CN118050452A - HPLC detection method for content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride - Google Patents
HPLC detection method for content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride Download PDFInfo
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Abstract
The application relates to the technical field of analytical chemistry, and particularly discloses an HPLC (high performance liquid chromatography) detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride. The HPLC detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride provided by the application comprises the following steps: (1) preparing a reference substance solution and a test substance solution; (2) Injecting the reference substance solution and the sample solution into a liquid chromatograph, and separating and detecting by adopting a chromatographic column filled with octadecylsilane chemically bonded silica gel and a mobile phase gradient elution method; (3) Recording the chromatogram, and calculating the content of 4-methyl-3-pentene-2 ketone in the sample according to the external standard method and peak area. The HPLC detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride provided by the application can be used for rapidly and accurately detecting the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride, and has the advantages of strong detection specificity, high sensitivity and good separation degree.
Description
Technical Field
The application relates to the technical field of analytical chemistry, in particular to an HPLC (high performance liquid chromatography) detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride.
Background
The lidocaine hydrochloride belongs to local anesthetics, has the characteristics of quick effect, strong dispersion, good anesthetic effect and the like, and has the dosage forms of injection, gel, ophthalmic gel, mucilage, local solution and the like, and has very wide clinical application. The chemical name of lidocaine hydrochloride is N- (2, 6-xylyl) -2- (diethylamino) acetamide hydrochloride monohydrate, and the structural formula is as follows:HCl，H2O
In the existing lidocaine hydrochloride synthetic route, the step of reacting lidocaine alkali with concentrated hydrochloric acid in acetone solvent is involved, because acetone can be subjected to self condensation under the catalysis of acidic conditions, so that 4-methyl-3-pentene-2 ketone impurities are generated, 4-methyl-3-pentene-2 ketone has an alpha, beta-unsaturated ketone warning structure, a compound with the warning structure is defined as potential genotoxic impurities by ICH (international coordination of human drug registration requirements), and because the genotoxic impurities can induce gene mutation at very low concentration and lead to chromosome breakage and rearrangement, and have potential carcinogenicity, the genotoxic impurities research has become one of the keys for ensuring the quality of medicines.
However, there is no report on the detection of 4-methyl-3-penten-2-one, a genotoxic impurity in lidocaine hydrochloride, at present. Therefore, it is needed to provide a detection method of 4-methyl-3-pentene-2 ketone with simple operation, high accuracy and high sensitivity to realize effective control of lidocaine hydrochloride quality, so as to ensure medication safety of human body.
Disclosure of Invention
In order to rapidly and accurately detect the content of 4-methyl-3-pentene-2 ketone as an impurity in lidocaine hydrochloride, the application provides an HPLC detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride.
The HPLC detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride provided by the application adopts the following technical scheme:
An HPLC detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride comprises the following steps:
(1) Preparing a reference substance solution and a test substance solution;
(2) Injecting the reference substance solution and the sample solution into a liquid chromatograph, and separating and detecting by adopting a chromatographic column filled with octadecylsilane chemically bonded silica gel and a mobile phase gradient elution method;
(3) Recording a chromatogram, and calculating the content of 4-methyl-3-pentene-2 ketone in the sample according to an external standard method and peak area;
The mobile phase gradient elution method comprises the following steps: 0-18min, mobile phase A is 100%, mobile phase B is 0%;18-45min, mobile phase A is reduced from 100% to 37.5%, and mobile phase B is increased from 0% to 62.5%;45-60min, mobile phase A of 37.5% and mobile phase B of 62.5%;60-63min, mobile phase A increased from 37.5% to 100%, mobile phase B decreased from 62.5% to 0%;63-80min, mobile phase A is 100%, mobile phase B is 0%.
In order to realize the content of genotoxic impurity 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride, the application provides a High Performance Liquid Chromatography (HPLC) detection method, and the detection method can rapidly and accurately detect the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride by controlling a mobile phase gradient elution method and related detection conditions, and has the advantages of strong detection specificity, high sensitivity and good separation degree; the detection method is used for the production of the lidocaine hydrochloride preparation, and can realize the effective control of the quality of the lidocaine hydrochloride preparation.
In the application, the chromatographic column can be a chromatographic column with octadecylsilane chemically bonded silica as a filler or a chromatographic column with equivalent efficacy.
