peak area of ​​N-nitrosodesmethylolopatadine in the test solution of olopatadine hydrochloride bulk drug; Ci is the concentration of the olopatadine hydrochloride bulk drug substance test solution, mg / mL; Ar is the peak area of ​​N-nitrosodesmethylolopatadine in the N-nitrosodesmethylolopatadine reference solution; Cr is the concentration of N-nitrosodesmethylolopatadine in the N-nitrosodesmethylolopatadine control solution, mg / mL.

[0027] In summary, the present application includes at least one of the following beneficial technical effects: The present application discloses a method for detecting the content of N-nitrosodesmethyl olopatadine in an olopatadine hydrochloride raw material. The method has high sensitivity, is simple and rapid, has strong specificity, good accuracy, high precision, good stability, and good linear relationship, and can be used to determine the content of N-nitrosodesmethyl olopatadine in an olopatadine hydrochloride raw material. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a sample test spectrum of the Olopatadine hydrochloride API, wherein the batch number of the Olopatadine hydrochloride API is CPC-058-M230502; Figure 2 This is a linear relationship diagram of the linear relationship detection of N-nitrosodesmethylolopatadine in the olopatadine hydrochloride raw material of this application; Figure 3 The graph is the limit of quantitation. DETAILED DESCRIPTION

[0029] The technical solution of the present application is further illustrated by specific embodiments below. The specific embodiments do not limit the protection scope of the present application; some non-essential modifications and adjustments made by others based on the concept of the present application still fall within the protection scope of the present application.

[0030] The raw materials involved in this application are all commercially available products, and the application is further described in detail below in conjunction with the embodiments and comparative examples.

[0031] Embodiment 1: A method for detecting the content of N-nitrosodesmethylolopatadine in olopatadine hydrochloride raw material medicine, comprising the following steps: 1.1. Reference substances and test substances: N-nitrosodesmethylolopatadine reference substance, batch number: 2310546O-YT-01, purity: 99.8%, source: China Standard Pharmaceutical Group Co., Ltd.; Olopatadine hydrochloride raw material, batch number: CPC-058-M230102, CPC-058-M230502, CPC-058-M230506, source: Changzhou Yabang Pharmaceutical Co., Ltd.

[0032] 1.2. Instruments and reagents: Liquid chromatography-mass spectrometry: Model: Agilent 1290-G6495, Manufacturer: Agilent; Chromatographic column, model: Infinitylab Porshell I20EC-C18, 2.1×50mm, 1.9μm, manufacturer: Agilent; Ultrasonic cleaning machine, model: KQ-500E, manufacturer: Kunshan Ultrasonic Instrument Co., Ltd.; Vortex instrument, model: G560E, manufacturer: Scientific Industries; Methanol, grade: HPLC, manufacturer: Shanghai Xingke, batch number: 0234240382D; Formic acid, grade: HPLC, manufacturer: Aladdin, batch number: I2321484; 1.3 Detection Method Chromatographic conditions: Chromatographic column: Infinitylab Porshell I20EC-C 18, 2.1×50 mm, 1.9 μm.

[0033] Mobile phase A: 0.1% formic acid aqueous solution; Preparation process of mobile phase A: measure 1 mL of formic acid and place it in a reagent bottle, add 1000 mL of pure water, mix well, and degas by ultrasonication.

[0034] Mobile phase B: methanol; The gradient elution method was used for elution. The elution procedure was as follows: Table 1 Elution program Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 2 70 30 6 30 70 8 30 70 8.1 70 30 10 70 30 The flow rate was 0.3 mL / min; the injection volume was 2 μL; and the column temperature was 35°C.

[0035] The mass spectrometry conditions were as follows: Detector: tandem mass spectrometry; Scan mode: mass spectrometry multiple reaction detection; Ion source: electrospray ion source; Polarity: positive mode; Valve switching: 4.5-10min ToMS, the rest of the time Towaste.

[0036] Among them, the detection ion pair information of N-nitrosodesmethylolopatadine is as follows: Table 2 Ion pair information of N-nitrosodesmethylolopatadine detection 1.4 Solution preparation The volume of all solutions can be enlarged or reduced to meet actual needs, provided that the target concentration and weighing accuracy meet the requirements.

[0037] 1.4.1. Blank solution: methanol; 1.4.2. Olopatadine hydrochloride API test solution: Accurately weigh 2 mg of the test sample, place it in a volumetric flask, add diluent methanol, and dilute it into a test sample solution containing 0.1 mg of olopatadine hydrochloride per 1 mL. Prepare two portions.

