Method for detecting nitrosamine impurities in metformin empagliflozin tablets

Abstract

The application provides a method for detecting nitrosamine impurities in metformin englitazin tablets, and belongs to the technical field of drug analysis. The application detects nitrosamine impurities in metformin englitazin tablets based on high performance liquid chromatography-tandem triple quadrupole mass spectrometer, strictly controls chromatographic and mass spectrometric parameters, and constructs a kind of exclusive, sensitive and efficient synchronous detection technology. In terms of sensitivity, the detection limit signal-to-noise ratio is greater than or equal to 3, the quantitative limit signal-to-noise ratio is greater than or equal to 10, the quantitative limit concentration is as low as ng / ml level, which fully meets the detection requirements of trace genetic toxic impurities, the quantitative limit repeatability precision RSD is less than or equal to 20%, the results are stable, the defects of the existing detection method can be effectively solved, and the strict requirements of trace detection of nitrosamine impurities in drug quality control are met.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for detecting nitrosamine impurities in metformin empagliflozin tablets. Background Technology

[0002] Metformin and empagliflozin tablets are a combination single-tablet formulation composed of metformin hydrochloride and empagliflozin, two hypoglycemic active ingredients with complementary mechanisms of action. It is indicated for adult patients with type 2 diabetes who require combined treatment with empagliflozin and metformin hydrochloride. This combination formulation, while exerting a synergistic hypoglycemic effect, can significantly reduce the number of tablets patients need to take daily, improving medication adherence, and is currently one of the mainstream hypoglycemic drugs commonly used in clinical practice.

[0003] Nitrosamines are highly genotoxic and potentially carcinogenic substances, listed as a "concern" in ICH M7(R1), and are a key target for quality control of nitrogen-containing organic pharmaceuticals. Following the detection of N-nitrosodimethylamine (NDMA) in valsartan raw material in 2018, domestic and international regulatory agencies have tightened regulations on genotoxic impurities. The "Technical Guidelines for Research on Nitrosamine Impurities in Chemical Drugs (Trial)" issued by the National Medical Products Administration and the "Guidelines for the Control of Genotoxic Impurities" in General Chapter 9306 of the 2025 edition of the Chinese Pharmacopoeia both clearly require full life-cycle control of nitrosamine impurities. Identified nitrosamine impurities in finished products must be included in quality standards. Detection methods must undergo comprehensive methodological validation, including specificity, sensitivity, and accuracy. The acceptable intake of a single impurity must be strictly calculated based on daily exposure (e.g., 96 ng / day for NDMA), and the total acceptable intake of multiple impurities must be managed according to treatment cycle levels.

[0004] Metformin contains a dimethylamine structure, which readily undergoes nitrosation in the presence of nitrite, generating NDMA and other nitrosamine impurities (such as NMBA, NDEA, NDIPA, NEIPA, NDBA, etc.). Studies have shown that the formation of nitrosamine impurities is closely related to the quality of raw and auxiliary materials, formulation processes (such as the high temperature and humidity environment during fluidized bed drying and wet granulation), and long-term storage degradation of the drug. Metformin empagliflozin tablets have a complex formulation, containing two active pharmaceutical ingredients and multiple excipients, and involve a moist heat treatment process in the manufacturing process. The formation and introduction pathways of nitrosamine impurities are more complex than in single-ingredient formulations, making this a core risk point for the quality control of this product.

[0005] However, existing methods for detecting nitrosamine impurities in metformin preparations still have significant shortcomings: Insufficient sensitivity: Conventional HPLC or GC-MS methods are difficult to meet the quantitation limit requirements for trace impurities at the ppm or even ppb level; Narrow coverage: Most methods can only detect NDMA as a single impurity and cannot simultaneously screen and quantify six common nitrosamines: NDMA, NMBA, NDEA, NDIPA, NEIPA, and NDBA. Poor specificity: The two main drugs and excipients of empagliflozin and metformin in the compound preparation are prone to interference with the nitrosamines to be tested under the test conditions, resulting in false positives or inaccurate quantification; Poor durability: Even small fluctuations in chromatographic conditions can affect peak shape and quantitative accuracy, making it difficult to meet the needs of enterprises for batch testing and routine quality control.