Optionally, the mobile phase A is monoammonium phosphate solution, methanol and acetonitrile with the volume ratio of (75-85): (8-12): (8-12); the mobile phase B is methanol and acetonitrile with the volume ratio of (0.8-1.2).
Optionally, the concentration of the ammonium dihydrogen phosphate solution is 11.5g/L, and the pH is 2.8-3.0.
In the application, the preparation method of the monoammonium phosphate solution comprises the following steps: weighing 11-12g of monoammonium phosphate, adding water for dissolution and dilution to 1L, and regulating the pH value to 2.9 by using phosphoric acid.
In some embodiments, the pH of the ammonium dihydrogen phosphate solution can be from 2.85 to 2.9 or from 2.9 to 2.95.
In a specific embodiment, the pH of the ammonium dihydrogen phosphate solution can also be 2.85, 2.9, or 2.95.
Optionally, in the separation detection process, the column temperature is 35-45 ℃, and the flow rate of the mobile phase is 0.7-0.9mL/min.
In some embodiments, the column temperature may be 35-40 ℃ or 40-45 ℃.
In a specific embodiment, the column temperature may also be 35 ℃, 40 ℃ or 45 ℃.
In some embodiments, the mobile phase may have a flow rate of 0.7 to 0.8mL/min or 0.8 to 0.9mL/min.
In a specific embodiment, the mobile phase flow rate may also be 0.7mL/min, 0.8mL/min, or 0.9mL/min.
Optionally, in the separation detection process, the sample injection amount is 18-22 mu L, and the detection wavelength is 208-212nm.
In some embodiments, the detection wavelength may be 208-210nm or 210-212nm.
In a specific embodiment, the detection wavelength may also be 208nm, 210nm or 212nm.
Optionally, the chromatographic column isXB-C18, specification 4.6X250 mm,5 μm.
Optionally, the preparation method of the reference substance solution comprises the following steps: taking 4-methyl-3-pentene-2 ketone reference substance, dissolving and diluting with mobile phase A to prepare reference substance solution with the concentration of 4-methyl-3-pentene-2 ketone of 9 mug/mL.
Optionally, the preparation method of the sample solution comprises the following steps: and dissolving lidocaine hydrochloride by using a mobile phase A, and quantitatively diluting to prepare a test solution containing lidocaine hydrochloride with the concentration of 3 mg/mL.
In the application, the content of 4-methyl-3-pentene-2 ketone in the sample can be obtained by detecting the peak areas of the sample solution and the reference solution through the following formula:
In the above formula, a Feed device : peak area of 4-methyl-3-pentene-2 ketone in the test solution; a For a pair of : peak area of the control; m For a pair of : weighing the reference substance (mg); p For a pair of : the content of the reference substance; v For a pair of : dilution volume of control (mL); v Feed device : sample dilution volume (mL); m Feed device : sample weight (mg) of test sample.
In summary, the application has the following beneficial effects:
1. The application provides a method for detecting genotoxic impurity 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride bulk drug by adopting high performance liquid chromatography, which is verified by methodologies such as system applicability, accuracy, quantitative limit and detection limit, linear range, precision, durability and the like, and the method is strong in specificity, high in sensitivity, good in accuracy, good in flow rate, column temperature, wavelength, mobile phase pH value and durability of chromatographic column, suitable for detecting genotoxic impurity 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride bulk drug, and capable of realizing effective control of quality of lidocaine hydrochloride bulk drug.
2. The HPLC detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride provided by the application has high detection sensitivity, the quantitative limit of 4-methyl-3-pentene-2 ketone is 0.013 mug/mL, and the detection limit is 0.0039 mug/mL.
3. The HPLC detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride provided by the application has good detection accuracy, the recovery rate of 4-methyl-3-pentene-2 ketone is in the range of 50% -150%, and the RSD of 9 parts of recovery rate is 1.5% and less than 6%.
Drawings
FIG. 1 is a detection spectrum obtained by detecting a control solution using the detection method provided in example 1;
FIG. 2 is a detection spectrum obtained by detecting a sample solution using the detection method provided in example 1;
FIG. 3 is a graph of 4-methyl-3-penten-2-one concentration versus peak area;
FIG. 4 is a quantitative HPLC chart of 4-methyl-3-penten-2-one;
FIG. 5 is a detection limit HPLC chart of 4-methyl-3-penten-2-one.