[0038] 1.4.3 System suitability solution: The preparation method of system suitability solution is shown in the following table: Table 3 System suitability solution preparation table 1.5. Sampling process: Take blank solution, system suitability solution and olopatadine hydrochloride bulk drug solution and inject it into liquid chromatography-mass spectrometry, among which, blank is injected once, system suitability solution: L-10, L-50, L-100, L-150, L-200 are injected once each, control solution (L-100 solution) is injected 5 times, test solution is prepared in parallel in two portions, each injected once, and the spectrum is recorded; the detection spectrum of olopatadine hydrochloride bulk drug sample (batch number: CPC-058-M230502) is as follows Figure 1 shown.

[0039] 1.6 System suitability test: Acceptance criteria: The blank solution should have no obvious interference at the target impurity peak, and the signal-to-noise ratio of the target impurity peak in the sensitivity (quantitation limit) solution should be S / N ≥ 10.0; the linear relationship within the relevant concentration range of the target impurity should be examined, and the relevant dilution r ≥ 0.990; the recovery rate of the five-shot L-100 solution (the recovery rate is the ratio of the five-shot L-100 solution to the first shot of the sequence L-100 solution) should meet 70%-130%.

[0040] Experimental results: See the table below for specific data results: Table 4 System suitability results Analysis of experimental results: According to Table 4, the blank has no obvious interference with the target impurity, and the signal-to-noise ratio of the sensitivity solution is ≥10.0; the linear relationship within the relevant concentration range of the target impurity is 0.9990, which meets the requirements; the ratio of the five-shot L-100 solution to the first shot L-100 solution in the sequence meets the requirement of 70%-130%.

[0041] The linear relationship diagram of the linear relationship test of N-nitrosodesmethyl olopatadine in olopatadine hydrochloride raw material is shown in the figure Figure 2 shown.

[0042] 1.7. Calculation of N-nitrosodesmethylolopatadine content The results of five consecutive injections of L-100 solution were used as the reference solution and substituted into the external standard method to calculate the impurity content in the test sample: the calculation formula for the content of N-nitrosodesmethylolopatadine is m = (Ai / Ar) × (Cr / Ci), Wherein, m is the test result of the impurity content of N-nitrosodesmethylolopatadine (ppm); Ai is the peak area of ​​N-nitrosodesmethylolopatadine in the test solution of olopatadine hydrochloride bulk drug; Ci is the concentration of the olopatadine hydrochloride bulk drug test solution (mg / mL); Ar is the peak area of ​​N-nitrosodesmethylolopatadine in the N-nitrosodesmethylolopatadine reference solution; Cr is the concentration of N-nitrosodesmethylolopatadine in the N-nitrosodesmethylolopatadine control solution (mg / mL).

[0043] 1.8. Limitation The limit standard stipulates that the limit of N-nitrosodesmethylolopatadine is 75ppm.

[0044] 1.9. The experimental results are shown in the following table Table 5 Experimental results Experimental conclusion: N-nitrosodesmethylolopatadine was not detected in any of the three batches of test samples.

[0045] Example 2: Validation of analytical method The analytical method for detecting the content of N-nitrosodesmethylolopatadine in the olopatadine hydrochloride raw material disclosed in the present application was verified, and it was proved that the established method was suitable for the corresponding detection requirements. The detection process is as follows: 2.1 System Applicability The experimental process, acceptance criteria and analysis of experimental results are the same as those in the system applicability section of Example 1.

[0046] 2.2 Exclusivity 2.2.1 Solution preparation and detection methods Blank solution: Take the blank solution under Solution Preparation 1.4 in Example 1.

[0047] Reference solution: Take L-100 under solution preparation item 1.4 in Example 1.

[0048] Test solution: Take the test solution of olopatadine hydrochloride raw material under the solution preparation item 1.4 in Example 1.

[0049] 100% spiked test solution: Take the spiked test solution Re-100-1 under 2.4 Accuracy in Example 2.

[0050] The above solution was injected into a high performance liquid chromatography-mass spectrometer, and the instrument parameters were the same as those of the detection method in 1.3 of Example 1.

[0051] 2.2.2 Experimental Results The experimental results show that the blank solution has no interference with the determination of the target impurities, and there are no adjacent peaks near the target impurity peaks in the test solution and the 100% spiked test solution, which meets the requirements. This method has good specificity for the determination of impurities, and there are no interfering peaks near the impurity chromatographic peaks.

