Detection method of beta-amino methyl crotonate

Abstract

The invention provides a method for detecting methyl beta-aminocrotonate, which adopts an HPLC (High Performance Liquid Chromatography) pre-column derivatization method for detection so as to realize the quality control of the methyl beta-aminocrotonate. The detection method provided by the invention has the advantages of simple operation, strong specificity, high sensitivity, high accuracy and good reproducibility.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and in particular relates to a method for detecting beta-aminocrotonic acid methyl ester by utilizing an HPLC pre-column derivatization method. Background Art

[0002] β-aminocrotonic acid methyl ester, also known as 3-aminocrotonic acid methyl ester or 3-amino-2-butenoic acid methyl ester, is an important intermediate for the synthesis of dipine drugs (used to treat hypertension, angina pectoris, arrhythmia and other cardiovascular and cerebrovascular diseases), and is used to synthesize felodipine, nitrendipine, nifedipine, amlodipine besylate, manidipine hydrochloride, etc. As a drug synthesis intermediate, β-aminocrotonic acid methyl ester may remain in the finished product. β-aminocrotonic acid methyl ester has an α, β unsaturated ketone warning structure. Compounds with this warning structure are defined as potential genotoxic impurities by ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) and have no clear PDE value. In recent years, the study of genotoxic impurities has become one of the keys to ensuring product quality.

[0003] Prior art CN111948306A discloses a method for detecting the content of β-aminocrotonic acid methyl ester, a genotoxic impurity, in amlodipine by using gas chromatography; Prior art CN118225956A discloses a method for detecting the content of β-aminocrotonic acid methyl ester in lacidipine by using gas chromatography-mass spectrometry; Although the prior art discloses a specific method for detecting β-aminocrotonic acid methyl ester, it still has certain defects. Therefore, it is of great significance to develop a new method for detecting β-aminocrotonic acid methyl ester. Summary of the invention

[0004] The present invention aims to provide a detection method for β-aminocrotonic acid methyl ester, which adopts HPLC pre-column derivatization method for detection, thereby realizing the quality control of β-aminocrotonic acid methyl ester. The detection method provided by the present invention has the advantages of simple operation, strong specificity, high sensitivity, high accuracy and good reproducibility. Specifically, the technical scheme of the present invention is as follows:

[0005] A HPLC pre-column derivatization detection method for β-aminocrotonic acid methyl ester comprises the following steps:

[0006] 1. Sample pretreatment: Mix the β-aminocrotonic acid methyl ester reference substance or the sample to be tested with the derivatization reagent evenly to carry out a derivatization reaction; then add the solvent and mix evenly to obtain a derivatized reference substance solution or a derivatized sample to be tested;

[0007] 2. HPLC detection: inject the derivatized reference substance or the derivatized sample solution into the liquid chromatograph and record the chromatogram;

[0008] In some embodiments, in the sample pretreatment step, the concentration of β-aminocrotonic acid methyl ester is 0.02-2.0 μg / ml, preferably, the concentration of β-aminocrotonic acid methyl ester is 0.1-1.0 μg / ml, and more preferably, the concentration of β-aminocrotonic acid methyl ester is 0.1-0.5 μg / ml;

[0009] In some embodiments, in the sample pretreatment step, the concentration of β-aminocrotonic acid methyl ester is 0.2 μg / ml;

[0010] In some embodiments, in the sample pretreatment step, the concentration of the derivatization reagent is 0.01 to 30 mg / ml, preferably, the concentration of the derivatization reagent is 0.1 to 10 mg / ml, and more preferably, the concentration of the derivatization reagent is 1 to 5 mg / ml;

[0011] In some embodiments, in the sample pretreatment step, the concentration of the derivatization reagent is 4 mg / ml;

[0012] In some embodiments, in the sample pretreatment step, the derivatization reagent is an alcohol; preferably, xanthanol;

[0013] In some embodiments, in the sample pretreatment step, the derivatization reaction is carried out under the action of an acid; the acid includes a protonic acid and a Lewis acid;

[0014] In some embodiments, in the sample pretreatment step, the protonic acid is one or more of hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, polyphosphoric acid, hydrofluoric acid, etc.;

[0015] In some embodiments, in the sample pretreatment step, the acid is hydrochloric acid;

[0016] In some embodiments, in the sample pretreatment step, the Lewis acid is one or more of aluminum trichloride, boron trifluoride, ferric chloride, titanium tetrachloride, zinc dioxide, etc.;

