Method for detecting content of nitrosamine impurity NMBA in cefepime hydrochloride for injection

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and relates to a method for detecting the content of a nitrosamine impurity N-nitroso-N-methyl-4-aminobutyric acid (hereinafter referred to as NMBA) in cefepime hydrochloride for injection. The method can accurately detect the content of the ppb-level NMBA impurity in the cefepime hydrochloride for injection, so that the safety of medication is ensured, and a prospective method is provided for improvement and innovation of raw material medicines and preparation processes of the cefepime hydrochloride for injection and further improvement of quality standards.

Description

[0001] This application claims the priority of a Chinese patent application filed with the China Patent Office on September 15, 2023, with application number 2023111965178 and invention name “A method for detecting the content of nitrosamine impurities NMBA in water-soluble drugs”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The invention belongs to the technical field of drug analysis, and specifically relates to a method for detecting the content of nitrosamine impurity N-nitroso-N-methyl-4-aminobutyric acid (hereinafter referred to as NMBA) in cefepime hydrochloride for injection. Background Art

[0003] NMBA is a nitrosamine compound, which belongs to the "cohort of concern" mentioned in the ICH M7 (R2) (Evaluation and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk) guideline. It is also a Class 2A carcinogen announced by the WHO, with a limit of 96ng / day, which is much smaller than the TTC limit of common genotoxic impurities (1.5μg / day). Common small molecule nitrosamine compounds (such as NDMA and NDEA, etc.) are generally detected by gas chromatography-mass spectrometry, but because NMBA contains a butyric acid group and has a high boiling point, it can only be controlled by liquid chromatography-mass spectrometry.

[0004] Major drug regulatory agencies have published some detection methods for the content of impurity NMBA in sartan drugs, such as USP General Chapter 1469 "NITROSAMINE IMPURITIES" and EP 2.5.42. "N-NITROSAMINES IN ACTIVE SUBSTANCES", etc. However, their LC-MS methods have certain defects (NMBA has double peaks and integration is difficult), and for some drugs with large daily doses, the limit of impurities will be extremely low, so the sensitivity of their methods is difficult to meet the detection requirements. For example, in cefepime hydrochloride for injection, the limit requirement for impurity NMBA is 8.4 ppb. Therefore, it is necessary to establish a detection method that can accurately detect the content of ppb-level NMBA impurities in cefepime hydrochloride for injection, so as to ensure the safety of medication. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for detecting the content of nitrosamine impurities NMBA in cefepime hydrochloride for injection, and the specific technical scheme is as follows:

[0006] (1) Instrument

[0007] High performance liquid chromatography-triple quadrupole mass spectrometry.

[0008] (2) Mass spectrometry parameters

[0009] Ionization was performed in positive ion electrospray mode and measurement was performed in multiple reaction monitoring mode. The collision gas pressure was 8 Psi, the nebulizer gas pressure was 50 Psi, the spray voltage was 5500 V, the curtain gas pressure was 35 Psi, the auxiliary gas pressure was 35 Psi, and the ion source temperature was 500 °C.

[0010] The ion pairs detected are shown in the table below:

[0011]

[0012] (3) Chromatographic conditions

[0013] Chromatographic column: hydrophilic bonded silica gel chromatographic column; chromatographic column particle size is 2-5 μm, preferably 5 μm; chromatographic column length is 50-250 mm, preferably 150 mm; chromatographic column inner diameter is 2.1-4.6 mm, preferably 2.1 mm. The preferred chromatographic column model is Waters Atlantis HILIC (2.1×150 mm, 5 μm).

[0014] The column temperature is 35°C to 45°C, preferably 40°C.

[0015] The flow rate is 0.3 ml / min to 0.5 ml / min, preferably 0.4 ml / min.

[0016] The injection volume is 10 μl to 30 μl, preferably 20 μl.

[0017] The mobile phase A is a mixed solution of water and acetonitrile, containing 80% to 90% acetonitrile, preferably 85% acetonitrile; and containing 2 to 6 mmol / L ammonium formate, preferably 4 mmol / L ammonium formate.

