CN114264762B - High performance liquid chromatography separation detection method for key intermediate enantiomer of tergolian praziquantel - Google Patents
High performance liquid chromatography separation detection method for key intermediate enantiomer of tergolian praziquantel Download PDFInfo
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Abstract
The application discloses a high performance liquid chromatography separation detection method of an enantiomer of a key tergoprazan intermediate, which uses an amylose derivative chiral column as a chromatographic column and a mixed solution of normal hexane and ethanol as a mobile phase, can realize separation and determination of the enantiomer of the key tergoprazan intermediate, and can rapidly and accurately separate an S configuration and an R configuration in the enantiomer of the key tergoprazan intermediate; furthermore, by limiting the chromatographic conditions of the separation detection method, the separation degree of the S configuration and the R configuration of the key intermediate enantiomer of the tergolian in the obtained high performance liquid chromatographic spectrogram can reach 1.84-5.52, the requirements of Chinese pharmacopoeia are met, the retention time is within 7-15min, and the purity detection of the key intermediate in the process of industrialized mass production of tergolian is facilitated, so that the quality of the final product tergolian in industrialized production can be ensured, and the method has important industrial value.
Description
Technical Field
The application belongs to the field of chiral structure detection, and relates to a high performance liquid chromatography separation detection method for key intermediate enantiomer of tergolian praecox.
Background
(S) -1-benzyl-4- ((5, 7-difluorochrome-4-yl) oxy) -N, N,2-trimethyl-1H-benzo [ d ]]Imidazole-6-carboxamide, designated (S) -1-benzoyl-4- ((5, 7-difluoro-chroman-4-yl) oxy) -N, N, 2-trimethyl-1H-benzol [ d ]]imidazole-6-carboxamide, no CAS number, chemical formula C 27 H 25 F 2 N 3 O 3 The structural formula is shown in the following figure, and is a key chiral intermediate for synthesizing tergolian praziram.
Tergolian is a competitive potassium acid blocker (P-CAB) and a hydrogen ion/potassium ion exchange atp enzyme (H+/K+ atpase) inhibitor, has quick response and can control the pH value of gastric juice for a long time. Tergolian was approved for the treatment of gastroesophageal reflux disease and erosive esophagitis by the korean food and drug safety division (MFDS) at month 7 of 2018. And (S) -1-benzyl-4- (5, 7-difluoro-4-yl) oxy) -N, N,2-trimethyl-1H-benzo [ d ] imidazole-6-carboxamide is used as a key intermediate for the synthesis of tergolian, and the optical purity of the intermediate directly influences the optical purity of the synthesized final product tergolian. Therefore, the separation and detection method of the enantiomer of the (S) -1-benzyl-4- (5, 7-difluoro chromium-4-yl) oxy) -N, N,2-trimethyl-1H-benzo [ d ] imidazole-6-formamide has important industrial value.
At present, the separation and detection method of chiral substance optical purity reported in literature is mainly a high performance liquid chromatography separation and detection method. Patent CN103344733B discloses a method for detecting bortezomib enantiomer by high performance liquid chromatography, which uses cellulose coating chiral column OZ-H chiral column as chromatographic column and n-hexane-methanol-ethanol-ethanolamine as mobile phase, wherein n-hexane: methanol: ethanol: the volume ratio of the ethanolamine is (88-92)%: 5%: (3-7)%: (0.08-0.12)%, and the separation degree is more than 1.5. Patent CN103698436B discloses a method for detecting enantiomers in pramipexole dihydrochloride and a method for detecting enantiomers in pramipexole dihydrochloride, wherein an amylose derivative chiral column is used as a chromatographic column, and n-hexane-isopropanol-amine solvent is used as a mobile phase, wherein n-hexane: isopropyl alcohol: the volume ratio of the amine solvent is (80-90)%: (10-20)%: (0.05-0.5)%, and the separation degree can reach 5.22-8.93. Patent CN111678998B discloses a method for separating and detecting enantiomer in raltitrexed, which uses octadecyl bonded silica gel column as chromatographic column, aqueous solution of cyclodextrin or aqueous solution of cyclodextrin derivative and acetonitrile as mobile phase, wherein the aqueous solution of cyclodextrin: acetonitrile in the volume ratio of (910-930)%: (70-90)%.
As is clear from the above, for the separation and detection of enantiomers of different chiral substances, the chromatographic column, mobile phase and other chromatographic conditions are selected to have large differences, and the chromatographic conditions are selected by combining factors such as the nature of a compound, the polarity of the mobile phase and the interaction of the compound, so that the detection conditions of the high performance liquid chromatography of different compounds have no determinable reference significance.
However, there is no published literature report on a method for detecting the separation of enantiomers of key intermediate (S) -1-benzyl-4- (5, 7-difluorochrome-4-yl) oxy) -N, 2-trimethyl-1H-benzo [ d ] imidazole-6-carboxamide of tergolian.
Disclosure of Invention
The application aims to solve the problems and provide a high performance liquid chromatography separation detection method for key intermediate enantiomers of tergoliprazan, which can rapidly and accurately separate and detect the retention time and the relative content of S configuration and R configuration in the key intermediate enantiomers of tergoliprazan, and the separation degree can reach 1.84-5.52.
