Method for detecting N-nitrosomeropenem impurity in meropenem
Through the LC-MS method and optimized chromatographic conditions, the problem of difficulty in detecting meropenem impurities N-nitrosotmeropenem in the existing technology is solved, and high sensitivity and high accuracy detection is achieved, satisfying the strictness of meropenem impurities Testing requirements.
Patent Information
- Application Number
- CN202411833833.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-13
- Publication Date
- 2025-01-17
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to effectively detect the meropenem impurity N-nitrosomeropenem, and its structure is similar to that of meropenem, resulting in difficulty in separation and low recovery rate, and lack of high sensitivity detection methods.
Using LC-MS method, using Waters Atlantis HILIC Silica liquid chromatography column, optimize mobile phase and gradient elution conditions, combine negative ion electrospray and multi-reacting ion monitoring mode to determine specific mass spectrometry detection parameters, and realize N-nitrosomei Effective separation and detection of Ropenem and Meropenem.
It realizes detection of high sensitivity, accuracy and precision, and can effectively separate and detect N-nitrosomeropenem impurities, meets strict detection requirements, has good linear relationships and strong applicability.
Smart Images
Figure CN119322133A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of pharmaceutical analytical chemistry, and specifically relates to a method for detecting a meropenem impurity N-nitrosomeropenem. Background Art
[0002] Meropenem is a synthetic broad-spectrum carbapenem antibiotic that produces antibacterial effects by inhibiting the synthesis of bacterial cell walls. Meropenem can easily penetrate the cell walls of most Gram-positive and Gram-negative bacteria and reach its target penicillin-binding protein (PBPS).
[0003] N-nitroso meropenem is produced in the synthesis step of meropenem, and the main process is as follows: protecting meropenem and removing the protecting group through catalytic hydrogenation to generate meropenem. After the protecting group protecting the amino group on the pyrrolidine ring of meropenem is removed, a secondary amino structure is generated. When the secondary amino structure encounters a nitrosating agent (nitrosating agents are introduced into the materials used in the preparation process of meropenem), it is easy to generate nitrosamines, namely N-nitroso meropenem.
[0004]
[0005] Nitrosamines are strong carcinogens, one of the most important chemical carcinogens, and one of the four major food contaminants. Nitrosamines are found in food, cosmetics, beer, and cigarettes. Smoked foods contain a large amount of nitrosamines. The incidence of certain digestive system tumors, such as esophageal cancer, is related to the amount of nitrosamines ingested in the diet. When smoked foods are consumed together with alcohol, the harm of nitrosamines to human health will increase exponentially.
[0006] As a type of nitrosamine impurity, N-nitrosomeropenem is also highly carcinogenic. As a broad-spectrum carbapenem antibiotic, the residue control requirements of N-nitrosomeropenem are very strict. Therefore, it is necessary to develop a highly specific method to accurately detect this impurity.
[0007] Since the FDA (U.S. Food and Drug Administration) proposed NDSRI (nitrosamines of drug-related impurities), research on NDSRI has become popular in the market. However, the characteristic of NDSRI is its low limit. The structure of N-nitrosomeropenem impurity is similar to that of meropenem, with only one nitroso group difference, which makes separation extremely difficult and the recovery rate is low. At present, there is no report on the detection of this impurity in the existing technology. Summary of the invention
[0008] In view of the deficiencies in the prior art, the present invention provides a method for detecting N-nitrosomeropenem, a meropenem impurity. The method of the present invention can effectively separate N-nitrosomeropenem impurities from meropenem, effectively detect N-nitrosomeropenem, a meropenem impurity, has strong specificity and applicability, high detection sensitivity, good linear relationship, and high precision and accuracy.
