Method for separating and detecting etomidate intermediate and enantiomer thereof

Abstract

The invention belongs to the field of pharmaceutical analysis, and particularly relates to a separation and detection method of an etomidate intermediate and an enantiomer thereof. According to the method, a polysaccharide derivative bonding type chiral chromatographic column with silica gel surface bonded amylose-tri (3-chloro-5-methyl phenyl carbamate) as a filler is adopted, a mixed solution of normal hexane, isopropanol and diethylamine is adopted as a mobile phase, the column temperature is 23-27 DEG C, the flow velocity is 0.9-1.1 ml / min, an ultraviolet detector is adopted, and the wavelength is 210-325 nm. According to the method, the etomidate intermediate and the enantiomer thereof can be directly and effectively separated, the detection result is good in accuracy and high in sensitivity, and therefore a good guarantee is provided for quality control of the intermediate and preparation of an etomidate finished product.

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 enantiomers thereof by using high performance liquid chromatography. Background Art

[0002] Etomidate is a short-acting, non-barbiturate intravenous anesthetic, which is currently widely used in clinical applications for induction of general anesthesia. Etomidate enhances the function of GABA receptors and inhibits excitatory nerve signaling by binding to specific binding sites related to Cl- on GABAA receptors. Etomidate has the characteristics of stable hemodynamics, low respiratory depression, brain protection, rapid onset of action 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-(+)-1-(Α-methylbenzyl)imidazole-2-thiol-5-carboxylate [ethyl (R)-2-mercapto-1-(1-phenylethyl)-1H-imidazole-5-carboxylate], with a molecular formula of C14H16N2O2S and a molecular weight of 276.35, has an absolute configuration of R configuration and can be used as an intermediate in the synthesis of etomidate. The 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 Etomidate intermediates and their enantiomer structures

[0007] By consulting Chinese and foreign literature and patents, it is found that there is no reported method for detecting the enantiomer of the intermediate of etomidate. Therefore, developing an efficient and convenient method that can separate the intermediate and its enantiomers and measure their contents plays a vital role in improving the quality control of the finished product of etomidate. Summary of the invention

[0008] The object of the present invention is to provide a separation and detection method for 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 separation and detection method of etomidate intermediate and its enantiomers of the present invention comprises a polysaccharide derivative bonded chiral chromatographic column with amylose-tris (3-chloro-5-methylphenylcarbamate) bonded to the surface of silica gel as filler, and a mixed solution of n-hexane, isopropanol and diethylamine as mobile phase.

[0011] Furthermore, the chromatographic column is CHIRALPAK IG (250 mm*4.6 mm, 5 μm).

[0012] Furthermore, in the above method of the present invention, the volume ratio of n-hexane-isopropanol-diethylamine in the mobile phase is (91-95):(5-9):(0.1-0.5), and the preferred volume ratio is 93:7:0.1.

[0013] Furthermore, in the above method of the present invention, the flow rate of the mobile phase is 0.9-1.1 ml / min.

[0014] Furthermore, in the above method of the present invention, the temperature of the chromatographic column is 23-27°C.

[0015] Furthermore, in the above method of the present invention, the detector is an ultraviolet detector, and the detection wavelength is 210~325nm, preferably 261nm.

[0016] Furthermore, in the above method of the present invention, the structures of the etomidate intermediate and the enantiomers of the etomidate intermediate are as shown in the following formula: .

[0017] All percentages involved in the present invention are volume percentages.

[0018] The method of the present invention realizes the effective separation and detection of etomidate intermediate and its enantiomers, and the normal phase chiral chromatography system used has the advantages of strong separation ability, no need for derivatization, simple operation process, etc., which can more conveniently realize the quality control of the product production process and improve the quality of the final product. The method of the present invention has high accuracy, good repeatability and short detection time, and provides a simpler, more stable and reliable analysis and detection method for product development and production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the HPLC chromatogram of the system suitability of Example 1; Figure 2 This is the HPLC chromatogram of the blank solution in the specificity experiment of Example 2; Figure 3 This is the HPLC chromatogram of the reference substance solution in the specificity experiment of Example 2; Figure 4 This is the HPLC chromatogram of the blank test solution in the specificity experiment of Example 2; Figure 5 This is a linear survey diagram of the enantiomers of the etomidate intermediate of Example 3; Figure 6 The HPLC chromatogram of the quantitative limit of the etomidate intermediate enantiomer of Example 4; Figure 7 HPLC chromatogram of Comparative Example 1; Figure 8 This is the HPLC chromatogram of Comparative Example 2. DETAILED DESCRIPTION

[0020] 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.