The technical scheme of the application is as follows:
the application discloses a high performance liquid chromatography separation detection method of an enantiomer of a key intermediate of tergoliprazan, which adopts an amylose derivative chiral column as a chromatographic column and a mixed solution of normal hexane and ethanol as an enantiomer of the key intermediate of tergoliprazan which flows relatively for separation detection;
the filler of the amylose derivative chiral column is a silica gel matrix, and the silica gel matrix is silica gel with the surface coated or covalently bonded with the amylose-phenyl carbamate derivative;
n-hexane in the mixed solution of n-hexane and ethanol: ethanol is (40-70)%: (60-30)%.
Chromatographic column with silica gel surface coated or covalently bonded with derivatives of amylose-phenylcarbamate as packing, n-hexane, according to the above technical scheme: ethanol is (40-70)%: and (60-30)% of a mixed solution of n-hexane and ethanol is used as a mobile phase, so that the effective separation of S configuration and R configuration in enantiomers of key tergorazan intermediates can be realized, and corresponding detection results can be obtained. The reason for this is probably due to the fact that the phenylcarbamate group in the packing of the chromatographic column has a steric structure resembling a helix, which is advantageous for steric matching and multipoint action when linked to amylose, so that the enantiomer of the key intermediate of tergolian forms stable combination with phenylcarbamate due to hydrogen bonding or dipole action, thereby facilitating separation of S configuration and R configuration; further, by coaction with a mobile phase of specific components and ratio, the S configuration and R configuration in enantiomer of key intermediate of tergolian can be identified by detector of high performance liquid chromatograph, and corresponding detection result is formed.
Preferably, the derivative of amylose-phenyl carbamate is amylose-tris (3, 5-dimethylphenyl carbamate), amylose-tris ((S) - α -methylphenyl carbamate) or amylose-tris (3-chlorophenyl carbamate).
By adopting the packing of the amylose derivative chiral column of the technical scheme, the enantiomer of the key intermediate of tergolian can be effectively separated and detected, so that the separation degree obtained by the separation detection of the high performance liquid chromatograph can meet the requirements of Chinese pharmacopoeia, namely, the separation degree is more than 1.5.
Preferably, the amylose derivative chiral column isAD-H 5μm 4.6mm*250mm、AS 5 μm 4.6mm 250mm or +.>ID 5μm 4.6mm*250mm。
By adopting the amylose derivative chiral column of the technical scheme, the separation detection result of the S configuration and the R configuration in the enantiomer of the key intermediate of tergoliprazan shows that the chiral column can obtain the separation effect with better retention time and separation degree, wherein the retention time t1 of the S configuration is within 7-20min, and the separation degree can reach 1.84-5.52.
Preferably, the n-hexane: ethanol is 40% by volume: 60%.
The volume ratio of the normal hexane to the ethanol can lead the retention time and the separation degree of the S configuration of the key intermediate enantiomer of the tergolian to reach the optimal values, namely the retention time t1 of the S configuration reaches 7-9min, and the separation degree can reach 2.17-5.52. The reason for this is probably due to the fact that as n-hexane increases, the polarity of the mobile phase decreases, so that the retention time of the sample in the column increases while also affecting the separation effect.
A high performance liquid chromatography separation detection method of key intermediate enantiomer of tergolian praziquantel, wherein the chromatographic conditions further comprise: the flow rate of the mobile phase is 0.5-0.8mL/min, the temperature of the chromatographic column is 20-40 ℃, the detection wavelength is 210-280nm, the sample injection amount is 5-20 mu L, and the detector is a VWD ultraviolet detector.
The detection wavelength of the technical scheme can enable the key intermediate enantiomer of tergolian to be in a range with a stronger detection signal, so that the spectrogram of the high performance liquid chromatography obtained by the method is clearer; furthermore, the chromatographic column temperature and the mobile phase flow rate adopting the technical scheme can ensure that the column pressure is below 60bar to maintain the column efficiency on the premise of ensuring that the retention time and the separation degree of the key intermediate enantiomer of tergore meet the requirements of industrial application, thereby prolonging the service time of equipment.
Preferably, the chromatographic conditions further comprise: the flow rate of the mobile phase is 0.6mL/min, the temperature of the chromatographic column is 30 ℃, the detection wavelength is 220nm, the sample injection amount is 5 mu L, and the detector is a VWD ultraviolet detector.
By adopting the chromatographic conditions of the technical scheme, the optimal detection result comprehensively considering the factors of retention time, separation degree and column pressure can be obtained, wherein the optimal detection result is that the retention time t1 of the S configuration of the key intermediate enantiomer of tergolian is 7.388min, the separation degree is 2.46 and the column pressure is 36.5bar. The separation degree can enable the target product to meet the requirements of Chinese pharmacopoeia (> 1.5), the retention time is suitable for applying the separation detection method to industrial scenes, and the column pressure is in a normal pressure range for ensuring the column effect of the amylose derivative chiral column, so that the separation detection method is suitable for industrial production and can reduce the use cost of equipment.
