CN111413427A

CN111413427A - Method for measuring imipenem content in blood plasma by ultrafiltration pretreatment high performance liquid chromatography

Info

Publication number: CN111413427A
Application number: CN202010270605.8A
Authority: CN
Inventors: 邹皓月; 蒙晋宁; 王贯宇
Original assignee: Suzhou Hehe Medical Test Co ltd
Current assignee: Suzhou Hehe Medical Test Co ltd
Priority date: 2020-04-08
Filing date: 2020-04-08
Publication date: 2020-07-14

Abstract

The invention discloses a method for determining imipenem content in plasma by ultrafiltration pretreatment high performance liquid chromatography, which comprises the following steps of 1) putting a plasma sample into an inner tube of an ultrafiltration centrifugal tube, adding an internal standard working solution and a stabilizer, performing vortex mixing to form a sample, 2) centrifuging the sample in the step 1), filtering out a filtrate, and performing sample injection detection on the filtrate, wherein the internal standard working solution in the step 1) is one selected from 5-hydroxyindole-3-acetic acid or 5-bromouracil.

Description

Method for measuring imipenem content in blood plasma by ultrafiltration pretreatment high performance liquid chromatography

Technical Field

The invention belongs to the technical field of in vivo drug analysis, and particularly relates to a method for determining the content of imipenem in blood plasma by ultrafiltration pretreatment and high performance liquid chromatography.

Background

Imipenem (IMP) is a carbapenem antibiotic with the chemical name of (5R,6S) -6- [ (1R) -1-hydroxyethyl ] -3- [ [2- [ (iminomethyl) amino ] ethyl ] thio ] -7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylic acid, has the characteristics of wide antibacterial spectrum, strong antibacterial effect and high stability to various β -lactamase enzymes, is mainly used for the treatment of multidrug-resistant gram-negative bacillus infection, mixed infection of severe aerobic bacteria and anaerobic bacteria and pathogen-undetermined severe infection, is one of the anti-infective drugs for critically infected patients, and has the structure shown in fig. 1.

The method for detecting imipenem has few domestic and foreign literature reports, and mainly adopts a hydrophilic interaction chromatography-mass spectrometry detection method and an HP L C-ultraviolet detection method, the mass spectrometry detection method has high requirements on instruments and is difficult to popularize at home, the reported HP L C-ultraviolet detection method has the defects of lack of a better protein treatment method or unstable baseline and the like, and the literature reports that imipenem has extremely high requirements on temperature and poor stability in plasma.

The above description is included in the technical recognition scope of the inventors, and does not necessarily constitute the prior art.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a method for determining the content of imipenem in blood plasma by using ultrafiltration pretreatment high performance liquid chromatography, which solves the problem of unstable baseline in the determination of the content of imipenem by using an HP L C-ultraviolet detection method in the prior art, ensures the stability of imipenem in blood plasma while improving the detection efficiency, prolongs the service life of a chromatographic column, and improves the accuracy and the repeatability of a determination result.

In order to achieve the aim, the invention provides a method for measuring the content of imipenem in blood plasma by ultrafiltration pretreatment high performance liquid chromatography, which is characterized by comprising the following steps:

1) putting the plasma sample into an inner tube of an ultrafiltration centrifugal tube, adding an internal standard working solution and a stabilizing agent, and performing vortex mixing to form a sample;

2) centrifuging the sample in the step 1), filtering out a filtrate, and carrying out sample injection detection on the filtrate;

wherein the internal standard working solution in the step 1) is selected from one of 5-hydroxyindole-3-acetic acid or 5-bromouracil.

Preferably, the internal standard working solution 5-bromouracil in the step 1) has a mass concentration of 0.5-3 mg/m L.

Preferably, the stabilizer described in step 1) comprises: according to volume ratio, MOPS solution: water: ethylene glycol 2: 1: 1.

further preferably, the MOPS solution has a molar concentration of 0.5 mol/L and a pH value ranging from 6.5 to 7.5, and the pH value of the MOPS solution is adjusted by using a NaOH solution with a mass fraction of 50%.

