CN112213402B - Method for detecting (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride and isomer thereof - Google Patents
Method for detecting (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride and isomer thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention discloses a method for detecting (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride and an enantiomer thereof, which comprises the steps of carrying out pre-column derivatization on 1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride by using a chiral derivatization reagent, namely isothiocyanate or isocyanate, and detecting by adopting a high performance liquid chromatography. The method realizes the separation of (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride and (3S, 4S) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride, has low detection cost and good sensitivity, specificity and accuracy, and is an effective detection method for controlling the quality of the starting raw material of tofacitinib citrate, thereby further finally ensuring the product quality of the tofacitinib citrate and the medication safety of patients.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a tofacitinib citrate starting material (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride (named as SM-B) and a detection method of an enantiomer thereof.
Background
Tofacitinib citrate was developed by spodumene usa and used for the treatment of patients with moderate to severe Rheumatoid Arthritis (RA) with adult active period and poor response to Methotrexate (MTX).
The mesocultural name of tofacitinib citrate is (3R, 4R) -4-methyl-3- (methyl-1H-pyrrolo [2, 3-d)]Pyrimidine-4-amino) -beta-oxo-1-piperidinepropanitrile, 2-hydroxy-1, 2, 3-propanetricarboxylic acid (1) 16 H 20 N 6 O·C 6 H 8 O 7 The structural formula is as follows:
the tofacitinib citrate has two chiral centers and has four optical isomers including RR, RS, SR and SS, wherein the RR configuration is a JAK-1 and-3 tyrosine kinase inhibitor, and the other configurations have no biological equivalence, so that strict quality control on the optical isomers is required in the process of drug synthesis.
1-benzyl-N, 4-dimethylpiperidin-3-amine dihydrochloride (also known as N, 4-dimethyl-1- (phenylmethyl) -3-piperidinamine hydrochloride) has two chiral centers and has four optical isomers of RR, RS, SR and SS, wherein only (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine dihydrochloride (also known as (3R, 4R) -N, 4-dimethyl-1- (phenylmethyl) -3-piperidinamine hydrochloride, CAS number 1062580-52-2) is a starting material for the efficient synthesis of RR-configuration halofantinib. In the prior art, commercially available (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride (SM-B) usually contains one or more optical isomer impurities such as RS, SR, SS and the like, and according to the principle of source control, the optical isomer of SM-B needs to be strictly quality-controlled so as to ensure the quality of tofacitinib citrate. In the prior art, separation of two diastereomers RS and SR of 1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride from RR configuration can be realized under conventional reverse phase chromatographic conditions, but SS configuration and RR configuration are a pair of enantiomers, which have the same physical properties, thermodynamic properties and chemical properties and show differences only in chiral environment, and separation of 1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride in SS configuration and RR configuration is difficult to realize under conventional reverse phase chromatographic conditions.
Disclosure of Invention
The invention aims to provide a method for separating and measuring tofacitinib citrate initial raw material (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride (SM-B) and an enantiomer thereof by using high performance liquid chromatography, and the content of chiral isomers in tofacitinib citrate is controlled from the initial raw material so as to ensure the safety and quality reliability of a medicament.
The structural formula of the (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride (SM-B) and the enantiomer thereof is as follows:
the specific technical scheme of the invention is as follows:
(3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride (SM-B) and enantiomers thereof are detected by performing pre-column derivatization on 1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride (the 1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride is SM-B which may contain an optical isomer in SS configuration or (3S, 4S) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride which contains an optical isomer in RR configuration) by using a chiral derivatization reagent, namely isothiocyanate or isocyanate, and separating the (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride and the (3S, 4S) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride by using high performance liquid chromatography.
Preferably, the chiral derivatizing agent is selected from 2,3,4, 6-tetra-O-acetyl-beta-D-glucopyranose isothiocyanate or (S) - (-) -1-phenylethyl isocyanate.
