Method for separating and detecting etomidate intermediate and enantiomer thereof

Abstract

According to the method, a polysaccharide derivative bonding type chiral chromatographic column with silica gel surface bonded amylose-tri (3, 5-dichlorophenyl carbamate) as a filler is adopted, a mixed solution of n-hexane and isopropanol is used as a mobile phase, the column temperature is 28-32 DEG C, the flow velocity is 1.1-1.3 ml / min, the column temperature is 20-30 DEG C, the column temperature is 20-30 DEG C, the column temperature is 20-30 DEG C, the column temperature is 20-30 DEG C, the column temperature is 20-30 DEG C, and the column temperature is 20-30 DEG C; an ultraviolet detector is adopted, the wavelength is 205-220 nm, and effective separation of the etomidate intermediate and the enantiomer thereof is achieved. The method is good in detection result accuracy and high in sensitivity, and compared with an existing analysis method, the method is better in separation effect, simpler in mobile phase composition, more durable in chromatographic column and lower in cost, so that a good guarantee is provided for quality control of the etomidate intermediate and etomidate.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis methods, and specifically relates to a method for separating and detecting etomidate intermediates and their enantiomers by high performance liquid chromatography. Background Art

[0002] Etomidate is a short-acting, non-barbiturate intravenous anesthetic that is currently widely used in clinical applications for induction of general anesthesia. - Related specific binding sites, enhance GABA receptor function, inhibit excitatory nerve signal conduction. Etomidate has the characteristics of stable hemodynamics, low respiratory depression, brain protection, rapid onset after single injection or continuous infusion, no accumulation, rapid awakening, less injection pain, high sedative potency, and a wide range of safety.

[0003] The chemical name of etomidate is (+)-1-(α-methylbenzyl)imidazole-5-carboxylic acid ethyl ester {Ethyl 1-[(1R)-1-phenylethyl]-1H -imidazole-5-carboxylate}, the molecular formula is C14H16N2O2, the molecular weight is 244.29, and the structural formula is shown in Table 1. Etomidate has a chiral center, and the configuration currently approved for clinical use is the R configuration. Since the R and S configurations show different efficacy and toxicity, the inspection of isomers in the production process of etomidate is of great significance for its safe use.

[0004] (R)-ethyl N-formyl-N-(1-phenylethyl)glycinate, molecular formula is C13H17NO3, molecular weight is 235.28, structural formula is shown in Table 2. Its absolute configuration is R configuration, and it can be used as etomidate intermediate, structural formula is shown in Table 2. Based on the QbD concept (quality by design), controlling or considering enantiomers in intermediates will be more conducive to ensuring the controllable quality of etomidate products.

[0005] Table 1 Structural formula of etomidate and its enantiomers

[0006] Table 2 Structural formula of etomidate intermediates and their enantiomers

[0007] At present, there are few documents reporting the use of high performance liquid chromatography to determine the enantiomers of the etomidate intermediate. In CN115932089A, Huang Ling et al. used a chiral chromatographic column (OJ-H) coated with cellulose-tri (4-methylbenzoate) on the surface of silica gel, and a mixed solvent of n-hexane-anhydrous ethanol-diethylamine as the mobile phase to determine the enantiomers of the etomidate intermediate. The literature uses a large number of examples to prove that only OJ-H with its mobile phase can effectively separate and determine the etomidate intermediate and its enantiomers. In order to broaden the separation and detection methods of the intermediate enantiomers, we have developed a new separation and detection method, using IE / IE-3 chiral chromatographic columns and n-hexane-isopropanol mobile phase system. Compared with the literature method, the separation effect is better, the mobile phase composition is simpler, the chromatographic column is more durable, and the cost is lower. Provide good guarantee for the quality control of etomidate intermediates and etomidate. Summary of the invention

[0008] The purpose of the present invention is to provide a method for separating and determining an etomidate intermediate and its enantiomers, which can effectively separate the etomidate intermediate and its enantiomers, accurately detect the enantiomers in the etomidate intermediate, and control the quality of the intermediate, thereby controlling the product quality of etomidate and ensuring its effectiveness and safety.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0010] The invention provides a separation and detection method for an etomidate intermediate and an enantiomer thereof, which is characterized by adopting a high performance liquid chromatography method and comprising the following chromatographic conditions: using a polysaccharide derivative bonded chiral chromatographic column with amylose-tris(3,5-dichlorophenylcarbamate) bonded to the surface of silica gel as a filler, and taking a mixed solution of n-hexane and isopropanol as a mobile phase, wherein the etomidate intermediate is (R)-ethyl N-formyl-N-(1-phenylethyl)glycine, and the corresponding isomer thereof is (S)-ethyl N-formyl-N-(1-phenylethyl)glycine.

