Detection method for simultaneously determining two nitrosamine impurities in fluoxetine hydrochloride bulk drug

Abstract

The invention belongs to the technical field of drug detection, and relates to a detection method for simultaneously determining two nitrosamine impurities in fluoxetine hydrochloride bulk drugs. Comprising the following steps: dissolving a fluoxetine hydrochloride bulk drug to obtain a test solution, and analyzing and detecting N-nitroso-fluoxetine and N-nitroso-fluoxetine EP impurity A in the test solution by adopting ultra-high performance liquid chromatography-mass spectrometry, in the analysis and detection process, a pentafluorophenyl propyl chromatographic column is adopted, a formic acid aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, and gradient elution is performed; triple quadrupole mass spectrometry is used as a detector, electrospray ionization source detection is adopted, and multi-reaction detection of MRM is carried out through positive ion mode scanning. The method is stronger in specificity and higher in sensitivity, detection can be completed only in 12 minutes, and the content of two nitrosamine impurities in a sample can be accurately and rapidly detected. The method is of great significance to quality control of fluoxetine hydrochloride bulk drugs and guarantee of medication safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug detection, and relates to a detection method for simultaneously detecting two nitrosamine impurities in fluoxetine hydrochloride bulk drug. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any country in the world.

[0003] In recent years, the risk control of nitrosamine genotoxic impurities in the field of global drug quality and safety supervision continues to upgrade. Such strong carcinogenic impurities can be introduced into the drug system through multiple paths: including but not limited to the residue of nitrosation reagent in the synthesis process of bulk drug, the interaction of formulation excipients, and the secondary reaction caused by improper storage conditions. It is particularly worth noting that the selective serotonin reuptake inhibitor antidepressant drug fluoxetine hydrochloride contains a secondary amine group in its molecular structure, which has significant nitrosation reactivity. The amine compounds commonly present in the synthesis starting materials, intermediate products and process by-products involved in the production process of the drug can react with nitrite substances under certain conditions to generate nitrosamine impurities such as N-nitroso-fluoxetine and N-nitroso-nitroso-fluoxetine EP impurity A, which have a clear carcinogenic risk.

[0004] According to the latest “Control of Nitrosamine Impurities in Human Drugs” issued by the US Food and Drug Administration (FDA), the acceptable daily intake (AI) of N-nitroso-fluoxetine is confirmed to be 100 ng through toxicological evaluation, and based on the maximum daily dose of 60 mg of the drug, the control standard of the bulk drug needs to be strictly controlled below 1.67 ppm; while N-nitroso-nitroso-fluoxetine EP impurity A, which has a higher toxicological risk, is classified as active warning structure 1 substance, and its AI value is further reduced to 25 ng / day, which corresponds to the ultra-trace detection level of 0.44 ppm of the control standard of the bulk drug. However, the existing pharmacopoeias of various countries (including USP, EP, ChP) have not established a special detection method for such nitrosamine impurities in the quality standard system of fluoxetine hydrochloride bulk drug and its preparations, resulting in a great safety hazard in the existing quality control system. Therefore, it is necessary to develop a rapid detection method with high sensitivity and high accuracy for nitrosamine impurities in fluoxetine hydrochloride bulk drug. SUMMARY

[0005] In order to solve the deficiencies of the prior art, the purpose of the present application is to provide a detection method for simultaneously detecting two nitrosamine impurities in fluoxetine hydrochloride bulk drug, which can rapidly and accurately detect the two nitrosamine impurities in fluoxetine hydrochloride bulk drug.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is: In a first aspect, a detection method for simultaneously detecting two nitrosamine impurities in fluoxetine hydrochloride bulk drug is provided, comprising the following steps: Dissolving the fluoxetine hydrochloride bulk drug to obtain a test solution with a concentration of 2.2-2.4 mg / mL, and using ultra-high performance liquid chromatography-mass spectrometry to analyze and detect N-nitroso-fluoxetine and N-nitroso-fluoxetine EP impurity A in the test solution. In the analysis and detection process, a pentafluorophenylpropyl chromatographic column is used, formic acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and gradient elution is performed; a triple quadrupole mass spectrometer is used as a detector, an electrospray ion source is used for detection, and a positive ion mode scan is used for multiple reaction monitoring MRM.