The pre-column derivatization method comprises the following steps of reacting a substance to be detected with a derivatization reagent according to a molar weight of 1.
Preferably, the separation analysis is performed using an octadecylsilane bonded silica chromatographic column.
According to the detection method, high performance liquid chromatography can adopt isocratic or gradient elution, and a mobile phase A is a buffer salt solution, wherein the pH value of the solution is 2-4, and the preferred pH value of the solution is 2.3-3.5; the mobile phase B is an organic solvent and is selected from one or more of methanol or a mixed solution of methanol and acetonitrile. Preferably, the buffered salt solution is selected from an ammonium acetate buffer or a phosphate buffer.
Preferably, the high performance liquid chromatography adopts isocratic elution, the volume of the mobile phase A and the mobile phase B is 7-2.
The detection method of the invention has the flowing speed of the mobile phase of the high performance liquid chromatography of 0.8 to 1.2ml/min, the temperature of the chromatographic column of 20 to 40 ℃, the ultraviolet detector as the detector and the detection wavelength of 200 to 400nm.
The invention uses pre-column derivatization-high performance liquid chromatography to analyze and detect the starting material SM-B of tofacitinib citrate, realizes the separation of (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride and (3S, 4S) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride, has low detection cost and good sensitivity, specificity and accuracy, is an effective detection method for controlling the enantiomer quality of the starting material SM-B of tofacib citrate, and further finally ensures the product quality of tofacib citrate and the medication safety of patients.
Drawings
Figure 1 separation of SM-B and its enantiomers by a conventional high performance liquid chromatography chiral column (n-hexane-isopropanol-triethylamine (95.
Figure 2 separation of SM-B and its enantiomers by a conventional high performance liquid chromatography chiral column (with potassium dihydrogen phosphate solution-acetonitrile (80).
FIG. 3 HPLC chromatogram with (S) - (+) - α -methoxy- α - (trifluoromethyl) phenylacetyl chloride as derivatizing agent. FIG. 4 HPLC chromatogram with (S) - (-) -1-phenylethyl isocyanate as derivatizing agent.
FIG. 5 HPLC profile with GITC as the derivatizing agent.
FIG. 6 HPLC profile of mobile phase A versus system suitability solution at different pH.
FIG. 7 HPLC chromatogram of system suitability solution and SM-B enantiomer solution with acetonitrile as mobile phase B.
FIG. 8 HPLC chromatogram of system suitability solution and SM-B enantiomer solution with methanol as mobile phase B.
FIG. 9 HPLC chromatogram of system suitability solution for example 4 of the present invention.
FIG. 10 HPLC comparative profile of each solution in inventive example 5.
Detailed Description
The present invention will be further described with reference to specific examples, comparative examples and test examples, which include but are not limited to the following examples.
EXAMPLE 1 separation of SM-B and its enantiomers by conventional high performance liquid chromatography chiral column
Test example 1
An experimental instrument: aglent 1260 high performance liquid chromatograph.
Chromatographic conditions are as follows: the column was CHIRALPAK AD-H (4.6X 250mm,5 μm); the column temperature was 35 ℃; adopting an ultraviolet detector, wherein the detection wavelength is 257nm; the flow rate of the mobile phase is 0.8ml/min; n-hexane-isopropanol-triethylamine (95.
The experimental steps are as follows:
solvent: n-hexane-isopropanol (50
SB-B enantiomer stock: 2.5mg of the suspension was taken and diluted to 50ml with a solvent.
System applicability solution: and (3) taking 10mg of SM-B, placing the SM-B into a 10ml measuring flask, adding 200 mu l of isomer stock solution, adding a solvent to dissolve and dilute the SM-B to a scale mark, and shaking up to obtain the isomer.