[0006] Therefore, developing a highly specific and sensitive method for the simultaneous quantification of multiple nitrosamine impurities in metformin empagliflozin tablets is of significant practical value for strictly controlling the risk of impurities in this product, ensuring the safety of clinical medication, and meeting the latest pharmacopoeia and regulatory compliance requirements. Summary of the Invention

[0007] The purpose of this invention is to provide a method for detecting nitrosamine impurities in metformin empagliflozin tablets. This method uses high performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-MS / MS) to achieve simultaneous separation and accurate quantification of six nitrosamine impurities. The method has been fully validated and is highly specific, sensitive, and robust. It can effectively solve the defects of existing detection methods and meet the stringent requirements for trace detection of nitrosamine impurities in drug quality control.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for detecting nitrosamine impurities in metformin empagliflozin tablets, the method comprising the following steps: (1) Prepare the test solution and a mixed reference solution containing metformin, empagliflozin and nitrosamine impurities; prepare the test solution, the spiked test solution and a mixed reference solution containing six nitrosamine impurities; S2. High-performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-MS / MS) is used for sample injection and detection. Chromatographic and mass spectrometric conditions are set. The sample is first mixed with a reference solution to determine the retention time and peak area of ​​each nitrosamine impurity. The test solution is then injected to determine the elution peak of each nitrosamine impurity by retention time, and the content of each nitrosamine impurity is calculated by external standard method.

[0009] Preferably, the nitrosamine impurities include N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodiisopropylamine (NDIPA), N-nitroso-N-ethylisopropylamine (NEIPA), and N-nitrosodi-n-butylamine (NDBA).

[0010] Preferably, the chromatographic conditions include: Chromatographic column: A chromatographic column packed with uncapped hydrophilic alkyl-bonded silica gel with side chain protection; Mobile phase A: 0.1%~0.2% formic acid aqueous solution; Mobile phase B: 100% methanol; Post-run time: 2-4 minutes; Flow rate: 0.3-0.5 ml / min; Column temperature: 30-50℃; Injection volume: 4~6 μL.

[0011] More preferably, the chromatographic column comprises a Poroshell 120 SB-Aq column.

[0012] More preferably, the chromatographic conditions include: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: methanol; Post-run time: 3 minutes; Flow rate: 0.4 ml / min; Column temperature: 40℃; Injection volume: 5 μL.

[0013] Preferably, the gradient elution method of the high-performance liquid chromatography is as follows:

[0014] Preferably, the mass spectrometry conditions include: Ion source: Atmospheric pressure chemical ionization (APCI) ion source; Atomizing gas, drying gas, and collision gas: nitrogen; Nebulizer gas pressure: 36.0~45.0 psi; Drying gas temperature: 300~350℃; Evaporation chamber temperature: 330~365℃; VCap voltage: 1250~1740V; Corona needle current: 5~7 μA; Polarity: Positive; Detection mode: Multiple reaction monitoring (MRM).

[0015] More preferably, the mass spectrometry conditions include: Nebulizer gas pressure: 40.0 psi; Drying gas temperature: 325℃; Evaporation chamber temperature: 350℃; VCap voltage: 1500V; Corona needle current: 6 μA.

[0016] The present invention also provides an application of the above-mentioned detection method in the detection of nitrosamine impurities in metformin empagliflozin tablets.

[0017] The beneficial effects of this invention compared to the prior art are as follows: (1) This invention utilizes high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-MS / MS) to detect nitrosamine impurities in metformin empagliflozin tablets. By strictly controlling chromatographic and mass spectrometric parameters, a specific, sensitive, and efficient simultaneous detection technique is constructed. In terms of sensitivity, the method has a detection limit signal-to-noise ratio (SNR) ≥3, a quantitation limit SNR ≥10, and a quantitation limit concentration as low as ng / ml, fully meeting the detection requirements for trace genotoxic impurities. The quantitation limit repeatability precision RSD ≤20%, and the results are stable. In terms of linearity, the linear correlation coefficients of the six impurities are all greater than 0.995 within the concentration range from the quantitation limit to 150% of the limit, and the ratio of the Y-axis intercept to the limit concentration response value is ≤20%, indicating high quantitative accuracy. In terms of stability and robustness, the repeatability and intermediate precision RSD are ≤15%, and the overall RSD of 12 samples is ≤20%. The spiked recovery rate is in the range of 75.0%~125.0%, with an RSD ≤15%, which meets the pharmaceutical quality control standards.