Preferably, in the step 1), a vortex instrument is adopted for vortex mixing, the rotating speed of the vortex instrument is 2000r/min, and the vortex mixing time is 0.1-5 min.

Preferably, in the step 2), the centrifugation is performed at a centrifugation speed of 8000rpm to 15000rpm for 5min to 15min at a centrifugation temperature of 2 ℃ to 25 ℃.

Preferably, the following components: step 1) and step 2) are to operate a plasma sample in an ice bath environment, wherein the temperature range of the plasma sample in the step 1) is 2-8 ℃.

Preferably, the sample injection detection in step 2) is performed by an ultraviolet detector, the ultraviolet detector comprises a chromatographic column, the chromatographic column is a C18 chromatographic column, and the mobile phase in the C18 chromatographic column consists of a liquid a and a liquid B, wherein the liquid a is an aqueous phase, the liquid B is an organic phase, the liquid a is selected from one of pure water, ammonium acetate water or an amine formate aqueous solution, and the liquid B is selected from one of methanol or acetonitrile.

More preferably, the liquid A in the mobile phase is 10 mmol/L ammonium acetate aqueous solution, the liquid B is methanol, gradient elution is adopted to enable the flow rate of the mobile phase to be 1m L/min, and the volume ratio of the liquid A to the liquid B is 90: 10-100: 0, preferably 97: 3.

Further preferably, the detection wavelength of the ultraviolet detector is 300nm, and the column temperature of the chromatographic column is 25 ℃.

The method for determining the content of imipenem in blood plasma by ultrafiltration pretreatment high performance liquid chromatography provided by the invention can bring the following beneficial effects:

1. the protein is separated by adopting the ultrafiltration tube pretreatment method, the operation is simple, the separation effect is good, the cost is low, and the problem that the detection requirements cannot be met by liquid-liquid extraction pretreatment and protein precipitation pretreatment is solved.

2. The pretreatment method of the ultrafiltration tube is adopted under the condition of optimizing the mobile phase (the solution A is 10 mmol/L ammonium acetate aqueous solution, the solution B is methanol), the base line is stable, the main peak has good shape, the main peak can be better separated from the impurity peak and the impurities, and the high water phase can wash the impurities in the sample.

3. The pretreatment is operated in an ice bath environment, the temperature of imipenem in a sample is ensured to be 2-8 ℃, and the problems of temperature sensitivity and instability of imipenem are solved.

4. The assay method of the present invention has excellent batch-to-batch and column-to-column reproducibility.

5. The measuring method of the invention takes 300nm as the detection wavelength, and can detect more impurities, thereby more comprehensively knowing and detecting the quality of the imipenem product.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a schematic diagram of the structure of imipenem;

FIG. 2 is an imipenem HP L C spectrum measured by liquid-liquid extraction pretreatment;

FIG. 3 is an imipenem HP L C spectrum measured by the method of the present invention using ultrafiltration tube pretreatment;

FIG. 4 is an Imipenem HP L C profile determined using a precipitated protein pretreatment protocol;

FIG. 5 is an imipenem HP L C spectrum measured using plastic cannula placement according to the methods of the present invention;

FIG. 6 is an Imipenem HP L C profile determined using glass cannula placement according to the methods of the present invention;

FIG. 7 is a graph of the method for determining imipenem using gradient elution I without high water phase washing of the chromatographic column of the present invention;

FIG. 8 is a graph of a method of the present invention for determining imipenem using gradient elution II, requiring high water phase washing of the chromatographic column;

FIG. 9 is a flow chart illustrating the use of an ultrafiltration tube in the method of the present invention.

Detailed description of the preferred embodiments

In order to more clearly illustrate the overall concept of the present invention, the present invention is further described in detail with reference to the following detailed description, and the experimental examples are given for the purpose of illustrating the present invention only and are not intended to limit the scope of the present invention. The experimental procedures in the following experimental examples are conventional ones unless otherwise specified. The instruments, reagents and consumables used in the following experimental examples are all commercially available products unless otherwise specified.