And (3) taking 10 mu l of the system applicability solution and the SM-B enantiomer stock solution respectively, injecting the solution into a liquid chromatograph, detecting by adopting the chromatographic conditions, and recording a chromatogram. The results are shown in fig. 1, and show that in the chromatographic condition, the SM-B peak at 5.2min and the SM-B enantiomer peak at 5.1min, the SM-B enantiomer and the SM-B enantiomer can not be effectively separated, and in the chromatographic condition, the baseline fluctuation is large, the peak response is weak, and the detection sensitivity is low.
Test example 2:
an experimental instrument: shimadzu LC-20AT high performance liquid chromatograph.
Chromatographic conditions are as follows: the chromatographic column is CHIRALPAK AD-RH (4.6 × 250mm,5 μm); the column temperature is 30 ℃; an ultraviolet detector is adopted, and the detection wavelength is 210nm; the flow rate of the mobile phase is 1.0ml/min; a0.01 mol/L potassium dihydrogen phosphate solution (pH 8.0) -acetonitrile (80) was used as a mobile phase, and the solution was collected at equal intervals for 30min.
The experimental steps are as follows:
SB-B enantiomer stock: 2.5mg was taken and diluted to 50ml with mobile phase.
System applicability solution: and (3) taking 10mg of SM-B, placing the SM-B into a 10ml measuring flask, adding 200 mu l of isomer stock solution, adding a mobile phase to dissolve and dilute the SM-B to a scale mark, and shaking up to obtain the isomer.
And (3) taking 10 mu l of the system applicability solution and the SM-B enantiomer stock solution respectively, injecting the solution into a liquid chromatograph, detecting by adopting the chromatographic conditions, and recording a chromatogram. The results are shown in fig. 2 and show that under this chromatographic condition, both SM-B and its enantiomer were severely smeared by a smear factor of 7.4 and could not be separated efficiently.
EXAMPLE 2 examination of different chiral derivatizing reagents
Test example 1: uses (S) - (+) -alpha-methoxy-alpha- (trifluoromethyl) phenylacetyl chloride as derivatization reagent
An experimental instrument: aglent 1260 high performance liquid chromatograph.
Chromatographic conditions are as follows: the column was Welch Ultimate XB-C18 (4.6 x 250mm,5 μm); the column temperature was 40 ℃; an ultraviolet detector is adopted, and the detection wavelength is 210nm; the flow rate of the mobile phase is 1.0ml/min; the mixture was extracted at an equal rate for 60min using 0.01mol/L potassium dihydrogenphosphate buffer (pH 3.0) -methanol (50).
The experimental steps are as follows:
0.1% triethylamine solution: and (3) taking 100 mu l of triethylamine, diluting the triethylamine to 100ml with acetonitrile, and shaking up to obtain the triethylamine.
Derivatization reagent diluent: taking 10mg of (S) - (+) -alpha-methoxy-alpha- (trifluoromethyl) phenylacetyl chloride, diluting the (S) - (+) -alpha-methoxy-alpha- (trifluoromethyl) phenylacetyl chloride to 10ml with acetonitrile, and shaking up to obtain the (S) - (+) -alpha-methoxy-alpha- (trifluoromethyl) phenylacetyl chloride.
SM-B enantiomer stock: 2.5mg of the suspension was diluted to 25ml with 0.1% triethylamine solution.
System applicability solution: placing SM-B2.5mg in a 5ml measuring flask, adding 250 μ l isomer stock solution, adding 2ml 0.1% triethylamine solution, adding 1ml derivatization reagent diluent, mixing, standing at room temperature for 30min, adding water to dilute to scale, and shaking uniformly to obtain the final product.
And (4) taking 10 mu l of the system applicability solution, injecting the solution into a liquid chromatograph, detecting by adopting the chromatographic conditions, and recording a chromatogram. FIG. 3 is an HPLC chromatogram obtained by detection of a system-compatible solution using (S) - (+) - α -methoxy- α - (trifluoromethyl) phenylacetyl chloride as a derivatizing agent. The results show that, under the modified chromatographic conditions, SM-B peaks at 10.2min and the SM-B enantiomer peaks at 11min, both are not completely separated, while other peaks interfere with the detection of the enantiomer.