[0018] (2) In terms of specificity, the blank solution and excipients and active pharmaceutical ingredients in the test sample of this invention do not interfere with the detection of the six nitrosamine impurities. The peaks of each impurity are well separated and have stable retention times, achieving accurate localization and quantification of impurities and effectively avoiding the risk of false positives or false negatives caused by matrix interference. In terms of system applicability, the separation and detection of the six nitrosamine impurities can be completed simultaneously with a single injection. The gradient elution procedure is reasonable, the detection cycle is short, and the detection throughput is significantly improved, making it suitable for routine quality monitoring of pharmaceuticals.

[0019] (3) In terms of stability, the reference solution, test solution and spiked test solution are stable at room temperature for 24 hours, with peak area RSD≤15%; in terms of robustness, the relative deviation of the detection results is ≤15% under column temperature ±2℃ and flow rate ±0.05ml / min fluctuation, indicating that the detection method of the present invention is highly adaptable and suitable for batch detection. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is the chromatogram of the blank solution in Example 1 of the present invention; Figure 2 This is the chromatogram of the NDMA localization solution in Example 1 of the present invention; Figure 3This is the chromatogram of the NDEA localization solution in Example 1 of the present invention; Figure 4 This is the chromatogram of the NMBA localization solution in Example 1 of the present invention; Figure 5 This is the chromatogram of the NEIPA localization solution in Example 1 of the present invention; Figure 6 This is the chromatogram of the NDIPA localization solution in Example 1 of the present invention; Figure 7 This is the chromatogram of the NDBA localization solution in Example 1 of the present invention; Figure 8 This is the chromatogram of the mixed reference solution in Example 1 of the present invention; Figure 9 This is the chromatogram of the test solution (batch number: 24040187) in Test Example 8 of the present invention; Figure 10 This is the chromatogram of the test solution (batch number: 24040287) in Test Example 8 of the present invention; Figure 11 This is the chromatogram of the test solution (batch number: 24040387) in Test Example 8 of the present invention; Figure 12 This is the chromatogram-mass spectrum of methanol in Comparative Example 1 of this invention; Figure 13 This is the chromatogram-mass spectrum of the mixed solution of control stock solution 2 in Comparative Example 1 of this invention; Figure 14 This is the chromatogram-mass spectrum of the mixed solution of control stock solution 2 in Comparative Example 2 of the present invention; Figure 15 This is the chromatogram-mass spectrum of metformin control in Comparative Example 2 of this invention. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] The reagents used in the following examples are as follows: 1. Stock solution of nitrosamine impurity reference standard: Accurately weigh appropriate amounts of NDMA, NMBA, NDEA, NDIPA, NEIPA, and NDBA reference standards, and quantitatively dilute them with methanol to prepare reference standard stock solutions containing 24 μg / ml NDMA, 2.4 μg / ml NMBA, 16 μg / ml NDEA, 16 μg / ml NDIPA, 16 μg / ml NEIPA, and 16 μg / ml NDBA per ml, respectively.

[0028] 2. Mixed reference solution: Accurately measure appropriate amounts of each reference stock solution and quantitatively dilute with solvent to prepare a mixed reference solution containing approximately 4.8 ng / ml NDMA, 4.8 ng / ml NMBA, 1.3 ng / ml NDEA, 1.3 ng / ml NDIPA, 1.3 ng / ml NEIPA, and 1.3 ng / ml NDBA per ml.

[0029] 3. Test solution: Take 20 metformin empagliflozin tablets, grind them into a fine powder, accurately weigh approximately 1.2g of the powder, place it in a 10ml volumetric flask, add an appropriate amount of solvent, sonicate for 10 minutes to dissolve, dilute to the mark with solvent, shake well, filter, and use the filtrate as the test solution; the concentration of metformin empagliflozin in the test solution should be 85-135mg / ml. The test solution should be freshly prepared before use or stored at 2-8℃ for no more than 24 hours to avoid changes in component content due to high temperature.

