CN113624894A

CN113624894A - Method for detecting nitrosamine impurities in biapenem

Info

Publication number: CN113624894A
Application number: CN202111189528.4A
Authority: CN
Inventors: 陈新国; 邱全玉; 邵广志; 黄军建; 陈亿展; 曹丹; 麦华凤; 蒋杰
Original assignee: Guangzhou Gb Inspection And Testing Co ltd
Current assignee: Guangzhou Gb Inspection And Testing Co ltd
Priority date: 2021-10-13
Filing date: 2021-10-13
Publication date: 2021-11-09
Anticipated expiration: 2041-10-13
Also published as: CN113624894B

Abstract

The application belongs to the technical field of drug analysis. The application provides a detection method of nitrosamine impurities in biapenem, which comprises the following steps: adding a sample to be tested into a methanol solution of an internal standard substance for dissolving, and then adding a mixed solvent for uniformly mixing to obtain a sample solution; respectively injecting the sample solution and the reference solution into a liquid chromatography-tandem mass spectrometer, and detecting the content of nitrosamine impurities according to an internal standard method; wherein the mixed solvent comprises methanol, formic acid and water. The method is developed according to the characteristics of the nitrosamine compound, so that the influence of the matrix effect can be effectively reduced, and the object to be detected is prevented from being inhibited. The detection method is verified from the aspects of specificity, linearity, range, detection limit, quantification limit, accuracy, stability and the like, and the method is confirmed to be suitable for simultaneously determining the contents of NDEA, EIPNA and DIPINA in biapenem.

Description

Method for detecting nitrosamine impurities in biapenem

Technical Field

The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a detection method of nitrosamine impurities in biapenem.

Background

Biapenem is a carbapenem synthetic antibiotic, and nitrosamine byproducts such as N-nitrosodiethylamine, N-nitrosoethyl isopropylamine and N-nitrosodiisopropylamine are generated in the production process. Nitrosamines are potent carcinogens, and not only can cause carcinogenesis in animals or humans in long-term small doses, but also can cause carcinogenesis in a single "shock" of higher doses.

At present, the detection method of nitrosamine impurities mainly comprises a gas chromatography thermal energy analysis method, a spectrophotometry method, a gas method and a liquid method, but the existing detection method lacks a detection method aiming at N-nitrosoethyl isopropylamine and N-nitrosodiisopropylamine. Meanwhile, the limit (0.022 ppm) of nitrosamine impurities in biapenem is extremely low, the requirement on detection sensitivity is high, and the requirement on quantitative monitoring of nitrosamine impurities in biapenem is difficult to realize by the existing detection method.

Disclosure of Invention

In view of the above, the present application provides a method for detecting nitrosamine impurities in biapenem, which can realize synchronous measurement of N-nitrosodiethylamine, N-nitrosoethylisopropylamine and N-nitrosodiisopropylamine, and has excellent sensitivity.

The specific technical scheme of the application is as follows:

a method for detecting nitrosamine impurities in biapenem is characterized by comprising the following steps:

s1: adding a sample to be tested into a methanol solution of an internal standard substance for dissolving, and then adding a mixed solvent for uniformly mixing to obtain a sample solution;

s2: respectively injecting the sample solution and the reference solution into a liquid chromatography-tandem mass spectrometer, and detecting the content of nitrosamine impurities according to an internal standard method;

wherein the mixed solvent comprises methanol, formic acid and water.

Preferably, the content of methanol in the methanol solution is 50-80%.

Preferably, the volume ratio of formic acid, methanol and water in the mixed solvent is 5:10: 85.

Preferably, the concentration of the internal standard substance in the methanol solution is 30-60 ng/ml.

Preferably, the volume ratio of the methanol solution to the mixed solvent is 1: 20-30.

Preferably, the concentration of the sample in the sample solution is 40-80 mg/ml, and the concentration of the reference solution is 3-130 ng/ml.

Preferably, the nitrosamine-type impurities include N-nitrosodiethylamine, N-nitrosoethylisopropylamine and/or N-nitrosodiisopropylamine.