[0011] The chromatographic column described in this method is CHIRALPAK® IE (250mm*4.6mm, 5μm), CHIRALPAK® IE-3 (250mm*4.6mm, 3μm), preferably CHIRALPAK® IE (250mm*4.6mm, 5μm).

[0012] Furthermore, in the above method of the present invention, the volume ratio of the mobile phase n-hexane-isopropanol is (88-92):(8-12), preferably 90:10.

[0013] Furthermore, in the above method of the present invention, the mobile phase flow rate is 1.1-1.3 ml / min, the chromatographic column temperature is 28-32° C., and the detection wavelength is 205-220 nm.

[0014] Furthermore, in the above method of the present invention, the flow rate of the mobile phase is 1.2 ml / min, the temperature of the chromatographic column is 30° C., and the detection wavelength is 210 nm.

[0015] In a specific embodiment, the method of the present invention comprises the following steps: Preparation of system suitability solution: Take an appropriate amount of etomidate intermediate and its enantiomer reference substance, add mobile phase to dissolve and dilute to prepare system suitability solution; Preparation of test solution: Take an appropriate amount of etomidate intermediate, add mobile phase to dissolve and dilute to prepare the test solution; Take the system suitability solution and the test solution, inject them into the high performance liquid chromatograph for detection, and record the chromatogram.

[0016] The method of the present invention realizes the effective separation and detection of etomidate intermediates and their enantiomers, and the normal phase chiral chromatography system used has the advantages of strong separation ability, no need for derivatization, simple operation process, etc., and can more conveniently realize the quality control of product production process links and improve the quality of the final product. The method has high accuracy, high sensitivity, and good repeatability, and has better separation effect, simpler mobile phase composition, more durable chromatographic column, and lower cost than the existing analytical method, thereby providing good guarantee for the quality control of the research and development and production process of etomidate intermediates and etomidate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the HPLC chromatogram of the system suitability of Example 5; Figure 2 This is the HPLC chromatogram of the blank solution in the specificity experiment of Example 5; Figure 3 This is the HPLC chromatogram of the reference substance solution in the specificity experiment of Example 5; Figure 4 This is the HPLC chromatogram of the blank test solution in the specificity experiment of Example 5; Figure 5 This is the linear investigation diagram of etomidate intermediate in Example 6; Figure 6 This is a linear survey diagram of etomidate intermediate enantiomers in Example 6; Figure 7 This is the HPLC chromatogram of the quantitative limit of the etomidate intermediate enantiomer in Example 7. DETAILED DESCRIPTION

[0018] The following examples are used to further illustrate and understand the spirit of the present invention, but are not intended to limit the scope of protection of the present invention in any way.

[0019] In the following examples, the instruments and reagents used are as follows: Instrument: Shimadzu LC-20AD high performance liquid chromatograph Reagents: n-hexane and isopropanol were of chromatography grade.

[0020] The etomidate intermediate enantiomer reference substance was prepared by Chongqing Yaoyou Pharmaceutical Co., Ltd., and its structure was confirmed and its content was calibrated by Southwest University.

[0021] Example 1 Screening of stationary phase Chromatographic columns: CHIRALPAK IG (250mm*4.6mm, 5μm), CHIRALCEL OZ-H (250mm*4.6mm, 5μm), CHIRALCEL OD-H (250mm*4.6mm, 5μm), CHIRALCEL OC-H (250mm*4.6mm, 5μm), CHIRALPAK IE (250mm*4.6mm, 5μm), CHIRALPAK AD-H (250mm*4.6mm, 5μm) and CHIRALPAK AS-H (250mm*4.6mm, 5μm) Detector and wavelength: PDA-210nm Flow rate: 1.2ml / min Injection volume: 10 μl Column temperature: 30°C Mobile phase: n-hexane-isopropanol (90:10, v / v) Solvent: Mobile phase System suitability solution: Take an appropriate amount of etomidate intermediate and its enantiomer reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 3 mg of intermediate and 0.045 mg of enantiomer per 1 ml, which is used as the system suitability solution.