The manufacturer of the imipenem Standard used in the experimental examples and comparative examples was EuropeanePharmacopeeiaReference Standard, and the manufacturer of the 5-olfactory uracil Standard was CATO, purchased by itself.

The instrument is a Thermo Fisher scientific U L timate 3000 high performance liquid chromatograph.

Column Sun Fire C18(5 μm, 150mm × 4.6.6 mm).

Factors such as pretreatment mode, mobile phase type, mobile phase gradient proportion, inner cannula selection and the like all influence the detection of imipenem. Therefore, the chromatographic conditions are optimized by the following experimental examples.

The method for determining the content of imipenem in plasma by ultrafiltration pretreatment high performance liquid chromatography adopts an ultrafiltration centrifugal tube to carry out pretreatment separation on plasma protein, and preferably adopts a 30K ultrafiltration centrifugal tube to carry out pretreatment separation on plasma protein; the pretreatment operation is carried out in an ice bath environment, and the plasma sample is kept at 2-8 ℃.

The method comprises the step of carrying out gradient elution on a system consisting of a liquid A and a liquid B, wherein the liquid A is pure water, an ammonium acetate aqueous solution with a molar concentration of 10 mmol/L, an ammonium acetate aqueous solution with a molar concentration of 30 mmol/L and an ammonium formate aqueous solution with a molar concentration of 10 mmol/L, preferably an ammonium acetate aqueous solution with a molar concentration of 10 mmol/L, and the liquid B is methanol or acetonitrile, preferably methanol.

When elution is started, the volume ratio of the solution A to the solution B is 90: 10-100: 0, preferably 97: 3, the gradient flow rate is 1m L/min, and the detection column temperature of the method is 20-40 ℃, preferably 25 ℃.

The chromatographic column used in the method is selected from Sun Fire C18 chromatographic column, Xbridge C18 chromatographic column and Acclaim^TMPolar Advantage II C18 chromatography column, more preferably Sun FireC18 chromatography column.

The detection wavelengths of the method are 254nm, 299nm, 203nm and 300nm, and more preferably 300 nm.

The method autosampler temperature is preferably 4 ℃; the chromatographic column is flushed by using a large proportion of high-water phase solution, so that the phenomenon that other impurities in a sample block the chromatographic column to cause the column efficiency to be reduced is prevented.

Plasma sample solutions were run as follows:

precisely transferring a 200 mu L plasma sample into an ultrafiltration centrifugal tube, adding 200 mu L stabilizer MOPS (3- (N-morpholinyl) propanesulfonic acid), adding 10 mu L of 5-bromouracil or-3-acetic acid internal standard working solution with the concentration of 0.5-3 mg/m L, preferably 1mg/m L, uniformly mixing by adopting 2000r/min vortex for 0.5min, centrifuging at 14000rpm for 5min, taking 100 mu L of filtrate, and putting the filtrate into a sample injection vial inner cannula for sample injection detection.

Preparation of a reference stock solution:

precisely weighing imipenem reference substance (not less than 2.070mg), correcting by correction factor, adding stabilizer MOPS by pipette to obtain 1mg/m L stock solution, and storing in-80 deg.C refrigerator.

Preparing a standard curve working solution of a reference substance:

transferring a proper amount of imipenem stock solution (1mg/m L) by a pipette, diluting the stock solution by a stabilizing agent MOPS step by step to prepare standard curve working solution with various concentrations, and storing the standard curve working solution in a refrigerator at the temperature of 80 ℃ below zero, wherein the specific dilution steps are shown in a table 1:

TABLE 1

The preparation of the working solution can proportionally enlarge or reduce the total volume according to actual conditions.