Preferably, the liquid chromatography conditions are: a chromatographic column: a C18 chromatography column; mobile phase A: 0.1% formic acid, mobile phase B: methanol; the column temperature is 38-42 ℃; the flow rate is 0.27-0.33 ml/min.

Preferably, the elution procedure of the liquid chromatography is: 0-3 min, A95% and B5%; 3.5-4.5 min, A90% and B10%; 5.5-6.5 min, A60% and B40%; 7-8 min, A20% and B80%; 8.1-9.5 min, A0% and B100%; 9.6-12 min, A95% and B5%.

Preferably, the mass spectrometry conditions are: ion source APCI source, scan mode MRM, ion pair comprising: 117.1/75.1, 131.1/89.1 and/or 103.1/47.0, and the collision energy is 15-29V.

To sum up, the application provides a method for detecting nitrosamine impurities in biapenem, which comprises the following steps: adding a sample to be tested into a methanol solution of an internal standard substance for dissolving, and then adding a mixed solvent for uniformly mixing to obtain a sample solution; respectively injecting the sample solution and the reference solution into a liquid chromatography-tandem mass spectrometer, and detecting the content of nitrosamine impurities according to an internal standard method; wherein the mixed solvent comprises methanol, formic acid and water. The method is developed according to the characteristics of the nitrosamine compound, so that the influence of the matrix effect can be effectively reduced, and the object to be detected is prevented from being inhibited. The detection method is verified from the aspects of specificity, linearity, range, detection limit, quantification limit, accuracy, stability and the like, and the method is confirmed to be suitable for simultaneously determining the contents of NDEA, EIPNA and DIPINA in biapenem.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive exercise.

FIG. 1 is a chromatogram of EIPNA specific for a control solution of example 2 of this application;

FIG. 2 is a chromatogram of a specific value of DIPINA in a control solution of example 2 of the present application;

FIG. 3 is a chromatogram of NDEA from a control solution of example 2 of the present application;

FIG. 4 is a chromatogram of EIPNA specific in a spiked solution according to example 2 of the present application;

FIG. 5 is a chromatogram of a specificity of DIPINA in a spiked solution according to example 2 of the present application;

FIG. 6 is a chromatogram of NDEA from a spiked solution according to example 2 of the present application;

FIG. 7 is a chromatogram of EIPNA in comparative example 1 of this application;

FIG. 8 is a chromatogram of DIPINA in comparative example 1 of the present application;

FIG. 9 is a chromatogram of NDEA from comparative example 1 of the present application;

FIG. 10 is a chromatogram of EIPNA in comparative example 4 of this application;

FIG. 11 is a chromatogram of DIPINA in comparative example 4 of the present application;

FIG. 12 is a chromatogram of NDEA from comparative example 4 of the present application;

FIG. 13 is a chromatogram of EIPNA in comparative example 6 of this application;

FIG. 14 is a chromatogram of DIPINA from comparative example 6 of the present application;

FIG. 15 is a chromatogram of NDEA from comparative example 6 of the present application;

FIG. 16 is a chromatogram of EIPNA in comparative example 8 of this application;

FIG. 17 is a chromatogram of DIPINA in comparative example 8 of the present application;

FIG. 18 is a chromatogram of NDEA from comparative example 8 of the present application.

Detailed Description

In order to make the objects, features and advantages of the present application more obvious and understandable, the technical solutions in the embodiments of the present application are clearly and completely described, and it is obvious that the embodiments described below are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The reagents and raw materials used in the examples of the present application are commercially available or self-made.

Example 1

1. Liquid chromatography conditions: a chromatographic column: waters ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm. times.100 mm); mobile phase A: 0.1% formic acid-water, mobile phase B: methanol; the column temperature is 40 ℃; the flow rate is 0.3 ml/min;

the elution procedure was: 0-3 min, A95% and B5%; 3.5-4.5 min, A90% and B10%; 5.5-6.5 min, A60% and B40%; 7-8 min, A20% and B80%; 8.1-9.5 min, A0% and B100%; 9.6-12 min, A95% and B5%.