[0018] The mobile phase B is a mixed solution of water and acetonitrile, containing 40% to 60% acetonitrile, preferably 50% acetonitrile; and 0.05% to 0.15% formic acid, preferably 0.1% formic acid.

[0019] Gradient elution program:

[0020] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 3 100 0 3.5 0 100 10 0 100 10.5 100 0 15 100 0

[0021] (4) Solution preparation

[0022] The solvent is acetonitrile. A certain concentration of inorganic salt solution is prepared, wherein the inorganic salt is preferably sodium chloride, and the concentration of the sodium chloride solution is 0.25 g / ml to 0.35 g / ml, preferably 0.30 g / ml.

[0023] Reference solution: Take an appropriate amount of impurity NMBA reference substance, weigh accurately, dissolve it in water and dilute it with acetonitrile to make a solution containing about 0.2 ng per 1 ml as the reference solution.

[0024] Test solution: Take about 240 mg of cefepime hydrochloride for injection, accurately weigh it, put it in a 15 ml centrifuge tube, add 1 ml of sodium chloride solution and shake it to dissolve it, then add 10 ml of acetonitrile, shake it to mix it evenly, centrifuge it at 4000 rpm for 5 minutes, and take the acetonitrile layer as the test solution.

[0025] (5) Solution detection

[0026] Accurately measure 20 μl of the reference solution and inject it into the HPLC-triple quadrupole mass spectrometer, record the ion flow diagram, and the relative standard deviation of the peak area of ​​the reference solution injected for 6 consecutive injections shall not exceed 10%. Accurately measure 20 μl of the test solution and inject it into the HPLC-triple quadrupole mass spectrometer, record the ion flow diagram.

[0027] (6) Result calculation

[0028]

[0029] In the formula:

[0030] A 样 is the peak area of ​​impurity NMBA in the test solution,

[0031] C 样 is the concentration of the drug in the test solution (mg / ml),

[0032] A 对 is the peak area of ​​impurity NMBA in the reference solution,

[0033] C 对 is the concentration of impurity NMBA in the reference solution (pg / ml).

[0034] Compared with the legal method, the present invention has the following important distinguishing features and beneficial improvements:

[0035] (1) Impurity NMNA has cis and trans isomers, which produce double peaks under the liquid phase conditions of USP and EP regulations, which is not conducive to integral quantitative analysis. However, under the conditions of the present invention, NMBA is a normal single peak.

[0036] (2) In the USP and EP regulatory methods, the minimum limit of quantification of NMBA is 1 ng / ml, while the present invention can reduce the limit of quantification to 19.63 pg / ml, see Table 4 of Experimental Example (3).

[0037] (3) In the USP and EP regulatory methods, the sample solution is simply dissolved and then sampled for analysis. Such a high concentration of the sample solution will not only cause greater detection interference and poor method recovery, but also contaminate the instrument after long-term use. The present invention uses acetonitrile to extract NMBA in the saturated sodium chloride layer, leaving a large amount of the sample in the saturated sodium chloride layer, which greatly improves the sensitivity and accuracy of the method.

[0038] (4) The detection method provided by the present invention has higher sensitivity than all methods published at the time of submission of the present invention, has strong specificity, and is fast in analysis. It can accurately determine extremely low levels of the impurity NMBA in cefepime hydrochloride for injection, and can provide a forward-looking method for the improvement and innovation of the raw material and preparation process of cefepime hydrochloride for injection, as well as the further improvement of quality standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Spectrum of impurity NMBA reference substance solution in Example 1

[0040] Figure 2 : Detection spectrum of impurity NMBA in the test solution in Example 1

[0041] Figure 3 : Detection spectrum of impurity NMBA in the spiked test solution in Example 1

[0042] Figure 4 : Spectrum of impurity NMBA reference substance solution in Comparative Example 1

[0043] Figure 5 : Spectrum of impurity NMBA reference substance solution in Comparative Example 2

[0044] Figure 6 : Spectrum of impurity NMBA reference substance solution in Comparative Example 3

[0045] Figure 7 : Sensitivity spectrum of impurity NMBA solution in Experimental Example (1) DETAILED DESCRIPTION