[0007] In the process of detecting nitrosamine impurities in fluoxetine hydrochloride bulk drug, it is found that the detection difficulty lies in: 1. Low impurity limit, high sensitivity requirement for the detection method, ordinary detectors such as UV cannot meet the sensitivity requirement; 2. Interfered by matrix components such as main components, the method recovery rate is difficult to meet the requirement; 3. N-nitroso-fluoxetine is interfered by other unknown ions during detection.

[0008] Therefore, the present application uses a triple quadrupole mass spectrometer as a detector and uses an MRM mode to enhance the detection sensitivity and improve the specificity of the detection method; at the same time, formic acid water is selected as the mobile phase to improve the ionization efficiency of the positive ion mode and thus improve the detection sensitivity. Secondly, the present application further increases the detection sensitivity and improves the method recovery rate by adjusting the concentration of the test solution to meet the detection requirements. Thirdly, the present application contains a pentafluorophenylpropyl chromatographic column in the chromatographic column, which can separate N-nitroso-fluoxetine from unknown impurities, thereby realizing rapid and accurate detection of two nitrosamine impurities in fluoxetine hydrochloride bulk drug.

[0009] The chemical structural formula of N-nitroso-fluoxetine in the present application is .

[0010] The chemical structural formula of N-nitroso-fluoxetine EP impurity A in the present application is .

[0011] In a second aspect, the above-mentioned detection method is applied to the quality evaluation of fluoxetine hydrochloride bulk drug.

[0012] The present application has the following advantages: The ultra-high performance liquid chromatography tandem mass spectrometry technology of the present application detects two nitrosamine impurities in fluoxetine hydrochloride bulk drug, has high selectivity, good method specificity, a limit of quantification as low as 10% of the limit, a detection limit as low as 3% of the limit, and can complete detection in only 12 minutes, has high sensitivity, and can quickly and accurately detect the content of the two nitrosamine impurities in the sample, effectively reducing the analysis cost. The present application experiment shows that, within the range of 50% to 150% of the limit concentration (N-nitrosamine-fluoxetine impurity is 1.67 ppm, N-nitrosamine-fluoxetine EP impurity A is 0.44 ppm), the recovery rate of the two nitrosamine impurities is between 80% and 120%, and good accuracy is achieved; the r of the two nitrosamine impurities in the range of the limit of quantification to 200% is greater than 0.990, and good linearity is achieved. BRIEF DESCRIPTION OF DRAWINGS

[0013] The accompanying drawings, which form a part of this application, are used to provide further understanding of the application and are incorporated in and constitute a part of this application. The schematic embodiments of the present application and the description thereof are used to explain the present application, and do not constitute an improper limitation on the present application.

[0014] Figure 1 It is the N-nitroso-fluoxetine blank solution spectrum of Example 1 of the present application; Figure 2 It is the N-nitroso-fluoxetine impurity control solution spectrum of Example 1 of the present application; Figure 3 It is the N-nitroso-fluoxetine spectrum in the test sample solution of Example 1 of the present application; Figure 4 It is the N-nitroso-fluoxetine spectrum in the 100% spiked solution of Example 1 of the present application; Figure 5 It is the N-nitroso-fluoxetine EP impurity A blank solution spectrum of Example 1 of the present application; Figure 6 It is the N-nitroso-fluoxetine EP impurity A impurity control solution spectrum of Example 1 of the present application; Figure 7 It is the N-nitroso-fluoxetine EP impurity A spectrum in the test sample solution of Example 1 of the present application; Figure 8 It is the N-nitroso-fluoxetine EP impurity A spectrum in the 100% spiked solution of Example 1 of the present application. DETAILED DESCRIPTION

[0015] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0016] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0017] The five-fluorophenyl propyl chromatographic column described in the present application refers to the five-fluorophenyl propyl contained in the stationary phase (or filler) in the chromatographic column; wherein the stationary phase (or filler) is generally silica gel, and the five-fluorophenyl propyl can be bonded with the silica gel through a siloxane bond (Si-O-Si).