[0017] In some embodiments, in the sample pretreatment step, the derivatization reaction time is 1 to 60 min; preferably, the derivatization reaction time is 10 to 45 min; more preferably, the derivatization reaction time is 15 to 30 min;

[0018] In some embodiments, in the sample pretreatment step, the derivatization reaction time is 15 min;

[0019] In some embodiments, in the sample pretreatment step, the derivatization reaction temperature is 20-45°C, preferably, the derivatization reaction temperature is 20-30°C; more preferably, the derivatization reaction temperature is 25°C;

[0020] In some embodiments, in the sample pretreatment step, β-aminocrotonic acid methyl ester and the derivatization reagent are mixed in a molar ratio of less than 1;

[0021] In some embodiments, in the sample pretreatment step, the derivatization reaction is performed under light-proof conditions;

[0022] In some embodiments, in the sample pretreatment step, the solvent is an organic solvent or an inorganic solvent; preferably, the solvent is one or more of water, acetonitrile, methanol, ethanol, propanol, n-butanol, etc.; more preferably, the solvent is acetonitrile;

[0023] In the present invention, the chromatographic column for HPLC detection is selected according to the sample to be tested;

[0024] In the present invention, the mobile phase for HPLC detection is selected according to the sample to be tested;

[0025] In the present invention, the chromatographic conditions for HPLC detection are selected according to the sample to be tested;

[0026] The present invention also provides a HPLC pre-column derivatization detection method for β-aminocrotonic acid methyl ester, comprising the following steps:

[0027] 1. Sample pretreatment: Mix the β-aminocrotonic acid methyl ester reference substance or the sample to be tested with the derivatization reagent xanthanol evenly, add hydrochloric acid to carry out the derivatization reaction; then add the solvent acetonitrile and mix evenly to obtain the derivatization reference substance solution or the derivatized sample to be tested respectively;

[0028] 2. HPLC detection: inject the derivatized reference substance or the derivatized sample solution into the liquid chromatograph and record the chromatogram;

[0029] In some embodiments, the β-aminocrotonic acid methyl ester reference substance or the test substance to be tested is mixed evenly with the derivatization reagent xanthanol under the action of hydrochloric acid to perform a derivatization reaction. The derivatization reaction formula is as follows:

[0030]

[0031] In some embodiments, in the HPLC, the chromatographic column is an XSelect chromatographic column;

[0032] In some embodiments, in the HPLC, the chromatographic column specifications are Waters Xselect HSS-T3150×4.6mm, 3.5μm;

[0033] In some embodiments, in the HPLC, the flow rate of the mobile phase is 1.0 ml / min;

[0034] In some embodiments, in the HPLC, the column temperature of the chromatographic column is 30°C;

[0035] In some embodiments, in the HPLC, the detection wavelength of the UV detector is 210 nm;

[0036] In some embodiments, in the HPLC, the UV detector injection volume is 10 μl;

[0037] In some embodiments, in the HPLC, the mobile phases are mobile phase A and mobile phase B, wherein mobile phase A is 0.1% phosphoric acid aqueous solution, and mobile phase B is acetonitrile;

[0038] In some embodiments, in the HPLC, the mobile phase adopts gradient elution, and the elution starts with 40% by volume of mobile phase A + 60% by volume of mobile phase B; 40% by volume of mobile phase A + 60% by volume of mobile phase B is used at 9 minutes; 5% by volume of mobile phase A + 95% by volume of mobile phase B is used at 10 minutes; 5% by volume of mobile phase A + 95% by volume of mobile phase B is used again at 13 minutes; 40% by volume of mobile phase A + 60% by volume of mobile phase B is used at 13.1 minutes; 40% by volume of mobile phase A + 60% by volume of mobile phase B is used at 18 minutes;

[0039] In the present invention, after pre-column derivatization HPLC detection, the content of β-aminocrotonic acid methyl ester in the sample to be tested is calculated using the external standard method.

[0040] The beneficial effects of the present invention are as follows: providing a HPLC pre-column derivatization detection method for β-aminocrotonic acid methyl ester, the pre-column derivatization method of the present invention has simple reaction conditions, high efficiency, high accuracy and high precision; the detection method provided by the present invention has the advantages of strong specificity, high sensitivity, high accuracy and good reproducibility, and can effectively realize the quality control of β-aminocrotonic acid methyl ester. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Liquid chromatogram of derivatization reagent;

[0042] Figure 2 Liquid chromatogram of derivatized β-aminocrotonic acid methyl ester reference solution;

[0043] Figure 3 Liquid chromatogram of sample solution;

[0044] Figure 4 Detection limit solution liquid chromatogram;

[0045] Figure 5 Liquid chromatogram of 50% concentration sample recovery solution;

[0046] Figure 6Liquid chromatogram of the solution with 100% concentration and recovery rate;

[0047] Figure 7 Liquid chromatogram of solution with 150% sample recovery rate; DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with specific implementation modes.