Test example 2: uses (S) - (-) -1-phenethyl isocyanate as a derivatization reagent
An experimental instrument: aglent 1260 high performance liquid chromatograph.
Chromatographic conditions are as follows: column Welch Ultimate XB-C18 (4.6X 250mm,5 μm); the column temperature was 40 ℃; an ultraviolet detector is adopted, and the detection wavelength is 210nm; the flow rate of the mobile phase is 1.0ml/min; the mixture was isocratically sampled for 60min using 0.01mol/L potassium dihydrogen phosphate buffer (pH 2.3) -methanol (50).
The experimental steps are as follows:
0.1% triethylamine solution: and (3) taking 100 mu l of triethylamine, diluting the triethylamine to 100ml with acetonitrile, and shaking up to obtain the triethylamine.
Derivatization reagent diluent: taking 50 mu l of (S) - (-) -1-phenethyl isocyanate, diluting to 10ml with acetonitrile, and shaking up to obtain the product.
SB-B enantiomer stock: 2.5mg of the suspension was diluted to 25ml with 0.1% triethylamine solution.
System applicability solution: placing SM-B2.5mg in a 5ml measuring flask, adding 250 μ l isomer stock solution, adding 2ml 0.1% triethylamine solution, adding 1ml derivatization reagent diluent, mixing, standing at room temperature for 30min, adding water to dilute to scale, and shaking uniformly to obtain the final product.
And (4) taking 10 mu l of the system applicability solution, injecting the system applicability solution into a liquid chromatograph, detecting by adopting the chromatographic conditions, and recording a chromatogram. FIG. 4 is an HPLC chromatogram obtained by detecting a solution applicable to a system using (S) - (-) -1-phenethyl isocyanate as a derivatization reagent. The results show that SM-B peaks at 19min and SM-B enantiomer peaks at 22min under the chromatographic conditions of the invention, both can be effectively separated, while the remaining hetero-peaks do not interfere with the detection of the enantiomers.
Experimental example 3 2,3,4,6-tetraacetyl- β -D-Glucopyranosyl Isothiocyanate (GITC) as a derivatization reagent
An experimental instrument: shimadzu LC-20AT high performance liquid chromatograph.
Chromatographic conditions are as follows: the column was YMC-Triart C18 (4.6X 250mm,5 μm); the column temperature is 30 ℃; an ultraviolet detector is adopted, and the detection wavelength is 250nm; the flow rate of the mobile phase is 1.0ml/min; the mixture was isocratically collected for 30min using 0.02mol/L ammonium acetate buffer (pH 3.5) -methanol (50).
The experimental steps are as follows:
0.1% triethylamine solution: and taking 100 mu l of triethylamine, diluting the triethylamine to 100ml by using a 50% acetonitrile solution, and shaking up to obtain the triethylamine.
2,3,4,6-tetraacetyl- β -D-Glucopyranosyl Isothiocyanate (GITC) solution: and taking 50mg of GITC, dissolving and diluting the GITC to 5ml by using a 50% acetonitrile solution, and shaking up to obtain the compound.
SB-B enantiomer stock: 2.5mg of the suspension was diluted to 25ml with 50% acetonitrile.
System applicability solution: and (3) placing SM-B2.5mg in a 5ml measuring flask, adding 250 mul of isomer stock solution, 0.5ml of 0.1% triethylamine solution and 1ml of GITC solution, mixing uniformly, standing at room temperature for 30min, adding 50% acetonitrile solution to dilute to a scale, and shaking uniformly to obtain the final product.
And (4) taking 10 mu l of the system applicability solution, injecting the solution into a liquid chromatograph, detecting by adopting the chromatographic conditions, and recording a chromatogram. FIG. 5 is an HPLC profile measured on a system-adapted solution using GITC as the derivatizing agent. The results show that, under the modified chromatographic conditions, SM-B peaked at 17min and the SM-B enantiomer peaked at 15.2min, both were effectively separated, while the remaining miscellaneous peaks did not interfere with the detection of the enantiomer.