[0021] In the following examples, the instruments and reagents used are as follows: Instrument: Shimadzu LC-20AD high performance liquid chromatograph Reagents: n-hexane, n-heptane, and isopropanol were all of chromatographic grade, and diethylamine was of analytical grade.

[0022] The etomidate intermediate enantiomer reference substance was prepared by the applicant, and its content was calibrated by the School of Chemistry and Chemical Engineering of Southwest University.

[0023] Example 1 System suitability investigation Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IG (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol-diethylamine (93:7:0.1, v / v / v) Detector and wavelength: PDA-261nm Flow rate: 1.0ml / min Injection volume: 10 μl Column temperature: 25°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 2 mg of intermediate and 0.006 mg of enantiomer per 1 ml as system suitability solution. Inject 10 μl, and the chromatographic results are as follows: Figure 1 shown.

[0024] Experimental results: The separation degree between the etomidate intermediate and its enantiomer is 3.2.

[0025] Example 2 Specificity Investigation Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IG (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol-diethylamine (93:7:0.1, v / v / v) Detector and wavelength: PDA-261nm Flow rate: 1.0ml / min Injection volume: 10 μl Column temperature: 25°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 0.009 mg of enantiomer per 1 ml as the reference solution. Inject 10 μl.

[0026] Blank test solution: Take an appropriate amount of etomidate intermediate, dissolve it in mobile phase and quantitatively dilute it to make a solution containing 2 mg of intermediate per 1 ml as blank test solution. Inject 10 μl, and the chromatographic results are as follows Figures 2~4 shown.

[0027] Conclusion: From Figure 2~Figure 4 The results showed that the blank solvent and blank test sample had no obvious interference with the detection.

[0028] Example 3 Linearity Investigation Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IG (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol-diethylamine (93:7:0.1, v / v / v) Detector and wavelength: PDA-261nm Flow rate: 1.0ml / min Injection volume: 10 μl Column temperature: 25°C Solvent: Mobile phase Quantitative method: Peak area external standard method Solution preparation: Enantiomer stock solution I: Take about 15 mg of the enantiomer reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and the solution is obtained (containing an enantiomer concentration of about 0.3 mg / ml).

[0029] Enantiomer stock solution II: Accurately pipette 2.5 ml of enantiomer stock solution I into a 50 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain (enantiomer concentration is about 0.015 mg / ml).

[0030] Accurately measure 1, 2, and 2 ml of the 15 μg / ml enantiomer stock solution and place it in 20, 25, and 10 ml volumetric bottles, respectively. Accurately measure 1, 1, and 1 ml of the 300 μg / ml enantiomer stock solution and place it in 50, 25, and 10 ml volumetric bottles, respectively. Add solvent to dilute to the scale, shake well, and prepare a series of standard solutions with concentrations of approximately 0.75, 1.2, 3, 6, 12, and 30 μg / 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 3, and the regression curve is shown in Figure 5 The regression equation was y = 1910.7x - 675.64 (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 0.71μg / ml~28.41μg / ml.

[0031] Table 3 Enantiomer standard curve

[0032] Example 4 Investigation of Quantitation Limit and Detection Limit Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IG (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol-diethylamine (93:7:0.1, v / v / v) Detector and wavelength: PDA-261nm Flow rate: 1.0ml / min Injection volume: 10 μl Column temperature: 25°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 0.00071 mg of 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 0.00035 mg of 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 4, and the quantitative limit chromatogram is shown in Figure 6 .

[0033] Table 4 Limit of quantitation and detection limit

[0034] Example 5 Accuracy Investigation Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IG (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol-diethylamine (93:7:0.1, v / v / v) Detector and wavelength: PDA-261nm Flow rate: 1.0ml / min Injection volume: 10 μl Column temperature: 25°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 2 mg of intermediate and 0.016 mg of enantiomer per 1 ml, as the 100% concentration level accuracy solution. Prepare 6 portions in parallel, inject them separately to calculate the enantiomer recovery rate in etomidate intermediate, and the results are shown in Table 5.

[0035] It can be seen from Table 5 that the recovery rate of enantiomers at the 100% level is in the range of 90% to 110%, and the recovery rate RSD is 2.1%. The accuracy of this method is good.

[0036] Table 5 Accuracy

[0037]

[0038] Example 6 Repeatability Study Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IG (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol-diethylamine (93:7:0.1, v / v / v) Detector and wavelength: PDA-261nm Flow rate: 1.0ml / min Injection volume: 10 μl Column temperature: 25°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 2 mg of intermediate and 0.016 mg of enantiomer per 1 ml as a repeatable solution. Prepare 6 portions in parallel, inject them separately to calculate the enantiomer content in etomidate intermediate, and the results are shown in Table 6.

[0039] As shown in Table 6, the detection method provided by the present invention has high precision, and the RSD value of the enantiomer content is 0.9%, which meets the requirements.