A high performance liquid chromatography separation detection method of key intermediate enantiomer of tergolian praziquantel comprises the following operation steps:
a) Mixing a tergolian key intermediate sample to be separated and detected with a mixed solution of n-hexane and ethanol to obtain a detection solution to be separated, wherein the concentration of the detection solution to be separated is 0.8-1.2 g/L;
b) Setting chromatographic conditions of a high performance liquid chromatograph;
c) And (3) injecting the detection solution to be separated into a high performance liquid chromatograph, and eluting a mobile phase to obtain the retention time and the relative content of the S configuration and the R configuration in the tergolian key intermediate sample.
By adopting the operation steps of the technical scheme, the retention time and the relative content of the S configuration and the R configuration in the enantiomer of the key intermediate of tergolian can be detected. The concentration of the detection solution to be separated is prepared to be 0.8-1.2g/L, and the detection solution is mainly based on the detection range of a detector of a high performance liquid chromatograph, if the sample concentration is too high, effective separation cannot be realized, and if the sample concentration is too low, the detection limit of the detector cannot be met.
Preferably, the high performance liquid chromatograph is Agilent 1220 info II.
By adopting the high performance liquid chromatograph of the technical scheme, the retention time and the relative content of the S configuration and the R configuration in the enantiomer of the key intermediate of tergolian can be detected under the chromatographic conditions and the operation steps.
In summary, the application has the following beneficial effects:
according to the high performance liquid chromatography separation detection method of the key intermediate enantiomer of tergolian, silica gel with the surface coated or covalently bonded with the derivative of amylose-phenyl carbamate is adopted as a chromatographic column and n-hexane of a filler: ethanol is (40-70)%: the mixed solution of (60-30)% of normal hexane and ethanol can be used as a mobile phase to effectively separate enantiomers of key intermediates (S) -1-benzyl-4- ((5, 7-difluoro chromium-4-yl) oxy) -N, N,2-trimethyl-1H-benzo [ d ] imidazole-6-formamide of tergolian and obtain detection results, so that the quality of the final product tergolian can be ensured, and the method has great significance to industrial mass production of tergolian and has strong practicability.
Further, the group linked by the silica gel matrix filler in the chromatographic column is amylose-tris (3, 5-dimethylphenylcarbamate), amylose-tris ((S) -alpha-methylphenyl carbamate) or amylose-tris (3-chlorophenyl carbamate), so that the enantiomer of the key intermediate of tergolian can be effectively separated and detected, and the separation degree obtained by the separation detection of the high performance liquid chromatograph can meet the requirements of Chinese pharmacopoeia, namely the separation degree>1.5. In particular, the amylose derivative chiral column is selectedAD-H 5μm 4.6mm*250mm、/>AS 5 μm 4.6mm 250mm or +.>The ID of 5 μm is 4.6mm.250mm, and the separation effect with better retention time and separation degree can be obtained, wherein the retention time t1 of the S configuration of the enantiomer of the key intermediate of tergolian is within 7-20min, and the separation degree can reach 1.84-5.52.
Further, under the conditions selected by the chromatographic column, setting chromatographic conditions as n-hexane in a mixed solution of n-hexane and ethanol: ethanol is 40% by volume: 60%, the flow rate of the mobile phase is 0.5-1.0mL/min, the temperature of a chromatographic column is 20-40 ℃, the detection wavelength is 210-280nm, the sample injection amount is 5-20 mu L, the retention time t1 of the S configuration of the key intermediate enantiomer of tergolian is 7-9min, the separation degree can reach 2.17-5.52, and the column pressure is 25.7-58.2bar. Therefore, the chromatographic conditions can maintain the column pressure to ensure the column efficiency of the chromatographic column on the premise of ensuring the retention time and the separation degree of the key intermediate enantiomer of the tergolian prasux to meet the industrialized application, thereby prolonging the service time of equipment.
Further, the chromatographic column is selectedAD-Setting chromatographic conditions as n-hexane in a mixed solution of n-hexane and ethanol under the conditions of H5 mu m 4.6mm×250mm: ethanol is 40% by volume: 60%, the flow rate of the mobile phase is 0.6mL/min, the temperature of the chromatographic column is 30 ℃, the detection wavelength is 220nm, and the sample injection amount is 5 mu L, so that the optimal detection effect under the comprehensive consideration of three factors of retention time, separation degree and column pressure can be obtained, the optimal value of the detection result is 7.388min for the retention time t1 of the S configuration of the key intermediate enantiomer of tergorazan, the separation degree is 2.46, and the column pressure is 36.5bar.