[0030] 5. Spiked solution for the test sample: Take 20 metformin empagliflozin tablets, grind them into a fine powder, weigh about 1.2g of the powder accurately, place it in a 10ml volumetric flask, add an appropriate amount of solvent, and sonicate for 10 minutes to dissolve it. Accurately measure 0.2ml of the reference solution stock solution and place it in the above volumetric flask, dilute to the mark with solvent, shake well, filter, and take the filtrate as the spiking solution for the test sample.

[0031] The water used in the preparation of the above solutions was all deionized water.

[0032] Example 1 Example 1 of this invention provides a method for determining the content of nitrosamine impurities in metformin empagliflozin tablets, the specific steps of which are as follows: (1) Reagent preparation Take the stock solution of nitrosamine impurity reference standards and dilute it separately to prepare mixed reference standard solutions containing 4.8 ng / ml NDMA, 4.8 ng / ml NMBA, 1.3 ng / ml NDEA, 1.3 ng / ml NDIPA, 1.3 ng / ml NEIPA, and 1.3 ng / ml NDBA per 1 ml.

[0033] Take 20 metformin empagliflozin tablets, grind them into a fine powder, accurately weigh about 1.2g of the powder, place it in a 10ml volumetric flask, add an appropriate amount of solvent, sonicate for 10 minutes to dissolve, dilute to the mark with solvent, shake well, filter, and take the filtrate as the test solution.

[0034] 0.1% formic acid aqueous solution: Accurately measure 1 ml of formic acid into a 1000 ml volumetric flask, add water to the mark, and mix well; (2) Sample injection detection Accurately measure the test solution and the mixed reference solution, inject them separately into the instrument, and record the chromatograms.

[0035] The chromatographic detection conditions are as follows: Chromatographic column: Poroshell 120 SB-Aq column, 3.0mm × 150mm, 2.7μm.

[0036] Mobile phase: Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is 100% methanol; Flow rate: 0.4 ml / min; Column temperature: 40℃; Injection volume: 5 μl.

[0037] The gradient elution method described in the liquid chromatograph is shown in Table 1: Table 1 Gradient elution parameters

[0038] Chromatogram as shown Figures 1-8 As shown.

[0039] If the chromatogram of the test solution contains a peak with the same retention time as the reference standard, calculated by peak area using the external standard method, the concentrations of NDMA and NMBA should not exceed 0.048 ppm, and the concentrations of NDEA, NDIPA, NEIPA, and NDBA should not exceed 0.013 ppm.

[0040] (2) Mass spectrometry was performed using a liquid chromatography-tandem triple quadrupole mass spectrometer. The mass spectrometry conditions were as follows: Ion source: Atmospheric pressure chemical ionization ion source (APCI); Atomizing gas, drying gas, and collision gas: nitrogen; Nebulizer gas pressure: 40.0 psi; Drying gas temperature: 325℃; Evaporation chamber temperature: 350℃; VCap voltage: 1500V; Corona needle current: 6μA; Polarity: Positive; Detection mode: Multiple response monitoring (MRM); Table 2. Monitoring ion parameters for each impurity (quantitative ion labeling*)

[0041] Experimental Example 1 Test Example 1 of this invention verified the specificity of the method in Example 1. The specific steps are as follows: The chromatographic and mass spectrometric conditions of Example 1 were used for injection detection. The injection sequence was: blank solution ≥ 1 injection → test solution 1 injection → each impurity localization solution 1 injection → mixed reference solution 1 injection. The interference of blank and the separation of each impurity were observed. The experimental results are shown in Table 3.

[0042] Table 3 Results of specificity verification experiments

[0043] In Table 3, the blank solution showed no chromatographic peaks at the retention times of each impurity, thus having no interference with detection. The retention times of the impurities in the localizing solutions and the mixed reference solutions were consistent. The six nitrosamine impurities mentioned above were not detected in the test solution, and there was no interference from any peak, indicating that the specificity met the requirements.