[0052] 2.3. Limit of detection and limit of quantification 2.3.1 Solution preparation and detection methods Quantitation limit solution: Prepare 6 portions of quantitation limit solution (L-10) according to 1.4 Solution Preparation in Example 1.

[0053] Detection limit solution: Measure 2.5 mL of quantitation limit solution into a 5 mL volumetric flask, add diluent methanol to dilute to the scale, shake well, mark as LOD, and prepare 1 aliquot.

[0054] The above solution was injected into a high performance liquid chromatography-mass spectrometer, and the instrument parameters were the same as those of the detection method in 1.3 of Example 1.

[0055] 2.3.2 Experimental Results The experimental results show that when the detection limit concentration of N-nitrosodemethylolopatadine is 0.3793 ng / mL (4 ppm), the signal-to-noise ratio of the detection limit solution is 7.6; when the concentration of the quantification limit solution is 0.7587 ng / mL (8 ppm), the signal-to-noise ratio of the quantification limit is greater than 10, indicating that this method has a high detection ability for impurities, meets the requirements, and can detect trace impurities in the sample; the detection spectrum of the quantification limit is shown in Figure 3 shown.

[0056] 2.4 Accuracy 2.4.1 Solution preparation and detection methods Test solution: prepare two solutions, the same as the Olopatadine hydrochloride API test solution under 1.4 Solution Preparation in Example 1; Prepare 3 portions of 10%, 100%, and 150% spiked test solution for accuracy. The preparation method is as follows: Spiked stock solution: same as stock 3 under 1.4 Solution preparation in Example 1.

[0057] Spiked test sample solution: weigh about 2 mg of the test sample and add it into a 20 mL volumetric flask, add an appropriate amount of spiked stock solution, add diluent methanol to the scale, shake well, and prepare as shown in Table 6.

[0058] Table 6 Accuracy solution - 10%, 100%, 150% spiked test solution preparation table The above solution was injected into a high performance liquid chromatography-mass spectrometer, and the instrument parameters were the same as those of the detection method 1.3 in Example 1.

[0059] 2.4.2 Experimental Results In the accuracy experiment, the recoveries of 10%, 100% and 150% spiked test solutions of N-nitrosodesmethylolopatadine were all between 70% and 130%, and the average recoveries of 9 spiked test solutions were 101%, and the RSDs of the recoveries were 3%, respectively, indicating that the impurity content measured using this method is very close to the actual value, and this method has good accuracy; the specific results are shown in the table below: Recovery calculation formula: Recovery rate = (measured amount - original amount) / theoretical added amount * 100%.

[0060] Table 7 Accuracy experimental results 2.5 Repeatability 2.5.1 Solution preparation and detection methods Spiked test solution: Take 100% of the spiked test solution Re-100-1 to 3 under 2.4 Accuracy in Example 2.

[0061] Three more spiked test solutions were prepared in parallel, using the same preparation method as the 100% spiked test solution under 2.4 accuracy in Example 2.

[0062] The above solution was injected into a high performance liquid chromatography-mass spectrometer, and the instrument parameters were the same as those of the detection method in 1.3 of Example 1.

[0063] 2.5.2 Experimental results The experimental results show that in the repeatability experiment, the recoveries of N-nitrosodesmethylolopatadine in 6 100% spiked test solutions were between 70% and 130%, the average recoveries were 100%, and the RSD was 3%, which met the requirements. The method has good repeatability. The specific results are shown in the table below: Recovery rate calculation formula: Recovery rate = (measured amount - original amount) / theoretical added amount * 100%.

[0064] Table 8 Repeatability test results 2.6 Solution stability 2.6.1 Solution preparation and detection methods Reference solution: Take L-100 in solution preparation item 1.4 in Example 1; Spiked test solution: Take the spiked test solution Re-100-1 under 2.4 Accuracy in Example 2; The above solution was injected into a high performance liquid chromatography-mass spectrometer at different time points, and the instrument parameters were the same as those of the detection method in 1.3 of Example 1.

[0065] 2.6.2 Experimental Results The experimental results show that under room temperature conditions, the reference solution remains stable for 9.7 hours, and the spiked test solution remains stable for 7.8 hours. The specific results are shown in the table below: Table 9 Solution stability test results The above method was used to verify the analytical method for the content of N-nitrosodemethylolopatadine in the olopatadine hydrochloride raw material disclosed in the present application, proving that the established method is suitable for the corresponding detection requirements. The experimental results show that the method can effectively detect the impurity.