Drawings
Fig. 1 shows a key intermediate sample of tergolian prazix on a CHIRLPAK AD-H5 μm 4.6mm x 250mm column, n-hexane: ethanol is 40% by volume: separating and detecting the HPLC diagram under the chromatographic conditions of 60% of mixed solution of normal hexane and ethanol as a mobile phase, the flow rate of the mobile phase being 0.6mL/min, the chromatographic column temperature being 30 ℃, the sample injection amount being 5 mu L and the detector being a DAD detector;
fig. 2 shows the S configuration of key intermediate enantiomer of tergolian in CHIRLPAK AD-H5 μm 4.6mm x 250mm as column, n-hexane: ethanol is 40% by volume: separating and detecting HPLC diagram under chromatographic conditions of mobile phase, mobile phase flow rate of 0.6mL/min, chromatographic column temperature of 30deg.C, detection wavelength of 220nm, sample injection amount of 5 μL and detector of VWD ultraviolet detector;
FIG. 3 is a sample of key intermediate of tergolian prazix in example 1, on CHIRLPAK AD-H5 μm 4.6mm by 250mm column, n-hexane: ethanol is 40% by volume: separating and detecting HPLC diagram under chromatographic conditions of mobile phase, mobile phase flow rate of 0.6mL/min, chromatographic column temperature of 30deg.C, detection wavelength of 220nm, sample injection amount of 5 μL and detector of VWD ultraviolet detector;
fig. 4 is a sample of key intermediate of tergolian prazix in example 2, chromatographic column, n-hexane at CHIRLPAK AS μm 4.6mm x 250 mm: ethanol is 40% by volume: separating and detecting HPLC diagram under chromatographic conditions of mobile phase, mobile phase flow rate of 0.6mL/min, chromatographic column temperature of 30deg.C, detection wavelength of 220nm, sample injection amount of 5 μL and detector of VWD ultraviolet detector;
fig. 5 is a sample of key intermediate of tergolian prazix in example 3, chromatographic column, n-hexane at CHIRLPAK IA μm 4.6mm x 250 mm: ethanol is 40% by volume: separating and detecting HPLC diagram under chromatographic conditions of mobile phase, mobile phase flow rate of 0.6mL/min, chromatographic column temperature of 30deg.C, detection wavelength of 220nm, sample injection amount of 5 μL and detector of VWD ultraviolet detector;
fig. 6 is a sample of key intermediate of tergolian prazix in example 4, chromatographic column, n-hexane at CHIRLPAK ID μm 4.6mm x 250 mm: ethanol is 40% by volume: separating and detecting HPLC diagram under chromatographic conditions of mobile phase, mobile phase flow rate of 0.6mL/min, chromatographic column temperature of 30deg.C, detection wavelength of 220nm, sample injection amount of 5 μL and detector of VWD ultraviolet detector;
fig. 7 shows key intermediate samples of tergolian in example 5 as chromatographic columns, n-hexane at a chip peak OD-H5 μm 4.6mm x 250 mm: ethanol is 40% by volume: separating and detecting HPLC diagram under chromatographic conditions of mobile phase, mobile phase flow rate of 0.6mL/min, chromatographic column temperature of 30deg.C, detection wavelength of 220nm, sample injection amount of 5 μL and detector of VWD ultraviolet detector;
fig. 8 shows key intermediate samples of tergolian in example 6 as chromatographic columns in n-hexane at a ratio of cherlpak OJ-H5 μm 4.6mm x 250 mm: ethanol is 40% by volume: separating and detecting HPLC diagram under chromatographic conditions of mobile phase, mobile phase flow rate of 0.6mL/min, chromatographic column temperature of 30deg.C, detection wavelength of 220nm, sample injection amount of 5 μL and detector of VWD ultraviolet detector;
fig. 9 is a sample of key intermediate of tergolian prazix in example 7, chromatographic column, n-hexane at CHIRLPAK IC μm 4.6mm x 250 mm: ethanol is 40% by volume: separating and detecting HPLC diagram under chromatographic conditions of mobile phase, mobile phase flow rate of 0.6mL/min, chromatographic column temperature of 30deg.C, detection wavelength of 220nm, sample injection amount of 5 μL and detector of VWD ultraviolet detector;
Detailed Description
The raw materials used are all commercially available except for key intermediates of tergolian praecox.
Tergolian prazan key intermediate, self-made by Shanghai Bai lion biotechnology Co.
High performance liquid chromatograph, agilent (G4290C) 1220Infinity II, including degasser, autosampler, column oven, VWD UV detector and workstation, agilent company, USA;
high performance liquid chromatograph, agilent (G4290C) 1220Infinity II, including degasser, autosampler, column oven, DAD detector and workstation, agilent company, USA;
branson Inlet ultrasonic cleaner, CHCPX8800H-C, must be trusted to ultrasonic (Shanghai) Inc.;
AD-H 5μm 4.6mm*250mm、/>IF 5μm 4.6mm*250mm、IA 5μm 4.6mm*250mm、/>ID 5μm 4.6mm*250mm、OD-H 5μm 4.6mm*250mm、/>OJ-H 5μm 4.6mm*250mm、IC 5 μm 4.6mm×250mm, chirality of the drug for major raceTechnology (Shanghai) Inc.
