Method for detecting N-nitrosomeropenem impurity in meropenem

Abstract

The invention provides a method for detecting N-nitrosomeropenem as an impurity in meropenem, and belongs to the field of pharmaceutical analytical chemistry. According to the detection method, an LC-MS method is adopted, and the chromatographic conditions are as follows: a Waters Atlants HILIC Silica liquid chromatographic column (150mm * 4.6 mm, 5 [mu] m) is adopted as a chromatographic column; a mobile phase A is ammonia water with the volume concentration of 0.04%-0.06%, and a mobile phase B is formic acid acetonitrile with the volume concentration of 0.05%-0.2%; and gradient elution. According to the method, the N-nitrosomeropenem impurity and meropenem can be effectively separated, the meropenem impurity N-nitrosomeropenem can be effectively detected, and the method is high in specificity and applicability, high in detection sensitivity, good in linear relation and high in precision and accuracy.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analytical chemistry, and particularly relates to a method for detecting the impurity N-nitrosomeropenem of meropenem. Background Art

[0002] Meropenem is a synthetic broad-spectrum carbapenem antibiotic that exerts 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 to reach its target penicillin-binding proteins (PBPs).

[0003] N-nitrosomeropenem is produced in the synthesis process of meropenem, and the main process is as follows: Protected meropenem generates meropenem by catalytic hydrogenation to remove the protecting group. After the protecting group of the amino group on the pyrrolidine ring of protected meropenem is removed, a secondary amine structure is formed. When this secondary amine structure encounters a nitrosating agent (which will be introduced in the materials used in the preparation process of meropenem), it is likely to form a nitrosamine, that is, N-nitrosomeropenem.

[0004]

[0005] Nitrosamines are strong carcinogens, one of the most important chemical carcinogens and one of the four major food contaminants. Nitrosamines are contained in food, cosmetics, beer, and cigarettes. In smoked and cured foods, there are a large number of nitrosamine substances. The incidence of certain digestive system tumors, such as esophageal cancer, is related to the amount of nitrosamines ingested in the diet. When smoked and cured foods are consumed together with alcohol, the harm of nitrosamines to human health will increase exponentially.

[0006] As a kind of nitrosamine impurity, N-nitrosomeropenem also has strong carcinogenicity. For meropenem, a broad-spectrum carbapenem antibiotic, the residual control requirements for N-nitrosomeropenem are very strict. Therefore, it is necessary to develop a highly specific method for accurate detection of this impurity.

[0007] Since the FDA (U.S. Food and Drug Administration) proposed NDSRI (Nitrosamines in Drug Substance Related Impurities), the research on NDSRI in the market has become very popular. However, the characteristic of NDSRI is its low limit. The impurity structure of N-nitrosomeropenem is similar to that of meropenem, differing only by a nitroso group, resulting in extremely difficult separation and low recovery rate. Currently, 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 the impurity N-nitrosomeropenem of meropenem. The method of the present invention can effectively separate the impurity N-nitrosomeropenem from meropenem, effectively detect the impurity N-nitrosomeropenem of meropenem, has strong specificity and applicability, high detection sensitivity, good linear relationship, high precision and accuracy.

[0009] The technical solution of the present invention is as follows:

[0010] A method for detecting the impurity N-nitrosomeropenem of meropenem, using the LC-MS method, and the chromatographic conditions are as follows:

[0011] Chromatographic column: Waters Atlantis HILIC Silica liquid chromatography column (150 mm × 4.6 mm, 5 μm); mobile phase A: ammonia water with a volume concentration of 0.04% - 0.06%; mobile phase B: acetonitrile formate with a volume concentration of 0.05% - 0.2%.

[0012] Gradient elution conditions: At 0 minute, the volume ratio of mobile phase A is 15 - 25%, and the volume ratio of mobile phase B is 75 - 85%; at 4 minutes, the volume ratio of mobile phase A is 15 - 25%, and the volume ratio of mobile phase B is 75 - 85%; at 4.1 minutes, the volume ratio of mobile phase A is 50%, and the volume ratio of mobile phase B is 50%; at 10 minutes, the volume ratio of mobile phase A is 50%, and the volume ratio of mobile phase B is 50%; at 10.1 minutes, the volume ratio of mobile phase A is 15 - 25%, and the volume ratio of mobile phase B is 75 - 85%; at 15 minutes, the volume ratio of mobile phase A is 15 - 25%, and the volume ratio of mobile phase B is 75 - 85%.