[0009] The technical solution of the present invention is as follows: A method for detecting N-nitrosomeropenem, an impurity of meropenem, using LC-MS method, and the chromatographic conditions are: Chromatographic column: Waters Atlantis HILIC Silica liquid chromatography column (150mm×4.6mm, 5μm); mobile phase A: 0.04% to 0.06% ammonia water by volume, mobile phase B: 0.05% to 0.2% formic acid acetonitrile by volume; Gradient elution conditions: at 0 minutes, the volume proportion of mobile phase A was 15-25%, and the volume proportion of mobile phase B was 75-85%; at 4 minutes, the volume proportion of mobile phase A was 15-25%, and the volume proportion of mobile phase B was 75-85%; at 4.1 minutes, the volume proportion of mobile phase A was 50%, and the volume proportion of mobile phase B was 50%; at 10 minutes, the volume proportion of mobile phase A was 50%, and the volume proportion of mobile phase B was 50%; at 10.1 minutes, the volume proportion of mobile phase A was 15-25%, and the volume proportion of mobile phase B was 75-85%; at 15 minutes, the volume proportion of mobile phase A was 15-25%, and the volume proportion of mobile phase B was 75-85%.
[0010] According to the preferred embodiment of the present invention, mobile phase A is 0.05% ammonia water by volume, and mobile phase B is 0.1% formic acid acetonitrile by volume. In the formic acid acetonitrile, formic acid is used as a solute, acetonitrile is used as a solvent, and the volume concentration of formic acid is 0.1%.
[0011] Preferably, according to the present invention, the gradient elution conditions are: at 0 minutes, the volume proportion of mobile phase A is 20%, and the volume proportion of mobile phase B is 80%; at 4 minutes, the volume proportion of mobile phase A is 20%, and the volume proportion of mobile phase B is 80%; at 4.1 minutes, the volume proportion of mobile phase A is 50%, and the volume proportion of mobile phase B is 50%; at 10 minutes, the volume proportion of mobile phase A is 50%, and the volume proportion of mobile phase B is 50%; at 10.1 minutes, the volume proportion of mobile phase A is 20%, and the volume proportion of mobile phase B is 80%; at 15 minutes, the volume proportion of mobile phase A is 20%, and the volume proportion of mobile phase B is 80%.
[0012] Preferably according to the present invention, the chromatographic conditions also include: injection volume: 2 μl; column temperature: 40°C; flow rate: 0.8 ml / min; analysis time: 15 min; ionization mode: negative ion electrospray ionization (AJS ESI-), multiple reaction ion monitoring (MRM) mode; full scan mode; curtain gas pressure (CUR): 45 psi; collision gas (CAD): Medium; ionization voltage (IS): 4500 V; ion source temperature (TEM): 550°C; spray gas pressure (GS1): 60 psi; auxiliary heating gas pressure (GS2): 60 psi.
[0013] According to the preferred mass spectrometry detection parameters of the present invention are: parent ion: 411m / Z; daughter ion: 198m / Z; declustering voltage (DP): -30V; entrance voltage (EP): -12V; dissociation energy (CE): -16.4eV; exit voltage (CXP): -17.9V.
[0014] Preferably according to the present invention, acetonitrile and water are used as solvents to prepare the test solution, wherein the volume ratio of acetonitrile to water is 4:1.
[0015] The technical features and beneficial effects of the present invention are as follows: 1. The impurity N-nitrosomeropenem has a similar structure to meropenem, differing only by one nitroso group, and has poor separation. Meropenem has a large polarity and a weak retention time. The water-resistant C18 column was used but separation could not be achieved, and the impurity peak shape was poor. Finally, the HILIC column of the present invention was selected to achieve the separation effect.
[0016] 2. In order to obtain better peak shape and response value, the mobile phase and elution gradient conditions were optimized. Formic acid water and ammonia water were used as mobile phase A, formic acid acetonitrile and acetonitrile were used as mobile phase B respectively; during the test, under acidic conditions, the ionization of N-nitroso meropenem was affected, making the response of N-nitroso meropenem low. When alkaline ammonia water was used, the response of N-nitroso meropenem was higher. Finally, the study found that when the mobile phase A and mobile phase B of the specific concentration and specific type of the present invention were used, the response value of N-nitroso meropenem was the highest.
[0017] 3. The method of the present invention has strong specificity and applicability, high detection sensitivity, good linear relationship, high precision and accuracy. The method of the present invention can effectively separate N-nitrosomeropenem impurities from meropenem and effectively detect the meropenem impurity N-nitrosomeropenem.