A method for determining the detection wavelength of high performance liquid chromatography of key intermediate enantiomer of tergolian comprises the following operation steps:
10mg of the key intermediate of tergolian was taken and put into a 10mL volumetric flask, and the volumetric flask was charged with n-hexane and ethanol at a volume ratio of 40% at room temperature: and (3) carrying out ultrasonic dissolution and volume fixing on the mixed solution of 60% of n-hexane-ethanol to obtain a detection solution to be separated.
Separating and detecting with high performance liquid chromatograph, wherein the detector is DAD detector, and chromatographic column is selectedAD-H5 μm 4.6mm.times.250mm, sample separation detection conditions are as follows: the flow rate of the mobile phase is 0.6mL/min, the temperature of the chromatographic column is 30 ℃, and the sample injection amount is 5 mu L.
The detection solution to be separated is injected into a high performance liquid chromatograph, and the mobile phase is eluted, so that an HPLC chart is shown as figure 1, and as can be seen from figure 1, the detection wavelength of the tergolian key intermediate sample is preferably in the range of 210-280 nm. Wherein, the electric signal obtained when the detection wavelength is 220nm is strongest, and the key intermediate of tergolian can be effectively and sensitively identified.
The application is exemplified by the detection wavelength of 220nm in chromatographic conditions, but does not affect the detection effect of other wavelengths in the range of 210-280 nm.
Examples
Example 1
A high performance liquid chromatography separation detection method of key intermediate enantiomer of tergolian praziquantel comprises the following operation steps: 10mg of the key intermediate of tergolian was taken and put into a 10mL volumetric flask, and the volumetric flask was charged with n-hexane and ethanol at a volume ratio of 40% at room temperature: and (3) carrying out ultrasonic dissolution and volume fixing on the mixed solution of 60% of n-hexane-ethanol to obtain a detection solution to be separated.
Separating and detecting by high performance liquid chromatography with VWD ultraviolet detector as detector and chromatographic column as detectorAD-H5 μm 4.6mm.times.250mm, sample separation detection conditions are as follows: the flow rate of the mobile phase is 0.6mL/min, the temperature of the chromatographic column is 30 ℃, the detection wavelength is 220nm, and the sample injection amount is 5 mu L.
The detection solution to be separated is injected into a high performance liquid chromatograph, mobile phase is eluted, and enantiomers of the key intermediate of tergorlazan are separated and detected, so that an HPLC diagram is shown in fig. 3, the S configuration of the key intermediate enantiomer of tergorzan with retention time t1= 7.388min is known from fig. 2 (HPLC diagram of S configuration of key intermediate enantiomer of tergorzan), the R configuration of the key intermediate enantiomer of tergorzan with retention time t2= 10.128min is calculated, and the degree of separation R is calculated to be 2.46.
Example 2
High performance liquid chromatography separation detection method for key intermediate enantiomer of tergolian prasugrel, and a chromatographic column is adopted asThe procedure was AS in example 1 except that AS was 5 μm 4.6mm 250mm. Enantiomers of key intermediates of tergolian were separated and detected to give HPLC patterns as shown in fig. 4 with retention times t1= 12.921, t2= 14.933, calculated as resolution R of 1.84.
Example 3
High performance liquid chromatography separation detection method for key intermediate enantiomer of tergolian prasugrel, and a chromatographic column is adopted asThe procedure of example 1 was followed except that IA was 5.mu.m 4.6mm.times.250 mm. Enantiomers of key intermediates of tergolian were separated and detected to give an HPLC diagram as shown in fig. 5 with retention times t1= 9.217, t2= 9.583, calculated as resolution R of 0.32.
Example 4
High performance liquid chromatography separation detection method for key intermediate enantiomer of tergolian prasugrel, and a chromatographic column is adopted asThe procedure of example 1 was followed except that ID was 5 μm 4.6mm 250mm. Enantiomers of key intermediates of tergolian were separated and detected to give an HPLC diagram as shown in fig. 6 with retention times t1= 19.428, t2= 23.033, calculated as resolution R of 2.15.
Example 5
High performance liquid chromatography separation detection method for key intermediate enantiomer of tergolian prasugrel, and a chromatographic column is adopted asThe same as in example 1, except that OD-H5 μm was 4.6mm.times.250mm. The enantiomers of the key intermediate of tergolipraziram were separated and detected to give an HPLC diagram as shown in fig. 7, and the S configuration and R configuration of the enantiomers of the key intermediate of tergolipraziram were not peaked.
Example 6
High performance liquid chromatography separation detection method for key intermediate enantiomer of tergolian prasugrel, and a chromatographic column is adopted asOJ-H5 μm 4.6mm 250mm, all other were the same as in example 1. Enantiomers of key intermediates of tergolian were separated and detected to give an HPLC diagram as shown in fig. 8 with retention time t1=9.009, r configuration without peaks.
Example 7
High performance liquid chromatography separation detection method for key intermediate enantiomer of tergolian prasugrel, and a chromatographic column is adopted asIC 5 μm 4.6mm 250mm, the same as in example 1. The enantiomers of the key intermediate of tergolipraziram were separated and detected to give an HPLC diagram as shown in fig. 9, and the S configuration and R configuration of the enantiomers of the key intermediate of tergolipraziram were not peaked.