[0013] According to the preference of the present invention, mobile phase A: ammonia water with a volume concentration of 0.05%; mobile phase B: acetonitrile formate with a volume concentration of 0.1%. Among them, in acetonitrile formate, formic acid is the solute and acetonitrile is the solvent, and the volume concentration of formic acid is 0.1%.

[0014] According to the preference of the present invention, gradient elution conditions: At 0 minute, the volume ratio of mobile phase A is 20%, and the volume ratio of mobile phase B is 80%; at 4 minutes, the volume ratio of mobile phase A is 20%, and the volume ratio of mobile phase B is 80%; at 4.1 minutes, the volume ratio of mobile phase A is 50%, and the volume ratio of mobile phase B is 50%; at 10 minutes, the volume ratio of mobile phase A is 50%, and the volume ratio of mobile phase B is 50%; at 10.1 minutes, the volume ratio of mobile phase A is 20%, and the volume ratio of mobile phase B is 80%; at 15 minutes, the volume ratio of mobile phase A is 20%, and the volume ratio of mobile phase B is 80%.

[0015] Preferably according to the present invention, the chromatographic conditions further include: injection volume: 2 μl; column temperature: 40 °C; flow rate: 0.8 ml / min; analysis time: 15 min; ionization mode: negative ion electrospray (AJS ESI-), multiple reaction 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; nebulizing gas pressure (GS1): 60 psi; auxiliary heating gas pressure (GS2): 60 psi.

[0016] Preferably according to the present invention, the mass spectrometry detection parameters are as follows: parent ion: 411 m / Z; daughter ion: 198 m / Z; declustering potential (DP): -30 V; entrance potential (EP): -12 V; collision energy (CE): -16.4 eV; exit potential (CXP): -17.9 V.

[0017] Preferably according to the present invention, acetonitrile and water are used as solvents to prepare the test solution, and the volume ratio of acetonitrile to water is 4:1.

[0018] The technical features and beneficial effects of the present invention are as follows:

[0019] 1. The impurity N-nitrosomeropenem is structurally similar to meropenem, differing only by a nitroso group, with poor resolution. Meropenem has a large polarity and a weak retention time. When using a water-resistant C18 chromatographic column, the impurity peak shape is poor and the peak response is low. Finally, the HILIC chromatographic column of the present invention is selected to achieve the separation effect.

[0020] 2. In order to obtain better peak shapes and response values, the mobile phase and elution gradient conditions are optimized. Formic acid water and ammonia water are used as mobile phase A, and formic acid acetonitrile and acetonitrile are used as mobile phase B respectively. During the test process, under acidic conditions, the ionization of N-nitrosomeropenem is affected, resulting in a low response of N-nitrosomeropenem. When using alkaline ammonia water, the response of N-nitrosomeropenem is higher. Finally, it is found that when using the mobile phase A and mobile phase B with specific concentrations and specific types of the present invention, the response value of N-nitrosomeropenem is the highest.

[0021] 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 the impurity N-nitrosomeropenem from meropenem and effectively detect the impurity N-nitrosomeropenem in meropenem.

[0022] 4. Determination of mass spectrometry conditions. Through optimization of positive and negative modes, it was found that in the commonly used positive mode, the response of impurity peaks was extremely small, and there was basically no response for impurities prepared according to the limit. Finally, a detection method was developed under the negative mode of the ESI source to meet the detection requirements. At the same time, the full-scan mode was adopted, and finally the mass spectrometry conditions were optimized: DP voltage -30 V, EP voltage -12 V, CE voltage -16.4 eV, CXP voltage: -17.9 V. Description of the Drawings

[0023] Figure 1 It is the mass spectrometry diagram of the blank test (diluent) in Example 1. Among them, the abscissa is time (min), and the ordinate is intensity, cps.

[0024] Figure 2 It is the mass spectrometry diagram of the reference substance in Example 1. Among them, the abscissa is time (min), and the ordinate is intensity, cps.

[0025] Figure 3 It is the mass spectrometry diagram of the test sample in Example 1. Among them, the abscissa is time (min), and the ordinate is intensity, cps.