Detailed Description
The application provides an HPLC detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride, which specifically comprises the following steps:
(1) Preparing a reference substance solution and a test substance solution;
(1-1) preparation of a control solution: taking 4-methyl-3-pentene-2 ketone reference substance, dissolving and diluting with mobile phase A to prepare reference substance solution with the concentration of 4-methyl-3-pentene-2 ketone of 9 mug/mL.
(1-2) Preparation of a test sample solution: and dissolving and diluting lidocaine hydrochloride by using a mobile phase A to prepare a test sample solution containing lidocaine hydrochloride with the concentration of 3 mg/mL.
(2) Injecting the reference substance solution and the sample solution into a liquid chromatograph, and separating and detecting by adopting a chromatographic column filled with octadecylsilane chemically bonded silica gel and a mobile phase gradient elution method; in the separation detection process, the column temperature is 35-45 ℃, the flow rate of the mobile phase is 0.7-0.9mL/min, the sample injection amount is 18-22 mu L, and the detection wavelength is 208-212nm.
(3) Recording a chromatogram, and calculating the content of 4-methyl-3-pentene-2 ketone in the sample according to an external standard method and peak area;
The mobile phase gradient elution method comprises the following steps: 0-18min, mobile phase A is 100%, mobile phase B is 0%;18-45min, mobile phase A is reduced from 100% to 37.5%, and mobile phase B is increased from 0% to 62.5%;45-60min, mobile phase A of 37.5% and mobile phase B of 62.5%;60-63min, mobile phase A increased from 37.5% to 100%, mobile phase B decreased from 62.5% to 0%;63-80min, mobile phase A is 100%, mobile phase B is 0%.
Wherein the mobile phase A is ammonium dihydrogen phosphate solution, methanol and acetonitrile with the volume ratio of (75-85): 8-12; the mobile phase B is methanol and acetonitrile with the volume ratio of (0.8-1.2). The concentration of the ammonium dihydrogen phosphate solution is 11-12g/L, and the pH value is 2.8-3.0.
The sources of the raw materials used in the present application are shown in the following table 1, and the rest raw materials, reagents, solvents, etc. are commercially available.
TABLE 1 sources of raw materials in the present application
The present application will be described in further detail with reference to examples, test experiments and accompanying drawings.
Example 1
Example 1 provides an HPLC detection method for 4-methyl-3-pentene-2 ketone content in lidocaine hydrochloride, which specifically comprises the following steps:
(1) Preparing a reference substance solution and a test substance solution;
(1-1) preparation of a control solution: taking about 9mg of 4-methyl-3-pentene-2 ketone reference substance, placing the reference substance into a 100mL measuring flask, adding a mobile phase A to dissolve and dilute the reference substance to a scale, shaking the reference substance uniformly, precisely measuring 1mL of the reference substance, placing the reference substance into a 10mL measuring flask, diluting the reference substance into the scale by using the mobile phase A, and shaking the reference substance uniformly to prepare a reference substance solution with the concentration of 9 mug/mL of 4-methyl-3-pentene-2 ketone.
(1-2) Preparation of a test sample solution: and (3) taking a proper amount of lidocaine hydrochloride, and dissolving and diluting the lidocaine hydrochloride by using the mobile phase A to prepare a test sample solution with the lidocaine hydrochloride concentration of 3 mg/mL.
(2) Respectively precisely measuring 20 mu L of blank solvent, reference substance solution and sample solution, injecting into a liquid chromatograph, and separating and detecting by mobile phase gradient elution method; in the separation and detection process, the chromatographic column is as follows: XB-C18, specification of 4.6X250mm, 5 μm, column temperature of 40deg.C, flow rate of mobile phase of 0.8mL/min, sample injection amount of 20 μl, detection wavelength of 210nm, and pH of 2.9;
The mobile phase gradient elution method comprises the following steps: 0-18min, mobile phase A is 100%, mobile phase B is 0%;18-45min, mobile phase A is reduced from 100% to 37.5%, and mobile phase B is increased from 0% to 62.5%;45-60min, mobile phase A of 37.5% and mobile phase B of 62.5%;60-63min, mobile phase A increased from 37.5% to 100%, mobile phase B decreased from 62.5% to 0%;63-80min, mobile phase A is 100%, mobile phase B is 0%;
The mobile phase A is ammonium dihydrogen phosphate solution, methanol and acetonitrile with the volume ratio of 80:10:10; the mobile phase B is methanol and acetonitrile with the volume ratio of 1:1;
the preparation method of the monoammonium phosphate solution comprises the following steps: weighing 11-12g of monoammonium phosphate, adding water for dissolution and dilution to 1L, and regulating the pH value to 2.9 by using phosphoric acid.