[0040] Table 6 Repeatability

[0041] Example 7 Intermediate Precision Investigation Chromatographic instruments: Two Shimadzu LC-20AD high performance liquid chromatographs with different internal numbers Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALPAK IG (250mm*4.6mm, 5μm) Mobile phase: n-hexane-isopropanol-diethylamine (93:7:0.1, v / v / v) Detector and wavelength: PDA-261nm Flow rate: 1.0ml / min Injection volume: 10 μl Column temperature: 25°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 2 mg of intermediate and 0.016 mg of enantiomer per 1 ml as a repeatable solution. Different experimenters used different instruments on different dates to parallelly determine the enantiomer detection amount in 6 repeatable solutions, and calculated the enantiomer detection amount 12 times. The results are shown in Table 7. The results show that the intermediate precision of this method is good, with an RSD of 1.2%.

[0042] Table 7 Intermediate precision

[0043] Example 8 Durability Study The durability 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, and the parameter range was: flow rate ±0.1ml / min, column temperature ±2℃, mobile phase ratio: n-hexane-isopropanol ratio was (95:5) and (91:9).

[0044] 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 2 mg of intermediate and 0.006 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 8.

[0045] Table 8 Durability

[0046] Comparative Example 1 Comparative experiment of chromatographic column with cellulose-carbamate derivative coated on silica gel surface Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALCEL® OD-H (5μm, 250×4.6mm) Mobile phase: n-hexane-isopropanol (98:2) Detector and wavelength: PDA-240nm Flow rate: 0.8ml / min Injection volume: 10 μl Column temperature: 25°C Solvent: Hexane: Isopropanol (90:10) 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 2 mg of intermediate and 0.03 mg of enantiomer per 1 ml.

[0047] Etomidate intermediate enantiomer localization solution: Take an appropriate amount of etomidate intermediate enantiomer reference substance, add mobile phase to dissolve and quantitatively dilute to prepare a solution containing 0.03 mg of enantiomer per 1 ml.

[0048] Under the above conditions, the system suitability solution and the etomidate intermediate enantiomer positioning solution were injected respectively, and the etomidate intermediate and the enantiomer could not be separated. The corresponding spectrum is shown in Figure 7 .

[0049] Comparative Example 2 Comparative Experiment of Silica Gel Surface Coated Cellulose-Carbamate Derivative Chromatographic Column Chromatographic instrument: Shimadzu LC-20AD high performance liquid chromatograph Chromatographic column: CHIRALCEL® OZ-H (5μm, 250×4.6mm) Mobile phase: n-hexane-isopropanol (98:2) Detector and wavelength: PDA-240nm Flow rate: 0.8ml / min Injection volume: 10 μl Column temperature: 25°C Solvent: Hexane: Isopropanol (90:10) 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 2 mg of intermediate and 0.03 mg of enantiomer per 1 ml.

[0050] Etomidate intermediate enantiomer localization solution: Take an appropriate amount of etomidate intermediate enantiomer reference substance, add mobile phase to dissolve and quantitatively dilute to prepare a solution containing 0.03 mg of enantiomer per 1 ml.

[0051] Under the above conditions, the system suitability solution and the etomidate intermediate enantiomer positioning solution were injected respectively, and the etomidate intermediate and the enantiomer could not be separated. The corresponding spectrum is shown in Figure 8 .

Claims

1. A method for separating and detecting an etomidate intermediate and its enantiomers, characterized in that: The invention comprises a polysaccharide derivative bonded chiral chromatographic column with amylose-tris (3-chloro-5-methylphenylcarbamate) bonded to the surface of silica gel as filler and a mixed solution of n-hexane, isopropanol and diethylamine as mobile phase.

2. The separation and detection method according to claim 1, wherein the chromatographic column is CHIRALPAK IG.

3. The separation and detection method according to claim 1, wherein the volume ratio of the mobile phase, n-hexane-isopropanol-diethylamine is (91-95): (5-9): (0.1-0.5).

4. The separation and detection method according to claim 3, wherein the volume ratio of the mobile phase, n-hexane-isopropanol-diethylamine, is 93:7:0.

1.

5. The separation and detection method according to claim 1, wherein the flow rate of the mobile phase is 0.9-1.1 ml / min.

6. The separation and detection method according to claim 1, wherein the temperature of the chromatographic column is 23-27°C.

7. The separation and detection method according to claim 1, wherein the detector is an ultraviolet detector and the detection wavelength is 210-325 nm. The separation and detection method according to claim 7 , wherein the detection wavelength is 261 nm.

9. The separation and detection method according to claim 1, wherein the chemical structure of the etomidate intermediate and its enantiomer is as shown below: 。

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