Examples 1-7 gave large differences in separation assay results under conditions of only the chromatographic column, where t1 is the retention time of the S configuration in the key intermediate enantiomer of tergolian and t2 is the retention time of the R configuration in the key intermediate enantiomer of tergolian, and the degree of separation R is calculated from the retention time and the half-width by the degree of separation, as shown in the following table:
as can be seen from the above table, the chromatographic column has a large difference in retention time and resolution for the S-configuration and R-configuration of the key intermediate enantiomer of tergolian under otherwise identical chromatographic conditions. Wherein the S and R configuration can be detected by high performance liquid chromatography if and only if the packing of the column is silica gel surface coated or covalently bonded with an amylose-phenylcarbamate derivative. The reason for this is probably due to the fact that the phenyl carbamate group has a steric structure similar to a helix, and when it is linked to amylose, it is advantageous for steric matching and multi-point action, so that the combination of the sample and phenyl carbamate can generate hydrogen bonding action or dipole action, and thus the S configuration and R configuration can be separated and the detection result can be obtained.
Further, when the above derivative of amylose-phenylcarbamate is amylose-tris (3, 5-dimethylphenylcarbamate), amylose-tris ((S) - α -methylphenylcarbamate) or amylose-tris (3-chlorophenyl carbamate), the degree of separation obtained by the separation detection by high performance liquid chromatography can be made to satisfy the requirements of the chinese pharmacopoeia, i.e., the degree of separation >1.5.
The following examples of the application are only toAD-H5 μm 4.6mm.times.250mm is exemplified as a chromatographic column, but the detection and separation effects of other chromatographic columns under the corresponding chromatographic conditions are not affected.
Example 8
A high performance liquid chromatography separation detection method of key intermediate enantiomer of tergolian praziquantel, which comprises the steps of removing n-hexane in a mobile phase: the volume ratio of the ethanol is 30 percent: the procedure of example 1 was repeated except for 70%. Enantiomers of key intermediates of tergolian were isolated and detected with retention times t1= 7.144, t2= 9.018, calculated as resolution R of 1.16.
Example 9
A high performance liquid chromatography separation detection method of key intermediate enantiomer of tergolian praziquantel, which comprises the steps of removing n-hexane in a mobile phase: the volume ratio of the ethanol is 50 percent: except for 50%, the procedure was the same as in example 1. Enantiomers of key intermediates of tergolian were isolated and detected with retention times t1=8.245, t2= 11.950, calculated as resolution R of 2.14.
Example 10
A high performance liquid chromatography separation detection method of key intermediate enantiomer of tergolian praziquantel, which comprises the steps of removing n-hexane in a mobile phase: the volume ratio of the ethanol is 60 percent: except for 40%, the procedure was the same as in example 1. Enantiomers of key intermediates of tergolian were isolated and detected with retention times t1= 9.245, t2= 14.780, calculated as resolution R of 2.20.
Example 11
A high performance liquid chromatography separation detection method of key intermediate enantiomer of tergolian praziquantel, which comprises the steps of removing n-hexane in a mobile phase: the volume ratio of the ethanol is 70 percent: except for 30%, the procedure was the same as in example 1. Enantiomers of key intermediates of tergolian were isolated and detected with retention times t1= 11.476, t2= 21.047, calculated as resolution R of 5.34.
The separation detection results obtained in examples 1, 8-11 under the conditions of different volume ratios of n-hexane to ethanol in the mobile phase have large differences, and the specific results are shown in the following table:
from the above table, it can be seen that the volume ratio of n-hexane to ethanol in the mobile phase has a certain effect on the retention time and degree of separation of the S-and R-configuration of the key intermediate enantiomer of tergolian under otherwise identical chromatographic conditions. Wherein the volume ratio of n-hexane to ethanol is between (40-70)% if and only if the column is: (60-30)% of the total separation degree can meet the requirements of Chinese pharmacopoeia; further, when the volume ratio of n-hexane to ethanol is (40-60)%: (60-40)% with increasing n-hexane ratio, the retention time increases gradually and the degree of separation decreases slightly, since with increasing n-hexane the polarity decreases, so that the retention time of the sample in the column increases and the separation effect is also affected. When the volume ratio of the normal hexane to the ethanol is 70 percent: at 30%, both retention time and separation increased significantly, probably due to the too small polarity of the mobile phase, resulting in a different increase in retention time for both the S and R configuration, with the difference in retention time increase resulting in a substantial increase in separation.
Thus, considering the retention time and the degree of separation of the S-configuration and the R-configuration of the key intermediate enantiomer of tergolian, when the volume ratio of n-hexane to ethanol is 40%: at 60%, the optimal retention time and separation degree can be obtained, so that the method is suitable for detecting the purity of the key intermediate in the process of industrially producing tergolian.
Example 12
A method for detecting key intermediate enantiomer of tergolian by high performance liquid chromatography separation is the same as in example 1 except that the chromatographic column temperature is 20 ℃. Isolation and detection of enantiomers of key intermediates of tergolian, with retention time t 1 ＝7.134，t 2 = 10.091, the degree of separation R was calculated to be 2.36.