[0018] 4. Determination of mass spectrometry conditions, optimization of positive and negative modes. It was found that under the commonly used positive mode, the impurity peak response was extremely small, and there was basically no response to the impurities prepared according to the limit. Finally, the detection method was developed under the ESI source negative mode to meet the detection requirements; at the same time, the full scan mode was adopted, and the mass spectrometry conditions were finally optimized: DP voltage -30V, EP voltage -12V, CE voltage -16.4eV, CXP voltage: -17.9V. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is the chromatogram of the blank test (diluent) in Example 1. The horizontal axis is time (min) and the vertical axis is intensity, cps.
[0020] Figure 2 This is the chromatogram of the reference substance in Example 1. The horizontal axis is time (min) and the vertical axis is intensity, cps.
[0021] Figure 3 This is the chromatogram of the test product in Example 1. The horizontal axis is time (min) and the vertical axis is intensity, cps.
[0022] Figure 4 It is the mass spectrum of the reference substance in Example 1. Wherein, the abscissa is m / z and the ordinate is intensity, cps.
[0023] Figure 5 It is the standard working curve in Test Example 2.
[0024] Figure 6 This is the chromatogram of the reference substance in Comparative Example 1. The horizontal axis is time (min) and the vertical axis is intensity, cps.
[0025] Figure 7 This is the chromatogram of the reference substance in Comparative Example 2. The horizontal axis is time (min) and the vertical axis is intensity, cps.
[0026] Figure 8 It is the mass spectrum of the reference substance in Comparative Example 3. Wherein, the abscissa is m / z and the ordinate is intensity, cps. DETAILED DESCRIPTION
[0027] The present invention is further described below by means of specific examples, but is not limited thereto.
[0028] Unless otherwise specified, the raw materials used in the examples are all conventional products; the methods used are all prior art unless otherwise specified.
[0029] Example 1 A method for detecting meropenem impurity N-nitrosomeropenem High performance liquid chromatography-mass spectrometry (1) Standards, reagents and instruments Standard: N-nitrosomeropenem Reagents: Acetonitrile (mass spectrometry grade, Merck); Formic acid (chromatographic grade, Sigma) Instruments: Shimadzu ultra-high performance liquid chromatograph (model: LC-20ADXR); AB mass spectrometer (model: Qtrap); Sartorius microbalance (model: MSA6.6S-0CE-DM) (2) Preparation of test solution: Weigh 1 g of meropenem sample, add 20 ml of water to dissolve it, then add acetonitrile to make up to 100 ml, dissolve it by ultrasonic, centrifuge at 10000 r / min for 10 min, and take the supernatant.
[0030] (3) Preparation of reference solution: Take an appropriate amount of N-nitrosomeropenem reference substance, weigh it accurately, dissolve it with a diluent (acetonitrile in water, the volume ratio of acetonitrile to water is 4:1) and quantitatively dilute it into a solution containing 2.2 ng N-nitrosomeropenem per 1 ml.
[0031] (4) Chromatographic conditions: Chromatographic column: Waters Atlantis HILIC Silica liquid chromatography column (150mm×4.6mm, 5μm); injection volume: 2μl; column temperature: 40℃; flow rate: 0.8ml / min; analysis time: 15min; ionization mode: negative ion electrospray ionization (AJSESI-), multiple reaction ion monitoring (MRM) mode; full scan mode; mobile phase A: 0.05% ammonia water by volume, mobile phase B: 0.1% formic acid acetonitrile by volume; curtain gas pressure (CUR): 45psi; collision gas (CAD): Medium; ionization voltage (IS): 4500V; ion source temperature (TEM): 550℃; spray gas pressure (GS1): 60psi; auxiliary heating gas pressure (GS2): 60psi.
[0032] Gradient elution conditions:
[0033] Mass spectrometry detection parameters:
[0034] (4) Determination method Accurately measure the diluent (acetonitrile in water, the volume ratio of acetonitrile to water is 4:1), reference solution, and test solution, inject them into the liquid chromatograph, and record the chromatogram.