Preparation of a standard curve sample:

respectively and precisely transferring 190 mu L of a blank plasma sample of a normal human body by using a pipette, placing the blank plasma sample into a 500 mu L ultrafiltration tube, respectively adding 10 mu L of imipenem working solution with 8 series of concentrations, and uniformly mixing the mixture by vortex for 0.5min to prepare a standard curve sample with the concentration range of 250-50000 ng/m L, wherein the standard curve sample needs to be prepared fresh during each measurement, and the specific dilution steps are shown in table 2:

TABLE 2

Preparing a quality control stock solution:

Preparing a quality control working solution:

transferring the imipenem stock solution (1mg/m L) by a liquid transfer machine, diluting the stock solution by the stabilizing agent MOPS step by step to prepare quality control working solutions with various concentrations, and storing the quality control working solutions in a refrigerator at the temperature of minus 80 ℃, wherein the specific dilution steps are shown in a table 3:

TABLE 3

Preparing a quality control sample:

precisely transferring a blank plasma sample 190 mu L by using a pipette, adding a quality control working solution corresponding to 10 mu L, uniformly mixing for 0.5min in a vortex manner to obtain quality control samples with the concentrations of 40000, 7500 and 750ng/m L respectively.

Preparing an internal standard working solution:

precisely weighing 5-olfactory uracil (not less than 2.070mg) or-3-acetic acid, adding stabilizer MOPS (mols) by using a pipette to prepare an internal standard working solution of 1mg/m L, and storing in a refrigerator at-20 ℃.

And (3) processing a standard curve sample and a quality control sample:

adding a stabilizer MOPS200 mu L into a prepared standard curve sample of 200 mu L, putting the standard curve sample into an ultrafiltration centrifugal tube, adding 5-olfactory uracil internal standard working solution with the concentration of 10 mu L being 0.5-3 mg/M L, preferably adding 1mg/M L of the 5-olfactory uracil internal standard working solution, uniformly mixing by adopting 2000r/min vortex for 0.5min, centrifuging at 14000rpm for 5min, taking 100 mu L of filtrate, and putting the filtrate into a sample injection vial inner cannula for sample injection detection.

Transferring a prepared quality control sample of 200 mu L, adding a stabilizer MOPS200 mu L, putting into an ultrafiltration centrifugal tube, adding 5-olfactory uracil internal standard working solution with the concentration of 10 mu L being 1mg/m L, uniformly mixing by vortex at 2000r/min for 0.5min, centrifuging at 14000rpm for 5min, taking filtrate of 100 mu L, and putting into a sample injection vial inner insert tube for sample injection detection.

As shown in fig. 2 to 4:

1. liquid-liquid extraction pretreatment is adopted.

The specific pretreatment method comprises the steps of transferring a blank plasma sample 190 mu L, adding 10 mu L standard substance, adding stabilizer MOPS200 mu L, adding 10 mu L internal standard solution, adding 400 mu L acetonitrile precipitant, placing the mixture into a 1.5m L plastic tube, uniformly mixing at 2000r/min in a vortex manner for 0.5min, centrifuging at 14000rpm for 5min, taking supernatant fluid 400 mu L, adding 400 mu L dichloromethane extractant, placing the mixture into a 1.5m L plastic tube, uniformly mixing at 2000r/min in a vortex manner for 0.5min, centrifuging at 14000rpm for 5min, taking supernatant fluid 100 mu L, placing the supernatant fluid into an insert tube of a sample injection vial for sample injection detection, and obtaining a detection result shown in figure 2.

Chromatographic column of Sun Fire C18(5 μm, 150mm, × 4.6.6 mm), column temperature of 25 deg.C, autosampler temperature of 4 deg.C, mobile phase of 10 mmol/L ammonium acetate water solution as liquid A and methanol as liquid B;

the gradient elution was performed as per the procedure of table 4:

TABLE 4

t(min)	0	4	5	7
					A(V/％)	97	80	97	97
B(V/％)	3	20	3	3

The flow rate is 1m L/min, and the detection wavelength is 300 nm.