[0018] In view of the fact that there is no detection method for nitrosamine impurities in fluoxetine hydrochloride bulk drug at present, thereby causing great safety hidden danger of fluoxetine hydrochloride bulk drug, the present application provides a detection method for simultaneously determining two kinds of nitrosamine impurities in fluoxetine hydrochloride bulk drug.

[0019] In a typical embodiment of the present application, a detection method for simultaneously determining two kinds of nitrosamine impurities in fluoxetine hydrochloride bulk drug is provided, which comprises the following steps: Dissolving the fluoxetine hydrochloride bulk drug to obtain a test solution with a concentration of 2.2-2.4 mg / mL, and using ultra-high performance liquid chromatography-mass spectrometry to analyze and detect N-nitroso-fluoxetine and N-nitroso-fluoxetine EP impurity A in the test solution; In the analysis and detection process, a five-fluorophenyl propyl chromatographic column is used, formic acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B for gradient elution; a triple quadrupole mass spectrometer is used as a detector, and a multi-reaction detection MRM is performed by scanning in a positive ion mode through an electrospray ion source.

[0020] In some embodiments, ultra-high performance liquid chromatography-mass spectrometry is used to analyze and detect N-nitroso-fluoxetine reference solution and N-nitroso-fluoxetine EP impurity A reference solution.

[0021] Specifically, the chromatogram of the test solution and the chromatogram of the reference solution are obtained through analysis and detection, respectively, and the content of the nitrosamine impurities in the fluoxetine hydrochloride bulk drug is calculated according to the peak area in the chromatogram.

[0022] In some embodiments, the solvent for dissolving fluoxetine hydrochloride bulk drug is an aqueous acetonitrile solution. The use of an aqueous acetonitrile solution not only has a higher solubility for fluoxetine hydrochloride bulk drug, but also has a good solubility for the two nitrosamine impurities, and can reduce interference, reduce matrix effect, and improve detection accuracy. Acetonitrile in the aqueous acetonitrile solution can be mixed with water in any ratio, and is preferably acetonitrile and water in a volume ratio of 48:52 to 52:48. Studies have shown that the stability of fluoxetine hydrochloride bulk drug and the two nitrosamine impurities in this solvent system is higher.

[0023] In some embodiments, the volume concentration of formic acid in the formic acid aqueous solution is 0.09-0.11%. That is, 0.9-1.1 mL of formic acid is contained in 1000 mL of solution.

[0024] In some embodiments, the elution program for gradient elution is, in terms of volume percentage: 0-8 min, mobile phase A decreases from 80% to 40%, mobile phase B increases from 20% to 60%; 8-8.1 min, mobile phase A decreases from 40% to 20%, mobile phase B increases from 60% to 80%; 8.1-10 min, mobile phase A 20%, mobile phase B 80%; 10-10.1 min, mobile phase A increases from 20% to 80%, mobile phase B decreases from 80% to 20%; 10.1-12 min, mobile phase A 80%, mobile phase B 20%.

[0025] Studies have shown that under these conditions, N-nitroso-fluoxetine and N-nitroso-fluoxetine EP impurity A can be better separated.

[0026] In some embodiments, the flow rate during the analysis and detection process is 0.35-0.45 mL / min, and is preferably 0.4 mL / min.

[0027] In some embodiments, the column temperature during the analysis and detection process is 25-35°C, and is preferably 30°C.

[0028] In some embodiments, the chromatographic column is a Phenomenex Kinetex F5, 100 mm x 3.0 mm, 2.6 μm.

[0029] In some embodiments, when mass spectrometry is performed, the mass spectrometry parameters for N-nitroso-fluoxetine are: Q1 is 339.1±0.5 Da, Q3 is 177.2±0.5 Da, DP is 96±0.5 V, and CE is 15±0.5 V.

[0030] In some embodiments, when mass spectrometry is used for detection, the mass spectrometry parameters of the N-nitroso-fluoxetine EP impurity A are: Q1 is 195.1±0.5 Da, Q3 is 117.1±0.5 Da, DP is 80±0.5 V, and CE is 10±0.5 V.