[0022] Table 3 Separation and detection of different stationary phases

[0023] Conclusion: The above results show that when the above chromatographic conditions are used to separate etomidate intermediates and their enantiomers, CHIRALPAK IE has the best separation effect and better peak shape, so CHIRALPAK IE chromatographic column is preferred.

[0024] Example 2 Screening of mobile phase Detection effect test of different mobile phase ratios Mobile phase 1: n-hexane-isopropanol (88:12, v / v) Mobile phase 2: n-hexane-isopropanol (90:10, v / v) Mobile phase 3: n-hexane-isopropanol (92:8, v / v) Chromatographic column: CHIRALPAK IE (250mm*4.6mm, 5μm) Detector and wavelength: PDA-210nm Flow rate: 1.2ml / min Injection volume: 10 μl Column temperature: 30°C Solvent: n-hexane-isopropanol (90:10, v / v) Table 4 Separation and detection results of different mobile phase ratios

[0025] Conclusion: The above results show that the enantiomeric separation of the three mobile phases investigated is greater than 1.5. Considering the comprehensive analysis time, when the mobile phase composition is n-hexane-isopropanol (90:10, v / v), both the separation and the analysis time can be taken into account.

[0026] Example 3 Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IE (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol (92:8, v / v) Detector and wavelength: PDA-210nm Flow rate: 1.2ml / min Injection volume: 10 μl Column temperature: 30°C Solvent: Mobile phase System suitability solution: Take an appropriate amount of etomidate intermediate and its enantiomer reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 3 mg of intermediate and 0.045 mg of enantiomer per 1 ml as system suitability solution. Inject 10 μl.

[0027] Conclusion: Under the chromatographic conditions, the separation degree of etomidate intermediate and its enantiomers was 2.5.

[0028] Example 4 Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IE-3 (250mm*4.6mm, 3μm) Mobile phase: n-hexane-isopropanol (90:10, v / v) Detector and wavelength: PDA-210nm Flow rate: 1.0ml / min Injection volume: 10 μl Column temperature: 30°C Solvent: Mobile phase System suitability solution: Take an appropriate amount of etomidate intermediate and its enantiomer reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 3 mg of intermediate and 0.045 mg of enantiomer per 1 ml as system suitability solution. Inject 10 μl.

[0029] Conclusion: Under the chromatographic conditions, the separation degree of etomidate intermediate and its enantiomers was 2.4.

[0030] Example 5 Specificity and system suitability experiment (1) System applicability Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IE (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol (90:10, v / v) Detector and wavelength: PDA-210nm Flow rate: 1.2ml / min Injection volume: 10 μl Column temperature: 30°C Solvent: Mobile phase System suitability solution: Take an appropriate amount of etomidate intermediate and its enantiomer reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 3 mg of intermediate and 0.045 mg of enantiomer per 1 ml as system suitability solution. Inject 10 μl, and the chromatographic results are as follows: Figure 1 shown.

[0031] (2) Exclusivity Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IE (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol (90:10, v / v) Detector and wavelength: PDA-210nm Flow rate: 1.2ml / min Injection volume: 10 μl Column temperature: 30°C Solvent: Mobile phase Reference solution: Take an appropriate amount of etomidate intermediate enantiomer reference, dissolve it in the mobile phase and quantitatively dilute it to make a solution containing 0.045 mg of etomidate intermediate enantiomer per 1 ml as the reference solution. Inject 10 μl.

[0032] Blank test solution: Take an appropriate amount of etomidate intermediate, dissolve it in mobile phase and quantitatively dilute it to make a solution containing 3 mg of etomidate intermediate per 1 ml as blank test solution. Inject 10 μl.

[0033] Conclusion: From Figure 2~Figure 4 The results showed that the blank solvent and blank test sample had no obvious interference with the detection.

[0034] Example 6 Linearity Experiment Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IE (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol (90:10, v / v) Detector and wavelength: PDA-210nm Flow rate: 1.2ml / min Injection volume: 10 μl Column temperature: 30°C Solvent: Mobile phase Quantitative method: Peak area normalization method (1) Etomidate intermediate linear Solution preparation: Etomidate intermediate reference substance stock solution: Weigh about 72 mg of etomidate intermediate reference substance, accurately weigh, place in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain (containing etomidate intermediate concentration of about 3.6 mg / ml).