[0046] The present invention is further described below through specific embodiments and in conjunction with the accompanying drawings. It should be understood that the following embodiments are only for further illustrating the features and advantages of the present invention, and that a person of ordinary skill in the art may make several modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0047] Example 1

[0048] A method for detecting impurity NMBA in cefepime hydrochloride for injection, the method is as follows:

[0049] (1) Liquid phase test conditions include: mobile phase A is water-acetonitrile (15:85) (containing 4 mmol / L ammonium formate), mobile phase B is water-acetonitrile (50:50) (containing 0.1% formic acid), the chromatographic column is a hydrophilic bonded silica column (Waters Atlantis HILIC, 2.1×150 mm, 5 μm), the column temperature is 40°C, the flow rate is 0.4 ml / min, the injection volume is 20 μl, and the gradient elution program is as follows:

[0050] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 3 100 0 3.5 0 100 10 0 100 10.5 100 0 15 100 0

[0051] (2) Mass spectrometry test conditions are as follows: positive ion electrospray (ESI+) mode ionization, multiple reaction monitoring (MRM) mode. Collision gas pressure is 8Psi, nebulizer gas pressure is 50Psi, spray voltage is 5500V, curtain gas pressure is 35Psi, auxiliary gas pressure is 35Psi, ion source temperature is 500℃, and the ion pair information of impurity mass spectrometry detection is as follows:

[0052]

[0053] (3) Solution preparation

[0054] Weigh about 15 g of sodium chloride, place it in a 50 ml volumetric flask, add water and shake to dissolve and dilute to the scale, shake well, and use this as the sodium chloride solution.

[0055] Take about 20 mg of the impurity NMBA reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add water, shake to dissolve and dilute to the scale, shake well; further accurately measure 1 ml, place it in a 100 ml volumetric flask, dilute to the scale with water, shake well; further accurately measure 1 ml, place it in a 100 ml volumetric flask, dilute to the scale with water, shake well, as the reference substance stock solution; further accurately measure 1 ml of the reference substance stock solution, place it in a 100 ml volumetric flask, dilute to the scale with acetonitrile, shake well, as the reference substance solution.

[0056] Accurately measure 1 ml of the reference solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well to prepare the sensitivity solution.

[0057] Take about 240 mg of cefepime hydrochloride for injection, accurately weigh it, put it in a 15 ml centrifuge tube, add 1 ml of sodium chloride solution and shake it to dissolve it, then add 10 ml of acetonitrile, shake it to mix it evenly, centrifuge it at 4000 rpm for 5 minutes, and take the acetonitrile layer as the test solution.

[0058] Take about 240 mg of cefepime hydrochloride for injection, weigh accurately, put it in a 15 ml centrifuge tube, add 1 ml of sodium chloride solution and shake to dissolve, then add 10 ml of reference solution, shake to mix evenly, centrifuge at 4000 rpm for 5 minutes, and take the acetonitrile layer as the spiked test solution.

[0059] (4) Solution detection

[0060] Accurately measure 20 μl of acetonitrile and inject it into the liquid-mass spectrometer, record the ion current graph, and determine the blank background.

[0061] Accurately measure 20 μl of the sensitivity solution and inject it into the liquid chromatography-mass spectrometer. Record the ion current graph. The signal-to-noise ratio (S / N) of the impurity peak must not be less than 10.

[0062] Accurately measure 20 μl of the reference solution and inject it into the liquid-mass spectrometer. Record the ion current graph. The relative standard deviation of the reference peak area for 6 consecutive injections shall not exceed 10%.

[0063] Accurately measure 20 μl of acetonitrile and inject it into the liquid-mass spectrometer, record the ion chromatogram, and confirm that there is no residue in the instrument.

[0064] Accurately measure 20 μl of the test solution and inject it into the liquid-mass spectrometer to record the ion current. Calculate the content of impurity NMBA using the external standard method.

[0065] Results: The spectrum of the impurity NMBA reference solution in Example 1 is shown in Figure 1 , the sample solution spectrum is shown in Figure 2 , the spectrum of the spiked test solution is shown in Figure 3 The method has high sensitivity and strong specificity and can achieve the expected detection effect. The specific methodological verification and results can be seen in the experimental examples.

