CN113295791B - Method for analyzing ethyl bromide in sulbactam sodium - Google Patents

Abstract

The invention provides an analysis method of ethyl bromide in sulbactam sodium, and relates to the technical field of chemical detection. The analysis method disclosed by the invention is specially used for detecting the content of the bromoethane in the sulbactam sodium, adopts a headspace sample injection gas chromatography, and uses a proper combined solvent to dissolve a sample, so that the dispersion systems of a test sample and a reference sample are consistent, and the technical problems of low detection sensitivity caused by large solubility difference and non-uniform dispersion systems among components are solved. The analysis method has the advantages of strong specificity, high sensitivity, high efficiency, convenience and strong applicability, and can be well applied to investigation and evaluation of the content of the bromoethane in the sulbactam sodium raw material and effective control of the product quality so as to ensure the medication safety.

Description

Method for analyzing ethyl bromide in sulbactam sodium

Technical Field

The invention belongs to the technical field of chemical detection, and particularly relates to an analysis method of ethyl bromide in sulbactam sodium.

Background

Sulbactam sodium is an irreversible competitive beta-lactamase inhibitor synthesized artificially, is white or white-like crystalline powder, is easy to dissolve in water, is usually combined with penicillin and cephalosporin medicaments, avoids the destruction of the penicillin and the cephalosporin medicaments by the beta-lactamase, and enhances the antibacterial activity of the medicaments. During the synthesis process, sulbactam sodium undergoes bromination reaction, and the generated by-product reacts with solvent ethanol to generate ethyl bromide.

The bromoethane is colorless oily liquid, has smell and burning taste similar to that of diethyl ether, gradually turns yellow when exposed to air or light, is easy to volatilize, is insoluble in water, and can be mixed and dissolved with ethanol, diethyl ether, trichloromethane and most organic solvents. Bromoethane has certain carcinogenicity, and is classified as a class 3 carcinogen in the carcinogen list of International cancer research institute of world health organization, so that the content of bromoethane needs to be detected before sulbactam sodium is marketed. At present, many relevant reports on the research of a bromoethane detection method exist.

As in the literature: in the determination of the residual amount of ethyl bromide in the tiogliptin hydrobromide by gas chromatography (Wu Fangning, etc., modern medicine and clinic, 2014,29 (10): 1109-1111), the determination of the residual amount of ethyl bromide in the tiogliptin hydrobromide by gas chromatography is disclosed, and the chromatographic conditions are set as follows: dima DM-624 column (30 m. Times.0.53 mm. Times.3.0 μm); an Electron Capture Detector (ECD); temperature programming: the initial column temperature is 40 ℃, the temperature is kept for 3min, the temperature is raised to 200 ℃ at the speed of 20 ℃/min, and the temperature is kept for 5min; sample inlet temperature: at 210 ℃; detector temperature: at 210 ℃; carrier gas: nitrogen gas; the split ratio is as follows: 3: 1; the sample size was 1. Mu.L. However, the method has the lowest detection limit of 0.26 mu g/mL and low sensitivity.

As in the literature: in the detection of bromoethane and other solvent residues in difenidol hydrochloride (Liu Shui Ping, zhongnan pharmacy 2018, 16 (2): 213-216), the determination of residual solvent bromoethane, ethanol and tetrahydrofuran in difenidol hydrochloride raw material medicines by adopting headspace Gas Chromatography (GC) is disclosed, and the chromatographic conditions are set as follows: DB-624 quartz capillary column; temperature rising procedure: the initial temperature is 50 ℃, the temperature is raised to 120 ℃ at the speed of 5 ℃/min, the temperature is maintained for 4min, the temperature is raised to 180 ℃ at the speed of 25 ℃/min, and the temperature is maintained for 2min; FID detector, temperature 250 ℃; the temperature of a sample inlet is 150 ℃; carrier gas: nitrogen gas; the flow rate is 3.0mL/min; taking water as a solvent, introducing a sample in a headspace, and balancing the temperature: 85 ℃, equilibration time: 20min; quantification of the Ring volume: lmL; the split ratio is as follows: 15: 1. The method has the following defects: the temperature of the sample inlet (150 ℃) is lower than the highest temperature (180 ℃) of the temperature programming, reverse air pressure is easy to generate in the detection process, so that gas flows back to the sample inlet from the chromatographic column, and pollution is caused; the headspace equilibrium temperature is too high, which easily causes the degradation of heat-labile substances (such as sulbactam sodium), interferes the detection of target substances, and pollutes a chromatographic column.