[0026] Figure 4 It is the mass spectrometry diagram of the reference substance in Example 1. Among them, the abscissa is m / z, and the ordinate is intensity, cps.

[0027] Figure 5 It is the mass spectrometry diagram and chromatogram of the 100% limit spiked solution in Test Example 1. Among them, the upper figure is the mass spectrometry diagram, and the lower figure is the chromatogram. In the chromatogram, the abscissa is time (min), and the ordinate is intensity, mAU; in the mass spectrometry diagram, the abscissa is time (min), and the ordinate is intensity, cps.

[0028] Figure 6 It is the standard working curve in Test Example 2.

[0029] Figure 7 It is the mass spectrometry diagram of the reference substance in Comparative Example 1. Among them, the abscissa is time (min), and the ordinate is intensity, cps.

[0030] Figure 8 It is the mass spectrometry diagram of the reference substance in Comparative Example 2. Among them, the abscissa is time (min), and the ordinate is intensity, cps.

[0031] Figure 9 It is the mass spectrometry diagram of the reference substance in Comparative Example 3. Among them, the abscissa is m / z, and the ordinate is intensity, cps. Detailed Implementation Modes

[0032] The present invention will be further described below through specific examples, but it is not limited thereto.

[0033] Unless otherwise specified, the raw materials used in the examples are all conventional products; unless otherwise specified, the methods used are all prior art.

[0034] Example 1

[0035] A detection method for meropenem impurity N-nitrosomeropenem

[0036] Using high performance liquid chromatography-mass spectrometry combined technology

[0037] (1) Standard products, reagents and instruments

[0038] Standard product: N-nitrosomeropenem

[0039] Reagents: Acetonitrile (mass spectrometry grade, Merck); Formic acid (chromatography grade, Sigma)

[0040] Instruments: Shimadzu ultra high performance liquid chromatograph (model: LC-20ADXR); AB mass spectrometer (model: Qtrap

[0041] );Sartorius one in a million balance (model: MSA6.6S-0CE-DM)

[0042] (2) Preparation of test solution:

[0043] Weigh 1 g of meropenem sample, first dissolve it in 20 ml of water, then add acetonitrile to make up to 100 ml, dissolve it by ultrasonic treatment, centrifuge at 10000 r / min for 10 min, and take the supernatant.

[0044] (3) Preparation of reference solution:

[0045] Take an appropriate amount of N-nitrosomeropenem reference substance, weigh it accurately, dissolve it with a diluent (aqueous solution of acetonitrile, the volume ratio of acetonitrile to water is 4:1) and quantitatively dilute it to a solution containing 2.2 ng of N-nitrosomeropenem per 1 ml.

[0046] (4) Chromatographic conditions:

[0047] Chromatographic column: Waters Atlantis HILIC Silica liquid chromatography column (150 mm × 4.6 mm, 5 μm); Injection volume: 2 μl; Column temperature: 40 °C; Flow rate: 0.8 ml / min; Analysis time: 15 min; Ionization mode: negative ion electrospray (AJSESI-), multiple reaction monitoring (MRM) mode; Full scan mode; Mobile phase A: 0.05% ammonia water by volume concentration, Mobile phase B: 0.1% formic acid acetonitrile by volume concentration; 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.

[0048] Gradient elution conditions:

[0049] Time (min) Mobile phase A (%) Mobile phase B (%) 0 20 80 4 20 80 4.1 50 50 10 50 50 10.1 20 80 15 20 80

[0050] Mass spectrometry detection parameters:

[0051]

[0052] (5) Determination method

[0053] Precisely measure the diluent (aqueous solution of acetonitrile, volume ratio of acetonitrile to water is 4:1), reference solution, and test solution, inject them into the liquid chromatograph and mass spectrometer, and record the chromatogram and mass spectrum.

[0054] The mass spectra of the blank test (diluent), reference substance, and test substance are respectively as Figure 1 、 2 and 3 shown, Figure 1 - 3 All are mass spectra in multiple reaction monitoring (MRM) mode, belonging to the quantitative mode. As can be seen from Figure 1 - 3 , in the method of the present invention, the response value of N-nitrosomeropenem is high and the peak shape is good; no impurity N-nitrosomeropenem is detected in the test substance.