[0049] Example 1

[0050] HPLC chromatographic conditions:

[0051] High performance liquid chromatograph: Agilent HPLC 1260

[0052] Column: Waters Xselect HSS-T3 150×4.6mm, 3.5μm

[0053] Mobile phase A: 0.1% phosphoric acid in water

[0054] Mobile phase B: acetonitrile

[0055] Flow rate: 1.0ml / min

[0056] Column temperature: 30°C

[0057] UV detector detection wavelength: 210nm

[0058] Injection volume: 10 μl

[0059] Gradient elution:

[0060]

[0061] 1. Preparation of acidic solution

[0062] Measure 13.5 ml of hydrochloric acid, place it in a 100 ml volumetric flask, add water to dilute to the mark, shake well, and obtain a 1.5 mol / L hydrochloric acid solution;

[0063] 2. Preparation of derivatization reagents

[0064] Weigh 400 mg of xanthanol, place it in a 100 ml volumetric flask, add solvent acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0065] 3. Take the derivatization reagent, inject and analyze according to the above chromatographic conditions, and record the chromatogram, as shown in the attached Figure 1 .

[0066] 4. Preparation of derivatized β-aminocrotonic acid methyl ester reference solution

[0067] 4.1 Preparation of β-aminocrotonic acid methyl ester reference stock solution ①

[0068] Take about 2 mg of β-aminocrotonic acid methyl ester, weigh it accurately, put it in a 100 ml volumetric flask, add acetonitrile, shake to dissolve and dilute to the scale, shake well. Accurately measure 10 ml, put it in a 100 ml volumetric flask, add solvent to dilute to the scale, shake well, and you have it.

[0069] 4.2 Preparation of β-aminocrotonic acid methyl ester reference stock solution ②

[0070] Accurately measure 10 ml of β-aminocrotonic acid methyl ester stock solution ①, place it in a 100 ml volumetric flask, add solvent acetonitrile to dilute to the scale, shake well, and obtain.

[0071] 4.3 Preparation of derivatized β-aminocrotonic acid methyl ester reference solution

[0072] Accurately measure 5 ml of the above β-aminocrotonic acid methyl ester stock solution ②, place it in a 10 ml volumetric flask, add 2 ml of the derivatization reagent, then add 0.5 ml of the above 1.5 mol / L hydrochloric acid solution, shake well, react at 25 ° C in the dark for 15 minutes, add solvent acetonitrile to dilute to the scale, shake well, and obtain the derivatized β-aminocrotonic acid methyl ester reference solution. Inject and analyze according to the above chromatographic conditions, and record the chromatogram, as shown in the attached figure. Figure 2 The spectrum shows that the β-aminocrotonic acid methyl ester derivative peak appears at 8.238min, and the derivative reagent peak does not interfere with it.

[0073] 5. Preparation of derivatized samples:

[0074] Take about 10 mg of amlodipine besylate sample, accurately weigh it, put it in a 10 ml volumetric flask, add 5 ml of acetonitrile, add 2 ml of derivatization reagent, and then add 0.5 ml of the above 1.5 mol / L hydrochloric acid solution, shake well, react at 25 ° C in the dark for 15 minutes, add solvent acetonitrile to dilute to the scale, shake well, and obtain the derivatized sample solution to be tested. Inject and analyze according to the above chromatographic conditions, and record the chromatogram, as shown in the attached figure. Figure 3 .

[0075] The results showed that no derivative peak of β-aminocrotonic acid methyl ester derivatization was detected in the amlodipine besylate sample, so the amlodipine besylate sample solution did not contain β-aminocrotonic acid methyl ester, and this method can achieve sample quality control.

[0076] Example 2 System Precision Investigation

[0077] Preparation of acidic solution: same as in Example 1

[0078] Preparation of derivatization reagent: same as in Example 1

[0079] Preparation of derivatized β-aminocrotonic acid methyl ester reference solution: same as in Example 1

[0080] Take the derivatized β-aminocrotonic acid methyl ester reference substance solution, inject it 5 times continuously, analyze it according to the chromatographic conditions in Example 1, and record the chromatogram;

[0081] The results are shown in Table 1 below. The results show that the derivatized β-aminocrotonic acid methyl ester reference solution was injected five times in succession, and the RSD of the derivatized β-aminocrotonic acid methyl ester peak area was 0.6%, which was less than 10.0%, indicating that the precision of the instrument injection was good.