[0031] In some embodiments, when mass spectrometry is used for detection, the IS voltage is 5500±50 V; the gas curtain GAS is 35±0.5 psi; the atomizer GAS1 is 50±0.5 psi; the auxiliary gas GAS2 is 50±0.5 psi; the ion source temperature is 550±5°C; and the collision gas is 8±0.5. The above mass spectrometry conditions are the preferred technical conditions in the technical solution of the present application, and under the conditions, the detection effect is better.

[0032] The second embodiment of the present application provides an application of the above-mentioned detection method in the quality evaluation of fluoxetine hydrochloride bulk drug.

[0033] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific embodiments.

[0034] In the following examples, the mass content of nitrosamine impurities in fluoxetine hydrochloride bulk drug can be calculated by external standard method, and the calculation formula is as follows: Nitrosamine impurities (%) = × 100 In the formula: A U --peak area of nitrosamine impurities in the test solution; A S --peak area of nitrosamine impurities in the control solution; C S --concentration of nitrosamine impurities in the control solution, ng / mL; C U --concentration of the test solution, ng / mL.

[0035] Example 1: Specificity Take 500 mL of acetonitrile and 500 mL of water and mix them evenly to obtain a diluent (blank solution).

[0036] Precisely weigh an appropriate amount of N-nitroso-fluoxetine and N-nitroso-fluoxetine EP impurity A impurity control, dissolve and dilute with a quantitative bottle to obtain a solution containing N-nitroso-fluoxetine and N-nitroso-fluoxetine EP impurity A at a concentration of 3.8 ng / mL and 1.0 ng / mL, respectively, i.e. a nitrosamine impurity control solution.

[0037] Take fluoxetine hydrochloride raw materials (manufacturer: Alembic Pharmaceuticals Ltd, batch number 2102018960) appropriate amount, add diluent to dissolve and use quantitative dilution to prepare a solution containing about 2.3 mg / mL of fluoxetine, which is the test solution.

[0038] Take appropriate amount of fluoxetine hydrochloride raw materials and appropriate amount of two nitrosamine impurities, dilute with diluent to prepare a mixed solution with a fluoxetine concentration of 2.3 mg / mL, an N-nitroso-fluoxetine concentration of 3.8 ng / mL, and an N-nitroso-fluoxetine EP impurity A concentration of 1.0 ng / mL, which is the system suitability solution.

[0039] The obtained nitrosamine impurity reference solution, test solution, and system suitability solution were injected into the ultra-high performance liquid chromatography-mass spectrometry instrument for analysis, and the chromatographic conditions and mass spectrometry conditions are as follows: Chromatographic conditions: Chromatographic column: Phenomenex Kinetex F5, 100 mm x 3.0 mm, 2.6 μm; Mobile phase: Mobile phase A is 0.1% (volume percent) formic acid aqueous solution; Mobile phase B is acetonitrile; Elution mode: Gradient elution program as shown in Table 1 Table 1 Gradient elution program

[0040] Flow rate 0.4 mL / min; Column temperature is 30°C; Injection volume is 10 μL.

[0041] Mass spectrometry conditions: Mass spectrometer: Triple quadrupole mass spectrometer; Ion source: ESI source, positive ion scanning; Scan mode: MRM acquisition mode; IS voltage: 5500 V (+); Gas curtain gas CUR is 35 psi; atomizing gas GAS1 is 50 psi; auxiliary gas GAS2 is 50 psi; ion source temperature is 550°C; collision gas: 8; wherein, the gas curtain gas, atomizing gas, auxiliary gas are all nitrogen.

[0042] Mass spectrometry parameters are shown in Table 2.