Example 3 screening of the Mobile phase for the detection method according to the invention
Test example 1: screening of mobile phase A pH value
The purpose of the experiment is as follows: selecting 0.01mol/L potassium dihydrogen phosphate water solution as a water phase, adjusting different pH values, inspecting the influence of the pH value of a mobile phase on the detection of the SM-B enantiomer, and selecting proper conditions for the detection of the SM-B enantiomer.
An experimental instrument: shimadzu LC-20AT high performance liquid chromatograph.
Chromatographic conditions are as follows: the column was Welch Ultimate XB-C18 (4.6 x 250mm,5 μm); the column temperature was 40 ℃; an ultraviolet detector is adopted, and the detection wavelength is 210nm; the flow rate of the mobile phase is 1.0ml/min; three different pH values (pH 2.3, pH3.0, and pH6.0, respectively) were adjusted using 0.01mol/L monopotassium phosphate buffer as mobile phase A, and mobile phase A-methanol (50) was isocratically collected for 60min.
The experimental steps are as follows:
0.1% triethylamine solution: and (3) taking 100 mu l of triethylamine, diluting the triethylamine to 100ml with acetonitrile, and shaking up to obtain the triethylamine.
Derivatization reagent diluent: taking 50 mu l of (S) - (-) -1-phenethyl isocyanate, diluting to 10ml with acetonitrile, and shaking up to obtain the product.
Stock SM-B enantiomer: 2.5mg of the suspension was diluted to 25ml with 0.1% triethylamine solution.
System applicability solution: placing SM-B2.5mg in a 5ml measuring flask, adding 250 μ l isomer stock solution, adding 2ml 0.1% triethylamine solution, adding 1ml derivatization reagent diluent, mixing, standing at room temperature for 30min, adding water to dilute to scale, and shaking uniformly to obtain the final product.
And (4) taking 10 mu l of the system applicability solution, injecting the solution into a liquid chromatograph, detecting under different mobile phase conditions respectively, and recording a chromatogram. FIG. 6 is a comparison HPLC chart obtained by using mobile phase A with different pH values to detect the system applicability solution. The results show that when the pH of mobile phase a is 6.0, no SM-B and its enantiomeric peaks are observed, which should not elute during the acquisition time; when the pH value of the mobile phase A is 2.3, the SM-B peak at 19min, the SM-B enantiomer peak at 22min, the SM-B peak and the SM-B enantiomer can be effectively separated, when the pH value of the mobile phase A is 3.0, the SM-B peak at 19.5min, the SM-B enantiomer peak at 23min, the SM-B enantiomer peak and the SM-B enantiomer can be effectively separated, and meanwhile, the rest miscellaneous peaks do not interfere with the detection of the enantiomers.
Test example 2: screening of Mobile phase B
The purpose of the experiment is as follows: and (3) investigating the influence of two organic solvents of methanol and acetonitrile on the detection of the SM-B enantiomer, and selecting proper conditions for the detection of the SM-B enantiomer.
An experimental instrument: shimadzu LC-20AT high performance liquid chromatograph.
Chromatographic conditions are as follows: column Welch Ultimate XB-C18 (4.6X 250mm,5 μm); the column temperature was 40 ℃; an ultraviolet detector is adopted, and the detection wavelength is 210nm; the flow rate of the mobile phase is 1.0ml/min; the mixture was collected at an equal temperature for 60min in a 0.01mol/L potassium dihydrogen phosphate buffer (pH 2.3) as mobile phase A, methanol or acetonitrile as mobile phase B, and mobile phase A-B (50).
The experimental steps are as follows:
0.1% triethylamine solution: and (3) taking 100 mu l of triethylamine, diluting the triethylamine to 100ml with acetonitrile, and shaking up to obtain the triethylamine.
Derivatization reagent diluent: taking 50 mu l of (S) - (-) -1-phenethyl isocyanate, diluting to 10ml with acetonitrile, and shaking up to obtain the product.