[0066] Comparative Example 1

[0067] A method for detecting impurity NMBA in cefepime hydrochloride for injection, the specific steps are the same as those in Example 1, except that mobile phase A is changed to water-acetonitrile (15:85) containing 0.1% formic acid.

[0068] Results: In Comparative Example 1, except that the mobile phase A is different from that in Example 1, the other test conditions are the same as those in Example 1. The spectrum of the impurity NMBA reference solution in Comparative Example 1 is shown in Figure 4 .Depend on Figure 4 It can be seen that when the reference solution of the same concentration is detected, the peak shape of the impurity NMBA under the method of Comparative Example 1 is significantly worse than that of Example 1. Figure 1 , the noise is also quite loud.

[0069] Comparative Example 2

[0070] A method for detecting impurity NMBA in drugs (refer to USP general chapter) <1469> The analytical method 3) is different from that in Example 1 in that:

[0071] (1) The liquid phase test conditions were modified as follows: mobile phase A was 0.1% formic acid solution, mobile phase B was 0.1% formic acid methanol solution, the chromatographic column was an octadecylsilane bonded silica column (Agilent Poroshell 120EC-C18, 3.0 mm × 150 mm, 2.7 μm), the column temperature was 60 °C, the flow rate was 0.5 ml / min, the injection volume was 20 μl, the sample chamber temperature was 18 °C, and the gradient elution program was as follows:

[0072] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 97 3 1.5 97 3 4.0 50 50 7.0 25 75 8.1 15 85 9.2 5 95 12.0 5 95 12.1 97 3 16 97 3

[0073] (2) The solvent used in the preparation of the reference solution was changed to 1% formic acid water.

[0074] (3) The concentration of the reference solution is about 2 ng / ml (about 0.2 ng / ml in Example 1).

[0075] (4) The mass spectrometer ion source was replaced with APCI source.

[0076] Results: Compared with Example 1, the liquid phase conditions, ion source and solvent used in the reference solution were changed in Comparative Example 2, and the Hilic method was changed to the C18 method. The spectrum of the impurity NMBA reference solution in Comparative Example 2 is shown in FIG. Figure 5 .Depend on Figure 5 It can be seen that the signal-to-noise ratio of the impurity NMBA is not as good as that of Example 1 when the reference solution with a concentration 10 times higher is detected. Figure 1 , the peak shape is also split into two peaks.

[0077] Comparative Example 3

[0078] A method for detecting impurity NMBA in a drug (refer to EP 2.5.42 analytical method A), compared with Example 1, the differences are: (1) the liquid phase test conditions are modified as follows: mobile phase A is 0.1% formic acid solution, mobile phase B is methanol, the chromatographic column is an octadecylsilane bonded silica gel chromatographic column (Agilent Zorbax Eclipse Plus C18, 4.6 mm×150 mm, 3.5 μm), the column temperature is 40°C, the flow rate is 0.5 ml / min, the injection volume is 20 μl, the sample chamber temperature is 5°C, and the gradient elution program is as follows:

[0079] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 1 80 20 6 55 45 14 55 45 16 5 95 35 5 95 35.1 80 20 45 80 20

[0080] (2) The solvent used in the preparation of the reference solution was changed to methanol.

[0081] (3) The concentration of the reference solution is about 2 ng / ml (about 0.2 ng / ml in Example 1).

[0082] (4) The mass spectrometer ion source was replaced with APCI source.

[0083] Results: Compared with Example 1, the liquid phase conditions, ion source and solvent used in the reference solution were changed in Comparative Example 2, and the Hilic method was changed to the C18 method. The spectrum of the impurity NMBA reference solution in Comparative Example 3 is shown in FIG. Figure 6 .Depend on Figure 6 It can be seen that the signal-to-noise ratio of the impurity NMBA is not as good as that of Example 1 when the reference solution with a concentration 10 times higher is detected. Figure 1 , the peak is front and split into two peaks.

[0084] Comparative Example 4

[0085] A method for detecting impurity NMBA in a drug, wherein the specific steps are the same as those in Example 1, except that the saturated sodium chloride solution is replaced with pure water.