For example, chinese patent application CN103926349B discloses a method for measuring ethyl bromide in ethyl triphenyl phosphonium bromide, which adopts headspace-capillary gas chromatography for measurement, and the chromatographic conditions are as follows: a chromatographic column: 30m × 0.32mm × 0.25 μm, stationary phase: succinic acid diethylene glycol polyester (DEGS); carrier gas flow rate: 1.2mL/min; a detector: an ECD; column temperature: 60 ℃, gasification chamber temperature: 200 ℃, detector temperature: 200 ℃; sample introduction amount: 50 mu L of the solution; stopping time: for 10min. The method uses absolute ethyl alcohol as a solvent, and is easy to cause incomplete dissolution of substances with good water solubility and poor fat solubility during solution preparation, so that a matrix effect is generated, for example, a target object is wrapped in an incompletely dissolved sample, so that the detection result is low, the risk to substances with low limit and high toxicity is high, and the quality control in actual production is not facilitated.

Further, as disclosed in chinese patent application CN110895264a, a method for measuring ethyl bromide in tenofovir alafenamide by direct injection method, the chromatographic conditions are as follows: a capillary column using 6% cyanopropylphenyl-94% dimethylpolysiloxane as a stationary liquid is started at 35-45 ℃, heated to 220 ℃ at the speed of 30 ℃/min and maintained for 5-8 minutes; the temperature of the detector is 250 ℃; the injection port temperature is 150-220 ℃, the split ratio is 2:1-5:1, and the column flow rate is 2-4mL/min. The method adopts direct injection method, which not only easily causes the degradation of the thermolabile drug (such as sulbactam sodium) and generates interference peak, but also has great harm to the chromatographic column, thus shortening the service life and increasing the cost and pollution.

The method for measuring the content of the ethyl bromide in the prior art also has the problems of high detection limit, low sensitivity, low detection result due to matrix effect, degradation of thermally unstable drugs due to high temperature in the detection process, pollution to a chromatographic column, influence on the service life of the chromatographic column and the like, and related reports on the detection of the ethyl bromide in the sulbactam sodium are few at present. Therefore, establishing a dedicated, sensitive, efficient and highly applicable analysis method for investigating and evaluating the content of bromoethane in the sulbactam sodium raw material and effectively controlling the product quality to ensure the medication safety is a problem which needs to be solved urgently by technical personnel in the field.

Disclosure of Invention

The invention provides a method for analyzing ethyl bromide in sulbactam sodium, aiming at the problems in the prior art. The analysis method has strong applicability and high sensitivity, is used for inspecting and evaluating the content of the bromoethane in the sulbactam sodium raw material, and can effectively control the product quality to ensure the medication safety.

In order to realize the purpose, the technical scheme adopted by the invention is as follows:

an analysis method of ethyl bromide in sulbactam sodium, which is characterized by comprising the following steps:

(1) Preparing a reference substance solution: dissolving bromoethane in an organic solvent, and diluting with water to obtain a reference substance solution;

(2) Preparing a test solution: dissolving sulbactam sodium in water, and adding an organic solvent for dilution to obtain a test solution;

(3) And (3) measuring the content by an external standard method: and (3) respectively placing the reference substance solution obtained in the step (1) and the test substance solution obtained in the step (2) into a headspace bottle for heating until the gas-liquid balance is achieved, injecting the gas at the upper part of the headspace bottle into a gas chromatograph, carrying out temperature programming, recording a chromatogram by a detector for sample analysis, and calculating the amount of bromoethane according to an external standard method.