[0066] The invention discloses a method for detecting the content of N-nitrosodesmethyl olopatadine in an olopatadine hydrochloride raw material medicine. The method has high sensitivity, is simple and rapid, has strong specificity, good accuracy, high precision, good stability and good linear relationship, and can be used for detecting the content of N-nitrosodesmethyl olopatadine in an olopatadine hydrochloride raw material medicine.

Claims

1. A method for detecting the content of N-nitrosodesmethylolopatadine in olopatadine hydrochloride raw material, characterized in that: The following steps are involved: Prepare blank solution, system suitability solution, and olopatadine hydrochloride API test solution; Take blank solution, system suitability solution, and olopatadine hydrochloride bulk drug test solution and inject them into high performance liquid chromatography-tandem mass spectrometry in sequence, and detect the content of N-nitrosodesmethylolopatadine according to the external standard method; The HPLC conditions include: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: methanol; Elution was performed using gradient elution; The gradient elution program is: 0-2 min, 30% B; 2-6 min, 30%-70% B; 6-8 min, 70% B; 8-8.1 min, 70%-30% B; 8.1-10 min, 30% B.

2. The method for detecting the content of N-nitrosodesmethylolopatadine in the olopatadine hydrochloride bulk drug according to claim 1, characterized in that: The high performance liquid chromatography conditions also include: the chromatographic column is Infinitylab PorshellI20 EC-C18, 2.1×50 mm, 1.9 μm.

3. The method for detecting the content of N-nitrosodesmethylolopatadine in the olopatadine hydrochloride bulk drug according to claim 1, characterized in that: The HPLC conditions also include: a flow rate of 0.2-0.4 mL / min; an injection volume of 1-3 μL; and a column temperature of 30-40° C.

4. The method for detecting the content of N-nitrosodesmethylolopatadine in the olopatadine hydrochloride bulk drug according to claim 3, characterized in that: The flow rate was 0.3 mL / min; the injection volume was 2 μL; and the column temperature was 35° C.

5. The method for detecting the content of N-nitrosodesmethylolopatadine in the olopatadine hydrochloride bulk drug according to claim 1, characterized in that: The mass spectrometry conditions include: Detector: tandem mass spectrometry; Scan mode: mass spectrometry multiple reaction detection; Ion source: electrospray ion source; Polarity: positive mode; Valve switching: 4.5-10min To MS, the rest of the time To waste.

6. The method for detecting the content of N-nitrosodesmethylolopatadine in the olopatadine hydrochloride bulk drug according to claim 5, characterized in that: The mass spectrometry conditions also include N-nitrosodesmethylolopatadine detection ion pair parameters, which are as follows:

7. The method for detecting the content of N-nitrosodesmethylolopatadine in the olopatadine hydrochloride bulk drug according to claim 1, characterized in that: The preparation method of the Olopatadine hydrochloride raw material test solution comprises the following steps: Accurately weigh the test sample, add diluent to dilute it, and prepare a test solution of olopatadine hydrochloride raw material with a mass concentration of 0.1 mg / mL.

8. The method for detecting the content of N-nitrosodesmethylolopatadine in the olopatadine hydrochloride bulk drug according to claim 1, characterized in that: The method for preparing the system suitability solution comprises the following steps: Accurately weigh N-nitrosodesmethyl olopatadine, add diluent to dissolve, and prepare system suitability solutions with concentrations of 0.75 ng / mL, 3.75 ng / mL, 7.5 ng / mL, 11.25 ng / mL, and 15 ng / mL, respectively.

9. The method for detecting the content of N-nitrosodesmethylolopatadine in the olopatadine hydrochloride bulk drug according to claim 7 or 8, characterized in that: The diluent is methanol.

10. The method for detecting the content of N-nitrosodesmethylolopatadine in the olopatadine hydrochloride bulk drug according to claim 1, characterized in that: The calculation formula of the content of N-nitrosodesmethylolopatadine is m=(Ai / Ar)×(Cr / Ci), Wherein, m is the detection result of N-nitrosodesmethylolopatadine, ppm, Ai is the peak area of ​​N-nitrosodesmethylolopatadine in the test solution of olopatadine hydrochloride bulk drug; Ci is the concentration of the olopatadine hydrochloride bulk drug test solution, mg / mL; Ar is the peak area of ​​N-nitrosodesmethylolopatadine in the N-nitrosodesmethylolopatadine reference solution; Cr is the concentration of N-nitrosodesmethylolopatadine in the N-nitrosodesmethylolopatadine control solution, mg / mL.

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