SM-B enantiomer stock: 2.5mg of the suspension was diluted to 25ml with 0.1% triethylamine solution.
System applicability solution: placing SM-B2.5mg in a 5ml measuring flask, adding 250 μ l isomer stock solution, adding 2ml 0.1% triethylamine solution, adding 1ml derivatization reagent diluent, mixing, standing at room temperature for 30min, adding water to dilute to scale, and shaking uniformly to obtain the final product.
SM-B enantiomer solution: taking 2ml of SM-B enantiomer stock solution, adding 1ml of derivatization reagent diluent, mixing uniformly, standing at room temperature for 30min, adding water to dilute to scale, and shaking uniformly to obtain the final product.
And taking 10 mu l of each of the system applicability solution and the SM-B enantiomer solution, injecting the solution into a liquid chromatograph, detecting under different mobile phase conditions respectively, and recording a chromatogram. FIG. 7 is an HPLC chromatogram obtained by detection of a system suitability solution and an SM-B enantiomer solution using acetonitrile as mobile phase B; FIG. 8 is an HPLC chromatogram obtained by detection of a system suitability solution and an SM-B enantiomer solution using methanol as mobile phase B. The result shows that when acetonitrile is used as the mobile phase B, the peak emergence time of SM-B and the enantiomer thereof is about 6min, the SM-B and the enantiomer thereof do not reach baseline separation, and a plurality of miscellaneous peaks exist before and after the peak emergence, which interfere with isomer detection; as shown in FIG. 3, when methanol was used as mobile phase B, SM-B peaked at 19min and the SM-B enantiomer peaked at 22min, both were effectively separated, while the remaining hetero-peaks did not interfere with the detection of the enantiomer.
Example 4 separation of SM-B and its enantiomers by the detection method of the invention
An experimental instrument: aglent 1260 high performance liquid chromatograph.
Chromatographic conditions are as follows: column Welch Ultimate XB-C18 (4.6X 250mm,5 μm); the column temperature is 40 ℃; an ultraviolet detector is adopted, and the detection wavelength is 210nm; the flow rate of the mobile phase is 1.0ml/min; the mixture was isocratically sampled for 60min using 0.01mol/L potassium dihydrogen phosphate buffer (pH 3.0) -methanol (50).
The experimental steps are as follows:
0.1% triethylamine solution: and (3) taking 100 mu l of triethylamine, diluting the triethylamine to 100ml with acetonitrile, and shaking up to obtain the triethylamine.
Derivatization reagent diluent: taking 50 mu l of (S) - (-) -1-phenethyl isocyanate, diluting to 10ml with acetonitrile, and shaking up to obtain the product.
SB-B enantiomer stock: 2.5mg of the suspension was diluted to 25ml with 0.1% triethylamine solution.
System applicability solution: placing SM-B2.5mg in a 5ml measuring flask, adding 250 μ l isomer stock solution, 2ml 0.1% triethylamine solution, adding 1ml derivatization reagent diluent, mixing, standing at room temperature for 30min, adding water to dilute to scale, and shaking to obtain the final product.
And (4) taking 10 mu l of the system applicability solution, injecting the system applicability solution into a liquid chromatograph, detecting by adopting the chromatographic conditions, and recording a chromatogram. FIG. 9 is an HPLC chromatogram obtained by detection of a system-compatible solution using the detection method of the present invention. The results show that, under the chromatographic conditions of the invention, SM-B peaked at 19.5min and the SM-B enantiomer peaked at 23min, both were able to be separated efficiently, while the remaining miscellaneous peaks did not interfere with the detection of the enantiomer.
Example 5 separation of SM-B and its enantiomers by the detection method of the invention
An experimental instrument: shimadzu LC-20AT high performance liquid chromatograph.