Further, the organic solvent in the step (1) is selected from one or more of methanol, ethanol, dimethylformamide (DMF), dimethyl sulfoxide and acetone;

further, the organic solvent in the step (2) is selected from one or more of alcohol, ethanol, dimethylformamide, dimethyl sulfoxide and acetone; the concentration of the sulbactam sodium in the test sample in the step (2) is 41mg/mL-63mg/mL; the volume ratio of the water to the organic solvent is 6:94-60:40.

Further, the volume ratio of the reference solution obtained in the step (1) to the test solution obtained in the step (2) added in the step (3) is 1:1; in the step (3), setting the balance temperature of the headspace bottle to be 58-62 ℃; the headspace heating time was 25-35 minutes.

Further, the chromatographic column of the gas chromatograph in the step (3) is one selected from a capillary column in which a fixing solution is 6% cyanopropylphenyl-94% dimethylpolysiloxane, 35% diphenyl-65% methylpolysiloxane, 50% diphenyl-50% dimethylpolysiloxane, 35% diphenyl-65% dimethylpolysiloxane, 14% cyanopropylphenyl-86% dimethylpolysiloxane, 50% phenyl-50% methylpolysiloxane, preferably in which a fixing solution is 6% cyanopropylphenyl-94% dimethylpolysiloxane or 50% phenyl-50% methylpolysiloxane.

Further, the chromatographic column specification of the gas chromatograph in step (3) is selected from 30m 0.32mm 1.8 μm or 30m 0.25mm 0.25 μm, preferably 30m 0.32mm 1.8 μm.

Further, the flow rate of the column carrier gas is set to be 0.8-1.2ml/min in the step (3).

Further, the gas chromatograph temperature programming operation in step (3) is: setting the initial temperature at 35-45 deg.C, maintaining for 3-5min, heating to 215-225 deg.C at 40-60 deg.C/min, and maintaining for 12-16min.

Further, setting the temperature of a sample inlet to be 220-240 ℃ in the step (3); the flow dividing ratio is 5-30; the detector temperature was set to 250-290 ℃.

Further, in the step (3), the detector is a hydrogen flame ionization detector.

Compared with the prior art, the invention has the following beneficial effects:

(1) The analysis method adopts the headspace sample injection gas chromatography to measure the content of the bromoethane in the sulbactam sodium bulk drug, has simple and quick operation and accurate and reliable measurement result, and can effectively control the quality of the sulbactam sodium bulk drug.

(2) The analysis method uses a proper combined solvent to dissolve the sample, so that the dispersion systems of the sample and the reference substance are consistent, the technical problem of low detection sensitivity caused by large solubility difference and non-uniform dispersion system among the components is solved, the method has high sensitivity and good accuracy, the product quality can be effectively controlled, and the product risk is reduced; the used solvent is simple and easy to obtain and has low price.

Drawings

FIG. 1 is a linear regression plot of bromoethane;

FIG. 2 is a gas chromatogram of ethyl bromide in sulbactam sodium;

Detailed Description

It should be noted that the raw materials used in the present invention are all common commercial products, and the sources thereof are not particularly limited.