[0086] Results: When the solvent of the test solution was replaced with pure water, the recovery rate of impurity NMBA determined by the method in Experimental Example (4) Method Accuracy and Method Precision was only about 30%. The recovery rate of impurity NMBA in Example 1 was between 70% and 80%, as shown in Table 5 of Experimental Example (4), which was significantly better than that in Comparative Example 3.

[0087] Experimental example

[0088] The detection method of Example 1 was methodologically validated according to high performance liquid chromatography (General Rules 0512 of Part IV of the Chinese Pharmacopoeia 2020 Edition)-mass spectrometry (General Rules 0431 of Part IV of the Chinese Pharmacopoeia 2020 Edition) and the guidelines for validation of analytical methods (Guidelines 9101 of Part IV of the Chinese Pharmacopoeia 2020 Edition), and the preparation methods of each solution and the liquid quality test conditions were the same as those in Example 1.

[0089] (1) System precision and system applicability

[0090] Accurately measure 20 μl of acetonitrile, sensitivity solution, and reference solution, respectively, and inject them into the LC-MS instrument to record the ion chromatogram.

[0091] Table 1 Impurity NMBA system precision test results

[0092]

[0093] Table 2 Impurity NMBA sensitivity test results

[0094] Impurities Blank noise Sensitivity solution peak height Signal-to-Noise Ratio (S / N) NMBA 35 499 14.3

[0095] As can be seen from Table 1 and Table 2, the impurity NMBA peak area RSD = 4.9%, the retention time RSD = 0.4%, and the signal-to-noise ratio of the impurity NMBA in the sensitivity solution is 14.3. The impurity NMBA sensitivity solution spectrum in the experimental example is shown in Figure 7 . The system precision and system applicability meet the requirements.

[0096] (2) Linear range

[0097] Accurately weigh 21.87 mg of impurity NMBA reference substance, place it in a 100ml volumetric flask, add water, shake to dissolve and dilute to the scale, shake well; accurately measure 1 ml, place it in a 100ml volumetric flask, dilute to the scale with water, shake well; accurately measure 1 ml, place it in a 100ml volumetric flask, dilute to the scale with water, shake well, as the reference substance stock solution; accurately measure 5 ml, place it in a 50ml volumetric flask, dilute to the scale with diluent, shake well, as the linear stock solution; accurately measure 1 ml, 3 ml, 5 ml, 5 ml, 15 ml, 5 ml of the linear stock solution, place them in 100 ml, 100 ml, 100 ml, 50 ml, 100 ml, 25 ml volumetric flasks respectively, dilute to the scale with diluent, shake well, as 10%, 30%, 50%, 100%, 150%, 200% linear determination solutions.

[0098] Accurately measure 20 μl of each linear solution and inject it into the liquid-mass spectrometer respectively, record the ion current diagram, make a regression curve based on the impurity concentration and the impurity peak area in the corresponding ion current diagram, and calculate the regression equation and the corresponding linear regression coefficient.

[0099] Table 3 Impurity NMBA linearity test results

[0100]

[0101] As shown in Table 3, the linear equation of the impurity NMBA is y=123x+947, r=0.9907 within the concentration range of 21.43 pg / ml to 428.66 pg / ml, which shows a good linear relationship.

[0102] (3) Limit of quantification and detection limit

[0103] Take acetonitrile as blank solvent, accurately measure 20 μl, inject three times in succession, record the instrument noise level within the peak range of five times half the peak width of the main peak of the analyte, and calculate the average noise.

[0104] Quantitation limit solution: Take 10% of the linear determination solution, which is the quantitation limit solution.

[0105] Detection limit solution: Take 3 ml of the quantitative limit solution and place it in a 10 ml volumetric flask. Dilute it to the scale with diluent and shake well. This is the detection limit solution.

[0106] Accurately measure 20 μl of acetonitrile, quantitative limit solution, and detection limit solution, respectively, and inject them into the LC-MS instrument to record the ion chromatogram.