[0035] Accurately measure 2, 2.5, 3, 4, and 5 ml of the 3.6 mg / ml etomidate intermediate reference stock solution and place them in 5, 5, 5, 5, and 5 ml volumetric bottles, add solvent to dilute to the scale, shake well, and make a standard series of solutions with concentrations of approximately 1.44, 1.8, 2.16, 2.88, and 3.6 mg / ml. Analyze and record the peak area according to the above chromatographic conditions, perform linear regression on the peak area and the concentration, and obtain a regression curve. The results are shown in Table 5, and the regression curve is shown in Figure 5 The regression equation was y=24933107x+2429199 (y was the peak area, x was the concentration in mg / ml), the correlation coefficient r was 0.9999, and the linearity was good in the concentration range of 1.40 mg / ml~3.51 mg / ml.

[0036] Table 5 Standard curve of etomidate intermediates

[0037] (2) Linearity of enantiomers of etomidate intermediates Solution preparation: Etomidate intermediate enantiomer reference substance stock solution: Weigh approximately 22.5 mg of etomidate intermediate enantiomer reference substance, accurately weigh, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain (containing etomidate intermediate enantiomer concentration of approximately 0.45 mg / ml).

[0038] Accurately measure 1, 3, and 2 ml of 0.45 mg / ml etomidate intermediate enantiomer reference stock solution, respectively, and place them in 10, 20, and 10 ml volumetric flasks, dilute to the mark with solvent, and shake well to prepare standard series solutions 3 to 5 with concentrations of approximately 45, 67.5, and 90 μg / ml; accurately measure 2 ml of standard series solution 3 and place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well to prepare standard series solution 2 with a concentration of approximately 9 μg / ml; accurately measure 3 ml of standard series solution 2 and place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well to prepare standard series solution 1 with a concentration of approximately 2.7 μg / ml.

[0039] According to the above chromatographic conditions, the peak area was analyzed and recorded, and a linear regression was performed on the peak area against the concentration to obtain a regression curve. The results are shown in Table 6 and the regression curve is shown in Figure 6 The regression equation was y=24691x+9829 (y was the peak area, x was the concentration in μg / ml), the correlation coefficient r was 0.9999, and the linearity was good in the concentration range of 2.84μg / ml~94.50μg / ml.

[0040] Table 6 Standard curve of etomidate intermediate enantiomers

[0041] Example 7 Quantitation Limit and Detection Limit Experiment Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IE (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol (90:10, v / v) Detector and wavelength: PDA-210nm Flow rate: 1.2ml / min Injection volume: 10 μl Column temperature: 30°C Solvent: Mobile phase Take an appropriate amount of etomidate intermediate enantiomer reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 0.0027 mg of etomidate intermediate enantiomer per 1 ml, as the quantitative limit solution. Take an appropriate amount of etomidate intermediate enantiomer reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 0.0013 mg of etomidate intermediate enantiomer per 1 ml, as the detection limit solution. At the same time, prepare 6 quantitative limit solutions in parallel for detection, calculate the relative standard deviation of the peak area, the results are shown in Table 7, and the quantitative limit chromatogram is shown in Figure 7 .

[0042] Table 7 Results of quantification limit and detection limit

[0043] Example 8 Accuracy Experiment Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IE (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol (90:10, v / v) Detector and wavelength: PDA-210nm Flow rate: 1.2ml / min Injection volume: 10 μl Column temperature: 30°C Solvent: Mobile phase Take an appropriate amount of etomidate intermediate and its enantiomer reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 3 mg of intermediate and 0.045 mg of enantiomer per 1 ml as the accuracy solution. Prepare 6 portions in parallel, inject them separately, and calculate the enantiomer content in etomidate intermediate by peak area normalization method. The results are shown in Table 8.

[0044] It can be seen from Table 8 that the recovery rate of etomidate intermediate enantiomers is in the range of 90% to 110%, and the RSD of the recovery rate is 0.4%. The accuracy of this method is good.