[0035] The chromatograms of blank test (diluent), reference substance and test substance are shown in Figure 1. Figure 1 , 2 and shown in 3. Depend on Figure 3 It can be seen that the meropenem impurity N-nitrosomeropenem can be effectively separated, and this method is suitable for the detection of the meropenem impurity N-nitrosomeropenem.
[0036] The mass spectrum of the reference substance is as follows Figure 4 shown.
[0037] Test Example 1 Specificity test (1) Preparation of test solution: Weigh 1 g of meropenem sample, add 20 ml of water to dissolve it, then add acetonitrile to make up to 100 ml, dissolve it by ultrasonic, centrifuge at 10000 r / min for 10 min, and take the supernatant.
[0038] (2) Preparation of reference solution: Take an appropriate amount of N-nitrosomeropenem reference substance, weigh it accurately, dissolve it with a diluent (acetonitrile in water, the volume ratio of acetonitrile to water is 4:1) and quantitatively dilute it into a solution containing 2.2 ng N-nitrosomeropenem per 1 ml.
[0039] The chromatographic conditions were the same as in Example 1.
[0040] Accurately measure the diluent (acetonitrile in water, the volume ratio of acetonitrile to water is 4:1), reference solution, and test solution, inject them into the liquid chromatograph, and record the chromatogram.
[0041] The chromatograms of blank test (diluent), reference substance and test substance are shown in Figure 1. Figure 1 , 2 As shown in Figures 3 and 4, N-nitrosomeropenem was well separated from adjacent impurities and there was no interference in the blank, which indicated that the method had good specificity.
[0042] Test Example 2 Standard working curve, detection limit, and quantitation limit test Take an appropriate amount of N-nitrosomeropenem reference substance, weigh it accurately, dissolve it with a diluent (acetonitrile in water, the volume ratio of acetonitrile to water is 4:1) and dilute it into solutions of 0.09 ng / ml (quantitative limit concentration), 1.32 ng / ml, 1.76 ng / ml, 2.2 ng / ml, 2.64 ng / ml, 3.3 ng / ml, and 4.4 ng / ml respectively.
[0043] The above solution was accurately measured and injected into a liquid chromatograph. The chromatographic conditions were the same as those in Example 1, and the chromatogram was recorded. The results are shown in Tables 1 and Figure 5 shown.
[0044] Table 1 Standard working curve, detection limit and quantification limit data
[0045] From the experimental results in the above table, we can see that N -Nitrosomeropenem had a good linear relationship between concentration and peak area in the range of 0.09 ng / ml to 4.4 ng / ml, and the minimum detection limit of this method was 0.03 ng / mL.
[0046] Test Example 3 Precision and accuracy testing At the same time, the same personnel conducted spike recovery experiments at three concentration levels: high (120% of the reference concentration), medium (reference concentration), and low (limit of quantitation concentration). The accuracy and precision data are shown in Tables 2 and 3, respectively.
[0047] Table 2 Accuracy experimental data
[0048] Table 3 Precision experimental data
[0049] From the experimental data in the above table, we can see that N -The accuracy and recovery of nitrosomeropenem were 87.5%-115.8%, both within the range of 80%-120%, and the precision RSD was 4.7%, less than 10%, indicating that this method has good accuracy and precision and is suitable for N -Detection of nitrosomeropenem.
[0050] Comparative Example 1 A method for detecting N-nitrosomeropenem, a meropenem impurity, is as described in Example 1, except that: mobile phase A is a formic acid aqueous solution with a volume concentration of 0.1%; other steps and conditions are the same as in Example 1.
[0051] The chromatogram of the reference substance is as follows Figure 6 As shown in the figure, it can be seen that under acidic conditions, the ionization of N-nitrosomeropenem is affected, resulting in a low response of N-nitrosomeropenem.
[0052] Comparative Example 2 A method for detecting N-nitrosomeropenem, a meropenem impurity, is as described in Example 1, except that the chromatographic column is a C18 liquid chromatography column (water-resistant type) (150 mm×4.6 mm, 5 μm); the other steps and conditions are the same as in Example 1.