[0055] The mass spectrum of the reference substance is as Figure 4 shown, Figure 4 which is a partial screenshot of the reference mass spectrometry flow chart in the full scan (Scan) negative mode, belonging to qualitative scanning, and monitoring all impurities in the range of mass-to-charge ratio from 100 to 1000.

[0056] Test example 1

[0057] Specificity test

[0058] (1) Preparation of test solution:

[0059] Weigh 1 g of meropenem sample, first dissolve it in 20 ml of water, then add acetonitrile to make up to 100 ml, dissolve it by ultrasonic treatment, centrifuge at 10000 r / min for 10 min, and take the supernatant.

[0060] (2) Preparation of reference solution:

[0061] Take an appropriate amount of N-nitrosomeropenem reference substance, weigh it accurately, dissolve it with diluent (aqueous solution of acetonitrile, volume ratio of acetonitrile to water is 4:1), and quantitatively dilute it to a solution containing 2.2 ng of N-nitrosomeropenem per 1 ml.

[0062] (3) 100% spiked solution:

[0063] Weigh 1 g of meropenem sample, add the reference solution to make up to 100 ml, dissolve it by ultrasonic treatment, centrifuge at 10000 r / min for 10 min, and take the supernatant.

[0064] The chromatographic conditions are the same as those in Example 1.

[0065] Precisely measure the diluent (aqueous solution of acetonitrile, volume ratio of acetonitrile to water is 4:1), reference solution, test solution, and 100% spiked solution, and inject them into the liquid chromatograph and mass spectrometer, and record the chromatogram and mass spectrum.

[0066] The mass spectra of the blank test (diluent), reference substance, test substance, and the chromatogram-mass spectrum of the 100% spiked solution are respectively as Figure 1 、 2 、3、5 shown, among which as Figure 5 shown in the chromatogram, the peak emergence time of N-nitrosomeropenem is 2.75 min, and the peak emergence time of meropenem is about 7.6 min, indicating that the method of the present invention can effectively separate the N-nitrosomeropenem peak and the meropenem peak, eliminate the interference of meropenem on N-nitrosomeropenem, and the results show that the method has good specificity.

[0067] Test Example 2

[0068] Standard working curve, detection limit, and quantitation limit tests

[0069] Take an appropriate amount of N-nitrosomeropenem reference substance, weigh it accurately, dissolve it with diluent (aqueous solution of acetonitrile, volume ratio of acetonitrile to water is 4:1), and respectively dilute it to solutions of 0.09 ng / ml (quantitation 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.

[0070] Precisely measure the above solution, inject it into a liquid chromatograph and a mass spectrometer. The chromatographic conditions are the same as those in Example 1, and record the chromatogram and mass spectrum. The test results according to the mass spectrum are shown in Table 1 and Figure 6 as shown.

[0071] Table 1 Standard working curve, detection limit, and quantification limit data

[0072]

[0073] From the experimental results in the above table, it can be seen that within the range of 0.09 ng / ml to 4.4 ng / ml, the concentration of N-nitrosomeropenem has a good linear relationship with the peak area of the mass spectrum, and the lowest detection limit of this method reaches 0.03 ng / mL.

[0074] Test Example 3

[0075] Precision and accuracy tests

[0076] At the same time, the same personnel respectively carried out spike recovery experiments at three concentration levels: high (120% of the reference substance concentration), medium (reference substance concentration), and low (quantification limit concentration). The accuracy and precision data are shown in Tables 2 and 3 below.

[0077] Table 2 Accuracy experimental data

[0078]

[0079] Table 3 Precision experimental data

[0080]

[0081] From the experimental data in the above table, it can be seen that the spike recovery results of the accuracy of N-nitrosomeropenem are: 87.5% - 115.8%, all within the range of 80% - 120%, and the precision RSD is 4.7%, less than 10%; indicating that the accuracy and precision of this method are good and suitable for the detection of N-nitrosomeropenem.

[0082] Comparative Example 1

[0083] A detection method for the impurity N-nitrosomeropenem of meropenem is as described in Example 1, except that: mobile phase A is an aqueous formic acid solution with a volume concentration of 0.1%; other steps and conditions are the same as in Example 1.

[0084] The mass spectrum of the reference substance is as Figure 7 shown. It can be seen from the figure that under acidic conditions, the ionization of N-nitrosomeropenem is affected, resulting in a low response of N-nitrosomeropenem, and the response value is only at the quantification limit level, and the content of N-nitrosomeropenem in meropenem cannot be effectively detected.