The detection result shows that the compound and the impurities cannot be completely separated by the liquid-liquid extraction pretreatment mode, and the compound and the impurities cannot reach a good separation degree; the liquid-liquid extraction mode is complex and the time is long; the imipenem is reported in documents to be sensitive to temperature change and unstable at normal temperature, and the treatment mode cannot meet the requirement; the lowest quantitative lower limit after liquid-liquid extraction can not meet the basic requirement of detection, so the pretreatment mode is not adopted.

2. And (4) adopting an ultrafiltration tube for pretreatment.

The pretreatment method comprises the steps of transferring a blank plasma sample of 190 mu L, adding a standard substance of 10 mu L, adding a stabilizer MOPS of 200 mu L, then placing the blank plasma sample into an ultrafiltration centrifugal tube, adding a 5-olfactory uracil internal standard working solution with the concentration of 10 mu L of 1mg/m L, selecting 2000r/min by a vortex instrument, carrying out vortex mixing for 0.5min, centrifuging by a centrifuge under the condition of 14000rpm and 5min, taking a filtrate of 100 mu L, transferring the filtrate into a sample injection vial by an automatic sample injector, inserting the vial into the sample injection vial for sample injection detection, and obtaining a detection result shown in figure 3.

In the preferred embodiment of the present invention, the stabilizer includes MOPS, water and ethylene glycol, wherein the volume ratio of MOPS to water to ethylene glycol is 2: 1: 1, and the molar concentration of MOPS is 0.5 mol/L, and the ultrafiltration centrifugal tube may be a 10K or 30K ultrafiltration centrifugal tube.

Furthermore, as a further preferable aspect of the present invention, the 0.5 mol/L MOPS solution is adjusted to have a pH value ranging from 6.5 to 7.5 by using a 50% NaOH solution.

Further, as a variation of the first embodiment of the present invention, the time range of the vortex kneading may be implemented to be 0.1min to 5 min. The centrifuge rotation speed range can be implemented to be 8000-15000 rpm. The centrifugal temperature range of the centrifugal machine is 2-25 ℃, and the preferred centrifugal temperature range is 4 ℃.

In the preferred embodiment of the present invention, the sample injection detection is performed by an ultraviolet detector, the ultraviolet detector comprises a chromatographic column, the chromatographic column is a C18 chromatographic column, and the mobile phase in the C18 chromatographic column consists of a liquid a and a liquid B, wherein the liquid a is an aqueous phase and the liquid B is an organic phase.

The liquid A in the mobile phase is 10 mmol/L ammonium acetate aqueous solution, the liquid B is methanol, gradient elution is adopted to enable the flow rate of the mobile phase to be 1m L/min, then an automatic sample injector is used for transferring the filtrate into the inner insertion tube of the sample injection vial for sample injection detection, a chromatographic column is Sun Fire C18(5 mu m, 150mm × 4.6.6 mm), the column temperature is 25 ℃, the temperature of the automatic sample injector is 4 ℃, the liquid A is 10 mmol/L ammonium acetate aqueous solution, and the liquid B is methanol;

the gradient elution was performed as per the procedure of table 5:

TABLE 5

t(min)	0	4	5	7
					A(V/％)	97	80	97	97
B(V/％)	3	20	3	3

The flow rate is 1m L/min, and the detection wavelength is 300 nm;

according to the experimental result, the compound can be separated from the impurities only by centrifugation in the experiment, a precipitator and a liquid-liquid extracting agent are not required to be added, the interference on the compound is small, and the operation is simple; carrying out experimental operation in an ice bath environment to meet the requirement of imipenem on sensitivity to temperature change; as can be seen from the chromatogram, the baseline is stable, the peak shape and the separation degree are good in the pretreatment mode, and the quantitative requirement is met.

3. Pre-treating the precipitated protein.

The specific pretreatment method comprises the steps of transferring a blank plasma sample of 190 mu L, adding a standard substance of 10 mu L, whirling at 2000r/min for 30s, adding an internal standard solution of 10 mu L, adding a acetonitrile precipitant of 400 mu L, whirling at 2000r/min for uniformly mixing for 3min, centrifuging at 14000rpm for 10min by a centrifuge, and taking a supernatant of 200 mu L to a sample injection vial for sample injection detection, wherein detection results are shown in figure 4.