(3) Recording a chromatogram, obtaining peak areas, and calculating the content of 4-methyl-3-pentene-2 ketone in the sample according to the following formula according to an external standard method:
In the above formula, a Feed device : peak area of 4-methyl-3-pentene-2 ketone in the test solution; a For a pair of : peak area of the control; m For a pair of : weighing the reference substance (mg); p For a pair of : the content of the reference substance; v For a pair of : dilution volume of control (mL); v Feed device : sample dilution volume (mL); m Feed device : sample weight (mg) of test sample.
(4) Detection result: the detection method is used for detecting the reference substance solution and the test substance solution, the detection spectrogram of the reference substance solution is shown in figure 1, and the detection spectrogram of the test substance solution is shown in figure 2. The detection results are shown in the following table 2.
TABLE 2 liquid chromatography detection results
According to the detection results of Table 2, the HPLC detection method for the content of 4-methyl-3-pentene-2 ketone can accurately detect 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride, and the separation degree between the 4-methyl-3-pentene-2 ketone and the adjacent chromatographic peaks in the obtained detection spectrogram is more than 1.5, so that the detection requirement is met.
Specificity and System applicability
(1) Preparing a reference substance solution, a test substance solution and a system applicability solution;
Preparing a system applicability solution: precisely weighing lidocaine reference substance 30mg, placing into 10mL measuring flask, measuring 4-methyl-3-pentene-2 ketone positioning solution 1mL, adding mobile phase A, dissolving, diluting to scale, and shaking.
(2) And precisely measuring 20 mu L of system applicability solution, injecting the solution into a liquid chromatograph, and recording a chromatogram.
(3) Detection result: the results of the detection of the system applicability solution are shown in the following table 3.
Table 3: methodological verification-systematic adaptability investigation results
From the detection results shown in tables 2 and 3, the separation degree between the 4-methyl-3-pentene-2 ketone and the adjacent chromatographic peak is larger than 1.5 in the sample solution and the system applicability solution, and the theoretical plate number meets the requirement. Therefore, the HPLC detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride provided by the application has good specificity and system applicability.
Precision of
(1) 20 Mu L of the reference solution was measured precisely, injected into a liquid chromatograph, detected by the method provided in example 1, sampled 6 times continuously, and the chromatogram was recorded.
(2) Detection result: the results of the precision measurement are shown in Table 4 below.
Table 4: methodological validation-precision investigation results
As can be seen from the test results shown in Table 4, the retention time RSD of 4-methyl-3-penten-2-one in the control solution was 0.03%, less than 1.0%, and the peak area RSD was 0.2%, less than 2.0% in the test results obtained by continuously feeding the sample 6 times. Therefore, the HPLC detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride provided by the application has good precision and meets the requirements.
Linear range
(1) Preparing a linear stock solution: about 9mg of 4-methyl-3-pentene-2 ketone reference substance is respectively taken, precisely weighed, placed in a 20mL measuring flask, added with mobile phase A for dissolution and dilution to scale, shaken well, precisely measured again for 5mL, placed in a 50mL measuring flask, diluted to scale with mobile phase A, shaken well and used as a linear stock solution.
Preparing a linear solution: the linear stock solutions were precisely measured and diluted with mobile phase A to obtain linear solutions having concentrations of 0.013. Mu.g/mL, 4.94. Mu.g/mL, 7.41. Mu.g/mL, 9.88. Mu.g/mL, 12.35. Mu.g/mL, 14.82. Mu.g/mL and 19.76. Mu.g/mL, respectively.