[0053] The chromatogram of the reference substance is as follows Figure 7 As shown in the figure, the peak width of N-nitrosomeropenem is relatively large and the peak shape is asymmetric, resulting in a low peak response, and it overlaps with meropenem and cannot be separated.
[0054] Comparative Example 3 A method for detecting the meropenem impurity N-nitrosomeropenem is as described in Example 1, except that: ionization mode: positive ion electrospray ionization (AJS ESI-), multiple reaction ion monitoring (MRM) mode; other steps and conditions are the same as in Example 1.
[0055] The mass spectrum of the reference substance is as follows Figure 8 As shown in the figure, under the commonly used positive mode, the impurity peak response is extremely small, and there is basically no response when the impurity is prepared according to the limit.
Claims
1. A method for detecting meropenem impurity N-nitrosomeropenem, characterized in that: The LC-MS method was used and the chromatographic conditions were: Chromatographic column: Waters Atlantis HILIC Silica liquid chromatography column (150mm×4.6mm, 5μm); mobile phase A: 0.04% to 0.06% ammonia water, mobile phase B: 0.05% to 0.2% formic acid acetonitrile; ionization mode: negative ion electrospray (AJS ESI-), multiple reaction ion monitoring (MRM) mode; Gradient elution conditions: at 0 minutes, the volume proportion of mobile phase A was 15-25%, and the volume proportion of mobile phase B was 75-85%; at 4 minutes, the volume proportion of mobile phase A was 15-25%, and the volume proportion of mobile phase B was 75-85%; at 4.1 minutes, the volume proportion of mobile phase A was 50%, and the volume proportion of mobile phase B was 50%; at 10 minutes, the volume proportion of mobile phase A was 50%, and the volume proportion of mobile phase B was 50%; at 10.1 minutes, the volume proportion of mobile phase A was 15-25%, and the volume proportion of mobile phase B was 75-85%; at 15 minutes, the volume proportion of mobile phase A was 15-25%, and the volume proportion of mobile phase B was 75-85%.
2. The method for detecting the meropenem impurity N-nitrosomeropenem according to claim 1, characterized in that: Mobile phase A: 0.05% ammonia water by volume, mobile phase B: 0.1% formic acid acetonitrile by volume.
3. The method for detecting the meropenem impurity N-nitrosomeropenem according to claim 1, characterized in that: Gradient elution conditions: at 0 min, the volume proportion of mobile phase A was 20%, and the volume proportion of mobile phase B was 80%; at 4 min, the volume proportion of mobile phase A was 20%, and the volume proportion of mobile phase B was 80%; at 4.1 min, the volume proportion of mobile phase A was 50%, and the volume proportion of mobile phase B was 50%; at 10 min, the volume proportion of mobile phase A was 50%, and the volume proportion of mobile phase B was 50%; at 10.1 min, the volume proportion of mobile phase A was 20%, and the volume proportion of mobile phase B was 80%; at 15 min, the volume proportion of mobile phase A was 20%, and the volume proportion of mobile phase B was 80%.
4. The method for detecting the meropenem impurity N-nitrosomeropenem according to claim 1, characterized in that: The chromatographic conditions also include: injection volume: 2 μl; column temperature: 40°C; flow rate: 0.8 ml / min; analysis time: 15 min; full scan mode; curtain gas pressure (CUR): 45 psi; collision gas (CAD): Medium; ionization voltage (IS): 4500 V; ion source temperature (TEM): 550°C; spray gas pressure (GS1): 60 psi; auxiliary heating gas pressure (GS2): 60 psi.
5. The method for detecting the meropenem impurity N-nitrosomeropenem according to claim 1, characterized in that: Mass spectrometry detection parameters: parent ion: 411m / Z; daughter ion: 198m / Z; declustering voltage (DP): -30V; entrance voltage (EP): -12V; dissociation energy (CE): -16.4eV; exit voltage (CXP): -17.9V.
6. The method for detecting the meropenem impurity N-nitrosomeropenem according to claim 1, characterized in that: Acetonitrile and water were used as solvents to prepare the test solution, wherein the volume ratio of acetonitrile to water was 4:1.