2. Mass spectrum conditions: ion source APCI source, scan mode MRM, ion pair: n-nitrosoethyl isopropylamine (EIPNA) 117.1/75.1, collision energy 15V; n-nitrosodiisopropylamine (DIPINA) 131.1/89.1, collision energy 15V; N-Nitrosodiethylamine (NDEA) 103.1/47.0, collision energy 29V; internal standard NDEA-d 4107.0/77.0, collision energy 16V; internal standard NDPA-d 14145.0/50.0, collision energy 19V.

3. Preparing a mixed solvent: 20ml of methanol is put into a reagent bottle, 170ml of water is added, 10ml of formic acid is added, and the mixture is shaken up.

4. Control solution: respectively taking EIPNA, DIPINA and NDEA standard substances, dissolving with 70% methanol as a solvent, performing gradient dilution with a mixed solvent as a diluent, and preparing a standard curve solution with the concentration of 0.066-2.639 ng/ml.

5. Internal standard solution: taking NDEA-d4 and NDPA-d14 internal standard substances, and using 70% methanol as a solvent to prepare a mixed internal standard solution with the concentration of 33 ng/ml.

6. Test solution: taking about 0.3g of biapenem raw material powder, placing the biapenem raw material powder in a 5ml volumetric flask, adding 0.2ml of internal standard solution, adding mixed solvent to a constant volume to a scale, uniformly mixing, and shaking up.

7. Adding a standard solution: taking about 0.3g of biapenem raw material powder, placing the biapenem raw material powder in a 5ml volumetric flask, adding 0.2ml of internal standard solution, then adding 0.1ml of control solution with the concentration of 66ng/ml, using mixed solvent to fix the volume to a scale, uniformly mixing, and shaking up.

And (3) performing analysis and detection by adopting an internal standard method, namely calculating the peak area ratio of the analytes (EIPNA, DIPINA and NDEA) to the internal standard substances (NDPA-d 14 and NDEA-d 4), and recording the chromatogram and the recovery rate.

Example 2

Referring to the test method of example 1, the test results are as follows:

1. specificity

Respectively taking a reference substance solution with the concentration of 1.319 ng/ml and a standard solution for sample loading test, and recording chromatograms as shown in figures 1-6. The figure shows that the peak position of the target peak in the 100% limit concentration standard sample solution has no other impurity interference, and the peak shape is similar to the peak shape of the 100% limit concentration reference sample solution, which indicates that the detection method has good specificity.

2. Linearity

A standard curve solution is taken for sample loading test, and a linear fitting equation is obtained through the relation between the peak area and the concentration, and is shown in the following table 1. In each concentration range of each impurity, the peak area ratio and the concentration have good linear relation, and the correlation coefficient r is not less than 0.995, thereby meeting the requirements.

TABLE 1

3. Detection limit and quantification limit

And (3) sampling and detecting the reference substance solution with the concentration of 0.066ng/ml for 3 times. The detection limit of NDEA is 0.066ng/ml, which is equivalent to 5% of the limit concentration and can be detected (namely the detection limit is 0.0011 ppm), and the signal-to-noise ratio is 18.1-23.8; the detection limit concentration of EIPNA is 0.066ng/ml, which is equivalent to 5% of the limit concentration and can be detected (namely the detection limit is 0.0011 ppm), and the signal-to-noise ratio is 62.8-65.2; the detection limit concentration of DIPINA is 0.066ng/ml, which is equivalent to 5% of the limit concentration and can be detected (namely the detection limit is 0.0011 ppm), and the signal-to-noise ratio is 23.2-28.8.