Chromatographic conditions are as follows: the column was Welch Ultimate XB-C18 (4.6 x 250mm,5 μm); the column temperature is 40 ℃; an ultraviolet detector is adopted, and the detection wavelength is 210nm; the flow rate of the mobile phase is 1.0ml/min; the mixture was extracted at an equal rate for 60min using 0.01mol/L potassium dihydrogenphosphate buffer (pH 2.3) -methanol (50).
The experimental steps are as follows:
0.1% triethylamine solution: and (3) taking 100 mu l of triethylamine, diluting the triethylamine to 100ml with acetonitrile, and shaking up to obtain the triethylamine.
Derivatization reagent diluent: taking 50 mu l of (S) - (-) -1-phenethyl isocyanate, diluting to 10ml with acetonitrile, and shaking up to obtain the product.
Stock SM-B enantiomer: 2.5mg of the suspension was diluted to 25ml with 0.1% triethylamine solution.
SM-B enantiomer control solution: and (3) taking 250 mu l of SM-B enantiomer stock solution, placing the stock solution in a 5ml measuring flask, adding 2ml of 0.1% triethylamine solution, adding 1ml of derivatization reagent diluent, uniformly mixing, standing at room temperature for 30min, adding water for diluting to a scale, and shaking uniformly to obtain the final product.
SM-B test solution: and (3) placing SM-B2.5mg in a 5ml measuring flask, adding 2ml of 0.1% triethylamine solution, adding 1ml of derivatization reagent diluent, uniformly mixing, standing at room temperature for 30min, adding water for diluting to a scale, and shaking uniformly to obtain the final product.
Recovery of the solution: placing SM-B2.5mg in a 5ml measuring flask, weighing 3 parts in parallel, adding 250 mul, 500 mul and 1000 mul of isomer stock solution, 2ml of 0.1% triethylamine solution and 1ml of derivatization reagent diluent respectively, mixing uniformly, placing at room temperature for 30min, adding water for diluting to scale, and shaking uniformly to obtain the final product.
Blank solution: taking 2ml of 0.1% triethylamine solution, placing in a 5ml measuring flask, adding 1ml of derivatization reagent diluent, mixing uniformly, standing at room temperature for 30min, adding water for diluting to a scale, and shaking uniformly to obtain the final product.
And (3) taking 10 mu l of blank solution, SM-B enantiomer reference solution, SM-B test sample solution and recovery solution respectively, injecting into a liquid chromatograph, detecting by adopting the chromatographic conditions, and recording a chromatogram. FIG. 10 is a comparative HPLC chart of the above solution detected by the detection method of the present invention. The result shows that under the chromatographic condition of the invention, the blank solution does not interfere with the detection of SM-B and the enantiomer thereof, the SM-B and the enantiomer thereof can be effectively separated, the recovery rate of the SM-B enantiomer is good, the recovery rates of 3 parts of recovery rate solution are respectively 96.8%, 93.7% and 99.4%, the detection sensitivity is high, and the signal-to-noise ratio of the SM-B enantiomer with the limit concentration (1%) is up to 100 and is far higher than the limit of quantitation.
Claims (1)
1. The detection method of (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride and enantiomers thereof is characterized in that a chiral derivatization reagent (S) - (-) -1-phenethyl isocyanate is used for carrying out pre-column derivatization on 1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride, the reaction is carried out according to the molar weight of a substance to be detected and a derivatization reagent being 1; detecting by high performance liquid chromatography with octadecylsilane chemically bonded silica chromatographic column; isocratic elution is adopted, the mobile phase A is a buffer salt solution, and the pH value of the solution is 2 to 4; the mobile phase B is an organic solvent and is selected from methanol or a mixed solution of methanol and acetonitrile, the volume of the mobile phase A and the mobile phase B is 7-2, the buffer salt solution is selected from an ammonium acetate buffer solution or a phosphate buffer solution, the flow rate of the mobile phase is 0.8 to 1.2ml/min, the column temperature of a chromatographic column is 20 to 40 ℃, the detector is an ultraviolet detector, and the detection wavelength is 200 to 400nm.
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