Example 13
A method for detecting key intermediate enantiomer of tergolian by high performance liquid chromatography separation is the same as in example 1 except that the chromatographic column temperature is 25 ℃. Isolation and detection of enantiomers of key intermediates of tergolian, with retention time t 1 ＝7.206，t 2 = 10.085, the degree of separation R was calculated to be 2.31.
Example 14
A method for detecting key intermediate enantiomer of tergolian by high performance liquid chromatography separation is the same as in example 1 except that the chromatographic column temperature is 35 ℃. Enantiomers of key intermediates of tergolian were isolated and detected with retention times t1= 7.317, t2= 10.133, calculated as resolution R of 2.38.
Example 15
A method for detecting key intermediate enantiomer of tergolian by high performance liquid chromatography separation is the same as in example 1 except that the chromatographic column temperature is 40 ℃. Enantiomers of key intermediates of tergolian were isolated and detected with retention times t1= 7.375, t2= 10.135, calculated as resolution R of 2.37.
The separation detection results obtained in examples 1, 12-15 under different conditions of chromatographic column temperatures are similar, and the specific results are shown in the following table:
as can be seen from the above table, the column temperature had less effect on retention time and resolution of S-configuration and R-configuration of key intermediate enantiomer of tergolian under otherwise identical chromatographic conditions. The optimum column temperature is 30 c taking the maximum degree of separation into account.
Example 16
A method for detecting the key intermediate enantiomer of tergolian by high performance liquid chromatography separation was the same as in example 1 except that the flow rate of the mobile phase was 0.5 mL/min. Enantiomers of key intermediates of tergolian were isolated and detected with retention times t1= 8.370, t2= 11.366, calculated as resolution R of 2.17.
Example 17
A method for detecting the key intermediate enantiomer of tergolian by high performance liquid chromatography separation was the same as in example 1 except that the flow rate of the mobile phase was 0.8 mL/min. Enantiomers of key intermediates of tergolian were isolated and detected with retention times t1= 6.594, t2= 9.940, calculated as resolution R of 4.75.
Example 18
A method for detecting the key intermediate enantiomer of tergolian by high performance liquid chromatography separation was the same as in example 1 except that the flow rate of the mobile phase was 1.0 mL/min. Enantiomers of key intermediates of tergolian were isolated and detected with retention times t1= 4.688, t2= 7.357, calculated as resolution R of 5.52.
The separation test results obtained in examples 1, 16-18 under different flow rates of the mobile phases are greatly different, and the specific results are shown in the following table:
| examples | Mobile phase flow rate/mL/min | t1/min | t2/min | R | Column pressure/bar |
| Example 16 | 0.5 | 8.370 | 11.366 | 2.17 | 25.7 |
| Example 1 | 0.6 | 7.388 | 10.128 | 2.46 | 36.5 |
| Example 17 | 0.8 | 6.594 | 9.940 | 4.75 | 58.2 |
| Example 18 | 1.0 | 4.688 | 7.357 | 5.52 | 77.3 |
As can be seen from the above table, the mobile phase flow rate has a greater effect on retention time, degree of separation, and system pressure of the S-and R-configuration of the tergolian key intermediate enantiomer, under otherwise unchanged chromatographic conditions. As the mobile phase flow rate increases, the retention time decreases and the degree of separation increases, and when the mobile phase flow rate is 1.0mL/min, the column pressure reaches 77.3bar, which can have a greater damage to the column efficiency of the chromatographic column, mainly due to the higher proportion of ethanol in the mobile phase, resulting in a substantial increase in pressure during separation detection. Therefore, the flow rate of the mobile phase is preferably selected to be 0.5-0.8 mL/min.
Therefore, three factors of the retention time, the separation degree and the column pressure of the S configuration and the R configuration of the key intermediate enantiomer of the tergolian are comprehensively considered, the separation effect can be achieved when the flow rate of the mobile phase is 0.6mL/min, and the service life of equipment is prolonged while the industrial application can be met.
In summary, according to the method for detecting the key intermediate enantiomer of tergolian by high performance liquid chromatography, silica gel with amylose-phenyl carbamate derivative coated or covalently bonded on the surface is used as a chromatographic column and n-hexane as a filler: ethanol is (40-70)%: the mixed solution of (60-30)% of normal hexane and ethanol can be used as a mobile phase to effectively separate enantiomers of key intermediates (S) -1-benzyl-4- ((5, 7-difluoro chromium-4-yl) oxy) -N, N,2-trimethyl-1H-benzo [ d ] imidazole-6-formamide of tergolian and obtain detection results, so that the quality of the final product tergolian can be ensured, and the method has great significance to industrial mass production of tergolian and has strong practicability.