[0043] Table 2 Mass spectrometry parameters

[0044] The detection results are as follows: the N-nitroso-fluoxetine spectrum in the blank solution is shown in Figure 1 , the N-nitroso-fluoxetine impurity control solution is shown in Figure 2 , the N-nitroso-fluoxetine in the test sample solution is shown in Figure 3 , the N-nitroso-fluoxetine in the 100% spiked test sample solution is shown in Figure 4 , the N-nitroso-fluoxetine EP impurity A in the blank solution is shown in Figure 5 , the N-nitroso-fluoxetine EP impurity A control solution is shown in Figure 6 , the N-nitroso-fluoxetine EP impurity A in the test sample solution is shown in Figure 7 , the N-nitroso-fluoxetine EP impurity A in the 100% spiked test sample solution is shown in Figure 8 The specificity results are summarized in Table 3. Figures 1-8

[0045] Table 3 Specificity results

[0046] Figures 1-8 The results of Table 1 and the above results show that the blank solvent and the test sample solution do not interfere with the detection of N-nitroso-fluoxetine and N-nitroso-fluoxetine EP impurity A, and the specificity of the N-nitroso-fluoxetine and N-nitroso-fluoxetine EP impurity A detection method is better.

[0047] Example 2: Linearity and range experiment A certain amount of nitrosamine impurity control was precisely weighed, and a series of linear solutions with concentrations of about 3% of the N-nitroso-fluoxetine limit (see Example 3) and about 10% of the N-nitroso-fluoxetine EP impurity A limit (see Example 3) to about 200% were prepared.

[0048] The above solution was precisely measured and injected into the ultra-high performance liquid chromatography-mass spectrometry instrument for detection (the chromatographic conditions and mass spectrometry conditions were the same as in Example 1), and the chromatogram was recorded. The results are shown in Table 4, and the linear regression equation was made with the concentration as the abscissa and the peak area as the ordinate.

[0049] Table 4 N-nitroso-fluoxetine impurity linear experiment results

[0050] Conclusion: Within the range of the quantitative limit (equivalent to 3% of the N-nitroso-fluoxetine limit and 10% of the N-nitroso-fluoxetine EP impurity A limit) to 200%, the linear relationship of each nitrosamine impurity is good, which meets the methodological requirements.

[0051] Example 3: Quantitative limit and detection limit ​The concentration at S / N = 3 was taken as the detection limit, and the concentration at S / N = 10 was taken as the quantification limit. The linear solution in Example 2 was diluted step by step.

[0052] The solution was precisely measured and injected into the ultra-high performance liquid chromatography-mass spectrometry instrument for detection (chromatographic conditions and mass spectrometric conditions were the same as in Example 1). The chromatogram was recorded, and the results are shown in Table 5.

[0053] Table 5 Test results of detection limit and quantification limit

[0054] Note: Impurity 1 is N-nitroso-fluoxetine, and impurity 2 is N-nitroso-fluoxetine EP impurity A.

[0055] Conclusion: The quantification limit of the two nitrosamine impurities is less than 10% of the impurity limit of the product, and the detection limit is less than 3% of the impurity limit of the product (fluoxetine hydrochloride raw material). The detection limit and quantification limit detected in this example meet the requirements for quantitative detection of the two nitrosamine impurities in the test product, and the sensitivity is high.

[0056] Example 4: Accuracy experiment The accuracy was investigated by recovery test, which covered a concentration range of 50% to 150% of the limit.

[0057] Prepare the diluent according to Example 1, i.e. 50% acetonitrile (abbreviated as 50% acetonitrile).

[0058] Precisely weigh an appropriate amount of N-nitroso-fluoxetine reference substance, dissolve and dilute with 50% acetonitrile to prepare a solution with a concentration of 100 μg / mL of N-nitroso-fluoxetine, which is the N-nitroso-fluoxetine impurity stock solution.

[0059] Take an appropriate amount of N-nitroso-isofluoxetine EP impurity A reference substance, precisely weigh and add 50% acetonitrile to dissolve and dilute to prepare a solution with a concentration of 100 μg / mL of N-nitroso-isofluoxetine EP impurity A, which is the N-nitroso-fluoxetine EP impurity A stock solution.

[0060] Precisely measure 3.8 mL of N-nitroso-fluoxetine impurity stock solution and 1.0 mL of N-nitroso-isofluoxetine EP impurity A stock solution into a 100 mL volumetric flask, dilute to the mark with 50% acetonitrile, shake well, and obtain a nitrosamine impurity mixed stock solution 1 with a concentration of 3.8 μg / mL of N-nitroso-fluoxetine and 1.0 μg / mL of N-nitroso-isofluoxetine EP impurity A.