[0044] Experimental Example 2 Test Example 2 of this invention verified the sensitivity of the method in Example 1. The specific steps are as follows: The mixed reference solution was serially diluted to prepare a detection limit solution with a signal-to-noise ratio of approximately 3 and a quantitation limit solution with a signal-to-noise ratio of approximately 10. The chromatographic and mass spectrometric conditions of Example 1 were used for injection detection. The quantitation limit solution was injected 6 times consecutively, and the peak area RSD value was calculated. The experimental results are shown in Table 4.

[0045] Table 4. Results of Sensitivity Verification Experiment

[0046] Table 4 shows the detection results. In the limit of detection solution, the signal-to-noise ratios (S / N) of the peaks of NDMA, NMBA, NDBA, NDIPA, NEIPA, and NDEA were 3, 4, 5, 10, 5, and 8, respectively, all greater than 3. In the limit of quantitation solution, the S / N of the peaks of NDMA, NMBA, NDBA, NDIPA, NEIPA, and NDEA were 25, 27, 24, 37, 25, and 31, respectively, all greater than 10. Furthermore, after six repeated injections of the limit of quantitation solution, the relative standard deviations of the peak areas of NDMA, NMBA, NDBA, NDIPA, NEIPA, and NDEA were 7.4%, 4.2%, 6.7%, 11.0%, 5.4%, and 17.4%, respectively, all below 20%, indicating high sensitivity.

[0047] Experimental Example 3 Experimental Example 3 of this invention verified the linearity and range of the method in Example 1. The specific steps are as follows: Linear series solutions with limit concentrations of 10%, 20%, 50%, 100%, and 150% were prepared and analyzed by chromatographic and mass spectrometric conditions as described in Example 1. A standard curve was plotted with concentration as the abscissa and peak area as the ordinate. The linear equation, correlation coefficient, and Y-axis intercept / limit concentration response value ratio were calculated. The results of the linearity and range verification experiments are shown in Table 5.

[0048] Table 5. Results of linearity and range verification experiments

[0049] The correlation coefficients of the linear equations for the NDMA, NMBA, NDBA, NDIPA, NEIPA, and NDEA peaks in Table 5 are 0.9995, 0.9998, 0.9991, 0.9995, 0.9993, and 0.9993, respectively, all greater than 0.995; the Y-axis intercept / limit concentration response values ​​are 0.27%, 0.055%, 1.6%, 1.4%, 1.4%, and 2.1%, respectively, all not exceeding 20%. This meets the requirements. The method exhibits good linearity and a reasonable quantitative range.

[0050] Test Example 4 Test Example 4 of this invention verified the precision of the method in Example 1. The specific steps are as follows: Six 100% limit concentration spiked test solutions were prepared in parallel from the same batch of samples and detected according to the chromatographic and mass spectrometric conditions of Example 1. The RSD of impurity content was calculated, and the results of the repeatability verification experiment are shown in Table 6.

[0051] Table 6 Results of Repeatability Verification Experiments

[0052] Table 6 shows that the relative standard deviations of the contents of NDMA, NMBA, NDBA, NDIPA, NEIPA, and NDEA in six parallel preparations of the same batch of samples were 1.2%, 3.0%, 5.6%, 3.4%, 1.5%, and 4.8%, respectively, all less than 15.0%. This meets the requirements.

[0053] Intermediate precision: Six spiked solutions of the same concentration were prepared by different experimenters on different dates, and the RSD of the content was calculated. The overall RSD was calculated by combining 12 data. The results of the intermediate precision experiment are shown in Table 7 and Table 8.

[0054] Table 7 Results of intermediate precision experiments (1)

[0055] Table 7 shows that the relative standard deviations of the contents of NDMA, NMBA, NDBA, NDIPA, NEIPA, and NDEA in six parallel preparations of the same batch of samples were 1.1%, 7.9%, 5.3%, 6.4%, 4.5%, and 7.9%, respectively, all less than 15.0%. This meets the requirements.

[0056] Table 8 Intermediate precision test results (2)

[0057] Table 8 shows the repeatability and intermediate precision of 12 spiked test solutions. The relative standard deviations of the impurities NDMA, NMBA, NDBA, NDIPA, NEIPA, and NDEA were 1.3%, 8.6%, 6.0%, 5.9%, 3.9%, and 6.5%, respectively, all less than 20.0%, which meets the requirements.