The following sources of raw materials are exemplary:

the fixative solution was a capillary column of 6% cyanopropylphenyl-94% dimethylpolysiloxane, agilent DB-624, 30m 0.32mm 1.8 μm;

example 1

(1) Preparing a reference substance solution: adding appropriate amount of DMF into bromoethane according to the proportion of 1:1 for dissolving to obtain reference substance solution stock solution, and diluting with water to obtain reference substance solution with concentration of 0.025 mg/ml;

(2) Preparing a test solution: dissolving sulbactam sodium in water, and adding DMF (dimethyl formamide) for dilution to obtain a test solution with the concentration of 50 mg/ml;

(3) And (3) measuring the content by an external standard method: and (3) heating the reference substance solution obtained in the step (1) and the test solution obtained in the step (2) in a headspace bottle until the gas-liquid balance is achieved, injecting 1.0ml of gas at the upper part of the headspace bottle into a gas chromatograph, recording a chromatogram as shown in figure 2, analyzing the sample, and calculating the amount of bromoethane according to an external standard method.

Wherein, the chromatographic conditions are set as follows:

head space temperature: 60 ℃;

headspace heating time: 30min;

a chromatographic column: the fixative solution was a capillary column of 6% cyanopropylphenyl-94% dimethylpolysiloxane, 30m 0.32mm 1.8 μm;

carrier gas: nitrogen gas;

temperature programming: maintaining at 40 deg.C for 3min, heating to 220 deg.C at 50 deg.C/min, and maintaining for 14min;

a detector: FID;

flow rate of carrier gas: 1.0ml/min;

the split ratio is as follows: 15: 1;

sample inlet temperature: 230 ℃;

detector temperature: 270 deg.C.

Examples of the experiments

Linear experiment

(1) Accurately weighing 99.82mg of bromoethane reference substance, adding a proper amount of DMF (dimethyl formamide) according to the proportion of 1:1 for dissolving, adding a proper amount of water to prepare a linear test solution, sealing by a gland, shaking uniformly, introducing a sample in a headspace, and recording a chromatogram;

(2) Taking the concentration of bromoethane as a horizontal coordinate x and the peak area as a vertical coordinate y, performing linear regression to obtain a linear equation: y =12721.0749x-4267.6191 ² =0.9992, the linear relationship is good. The results are shown in table 1 and fig. 1.

TABLE 1

Serial number	Concentration (μ g/ml)	Content (ppm)	Peak area (fA S)
				1	48.97	979	625357
2	24.49	490	298907
				3	12.24	245	141293
4	2.449	49	27112
				5	0.2449	5	2458
6	0.1469	3	1351
				7	0.09794	2	1228

Sensitivity test

(1) Accurately weighing 99.82mg of bromoethane reference substance, adding a proper amount of DMF (dimethyl formamide) according to the proportion of 1:1 for dissolving, adding a proper amount of water to prepare a quantitative limiting solution, sealing by a gland, shaking uniformly, introducing a sample in a headspace, and recording a chromatogram;

(2) Precisely transferring the quantitative limiting solution, adding a proper amount of DMF (dimethyl formamide) for dilution according to the ratio of 1:1, adding a proper amount of water to prepare a detection limiting solution, sealing by a gland, shaking uniformly, introducing a sample in a headspace, and recording a chromatogram.

The results of the sensitivity experiments are shown in table 2:

TABLE 2

And (4) conclusion: the detection limit of the analytical method for bromoethane is 0.02938 mug/ml, the sensitivity is 0.59ppm, and the method has high sensitivity.

Precision test

Accurately weighing 100.95mg of bromoethane reference substance, adding a proper amount of DMF (dimethyl formamide) according to the proportion of 1:1 to dissolve, adding a proper amount of water to prepare a test solution, sealing by a gland, shaking uniformly, injecting sample in a headspace, and recording a chromatogram.

The results of the precision measurements are shown in table 3:

TABLE 3

Serial number	Peak area of bromoethane in control solution	Number of theoretical plate	Degree of separation
				1	305440	67067	47.9
2	321157	64478	47.3
				3	291684	65298	47.7
4	305548	62930	47.0
				5	306899	66057	47.4
6	308181	66047	47.7

And (4) conclusion: the RSD of the peak area of bromoethane measured by the analysis method is 3.1%, and the precision of the instrument is good.