[0082] Table 1 System precision results

[0083]

[0084] Example 3 Standard curve and correlation coefficient investigation

[0085] Preparation of acidic solution: same as in Example 1

[0086] Preparation of derivatization reagent: same as in Example 1

[0087] Preparation of β-aminocrotonic acid methyl ester reference substance stock solution ①: same as in Example 1

[0088] Accurately measure 2 ml, 5 ml, 10 ml, 15 ml, and 20 ml of the reference stock solution of β-aminocrotonic acid methyl ester, respectively, and place them in a 100 ml volumetric flask, dilute them to the mark with acetonitrile, and shake well to prepare linear stock solutions of different concentrations. Accurately measure 5 ml of linear stock solutions of different concentrations, place them in a 10 ml volumetric flask, accurately add 2 ml of the derivatization reagent, and 0.5 ml of 1.5 mol / L hydrochloric acid solution, react at 25 ° C in the dark for 15 min, add the solvent acetonitrile to dilute to the mark, and shake well. Inject and analyze according to the chromatographic conditions of Example 1, and record the chromatogram.

[0089] The results are shown in Table 2 below. The results show that the chromatographic peak area (A) and concentration (C) of the derivatized β-aminocrotonic acid methyl ester are in the range of 0.0382 to 0.3819 μg / ml, and the regression equation is A=63.3551C+0.0571, with a correlation coefficient of r=1.0000. The concentration and peak area of ​​β-aminocrotonic acid methyl ester are in the range of 0.0382 to 0.3819 μg / ml, and a good linear relationship is formed.

[0090] Table 2 Linearity test results

[0091]

[0092] Example 4 Detection Limit Investigation

[0093] Preparation of acidic solution: same as in Example 1

[0094] Preparation of derivatization reagent: same as in Example 1

[0095] β-aminocrotonic acid methyl ester reference substance stock solution ① Preparation: Same as Example 1

[0096] β-aminocrotonic acid methyl ester reference substance stock solution ② Preparation: Same as Example 1

[0097] Accurately measure 1 ml of reference stock solution ②, place in a 25 ml volumetric flask, add acetonitrile to dilute to scale, shake well, and prepare the detection limit stock solution. Accurately measure 5 ml of detection limit stock solution, place in a 10 ml volumetric flask, add 2 ml of derivatization reagent, then add 0.5 ml of 1.5 mol / L hydrochloric acid solution, shake well, react at 25 ° C in the dark for 15 min, add acetonitrile to dilute to scale, shake well, and analyze according to the chromatographic conditions of Example 1, and record the chromatogram, as shown in the attached figure. Figure 4 .

[0098] The results showed that the detection limit of this method for derivatized β-aminocrotonic acid methyl ester was 0.0095 μg / ml.

[0099] Example 5: Sample recovery rate study

[0100] Acidic solution preparation: same as Example 1

[0101] Preparation of derivatization reagent: same as in Example 1

[0102] Take about 2 mg of β-aminocrotonic acid methyl ester, accurately weigh it, put it in a 100 ml volumetric flask, add an appropriate amount of acetonitrile to dissolve and dilute to the scale, shake well, and prepare a reference stock solution. Accurately measure 2 ml, 5 ml, and 15 ml of the reference stock solution respectively, put it in a 100 ml volumetric flask, dilute to the scale with acetonitrile, shake well, and prepare reference stock solutions of different concentrations. Take about 10 mg of amlodipine besylate sample, accurately weigh it, put it in a 10 ml volumetric flask, accurately add 5 ml of reference stock solution of different concentrations, accurately add 2 ml of derivatization reagent and 0.5 ml of 1.5 mol / L hydrochloric acid solution respectively, react at 25 ° C in the dark for 15 minutes, dilute to the scale with acetonitrile, and shake well. Prepare 3 parallel portions of each loading level solution, for a total of 9 loading recovery solutions. Sample and analyze according to the chromatographic conditions of Example 1, and record the chromatograms, as shown in the attached Figure 5-7 ; Calculate the ratio of the measured amount to the added amount by the external standard method and calculate the recovery rate.

[0103] The results are shown in Table 3 below. The results show that the average recovery rate of β-aminocrotonic acid methyl ester is 100.0%, within the range of 80% to 110%, and its RSD is 2.1% (n=9), which is less than 10.0%, indicating that the accuracy of this method for determining β-aminocrotonic acid methyl ester is good.