0.132ng/ml of control solution is injected and tested for 6 times. The limit concentration of NDEA is 0.132ng/ml, which is equal to 10% of the limit concentration (namely the limit of quantitation is 0.0022 ppm), the signal-to-noise ratio is 32.5-41.4, and the RSD value of the peak area ratio of 6 continuous pins of limit solution is 6.6%; the limit concentration of EIPNA is 0.132ng/ml, which is equivalent to 10% of the limit concentration (namely the limit of quantitation is 0.0022 ppm), the signal-to-noise ratio is 81.3-120.5, and the RSD value of the peak area ratio of 6 continuous pins of limit solutions is 6.0%; the limit concentration of the DIPINA is 0.132ng/ml, which is equivalent to 10% of the limit concentration (namely the limit of the quantification is 0.0022 ppm), the signal-to-noise ratio is 32.4-48.6, and the RSD value of the peak area ratio of 6 continuous pins of the limit solution is 6.5%, thus meeting the requirement.

4. Accuracy of

Taking 3 parts of different standard solutions (corresponding to standard sample solutions with the limit concentrations of each impurity of 50%, 100% and 150%), respectively injecting samples for 1 time, recording chromatograms, and calculating the recovery rate as shown in the following tables 2-4. The recovery rates of NDEA, EIPNA and DIPINA in 9 parts of accuracy solutions are respectively in the ranges of 74.7-97.4%, 72.6-91.2% and 75.0-99.2%, the RSD values are respectively 10.2%, 8.1% and 7.3%, and the accuracy results meet the regulations.

TABLE 2

TABLE 3

TABLE 4

5. Stability of

Respectively taking a reference substance solution and a standard solution with the concentration of 1.319 ng/ml, respectively placing the reference substance solution and the standard solution for at least 24 hours at room temperature (10-30 ℃) in a dark condition, carrying out sample injection analysis at different time points, and recording a chromatogram and the recovery rate, wherein the results are shown in the following table 5-6. The determination concentrations of NDEA, EIPNA and DIPINA in the 100% limit concentration reference substance solution at different times are respectively 94.3% -107.9%, 85.0% -99.0% and 85.8% -109.7% of the initial concentration. The determination concentrations of NDEA, EIPNA and DIPINA in the standard sample solution with 100% limit concentration are respectively 82.5% -96.7%, 79.1% -93.8% and 92.8% -104.1% of the initial concentration. The results show that the impurities NDEA, EIPNA and DIPINA are stable for at least 48h in the reference solution with 100% limit concentration and the standard solution with 100% limit concentration.

TABLE 5

TABLE 6

Comparative example 1

Referring to the detection method of example 1, the difference is that no internal standard solution is added to the spiked solution, and the spiked solution is detected by an external standard method, i.e., peak areas of the recovery rate solution of the analyte (EIPNA, DIPINA, NDEA) and the control solution are calculated, and the average value is taken by repeating 3 times to record the chromatogram and the recovery rate.

Chromatograms of EIPNA, DIPINA and NDEA in the standard solution are shown in FIGS. 7-9, and the recovery rates are 77.2%, 81.5% and 38.9%, respectively. In the recovery rate solution, other components are eluted before the peak emergence of NDEA, the spectrum shows a cracked peak and has a matrix inhibition effect on NDEA, so that the external standard method is not suitable for detecting nitrosamine impurities in biapenem.

Comparative example 2

Referring to the test method of example 1, the only difference was that the mixed solvent in both the control solution and the spiked solution was replaced with 70% methanol, and it was observed that the sample powder was not completely dissolved, the spiked solution had poor solubility, and the recovery rate was unsatisfactory.

Comparative example 3

Referring to the test method of example 1, the difference is only that the mixed solvent in both the control solution and the spiked solution was replaced with 70% methanol, and 0.5ml DMSO was added, and the test was repeated 3 times, and the recovery rate of the spiked solution was recorded.

The recovery rates of EIPNA, DIPINA and NDEA in the standard solution are 90.7-96.5%, 90.7-96.5% and 85.3-101.3% respectively, and are qualified, but the standard solution is precipitated after being placed for 4 hours, so that the sample introduction repeatability and the detection stability are influenced, and the standard solution is not qualified.

Comparative example 4

Referring to the test method of example 1, the difference is that the mixed solvent in both the control solution and the spiked solution was replaced with 10% methanol-2% formic acid solution, the measurement was repeated 3 times to obtain an average value, and the recovery rate of the spiked solution was recorded.