Standard recovery rate experiment

Precisely weighing 9 parts of sulbactam sodium sample, adding a proper amount of water according to the proportion of 1:1 for dissolving, respectively adding the bromoethane reference solutions obtained in the step (1) in the example 1 to prepare standard adding solutions with different concentrations, preparing 3 parts of each solution with different concentrations, sealing by a gland, shaking uniformly, introducing sample in a headspace, and recording a chromatogram.

The results of the experiment are shown in table 4:

TABLE 4

And (4) conclusion: the recovery rate of the three concentration points of the bromoethane in the method is 95.56-98.28%, the average recovery rate is 97.37%, and the RSD is 1.1%, so that the method has good recovery rate and high accuracy.

Comparative example 1

The difference from example 1 is that in the preparation of the control solution in step (1) and the preparation of the test solution in step (2), the same applies to water or DMF alone instead of the combined solvent of water and DMF, and the recovery rates of the spiked samples were measured, and the results are shown in Table 5

TABLE 5

Solvent(s)	Water (I)	DMF
			Recovery (%) on standard	54.7％	56.9％

Comparative example 2

The difference from example 1 is that in the preparation of the reference solution in step (1) and the preparation of the test solution in step (2), diethyl ether, n-hexane and petroleum ether were used as organic solvents instead of DMF, and the results are shown in Table 6.

TABLE 6

Extraction solvent	Ether, diethyl ether	N-hexane	Petroleum ether
				Recovery (%)	74.33％	68.32％	50.84％

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.

Claims (7)

1. An analysis method of ethyl bromide in sulbactam sodium, which is characterized by comprising the following steps:

(1) Preparing a reference substance solution: dissolving bromoethane in an organic solvent, and diluting with water to obtain a reference substance solution;

(2) Preparing a test solution: dissolving sulbactam sodium in water, and adding an organic solvent for dilution to obtain a test solution;

(3) And (3) measuring the content by an external standard method: respectively placing the reference substance solution obtained in the step (1) and the test solution obtained in the step (2) in a headspace bottle for heating until gas-liquid balance is achieved, injecting gas at the upper part of the headspace bottle into a gas chromatograph, carrying out temperature programming, recording a chromatogram by a detector for sample analysis, and calculating the amount of bromoethane according to an external standard method;

the volume ratio of the water to the organic solvent in the step (2) is 6-60;

the organic solvent in the step (1) is dimethylformamide;

in the step (2), the organic solvent is dimethylformamide;

the chromatographic column of the gas chromatograph in the step (3) is selected from a capillary column of which the stationary liquid is 6 percent of cyanopropylphenyl-94 percent of dimethylpolysiloxane;

in the step (3), the temperature-programmed operation of the gas chromatograph is as follows: setting the initial temperature at 35-45 deg.C, maintaining for 3-5min, heating to 215-225 deg.C at 40-60 deg.C/min, and maintaining for 12-16min.

2. The analytical method of claim 1, wherein: the concentration of the sulbactam sodium in the test solution in the step (2) is 41-63mg/mL.

3. The analytical method of claim 1, wherein: the volume ratio of the reference substance solution obtained in the step (1) to the test substance solution obtained in the step (2) added in the step (3) is 1:1.

4. The analytical method of claim 1, wherein: in the step (3), setting the balance temperature of the headspace bottle to be 58-62 ℃; heating in the headspace for 25-35min.

5. The analytical method of claim 1, wherein: the chromatographic column specification of the gas chromatograph in step (3) is selected from 30m0.32mm 1.8 μm or 30m0.25mm 0.25 μm; the flow rate of the column carrier gas is set to be 0.8-1.2ml/min.

6. The analytical method of claim 1, wherein: setting the temperature of a sample inlet to be 220-240 ℃ in the step (3); the flow dividing ratio is 5-30; the detector temperature was set to 250-290 ℃.

7. The analytical method of claim 1, wherein: and (4) selecting a hydrogen flame ionization detector as the detector in the step (3).

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