Further, the group linked by the silica gel matrix filler in the chromatographic column is amylose-tris (3, 5-dimethylphenylcarbamate), amylose-tris ((S) -alpha-methylphenyl carbamate) or amylose-tris (3-chlorophenyl carbamate), so that the enantiomer of the key intermediate of tergolian can be effectively separated and detected, and the separation degree obtained by the separation detection of the high performance liquid chromatograph can meet the requirements of Chinese pharmacopoeia, namely the separation degree>1.5. In particular, the amylose derivative chiral column is selectedAD-H 5μm 4.6mm*250mm、/>AS 5 μm 4.6mm 250mm or +.>The ID of 5 μm is 4.6mm.250mm, and the separation effect with better retention time and separation degree can be obtained, wherein the retention time t1 of the S configuration of the enantiomer of the key intermediate of tergolian is within 7-20min, and the separation degree can reach 1.84-5.52.
Further, under the conditions selected by the chromatographic column, setting chromatographic conditions as n-hexane in a mixed solution of n-hexane and ethanol: ethanol is 40% by volume: 60%, the flow rate of the mobile phase is 0.5-1.0mL/min, the temperature of a chromatographic column is 20-40 ℃, the detection wavelength is 210-280nm, the sample injection amount is 5-20 mu L, the retention time t1 of the S configuration of the key intermediate enantiomer of tergolian is 7-9min, the separation degree can reach 2.17-5.52, and the column pressure is 25.7-58.2bar. Therefore, the chromatographic conditions can maintain the column pressure to ensure the column efficiency of the chromatographic column on the premise of ensuring the retention time and the separation degree of the key intermediate enantiomer of the tergolian prasux to meet the industrialized application, thereby prolonging the service time of equipment.
Further, the chromatographic column is selectedSetting chromatographic conditions as n-hexane in a mixed solution of n-hexane and ethanol under the conditions of AD-H5 mu m 4.6mm×250mm: ethanol is 40% by volume: 60%, the flow rate of the mobile phase is 0.6mL/min, the temperature of the chromatographic column is 30 ℃, the detection wavelength is 220nm, and the sample injection amount is 5 mu L, so that the optimal detection effect under the comprehensive consideration of three factors of retention time, separation degree and column pressure can be obtained, the optimal value of the detection result is 7.388min for the retention time t1 of the S configuration of the key intermediate enantiomer of tergorazan, the separation degree is 2.46, and the column pressure is 36.5bar.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Claims (7)
1. A high performance liquid chromatography separation detection method for key intermediate enantiomer of tergolian is characterized in that an amylose derivative chiral column is adopted as a chromatographic column, and a mixed solution of normal hexane and ethanol is adopted as a flowing enantiomer of key intermediate of tergolian for separation detection;
the filler of the amylose derivative chiral column is a silica gel matrix, and the silica gel matrix is silica gel with the surface coated or covalently bonded with the amylose-phenyl carbamate derivative;
n-hexane in the mixed solution of n-hexane and ethanol: ethanol is (40-70)%: (60-30)%;
and the derivative of amylose-phenyl carbamate is amylose-tris (3, 5-dimethylphenyl carbamate) or amylose-tris (3-chlorophenyl carbamate);
the structural formula of the tergolian prazan key intermediate is as follows。
2. The method for high performance liquid chromatography separation and detection of key intermediate enantiomers of tergolian as claimed in claim 1, wherein said chiral column of amylose derivatives is cherllpak_ad-H5 μm4.6mm x 250mm or cherllpak_id5_μm4.6mm x 250mm.
3. The method for high performance liquid chromatography separation and detection of key intermediate enantiomer of tergolian prazan according to claim 2, wherein n-hexane in the mixed solution of n-hexane and ethanol is as follows: ethanol is 40% by volume: 60%.
4. The method for high performance liquid chromatography separation detection of key intermediate enantiomer of tergolian as claimed in claim 3, wherein the chromatographic conditions further comprise:
the flow rate of the mobile phase is 0.5-0.8mL/min,
the temperature of the chromatographic column is 20-40 ℃,
the detection wavelength is 210-280nm,
the sample injection amount is 5-20 mu L,
the detector is a VWD ultraviolet detector.
5. The method for high performance liquid chromatography separation detection of key intermediate enantiomer of tergolian as claimed in claim 4, wherein the chromatographic conditions further comprise: the flow rate of the mobile phase is 0.6mL/min, the temperature of the chromatographic column is 30 ℃, the detection wavelength is 220nm, the sample injection amount is 5 mu L, and the detector is a VWD ultraviolet detector.
6. The method for high performance liquid chromatography separation and detection of key intermediate enantiomers of tergolian as claimed in any one of claims 1 to 5, characterized in that it comprises the following operative steps:
a) Mixing a tergolian key intermediate sample to be separated and detected with a mixed solution of n-hexane and ethanol to obtain a detection solution to be separated, wherein the concentration of the detection solution to be separated is 0.8-1.2 g/L;
b) Setting chromatographic conditions of a high performance liquid chromatograph;
c) And (3) injecting the detection solution to be separated into a high performance liquid chromatograph, and eluting a mobile phase to obtain the retention time and the relative content of the S configuration and the R configuration in the tergolian key intermediate sample.
7. The method for detecting the high performance liquid chromatography of the key intermediate enantiomer of tergolian praecox according to claim 6, wherein the high performance liquid chromatography is Agilent 1220 property II.
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