Chromatographic column of Sun Fire C18(5 μm, 150mm, × 4.6.6 mm), column temperature of 25 deg.C, autosampler temperature of 4 deg.C, mobile phase of 10 mmol/L ammonium acetate water solution as liquid A and methanol solution as liquid B;

upon trial, the final elution procedure was determined to be shown in table 6 below:

TABLE 6

t(min)	0	4	5	7
					A(V/％)	97	80	97	97
B(V/％)	3	20	3	3

The flow rate is 1m L/min, and the detection wavelength is 300 nm;

through the pretreatment mode, the precipitant has partial dilution effect on the sample and cannot meet the requirement of quantitative lower limit; and the liquid phase method finds that the compound can not be separated from impurities, the baseline fluctuation is large, and the quantitative requirement can not be met.

As shown in fig. 5 and 6:

the chromatographic column comprises Sun Fire C18(5 mu m, 150mm × 4.6.6 mm), the column temperature is 25 ℃, the autosampler temperature is 4 ℃, and the mobile phases comprise A liquid which is pure water, 10 mmol/L ammonium acetate aqueous solution, 30 mmol/L ammonium acetate aqueous solution and 10 mmol/L ammonium formate aqueous solution, and B liquid which is methanol or acetonitrile;

the gradient elution was performed according to the following procedure 7:

TABLE 7

t(min)	0	4	5	7
					A(V/％)	97	80	97	97
B(V/％)	3	20	3	3

The flow rate is 1m L/min, and the detection wavelength is 300 nm;

the mobile phase selects water phase (pure water, 10 mmol/L ammonium acetate aqueous solution, 30 mmol/L ammonium acetate aqueous solution and 10 mmol/L ammonium formate aqueous solution), organic phase (methanol and acetonitrile) to carry out the method groping, and the specific experimental results are shown in the following table 8:

TABLE 8

the gradient elution was performed according to the following procedure in table 9:

TABLE 9

t(min)	0	4	5	7
					A(V/％)	97	80	97	97
B(V/％)	3	20	3	3

The flow rate is 1m L/min, the detection wavelength is 300nm, and the specific spectrum is 2 and 3.

Through a series of adjustment and analysis, a glass container is found to have a certain adsorption effect on imipenem, the obtained sample is directly placed in a brown glass liquid phase vial in the early stage, repeated sample injection is carried out, the chromatographic peak area of imipenem is found to be irregularly reduced, and the internal standard peak area is in a stable state; then, comparison is carried out on the stock solution in the glass vial, and the peak area of the stock solution is found to be reduced compared with that of the stock solution just prepared, so that certain adsorption effect on the stock solution is presumed to be generated by glass in a liquid state in an MOPS system.

In addition, as shown in FIG. 7, FIG. 8 and FIG. 9, the column was Sun Fire C18(5 μm, 150mm × 4.6.6 mm), the column temperature was 25 ℃ and the autosampler temperature was 4 ℃ and the mobile phase was 10 mmol/L ammonium acetate aqueous solution as solution A and methanol as solution B;

the gradient elution i was performed according to the procedure of table 10 below:

watch 10

t(min)	0	4	5	7
					A(V/％)	97	80	97	97
B(V/％)	3	20	3	3

The flow rate is 1m L/min, the detection wavelength is 300nm, and the specific figure is 7.

After long-time sample treatment, the liquid phase gradient elution is found to be short in time, but has great damage to the column, because a large amount of impurities in the sample cannot be completely eluted, the column effect of the chromatographic column is irreversibly influenced, and even after a complete analysis batch, the influence on the chromatographic column cannot be reduced by adding a long-time column washing step.

Thus, in a first embodiment of the invention, the liquid phase gradient was optimized by adding a long elution schedule after each needle of the liquid phase method, which is the optimized liquid phase elution schedule, gradient elution ii, as shown in table 11 below:

TABLE 11

t(min)	0	7.5	8.5	10	11	15
							A(V/％)	97	97	10	10	97	97
B(V/％)	3	3	90	90	3	3

See in particular fig. 8.