[0061] Accurately pipette 1 mL of the nitrosamine impurity stock solution 1 into a 100 mL volumetric flask, dilute to the mark with 50% acetonitrile, shake well, and obtain a nitrosamine impurity mixed stock solution 2 with a N-nitroso-fluoxetine concentration of 38 ng / mL and a N-nitroso-isofluoxetine EP impurity A concentration of 10 ng / mL.

[0062] Accurately pipette 1 mL of the nitrosamine impurity mixed stock solution 2 into a 10 mL volumetric flask, dilute to the mark with 50% acetonitrile, shake well, and obtain the reference solution.

[0063] Accurately weigh 26 mg of fluoxetine hydrochloride into a 10 mL volumetric flask, accurately pipette 0.5 mL, 1.0 mL, and 1.5 mL of the nitrosamine impurity stock solution 2, respectively, dissolve and dilute to the mark with 50% acetonitrile, shake well, and obtain accuracy solutions with different spiked concentrations, with 3 replicates for each spiked concentration level.

[0064] Accurately pipette the above solutions into an ultra-high performance liquid chromatograph-mass spectrometer for detection (chromatographic conditions and mass spectrometric conditions are the same as in Example 1), record the chromatogram, and calculate the peak area by external standard method. The accuracy results are shown in Tables 6 and 7.

[0065] Table 6: N-nitroso-fluoxetine accuracy detection results

[0066] Table 7: N-nitroso-fluoxetine EP impurity A accuracy detection results

[0067] Conclusion: In the 50%, 100%, and 150% accuracy solutions, the recoveries of the three nitrosamine impurities are all between 80% and 120%, and the RSD values are all less than 10%. The detection results show that the detection method of this example has good accuracy.

[0068] Example 5: Precision experiment Prepare the diluent according to the method of Example 1.

[0069] Accurately weigh 26 mg of fluoxetine hydrochloride into a 10 mL volumetric flask, add 1.0 mL of the N-nitrosamine impurity mixed stock solution 2 prepared in Example 4, dissolve and dilute to the mark with the diluent, shake well, and prepare 6 replicates.

[0070] Prepare the reference solution according to the method of Example 4.

[0071] Accurately pipette the above solutions into an ultra-high performance liquid chromatograph-mass spectrometer for detection (chromatographic conditions and mass spectrometric conditions are the same as in Example 1), record the chromatogram, and calculate the impurity content. The precision results are shown in Table 8.

[0072] Table 8 Precision Test Results

[0073] Conclusion: The RSD values ​​of the two nitrosamine impurities were less than 10% in the six parallel prepared reproducible sample solutions, proving that the detection method in this embodiment has good repeatability.

[0074] Example 6: Solution Stability Experiment Take the test solution and the reference solution from Example 1, place them at room temperature, and obtain the injection solutions at different time points.

[0075] Accurately measure the above solution and inject it into an ultra-high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) instrument for detection (chromatographic and mass spectrometry conditions are the same as in Example 1). Record the chromatogram and the solution stability results are shown in Table 9.

[0076] Table 9 Results of solution stability test

[0077] Conclusion: Table 9 shows that the peak areas of the two nitrosamine impurities did not change significantly after the test solution and the control solution were left at room temperature for 18 hours, indicating that the test solution and the control solution were stable within 18 hours.

[0078] Example 7: Sample Testing The diluent was prepared according to the method in Example 1.

[0079] Accurately weigh 26 mg of fluoxetine hydrochloride raw material from different batches (manufacturer: Alembic Pharmaceuticals Ltd, batch numbers 2102018960 and 1902002724 respectively) and place them in 10 mL volumetric flasks. Dissolve and dilute to the mark with diluent and shake well.

[0080] The control solution was prepared according to the method in Example 4.

[0081] Accurately measure the above solution and inject it into an ultra-high performance liquid chromatography-mass spectrometry instrument for detection (chromatographic and mass spectrometry conditions are the same as in Example 1). Record the chromatogram, calculate the impurity content, and the precision results are shown in Table 10.

