Isomer detection method of remazolam key intermediate
By using Lux-I-AMYLOSE-3 hand-culinary elution method in chromatography, the problem of detection problems in the intermediate heterogeneous body heterogeneous in the intermediate body of the pharmaceutical quality is solved, and the quality of the drug quality is improved and the detection effect is improved.
Patent Information
- Application Number
- CN202411329825.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-24
- Publication Date
- 2025-01-24
Smart Images
Figure CN119355151A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of drug detection, and in particular to a method for detecting isomers of a key intermediate of remimazolam. Background Art
[0002] Remimazolam besylate was officially approved for marketing in July 2020 and is used in the fields of painless diagnosis and treatment sedation, general anesthesia, ICU sedation, and local anesthesia sedation. Remimazolam is a new type of ultra-short-acting benzodiazepine anesthetic, which has the characteristics of predictable sedation time and rapid recovery in painless gastrointestinal endoscopy. Compound A is an important intermediate in the synthesis of remimazolam, and the quality of compound A will have a great impact on the subsequent quality of remimazolam. The structural formula is as follows:
[0003] As an important drug precursor or active ingredient, remimazolam has broad application prospects in the pharmaceutical industry. The key intermediate of remimazolam, (3S)-7-bromo-2,3-dihydro-2-oxo-5-(2-pyridyl)-1H-1,4-benzodiazepine-3-propionic acid methyl ester and its isomer, (3R)-7-bromo-2,3-dihydro-2-oxo-5-(2-pyridyl)-1H-1,4-benzodiazepine-3-propionic acid methyl ester, the quality control of this intermediate is crucial to ensure the safety and effectiveness of the final drug. Therefore, it is of great significance to develop an analytical detection method to ensure the quality and safety of subsequent remimazolam. Summary of the invention
[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for detecting isomers of a key intermediate of remimazolam. The present invention is achieved through the following technical solutions: On the one hand, a method for detecting isomers of a key intermediate of remimazolam is provided, comprising the following steps: Step 1) using an amylose filler as a chiral separation chromatographic column; Step 2) using acid water as mobile phase A and organic solvent as mobile phase B; Step 3) The mobile phase adopts a gradient elution method, the mobile phase flow rate is 0.5~2.0mL / min, the detection wavelength is 200nm~280nm, and the column temperature is 20~45°C; wherein the detection wavelength is preferably 200nm~254nm, more preferably 230nm; the flow rate is preferably 0.5~1.5mL / min, more preferably 1.0 mL / min; the column temperature of the chromatographic column is preferably 30°C~40°C, more preferably 30°C; and a diluent is also included, and the diluent is methanol.
[0005] The gradient elution method of the mobile phase is as follows:
[0006] Wherein, the chiral separation chromatographic column is Lux-i-Amylose-3, with specifications of 250mm*4.6mm, 5μm.
[0007] Furthermore, the key intermediate of remimazolam is (3S)-7-bromo-2,3-dihydro-2-oxo-5-(2-pyridyl)-1H-1,4-benzodiazepine-3-propionic acid methyl ester (A); the enantiomer is (3R)-7-bromo-2,3-dihydro-2-oxo-5-(2-pyridyl)-1H-1,4-benzodiazepine-3-propionic acid methyl ester (B), and the structural formula is shown below: .
[0008] Furthermore, the detection method further comprises the following steps: Step 4) Preparation of the test solution: Take an appropriate amount of the test sample, add an appropriate amount of diluent to dissolve and dilute to prepare a sample solution containing 5-30 mg of remimazolam intermediate per mL as the test solution; Step 5) Preparation of reference solution: Accurately weigh an appropriate amount of isomer, add an appropriate amount of diluent to dissolve and dilute to make a solution with a concentration of 0.1%~1.5% of the test sample concentration; 0.5% is preferred as the reference solution.