(2) 20. Mu.L of each of the above-mentioned linear solutions was measured precisely, and injected into a liquid chromatograph, and the linear solutions were examined in the manner provided in example 1, and a chromatogram was recorded, and a graph of the linear relationship between the concentration of 4-methyl-3-penten-2-one and the peak area was shown in FIG. 3.
(3) Detection result: the concentration and the main peak area were linearly regressed, and the detection results are shown in table 5 below.
TABLE 5 Linear test detection results
As is clear from the results of the measurements shown in Table 5, the linear correlation coefficient r of 4-methyl-3-penten-2-one was 0.9987, which was greater than 0.990, and was satisfactory in terms of the linear relationship between the 4-methyl-3-penten-2-one concentration and the peak area in the proposed range.
Limit of quantification and limit of detection
(1) Preparing detection limit and quantitative limit solution: precisely weighing a proper amount of 4-methyl-3-pentene-2-one reference substance, and taking the reference substance as a quantitative limit when the reference substance is diluted by a mobile phase until the signal to noise ratio S/N is about 10:1; the signal to noise ratio S/N is about 3:1.
(2) 20. Mu.L of each of the quantitative limit solution and the detection limit solution was measured precisely, injected into a liquid chromatograph, detected by the method of example 1, and a chromatogram was recorded.
(3) The quantitative limit HPLC detection chart of 4-methyl-3-pentene-2 ketone is shown in FIG. 4, the detection limit HPLC detection chart of 4-methyl-3-pentene-2 ketone is shown in FIG. 5, and the quantitative limit and detection limit examination results are shown in Table 6 below.
TABLE 6 methodological validation-quantitative limit and detection limit test results
According to the detection results of Table 6, the quantitative limit of 4-methyl-3-penten-2-one was 0.013. Mu.g/mL, and the detection limit was 0.0039. Mu.g/mL. Therefore, the HPLC detection method for detecting the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride provided by the application has high detection sensitivity, and can accurately detect the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride.
Accuracy of
(1) Preparing a recovery rate stock solution: about 9mg of 4-methyl-3-pentene-2 ketone reference substance is taken and placed in the same 100mL measuring flask, mobile phase A is added for dissolution and dilution to scale, shaking is carried out uniformly, and the concentration is 90 mug/mL as a recovery rate stock solution.
Preparing a reference substance solution: precisely weighing about 9mg of 4-methyl-3-pentene-2-one reference substance, placing in a 100mL measuring flask, adding mobile phase A for dilution to scale, shaking uniformly, precisely weighing 1mL, placing in a 10mL measuring flask, diluting to scale with mobile phase A, shaking uniformly, and preparing 2 parts in parallel, wherein the concentration is 9 mug/mL.
Preparing a background solution: about 60mg of lidocaine hydrochloride is precisely weighed, placed in a 20mL measuring flask, added with mobile phase A for dissolution and dilution to scale, and shaken uniformly to serve as background solution.
Preparing a recovery rate solution: 60mg of lidocaine hydrochloride is taken and placed in a 20mL measuring flask, 1mL,2mL and 3mL of recovery stock solution are respectively added, the stock solution is diluted to a scale by using a mobile phase A, and the stock solution is uniformly shaken to prepare 3 parts of recovery solution for each concentration in parallel.
(2) Each 20 μl of the above solutions was precisely measured, poured into a liquid chromatograph, and detected by the method provided in example 1, and a chromatogram was recorded.
(3) The recovery rate of 4-methyl-3-penten-2-one was calculated as peak area by external standard method according to recovery rate= (measured amount-content in sample)/addition amount, and the results are shown in table 7.
TABLE 7 methodological verification-accuracy test results
As is clear from the results of the measurements in Table 7, the recovery rate of 4-methyl-3-penten-2-one was in the range of 50% to 150%, and the RSD at 9 parts of recovery rate was 1.5% and less than 6%. Therefore, the HPLC detection method for determining the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride provided by the application is good in accuracy.
Repeatability of
(1) Preparing a test solution: accurately weighing lidocaine hydrochloride about 30mg, placing into a10 mL measuring flask, adding mobile phase A, dissolving, diluting to scale, and shaking. 6 parts of the mixture were prepared in the same manner.
(2) 20. Mu.L of each of the above solutions was measured precisely, poured into a liquid chromatograph, and detected by the method of example 1, and a chromatogram was recorded.
(3) The results of the repeatability test are shown in Table 8.