[0104] Table 3 Sample recovery results

[0105]

[0106] Example 5 Repeatability Study

[0107] The solution with 100% recovery rate in Example 4 was selected for repeatability test, and the repeatability of the method was evaluated by calculating the RSD value of the content of β-aminocrotonic acid methyl ester.

[0108] Six 100% recovery rate solutions were prepared in parallel according to the method of Example 4, and were injected and analyzed according to the chromatographic conditions of Example 1, and the chromatograms were recorded.

[0109] The specific results are shown in Table 4. The spiked sample solution was used to continuously measure 6 portions. The RSD value of the β-aminocrotonic acid methyl ester content was 2.9% and less than 10.0%, and the average recovery rate was 100.4%, indicating that the method had good repeatability.

[0110] Table 4 Repeatability results

[0111]

[0112] Example 6 Solution Stability Study

[0113] Take the derivatized β-aminocrotonic acid methyl ester reference solution in Example 1 and the 100% sample recovery solution in Example 4, respectively, place them in the sample chamber at 25°C, inject samples at different time periods, and calculate the absolute value of the relative deviation between the peak area at each time point and the peak area at 0h. The specific data are shown in Table 5.

[0114] Table 5 Solution stability results

[0115]

[0116] The experimental results show that, under 25°C conditions, the absolute values ​​of the relative deviations of the corresponding β-aminocrotonate methyl ester peak areas at each time point in the derivatized β-aminocrotonate methyl ester reference solution and the 100% sample recovery solution within 14 hours were all less than 10.0%, compared with the peak area at 0h. This indicates that under room temperature conditions, the derivatized β-aminocrotonate methyl ester reference solution and the 100% sample recovery solution remain stable within 14 hours and can be used to accurately determine the β-aminocrotonate content in samples.

Claims

1. A method for detecting β-aminocrotonic acid methyl ester by HPLC pre-column derivatization, characterized in that: Sample preparation steps include: The β-aminocrotonic acid methyl ester reference substance or the sample to be tested is mixed evenly with the derivatization reagent to carry out a derivatization reaction; then a solvent is added and mixed evenly to obtain a derivatized reference substance solution or a derivatized sample to be tested.

2. The detection method according to claim 1, characterized in that: The concentration of the β-aminocrotonic acid methyl ester is 0.02-2.0 μg / ml; preferably, the concentration of the β-aminocrotonic acid methyl ester is 0.1-1.0 μg / ml; more preferably, the concentration of the β-aminocrotonic acid methyl ester is 0.1-0.5 μg / ml.

3. The detection method according to claim 1, characterized in that: The concentration of the derivatization reagent is 0.01-30 mg / ml, preferably, the concentration of the derivatization reagent is 0.1-10 mg / ml, and more preferably, the concentration of the derivatization reagent is 1-5 mg / ml.

4. The detection method according to claim 1, characterized in that: The derivatization agent is an alcohol, preferably xanthanol.

5. The detection method according to claim 1, characterized in that: In the sample pretreatment step, the derivatization reaction is carried out under the action of an acid; the acid includes a protonic acid and a Lewis acid.

6. The detection method according to claim 5, characterized in that: In the sample pretreatment step, the protonic acid is one or more of hydrochloric acid, sulfuric acid, acetic acid, nitric acid, phosphoric acid, polyphosphoric acid, hydrofluoric acid, etc., preferably, hydrochloric acid; The Lewis acid is one or more of aluminum trichloride, boron trifluoride, ferric chloride, titanium tetrachloride, zinc dioxide and the like.

7. The detection method according to claim 1, characterized in that: In the sample pretreatment step, the derivatization reaction is carried out under light-proof conditions.

8. The detection method according to claim 1, characterized in that: In the sample pretreatment step, the derivatization reaction time is 1 to 60 min, preferably, the derivatization reaction time is 10 to 45 min, and more preferably, the derivatization reaction time is 15 to 30 min.

9. The detection method according to claim 1, characterized in that: In the sample pretreatment step, the derivatization reaction temperature is 20-45°C, preferably, the derivatization reaction temperature is 20-30°C; more preferably, the derivatization reaction temperature is 25°C.

10. The detection method according to claim 1, characterized in that: In the sample pretreatment step, the solvent is an organic solvent or an inorganic solvent; preferably, the solvent is one or more of water, acetonitrile, methanol, ethanol, propanol, n-butanol, etc.; more preferably, the solvent is acetonitrile.

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