[0009] Step 6) Set the injection volume to 5-30 μL, accurately measure equal volumes of the test solution and reference solution, inject them into the liquid chromatograph, and record the chromatogram.
[0010] Furthermore, in step 2), the acid water is selected from one or more of phosphoric acid, formic acid, and trifluoroacetic acid; and the organic solvent is selected from one or more of methanol, ethanol, and acetonitrile.
[0011] Furthermore, the acid water is trifluoroacetic acid; the concentration of the trifluoroacetic acid is 0.01% to 0.15% V / V, preferably 0.1% V / V.
[0012] Furthermore, the retention time (RT) of the peak position of the remimazolam intermediate is 10.5-11.5 min, the RT of its enantiomer is 9.5-10.2 min, and the separation degree is ≥1.5.
[0013] Further, the specific chromatographic conditions are as follows: Chromatographic column: Lux-i-Amylose-3250*4.6mm, 5μm; Wavelength: 230nm Column temperature: 30°C Flow rate: 1.0ml / min Injection volume: 10ul Mobile phase A: 0.1% trifluoroacetic acid in water Mobile phase B: acetonitrile The gradient elution method of the mobile phase is as follows:
[0015] Furthermore, the remimazolam isomer was prepared into a 0.4 μg / ml solution, and under this concentration condition, the signal-to-noise ratio of the chromatographic peak was 13.2.
[0016] Beneficial Effects The analytical detection method of the present invention can realize the detection of remimazolam intermediates and isomers thereof, improves the quality of remimazolam, provides quality assurance for the subsequent synthesis of remimazolam, and has the advantages of high accuracy, good precision, high sensitivity, etc.
[0017] The present invention uses a Lux-i-Amylose-3 chiral chromatographic column (250mm*4.6mm, 5μm) as a separation chromatographic column, uses a 0.1% trifluoroacetic acid aqueous solution as a mobile phase A, and uses acetonitrile as a mobile phase B. By adopting a gradient elution method, a remimazolam intermediate and its enantiomers can be baseline separated, and the content of its corresponding isomers in the remimazolam intermediate can be simply, quickly, and accurately controlled. The present invention uses Lux-i-Amylose-3 as a chiral chromatographic column, the retention time (RT) of the peak position of the remimazolam intermediate is about 11.2min, the RT of its enantiomer is about 10.1min, the separation degree reaches more than 1.5, meets the requirements of the pharmacopoeia, and the peak position is stable, thereby realizing the separation and determination of the remimazolam intermediate and its enantiomers. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 : Liquid chromatogram of blank solution; Figure 2 : Liquid chromatogram of system suitability solution of remimazolam intermediate and its enantiomers; Figure 3 : Liquid chromatogram of the sensitive solution (0.1%) of remimazolam intermediate; Figure 4 : Liquid chromatogram of remimazolam intermediate test solution.