Chromatograms of EIPNA, DIPINA and NDEA in the standard solution are shown in figures 10-12, the standard solution is good in solubility, the recovery rates of EIPNA, DIPINA and NDEA in the standard solution are respectively 98.2%, 81.6% and 86.5%, the recovery rates basically meet requirements, and the recovery rate of DIPINA is low. In addition, the labeling solution is placed under a refrigeration condition, so that precipitation is generated, and the sample introduction repeatability and the detection stability are influenced.

Comparative example 5

Referring to the test method of example 1, the difference was that the mixed solvent in both the control solution and the spiked solution was replaced with 10% methanol-2% formic acid solution, and 0.5ml ammonia water was added for neutralization, and the measurement was repeated 3 times to record the recovery rate of the spiked solution.

The experimental result shows that the peak shape of the control solution is not affected, but the object to be tested is inhibited in the standard solution, wherein the NDEA recovery rate is 0.

Comparative example 6

Referring to the detection method of example 1, the only difference is that the mixed solvent in the control solution and the spiked solution was replaced with 10% methanol-2% formic acid solution, and the elution procedure was adjusted to: 0-1 min, A95% and B5%; 5-7 min, A90% and B10%; 7.7-10 min, A60% and B40%. The assay was repeated 3 times and the recovery of the spiked solution was recorded.

Experimental results show that the three compounds of EIPNA, DIPINA and NDEA in the standard solution are severely inhibited by the matrix (see figures 13-15), namely the substance to be detected and the main drug are mixed and flow out, and the recovery rate is poor.

Comparative example 7

Referring to the detection method of example 1, the only difference is that the elution program will be adjusted to: 0-1 min, A95% and B5%; 5-7 min, A90% and B10%; 7.7-10 min, A60% and B40%. The assay was repeated 3 times and the recovery of the spiked solution was recorded.

The experimental result shows that three impurities in the standard solution are still inhibited, the recovery rate is low, and the detection requirement cannot be met.

Comparative example 8

Referring to the detection method of example 1, the only difference is that the elution program will be adjusted to: 0-4 min, A90% and B10%; 5-7 min, A5% and B95%; 7.1-10 min, A90% and B10%. The assay was repeated 3 times and the recovery of the spiked solution was recorded.

The experimental result shows that after the initial equilibrium proportion is prolonged, three impurities in the standard solution are inhibited, the recovery rate is poor (see fig. 16-18), and the detection requirement cannot be met.

The above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions in the embodiments of the present application.

Claims

1. A method for detecting nitrosamine impurities in biapenem is characterized by comprising the following steps:

in the mixed solvent, the volume ratio of formic acid to methanol to water is 5:10: 85;

the liquid chromatography conditions are as follows: a chromatographic column: a C18 chromatography column; mobile phase A: 0.1% formic acid, mobile phase B: methanol; the column temperature is 38-42 ℃; the flow rate is 0.27-0.33 ml/min;

the elution procedure for the liquid chromatography was: 0-3 min, A95% and B5%; 3.5-4.5 min, A90% and B10%; 5.5-6.5 min, A60% and B40%; 7-8 min, A20% and B80%; 8.1-9.5 min, A0% and B100%; 9.6-12 min, A95% and B5%;

the nitrosamine impurities include N-nitrosodiethylamine, N-nitrosoethylisopropylamine and/or N-nitrosodiisopropylamine.

2. The detection method according to claim 1, wherein the content of methanol in the methanol solution is 50 to 80%.

3. The detection method according to claim 1, wherein the concentration of the internal standard substance in the methanol solution is 30 to 60 ng/ml.

4. The detection method according to claim 1, wherein the volume ratio of the methanol solution to the mixed solvent is 1:20 to 30.

5. The detection method according to claim 1, wherein the concentration of the sample solution is 40 to 80mg/ml, and the concentration of the control solution is 3 to 130 ng/ml.

6. The detection method according to claim 1, wherein the mass spectrometry conditions are: ion source APCI source, scan mode MRM, ion pair comprising: 117.1/75.1, 131.1/89.1 and/or 103.1/47.0, and the collision energy is 15-29V.