Table 8 methodological validation-repeatability test results
| Sample name | 4-Methyl-3-penten-2-one content (%) |
| 1 | 0.0052 |
| 2 | 0.0047 |
| 3 | 0.0057 |
| 4 | 0.0048 |
| 5 | 0.0052 |
| 6 | 0.0051 |
| Average (%) | 0.0051 |
| Extremely poor | 0.0010 |
| Very poor/limit (%) | 3.33 |
As can be seen from the results of the test in Table 8, the content of 4-methyl-3-penten-2-one in the 6-part sample solution was extremely small, 3.33% or less than 5%. Therefore, the HPLC detection method for the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride provided by the application is good in repeatability.
Durability of
Examples 2 to 10
Examples 2-10 provide an HPLC detection method for the content of 4-methyl-3-penten-2-one in lidocaine hydrochloride, respectively.
The above embodiment differs from embodiment 1 in that: the detection conditions of the high performance liquid chromatography are shown in Table 9 below.
Precisely measuring 20 mu L of each of the blank solvent, the reference substance solution and the test substance solution, injecting into a liquid chromatograph, recording a chromatogram, and then calculating the content of 4-methyl-3-pentene-2 ketone in the test substance solution according to a formula.
TABLE 9 conditions for HPLC detection in the detection methods provided in examples 1-10
As is clear from the results of the tests in Table 9, the difference between the results of the tests conducted on the test solutions under the test conditions of examples 2 to 10 and the results of the tests conducted in example 1 was less than 5% of the limit, and the results were satisfactory. Therefore, the HPLC detection method for the content of 4-methyl-3-pentene-2 ketone in the lidocaine hydrochloride provided by the application has good durability, and can meet the detection requirement of 4-methyl-3-pentene-2 ketone in the lidocaine hydrochloride.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (8)
1. An HPLC method for detecting the content of 4-methyl-3-pentene-2 ketone in lidocaine hydrochloride, which is characterized by comprising the following steps:
(1) Preparing a reference substance solution and a test substance solution;
(2) Injecting the reference substance solution and the sample solution into a liquid chromatograph, and separating and detecting by adopting a chromatographic column filled with octadecylsilane chemically bonded silica gel and a mobile phase gradient elution method;
(3) Recording a chromatogram, and calculating the content of 4-methyl-3-pentene-2 ketone in the sample according to an external standard method and peak area;
The mobile phase gradient elution method comprises the following steps: 0-18min, mobile phase A is 100%, mobile phase B is 0%;18-45min, mobile phase A is reduced from 100% to 37.5%, and mobile phase B is increased from 0% to 62.5%;45-60min, mobile phase A of 37.5% and mobile phase B of 62.5%;60-63min, mobile phase A increased from 37.5% to 100%, mobile phase B decreased from 62.5% to 0%;63-80min, mobile phase A is 100%, mobile phase B is 0%.
2. The method according to claim 1, wherein the mobile phase A is a monoammonium phosphate solution, methanol and acetonitrile in a volume ratio of (75-85): (8-12): (8-12); the mobile phase B is methanol and acetonitrile with the volume ratio of (0.8-1.2).
3. The method according to claim 2, wherein the concentration of the ammonium dihydrogen phosphate solution is 11-12 g/L and the pH is 2.8-3.0.
4. The method according to claim 1, wherein the column temperature is 35-45 ℃ and the flow rate of the mobile phase is 0.7-0.9mL/min during the separation and detection.
5. The detection method according to claim 1, wherein in the separation detection process, the sample injection amount is 18-22 μl, and the detection wavelength is 208-212nm.
6. The method according to claim 1, wherein the chromatographic column is Ultimate XB-C18, the specification is 4.6X250 mm,5 μm.
7. The method according to any one of claims 1 to 6, wherein the control solution is formulated by: taking 4-methyl-3-pentene-2 ketone reference substance, dissolving and diluting with mobile phase A to prepare reference substance solution with the concentration of 4-methyl-3-pentene-2 ketone of 9 mug/mL.
8. The method according to any one of claims 1 to 6, wherein the method for preparing the sample solution comprises: and dissolving and diluting lidocaine hydrochloride by using a mobile phase A to prepare a test sample solution containing lidocaine hydrochloride with the concentration of 3 mg/mL.
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