[0107] Table 4 Impurity NMBA quantitative limit and detection limit test results

[0108]

[0109] As shown in Table 4, the limit of quantification of impurity NMBA is 0.82ppb, the signal-to-noise ratio is 10.8, and the peak height RSD is 3.3%. The limit of detection of impurity NMBA is 0.25ppb, and the signal-to-noise ratio is 3.4. Both the limit of quantification and the limit of detection meet the requirements.

[0110] (4) Method accuracy and method precision

[0111] Accurately measure 20 μl of the reference solution, test solution, and spiked test solution, respectively, and inject them into the LC-MS instrument to record the peak area.

[0112] Table 5 Impurity NMBA method accuracy and method precision test results

[0113]

[0114] As shown in Table 5, the recovery of impurity NMBA was between 70% and 80%, and the RSD was 3.7%. The accuracy and precision of the method were good.

[0115] It can be seen from the above embodiments, comparative examples and experimental examples that the detection method provided by the present invention has higher sensitivity than all the methods published when the present invention was submitted, has strong specificity and rapid analysis, and can accurately determine the extremely low content of impurity NMBA in cefepime hydrochloride for injection. It can provide a forward-looking method for the improvement and innovation of the raw material and preparation process of cefepime hydrochloride for injection, as well as the further improvement of quality standards.

Claims

1. A method for determining the content of N-nitroso-N-methyl-4-aminobutyric acid in cefepime hydrochloride for injection, characterized in that: (1) Preparation of the test solution: Dissolve cefepime hydrochloride for injection in sodium chloride solution, then add acetonitrile, centrifuge and take the acetonitrile layer as the test solution; (2) Instruments and chromatographic conditions: The instrument used was a high performance liquid chromatography-triple quadrupole mass spectrometer, the liquid chromatography column used hydrophilic bonded silica gel as the stationary phase, mobile phase A was an acetonitrile-water mixed solution containing ammonium formate and mobile phase B was an acetonitrile-water mixed solution containing formic acid, and gradient elution was adopted; the mass spectrometer used positive ion electrospray mode ionization and multiple reaction monitoring mode for determination; (3) Detection of the test solution: The test solution is injected into the high performance liquid chromatography-triple quadrupole mass spectrometer to complete the determination of the content of NMBA in cefepime hydrochloride for injection.

2. The method according to claim 1, characterized in that: The concentration of the sodium chloride solution is 0.25 g / ml to 0.35 g / ml, preferably 0.30 g / ml.

3. The method according to claim 1, characterized in that: The HPLC column is a hydrophilic bonded silica gel column Waters Atlantis HILIC, with a particle size of 2-5 μm, a length of 50-250 mm, and an inner diameter of 2.1-4.6 mm, preferably a particle size of 5 μm, a length of 150 mm, and an inner diameter of 2.1 mm.

4. The method according to claim 1, characterized in that: The mobile phase A contains an acetonitrile-water mixed solution of ammonium formate, the concentration of ammonium formate is 2-6 mmol / L, preferably 4 mmol / L; the concentration of acetonitrile is 80%-90%, preferably 85%.

5. The method according to claim 1, characterized in that: The concentration of formic acid in the acetonitrile-water mixed solution containing formic acid in the mobile phase B is 0.05% to 0.15%, preferably 0.1%.

6. The method according to claim 1, characterized in that: The concentration of acetonitrile in the acetonitrile-water mixed solution containing formic acid in the mobile phase B is 40% to 60%, preferably 50%.

7. The method according to claim 1, characterized in that: The gradient elution is performed as follows: 。 8. The method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.3 ml / min to 0.5 ml / min, preferably 0.4 ml / min.

9. The method according to claim 1, characterized in that: The column temperature of the liquid chromatography column is 35° C. to 45° C., preferably 40° C.; the injection volume of the test solution is 10 μl to 30 μl, preferably 20 μl.

10. The method according to claim 1, characterized in that: The mass spectrometer collision gas pressure is 8 Psi, the nebulizer gas pressure is 50 Psi, the spray voltage is 5500 V, the curtain gas pressure is 35 Psi, the auxiliary gas pressure is 35 Psi, and the ion source temperature is 500°C.

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