[0058] Experimental Example 5 Test Example 5 of this invention verified the accuracy of the method in Example 1. The specific steps are as follows: Spiked test solutions with limit concentrations of 50%, 100%, and 150% were prepared, with three replicates for each concentration. The solutions were tested according to the prescribed method, and the recovery rate and RSD of each impurity were calculated. The results of the accuracy verification experiment are shown in Table 9.

[0059] Table 9. Results of Accuracy Verification Experiment

[0060] Table 9 shows that the recoveries of NDMA, NMBA, NDBA, NDIPA, NEIPA, and NDEA in the solutions with different recovery rates were 103.0%, 111.0%, 105.5%, 107.4%, 101.0%, and 105.4%, respectively, all within the range of 75% to 125%. The relative standard deviations of each impurity were 2.3%, 6.5%, 5.7%, 5.9%, 2.7%, and 5.6%, respectively, all less than 15.0%, which meets the requirements.

[0061] Experimental Example 6 Test Example 6 of this invention verified the stability of the solution from Example 1. The specific steps are as follows: Take the mixed reference solution, the test solution, and the spiked test solution, and place them at room temperature. Inject samples at 0h, 4h, 8h, 12h, 16h, 20h, and 24h, respectively, and calculate the peak area RSD value at each time point. The results of the solution stability verification experiment are shown in Table 10.

[0062] Table 10 Results of solution stability experiments

[0063] In the reference solution, the RSDs of the peak areas of impurities NDMA, NMBA, NDBA, NDIPA, NEIPA, and NDEA were 1.5%, 4.1%, 3.3%, 2.7%, 3.2%, and 7.3%, respectively, all below 15.0%. In the test solution, none of the impurities were detected. In the spiked test solution, the RSDs of the peak areas of impurities NDMA, NMBA, NDBA, NDIPA, NEIPA, and NDEA were 1.8%, 1.6%, 5.1%, 4.2%, 3.8%, and 6.4%, respectively, all below 15.0%. This meets the requirements.

[0064] Experimental Example 7 Test Example 7 of this invention verified the robustness of the method in Example 1. The specific steps are as follows: To investigate the effect of small fluctuations in chromatographic conditions on the detection results, column temperatures were set at 38℃ and 42℃ (normal 40℃), and flow rates were set at 0.35 ml / min and 0.45 ml / min (normal 0.4 ml / min), with other conditions remaining unchanged. The spiked test solution was detected, and the relative deviation (RD) from the results under normal conditions was calculated. The robustness chromatographic conditions are shown in Table 11, and the robustness test results are shown in Table 12.

[0065] Table 11 Robust Chromatographic Conditions

[0066] Table 12 Durability Test Results

[0067] Table 12 shows that the RD values ​​of the contents of NDMA, NMBA, NDBA, NDIPA, NEIPA and NDEA in the spiked test solution compared with the contents of each impurity under normal conditions did not exceed 15.0%, which meets the requirements.

[0068] Experimental Example 8 Test Example 8 of this invention verified the specificity of the method in Example 1. The specific steps are as follows: The method of this invention was used to detect three batches of metformin empagliflozin tablets (24040187, 24040287, and 24040387). The detection results are shown in Table 13, and the chromatograms are shown in [the original text]. Figures 9-11 .

[0069] Table 13 Detection results of nitrosamine impurities in samples

[0070] As can be seen from the data in Table 11, the nitrosamine impurities in the three batches of metformin empagliflozin tablets, namely 24040187, 24040287, and 24040387, all meet the limit requirements.

[0071] Comparative Example 1 The method of Example 1 of this invention was used, employing an InfinityLab Poroshell PFP column (2.1*100mm, 1.9μm). Other conditions were as described in Example 1 for detection. The results are as follows: Figure 12 , 13 As shown.

[0072] Figure 12 , 13 The results showed that methanol exhibited interference and large baseline fluctuations within 0.5-3 minutes, while the reference solution showed a low impurity response.

[0073] Comparative Example 2 Using the method of Example 1 of this invention, the flow rate in the above method was changed to 0.3 ml / min, and other conditions were selected according to the method in Example 1 for detection. The results are as follows. Figure 14 As shown.