[0082] Table 10 Sample Test Results

[0083] Conclusion: Table 10 shows that N-nitroso-fluoxetine was detected in both batches of samples produced by Alembic Pharmaceuticals Ltd, while N-nitroso-fluoxetine EP impurity A was not detected.

[0084] Based on the above experimental results, the detection method of this invention was successfully applied to detect two nitrosamine impurities in fluoxetine hydrochloride raw material. The blank solvent and main components did not interfere with the detection of these impurities. The method exhibits high specificity, high sensitivity, and accurate quantification. This method is of great significance for ensuring the quality control of fluoxetine hydrochloride and its preparations, guaranteeing medication safety.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for simultaneously determining two nitrosamine impurities in fluoxetine hydrochloride raw material, characterized in that, Includes the following steps: Fluoxetine hydrochloride raw material was dissolved to obtain a test solution with a concentration of 2.2~2.4 mg / mL. The N-nitroso-fluoxetine and N-nitroso-fluoxetine EP impurity A in the test solution were analyzed and detected by ultra-high performance liquid chromatography-mass spectrometry. In the analytical detection process, a pentafluorophenylpropyl column was used with formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B for gradient elution; a triple quadrupole mass spectrometer was used as the detector, and multiple reaction detection (MRM) was performed by electrospray ionization source scanning in positive ion mode.

2. The detection method as described in claim 1, characterized in that, Ultra-high performance liquid chromatography-mass spectrometry was used to analyze and detect N-nitrosofluoxetine reference solution and N-nitrosofluoxetine EP impurity A reference solution.

3. The detection method as described in claim 2, characterized in that, The chromatograms of the test solution and the reference solution were obtained by analysis and detection. The content of nitrosamine impurities in fluoxetine hydrochloride raw material was calculated based on the peak area in the chromatograms.

4. The detection method as described in claim 1, characterized in that, The solvent for dissolving fluoxetine hydrochloride raw material is an aqueous solution of acetonitrile, preferably, the volume ratio of acetonitrile to water is 48:52 to 52:

48.

5. The detection method as described in claim 1, characterized in that, The volume concentration of formic acid in the formic acid aqueous solution is 0.09~0.11%.

6. The detection method as described in claim 1, characterized in that the elution procedure, expressed as a volume percentage, is as follows: From 0 to 8 minutes, mobile phase A decreased from 80% to 40%, while mobile phase B increased from 20% to 60%. Within 8 to 8.1 minutes, mobile phase A decreased from 40% to 20%, while mobile phase B increased from 60% to 80%. 8.1~10 min, mobile phase A 20%, mobile phase B 80%; Over 10~10.1 min, mobile phase A increased from 20% to 80%, while mobile phase B decreased from 80% to 20%. 10.1~12 min, mobile phase A 80%, mobile phase B 20%.

7. The detection method as described in claim 1, characterized in that... During the analysis and detection process, the flow rate is 0.35~0.45 mL / min, preferably 0.4 mL / min; Alternatively, during the analysis and detection process, the column temperature is 25~35℃, preferably 30℃; Alternatively, the chromatographic column is a Phenomenex Kinetex F5, 100 mm × 3.0 mm, 2.6 μm.

8. The detection method as described in claim 1, characterized in that, During mass spectrometry detection, the mass spectrometry parameters for N-nitroso-fluoxetine were: Q1 339.1±0.5 Da, Q3 177.2±0.5 Da, DP 96±0.5 V, and CE 15±0.5 V. Alternatively, during mass spectrometry detection, the mass spectrometry parameters for N-nitroso-fluoxetine EP impurity A are: Q1 = 195.1 ± 0.5 Da, Q3 = 117.1 ± 0.5 Da, DP = 80 ± 0.5 V, and CE = 10 ± 0.5 V.

9. The detection method as described in claim 1, characterized in that, During mass spectrometry detection, the IS voltage was 5500±50V; the curtain gas CUR was 35±0.5psi; the nebulizer GAS1 was 50±0.5psi; the auxiliary gas GAS2 was 50±0.5psi; the ion source temperature was 550±5°C; and the collision gas was 8±0.

5.

10. The application of the detection method according to any one of claims 1 to 9 in the quality assessment of fluoxetine hydrochloride raw material.

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