In summary, the following steps: the detection method can realize the good separation of imipenem from impurities and impurities, and the combination of imipenem and cilastatin can avoid the destruction of imipenem by renal dehydrogenation coenzyme I, and the experimental method can avoid the interference of cilastatin on an imipenem chromatogram map, can better quantify imipenem in a sample, and plays a guiding role in the medication of patients.

The embodiments in the present specification are described in a progressive manner, and the same and similar parts among the embodiments are referred to each other, and each embodiment focuses on the differences from the other embodiments. In particular, for the system embodiment, since it is substantially similar to the method embodiment, the description is simple, and for the relevant points, reference may be made to the partial description of the method embodiment.

The above description is only an example of the present invention, and is not intended to limit the present invention. Various modifications and alterations to this invention will become apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for measuring the content of imipenem in blood plasma by ultrafiltration pretreatment high performance liquid chromatography is characterized by comprising the following steps:

2. The method for determining the content of imipenem in plasma by pre-ultrafiltration high performance liquid chromatography according to claim 1, wherein the mass concentration of the internal standard working solution 5-bromouracil in the step 1) is 0.5-3 mg/m L.

3. The method for determining the content of imipenem in plasma by pre-ultrafiltration high performance liquid chromatography as claimed in claim 1, wherein the stabilizer in step 1) comprises: according to volume ratio, MOPS solution: water: ethylene glycol 2: 1: 1.

4. the method for determining the content of imipenem in plasma by pre-ultrafiltration treatment high performance liquid chromatography as claimed in claim 3, wherein the MOPS solution has a molar concentration of 0.5 mol/L and a pH value ranging from 6.5 to 7.5, and the pH value of the MOPS solution is adjusted by using a NaOH solution with a mass fraction of 50%.

5. The method for determining the imipenem content in the plasma by ultrafiltration pretreatment high performance liquid chromatography according to claim 1, wherein a vortex mixer is adopted to carry out vortex mixing in the step 1), the rotation speed of the vortex mixer is 2000r/min, and the vortex mixing time is 0.1-5 min.

6. The method for determining the content of imipenem in plasma by pre-ultrafiltration treatment high performance liquid chromatography as claimed in claim 1, wherein the centrifugation in step 2) is performed at 8000rpm to 15000rpm for 5min to 15min at a temperature of 2 ℃ to 25 ℃.

7. The method for determining the content of imipenem in plasma by pre-ultrafiltration high performance liquid chromatography as claimed in claim 1, wherein the steps 1) and 2) are performed by operating a plasma sample in an ice bath environment, wherein the temperature of the plasma sample in the step 1) is in the range of 2-8 ℃.

8. The method for determining imipenem content in plasma by pre-ultrafiltration treatment high performance liquid chromatography as claimed in claim 1, wherein the sample injection detection in step 2) is performed by an ultraviolet detector, the ultraviolet detector comprises a chromatographic column, the chromatographic column is a C18 chromatographic column, the mobile phase in the C18 chromatographic column consists of solution A and solution B, wherein the solution A is an aqueous phase, the solution B is an organic phase, the solution A is one selected from pure water, ammonium acetate water or ammonium formate water solution, and the solution B is one selected from methanol or acetonitrile.

9. The method for determining the content of imipenem in plasma by pre-ultrafiltration high performance liquid chromatography as claimed in claim 8, wherein the A liquid in the mobile phase is 10 mmol/L ammonium acetate aqueous solution, the B liquid is methanol, and gradient elution is adopted to enable the flow rate of the mobile phase to be 1m L/min, and the volume ratio of the A liquid to the B liquid is 90: 10-100: 0, preferably 97: 3.

10. The method for determining the content of imipenem in plasma by pre-ultrafiltration high performance liquid chromatography as claimed in claim 8, wherein the detection wavelength of the ultraviolet detector is 300nm, and the column temperature of the chromatographic column is 25 ℃.