[0074] Figure 14 The reference solution showed a low impurity response.

[0075] Comparative Example 3 Using the method of Example 1 of this invention, the liquid chromatography gradient elution conditions were changed as shown in Table 14, and other conditions were selected from the method in Example 1 for detection; the mass spectrometry conditions were simultaneously changed as follows: Ion source: Atmospheric pressure chemical ionization ion source (APCI); Atomizing gas, drying gas, and collision gas: nitrogen; Nebulizer gas pressure: 50 psi; Drying gas temperature: 300℃; Evaporation chamber temperature: 350℃; VCap voltage: 1500V; Corona needle current: 6μA; Polarity: Positive; Detection mode: Multiple response monitoring (MRM) Liquid chromatography gradient elution conditions: Table 14 Gradient elution conditions for liquid chromatography

[0076] Figure 15 The results show that some impurity peaks in the gradients in Table 14 did not appear, indicating that the gradients were inappropriate.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting nitrosamine impurities in metformin empagliflozin tablets, characterized in that, The detection method includes the following steps: (1) Prepare the test solution and a mixed reference solution containing metformin, empagliflozin and nitrosamine impurities; prepare the test solution, the spiked test solution and a mixed reference solution containing six nitrosamine impurities; S2. High-performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-MS / MS) is used for sample injection and detection. Chromatographic and mass spectrometric conditions are set. The sample is first mixed with a reference solution to determine the retention time and peak area of ​​each nitrosamine impurity. The test solution is then injected to determine the elution peak of each nitrosamine impurity by retention time, and the content of each nitrosamine impurity is calculated by external standard method.

2. The method for detecting nitrosamine impurities in metformin empagliflozin tablets according to claim 1, characterized in that, The nitrosamine impurities include N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodiisopropylamine (NDIPA), N-nitroso-N-ethylisopropylamine (NEIPA), and N-nitrosodi-n-butylamine (NDBA).

3. The method for detecting nitrosamine impurities in metformin empagliflozin tablets according to claim 1, characterized in that, The chromatographic conditions include: Chromatographic column: A chromatographic column packed with uncapped hydrophilic alkyl-bonded silica gel with side chain protection; Mobile phase A: 0.1%~0.2% formic acid aqueous solution; Mobile phase B: 100% methanol; Post-run time: 2-4 minutes; Flow rate: 0.3-0.5 ml / min; Column temperature: 30-50℃; Injection volume: 4~6 μL.

4. The method for detecting nitrosamine impurities in metformin empagliflozin tablets according to claim 2, characterized in that, The chromatographic column includes a Poroshell 120 SB-Aq column.

5. The method for detecting nitrosamine impurities in metformin empagliflozin tablets according to claim 1, characterized in that, The chromatographic conditions include: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: methanol; Post-run time: 3 minutes; Flow rate: 0.4 ml / min; Column temperature: 40℃; Injection volume: 5 μL.

6. The method for detecting nitrosamine impurities in metformin empagliflozin tablets according to claim 1, characterized in that, The gradient elution method for high performance liquid chromatography is as follows: 。 7. The method for detecting nitrosamine impurities in metformin empagliflozin tablets according to claim 1, characterized in that, The mass spectrometry conditions include: Ion source: Atmospheric pressure chemical ionization (APCI) ion source; Atomizing gas, drying gas, and collision gas: nitrogen; Nebulizer gas pressure: 36.0~45.0 psi; Drying gas temperature: 325℃; Evaporation chamber temperature: 330~365℃; VCap voltage: 1250~1740V; Corona needle current: 5~7 μA; Polarity: Positive; Detection mode: Multiple reaction monitoring (MRM).

8. The method for detecting nitrosamine impurities in metformin empagliflozin tablets according to claim 7, characterized in that, The mass spectrometry conditions include: Nebulizer gas pressure: 40.0 psi; Drying gas temperature: 325℃; Evaporation chamber temperature: 350℃; VCap voltage: 1500V; Corona needle current: 6 μA.

9. The application of the detection method according to any one of claims 1 to 8 in the detection of nitrosamine impurities in metformin empagliflozin tablets.

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