[0085] Comparative Example 2

[0086] A detection method for meropenem impurity N-nitroso meropenem, as described in Example 1, except that: the chromatographic column is a C18 liquid chromatography column (water-tolerant type) (150 mm × 4.6 mm, 5 μm); other steps and conditions are the same as those in Example 1.

[0087] The mass spectrum of the reference substance is as Figure 8 shown. It can be seen from the figure that the peak width of N-nitroso meropenem is relatively large, the peak shape is asymmetric, the peak is split, and the peak response is relatively low.

[0088] Comparative Example 3

[0089] A detection method for meropenem impurity N-nitroso meropenem, as described in Example 1, except that: ionization mode: positive ion electrospray (AJS ESI+), multiple reaction monitoring (MRM) mode; other steps and conditions are the same as those in Example 1.

[0090] The mass spectrum of the reference substance is as Figure 9 shown, and this figure is a partial screenshot of the reference mass spectrum in the full scan (Scan) positive mode. It can be seen from the figure that in the case of the commonly used positive mode, the response of the impurity peak is extremely small, and there is basically no response for the impurity prepared according to the limit.

Claims

1. A method for detecting meropenem impurity N-nitroso meropenem, characterized in that, The LC-MS method was adopted, and the chromatographic conditions were as follows: Chromatographic column: Waters Atlantis HILIC Silica liquid chromatography column (150 mm × 4.6 mm, 5 μm); Mobile phase A: ammonia water with a volume concentration of 0.04% - 0.06%; Mobile phase B: acetonitrile formate with a volume concentration of 0.05% - 0.2%; Ionization mode: negative ion electrospray (AJS ESI-), multiple reaction ion monitoring (MRM) mode; Gradient elution conditions: At 0 minute, the volume ratio of mobile phase A was 15 - 25%, and the volume ratio of mobile phase B was 75 - 85%; At 4 minutes, the volume ratio of mobile phase A was 15 - 25%, and the volume ratio of mobile phase B was 75 - 85%; At 4.1 minutes, the volume ratio of mobile phase A was 50%, and the volume ratio of mobile phase B was 50%; At 10 minutes, the volume ratio of mobile phase A was 50%, and the volume ratio of mobile phase B was 50%; At 10.1 minutes, the volume ratio of mobile phase A was 15 - 25%, and the volume ratio of mobile phase B was 75 - 85%; At 15 minutes, the volume ratio of mobile phase A was 15 - 25%, and the volume ratio of mobile phase B was 75 - 85%.

2. The detection method of meropenem impurity N-nitroso meropenem according to claim 1, characterized in that, Mobile phase A: ammonia water with a volume concentration of 0.05%; Mobile phase B: acetonitrile formate with a volume concentration of 0.1%.

3. The detection method of meropenem impurity N-nitroso meropenem according to claim 1, wherein Gradient elution conditions: At 0 minute, the volume ratio of mobile phase A was 20%, and the volume ratio of mobile phase B was 80%; At 4 minutes, the volume ratio of mobile phase A was 20%, and the volume ratio of mobile phase B was 80%; At 4.1 minutes, the volume ratio of mobile phase A was 50%, and the volume ratio of mobile phase B was 50%; At 10 minutes, the volume ratio of mobile phase A was 50%, and the volume ratio of mobile phase B was 50%; At 10.1 minutes, the volume ratio of mobile phase A was 20%, and the volume ratio of mobile phase B was 80%; At 15 minutes, the volume ratio of mobile phase A was 20%, and the volume ratio of mobile phase B was 80%.

4. The detection method of meropenem impurity N-nitroso meropenem according to claim 1, characterized in that, The chromatographic conditions also included: 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 detection method of meropenem impurity N-nitroso meropenem according to claim 1, characterized in that, Mass spectrometry detection parameters: Parent ion: 411 m / Z; Daughter ion: 198 m / Z; Declustering potential (DP): -30 V; Entrance potential (EP): -12 V; Collision energy (CE): -16.4 eV; Exit potential (CXP): -17.9 V.

6. The detection method of meropenem impurity N-nitroso meropenem according to claim 1, characterized in that, Acetonitrile and water were used as solvents to prepare the test solution, and the volume ratio of acetonitrile to water was 4:1.

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