[0019] Example 1 1. Instruments and conditions Agilent 1260 Infinity II HPLC CPA225D electronic analytical balance (Sartorius) Acetonitrile and trifluoroacetic acid were chromatographically pure 2. Experimental Methods and Results Column 1: CHIRALPAK AS-RH chiral column (250*4.6,5μm); Column 2: Lux-i-Amylose-3 chiral column (250*4.6,5μm); Column 3: CHIRALPAK IC chiral column (250*4.6,5μm); Detection wavelength: 230nm; Column temperature: 30-40°C; Injection volume: 10 μL; Flow rate: 0.6~1.0mL / min; Diluent: methanol; System suitability solution: Take about 40 mg of the remimazolam intermediate isomer reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add an appropriate amount of diluent to dissolve it by ultrasound, add diluent to dilute to the scale, shake well, and use it as a 0.4 mg / ml isomer reference substance stock solution. Take about 40 mg of the remimazolam intermediate reference substance, accurately weigh it, place it in a 100 ml volumetric flask, accurately measure 1 ml of the isomer reference substance stock solution and place it in the same 100 ml volumetric flask, add an appropriate amount of diluent to dissolve it by ultrasound, add diluent to dilute to the scale, shake well, and you have it. (Remimazolam intermediate concentration 0.40 mg / ml, isomer concentration 4ug / ml) The flow rate, column temperature, mobile phase ratio and chromatographic column in the chromatographic conditions were changed respectively, and other conditions and parameters remained unchanged to investigate the separation of the remimazolam intermediate and its enantiomers. The results are shown in Table 1 Table 1: Baseline separation status after changing test parameters
[0020]
[0021]
[0022] In summary, the optimal chromatographic conditions are as follows: Chromatographic column: Lux-i-Amylose-3250*4.6mm, 5μm; Wavelength: 230nm Column temperature: 30°C Flow rate: 1.0ml / min Injection volume: 10ul Mobile phase A: 0.1% trifluoroacetic acid in water Mobile phase B: acetonitrile Gradient elution:
[0023] Example 2 Validation of analytical methods 1. Instruments and conditions Agilent 1260 Infinity II HPLC CPA225D electronic analytical balance (Sartorius) Acetonitrile and trifluoroacetic acid were chromatographically pure 2. Experimental Methods and Results 1. Solution Preparation Diluent: Methanol Isomer reference stock solution: Take 40 mg of remimazolam intermediate isomer, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute it to the scale, shake well, and use it as the isomer reference stock solution (0.40 mg / ml).
[0024] Isomer reference solution: Accurately measure 1 ml of isomer reference stock solution and place it in a 10 ml volumetric flask, add diluent to dissolve and make up to volume (0.04 mg / ml).
[0025] System suitability solution: Take about 40 mg of the remimazolam intermediate reference substance, accurately weigh it, and place it in a 100 ml volumetric flask. Accurately measure 1 ml of the isomer reference substance stock solution and place it in the same 100 ml volumetric flask. Add an appropriate amount of diluent and ultrasonically dissolve it. Add diluent to dilute to the scale, shake well, and you have it. (Remimazolam intermediate concentration 0.40 mg / ml, isomer concentration 4 ug / ml) Sensitivity solution (0.1%): 40 mg of remimazolam intermediate is placed in a 100 ml volumetric flask. Accurately measure 1 ml of isomer reference solution and place it in the same volumetric flask. Add diluent to dissolve and dilute into a solution containing 0.40 mg of remimazolam intermediate and 0.4 μg of isomer per 1 ml, which is used as the sensitivity solution.
[0026] Test solution: Take about 40 mg of the remimazolam intermediate test sample, accurately weigh it, put it in a 100 ml volumetric flask, dissolve it with a diluent and dilute it to the scale, shake it well, and use it as the test solution. (0.40 mg / ml) 2. Chromatographic conditions Chromatographic column: Lux-i-Amylose-3250*4.6mm, 5μm; Wavelength: 230nm Column temperature: 30°C Flow rate: 1.0ml / min Injection volume: 10ul Mobile phase A: 0.1% trifluoroacetic acid in water Mobile phase B: acetonitrile Gradient elution:
[0027] 3. System suitability investigation The diluent, system suitability solution, and test solution were sampled and tested respectively. The results showed that the baselines of the intermediate and isomer of remimazolam were completely separated, and the blank did not interfere with the determination. The blank solution chromatogram is shown in Figure 1 , system suitability chromatograms are shown in Figure 2 .
[0028]
[0029] 4. Sensitivity test The remimazolam intermediate isomer was prepared into a 0.4 μg / ml solution (i.e. 0.1%). The results showed that under this concentration condition, the chromatographic peak signal-to-noise ratio was 13.2, with high sensitivity. See the chromatogram of the sensitive solution for details. Figure 3 .
[0030] 5. Determination of isomer content in products Take an appropriate amount of the test sample, prepare the test sample solution and isomer solution according to the above method, inject them for measurement respectively, and record the chromatogram.