[0045] Table 8 Accuracy test results

[0046] Example 9 Repeatability Experiment

[0047] Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IE (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol (90:10, v / v) Detector and wavelength: PDA-210nm Flow rate: 1.2ml / min Injection volume: 10 μl Column temperature: 30°C Solvent: Mobile phase Take an appropriate amount of etomidate intermediate and its enantiomer reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 3 mg of intermediate and 0.045 mg of enantiomer per 1 ml as a repeatable solution. Prepare 6 portions in parallel, inject samples separately, and calculate the enantiomer content in etomidate intermediate by peak area normalization method. The results are shown in Table 9.

[0048] As can be seen from Table 9, the detection method provided by the present invention has high precision, and the RSD value of the enantiomer content of the etomidate intermediate is 2.1%, which meets the requirements.

[0049] Table 9 Repeatability test results

[0050] Example 10 Intermediate Precision Experiment

[0051] Chromatographic instruments: Two Shimadzu LC-20AD high performance liquid chromatographs with different internal numbers Chromatographic column: CHIRALPAK IE (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol (90:10, v / v) Detector and wavelength: PDA-210nm Flow rate: 1.2ml / min Injection volume: 10 μl Column temperature: 30°C Solvent: Mobile phase Take an appropriate amount of etomidate intermediate and its enantiomer reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 3 mg of intermediate and 0.045 mg of enantiomer per 1 ml as a repeatable solution. Different experimenters used different instruments on different dates to parallelly determine the detection amount of etomidate intermediate enantiomers in 6 repeatable solutions, and calculated the detection amount of etomidate intermediate enantiomers 12 times. The results are shown in Table 10. The results show that the intermediate precision of this method is good, with an RSD of 1.6%.

[0052] Table 10 Intermediate precision test results

[0053] Example 11 Durability Test The robustness of the method was investigated by changing parameters such as flow rate, column temperature, and mobile phase ratio. Only one parameter was changed each time, with the following parameter ranges: flow rate ±0.1 ml / min, column temperature ±2 °C, mobile phase ratio: n-hexane-isopropanol ratio (88:12), and the remaining chromatographic conditions were the same as those in Example 5 under System Suitability.

[0054] System suitability solution: Take an appropriate amount of etomidate intermediate and its enantiomer reference substance, add mobile phase to dissolve and quantitatively dilute to make a solution containing 3 mg of intermediate and 0.0045 mg of enantiomer per 1 ml. Under the above conditions, the system suitability solution was injected separately, and the chromatographic peak separation of etomidate intermediate and enantiomer was greater than 1.5. The results are shown in Table 11.

[0055] Table 11 Durability test results

Claims

1. A method for separating and detecting an etomidate intermediate and its enantiomers, characterized in that: The high performance liquid chromatography method comprises the following chromatographic conditions: a polysaccharide derivative bonded chiral chromatographic column with amylose-tris (3, 5-dichlorophenylcarbamate) bonded to the surface of silica gel as filler, a mixed solution of n-hexane and isopropanol as mobile phase, and the intermediate is (R)-ethyl N-formyl-N-(1-phenylethyl)glycine.

2. According to the separation and detection method according to claim 1, the chromatographic column is CHIRALPAK® IE (250mm*4.6mm, 5μm) or CHIRALPAK® IE-3 (250mm*4.6mm, 3μm).

3. According to the separation and detection method according to claim 2, the chromatographic column is CHIRALPAK® IE (250mm*4.6mm, 5μm).

4. The separation and detection method according to claim 1, wherein the volume ratio of n-hexane to isopropanol in the mobile phase is (88-92): (8-12), preferably 90:

10.

5. The separation and detection method according to claim 1, wherein the flow rate of the mobile phase is 1.1-1.3 ml / min, preferably 1.2 ml / min.

6. The separation and detection method according to claim 1, wherein the temperature of the chromatographic column is 28-32°C.

7. The separation and detection method according to claim 6, wherein the temperature of the chromatographic column is 30°C.

8. The separation and detection method according to claim 1, wherein the detection wavelength is 205-220 nm.

9. The separation and detection method according to claim 8, wherein the detection wavelength is 210 nm.

10. The method according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: 1) Preparation of system suitability solution: Take an appropriate amount of etomidate intermediate and its enantiomer reference substance, add mobile phase to dissolve and dilute to prepare system suitability solution; 2) Preparation of test solution: Take an appropriate amount of etomidate intermediate, add mobile phase to dissolve and dilute to prepare the test solution; 3) Take the system suitability solution and the test sample solution, inject them into the high performance liquid chromatograph for detection, and record the chromatogram.

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