[0031] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. DETAILED DESCRIPTION
[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0033] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods without specific conditions in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, all percentages, ratios, proportions or parts are by weight.
[0034] Unless otherwise specified, the reagents and raw materials used in the examples and comparative examples of the present invention can be obtained through commercial channels.
[0035] Agilent 1260 high performance liquid chromatograph with UV detector; CPA225D electronic analytical balance (Sartorius); remimazolam intermediates and their enantiomers were all homemade; trifluoroacetic acid, acetonitrile and methanol were all chromatographic grade.
Claims
1. A method for detecting isomers of a key intermediate of remimazolam, characterized in that: The following steps are involved: Step 1) using an amylose filler as a chiral separation chromatographic column; Step 2) using acid water as mobile phase A and organic solvent as mobile phase B; Step 3) The mobile phase adopts a gradient elution method, the mobile phase flow rate is 0.5~2.0mL / min, the detection wavelength is 200nm~280nm, and the column temperature is 20~45℃; The gradient elution method of the mobile phase is as follows: Wherein, the chiral separation chromatographic column is Lux-i-Amylose-3, with specifications of 250mm*4.6mm, 5μm.
2. The isomer detection method of the key intermediate of remimazolam according to claim 1, characterized in that: The key intermediate of remimazolam is (3S)-7-bromo-2,3-dihydro-2-oxo-5-(2-pyridyl)-1H-1,4-benzodiazepine-3-propionic acid methyl ester (A); the enantiomer is (3R)-7-bromo-2,3-dihydro-2-oxo-5-(2-pyridyl)-1H-1,4-benzodiazepine-3-propionic acid methyl ester (B), and the structural formula is shown below: 。 3. The isomer detection method of the key intermediate of remimazolam according to claim 1 or 2, characterized in that: The detection method further comprises the following steps: Step 4) Preparation of the test solution: Take an appropriate amount of the test sample, add an appropriate amount of diluent to dissolve and dilute to prepare a sample solution containing 5-30 mg of remimazolam intermediate per mL as the test solution; Step 5) Preparation of reference solution: Accurately weigh an appropriate amount of isomer, add an appropriate amount of diluent to dissolve and dilute to make a solution with a concentration of 0.1% to 1.5% of the test sample concentration; Step 6) Set the injection volume to 5-30 μL, accurately measure equal volumes of the test solution and reference solution, inject them into the liquid chromatograph, and record the chromatogram.
4. The isomer detection method of the key intermediate of remimazolam according to claim 1, characterized in that: In step 2), the acid water is selected from one or more of phosphoric acid, formic acid, and trifluoroacetic acid; and the organic solvent is selected from one or more of methanol, ethanol, and acetonitrile.
5. The isomer detection method of the key intermediate of remimazolam according to claim 4, characterized in that: The acid water is trifluoroacetic acid; the concentration of the trifluoroacetic acid is 0.01%~0.15%V / V.
6. The isomer detection method of the key intermediate of remimazolam according to claim 4, characterized in that: The retention time (RT) of the peak position of the remimazolam intermediate is 10.5-11.5 min, the RT of its enantiomer is 9.5-10.2 min, and the separation degree is ≥1.
5.
7. The isomer detection method of the key intermediate of remimazolam according to claim 1, characterized in that: The specific chromatographic conditions are as follows: Chromatographic column: Lux-i-Amylose-3250*4.6mm, 5μm; Wavelength: 230nm Column temperature: 30°C Flow rate: 1.0ml / min Injection volume: 10ul Mobile phase A: 0.1% trifluoroacetic acid in water Mobile phase B: acetonitrile The gradient elution method of the mobile phase is as follows:
8. The isomer detection method of the key intermediate of remimazolam according to claim 3, characterized in that: The remimazolam isomers were prepared into a 0.4 μg / ml solution. Under this concentration condition, the signal-to-noise ratio of the chromatographic peak was 13.2.