CN110361472B

CN110361472B - HPLC detection method of mikui ammonium chloride intermediate MIV-G isomer

Info

Publication number: CN110361472B
Application number: CN201910686614.2A
Authority: CN
Inventors: 吕小倍
Original assignee: Wuhan Jianuokang Pharmaceutical Technology Co ltd
Current assignee: Wuhan Jianuokang Pharmaceutical Technology Co ltd
Priority date: 2019-07-29
Filing date: 2019-07-29
Publication date: 2022-04-29
Anticipated expiration: 2039-07-29
Also published as: CN110361472A

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly discloses an HPLC (high performance liquid chromatography) detection method of mikul ammonium chloride intermediate isomers. The invention adopts a polysaccharide derivative coated chiral chromatographic column (normal phase) as a chiral chromatographic column of a filling agent, and takes a mixed solution of normal hexane and an alcohol compound as a flowing phase to carry out separation and determination on four chiral configurations of the mikul ammonium chloride intermediate MIV-G, thereby realizing the effective separation and detection of four isomers of the mikul ammonium chloride intermediate MIV-G, better realizing the quality control of the mikul ammonium chloride intermediate MIV-G and a finished product mikul ammonium chloride, improving the quality of a final product and ensuring the safety and effectiveness of clinical medication.

Description

HPLC detection method of mikui ammonium chloride intermediate MIV-G isomer

Technical Field

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to an HPLC (high performance liquid chromatography) detection method of mikui ammonium chloride intermediate isomers.

Background

Micstore ammonium chloride, the least effective non-depolarizing neuromuscular blocker at present, was marketed in the united states in 1992 under the name Mivacron and is commonly used for maintaining muscle relaxation during tracheal intubation and surgery. The effect of the composition is similar to that of the closantine, no obvious accumulation exists under clinical dosage, the histamine release promoting effect is small, no adverse effect is caused on intracranial pressure and intraocular pressure, the concentration and range of muscle relaxation are easy to control, and the postoperative recovery is fast. The micstore ammonium chloride can be flexibly applied to various operations, and can be applied to patients with neuromuscular diseases and hyperkalemia besides general patients; especially, the cardiovascular disease of children is rarely influenced in pediatric operations, and the medicine is regarded as a substitute medicine of the succinylcholine. Due to the good clinical manifestations of mickojim, more and more attention is paid to anesthesia surgery.

The intermediate (code MIV-G) is a key intermediate in the synthesis process of mikul ammonium chloride, the Chinese chemical name is (1R) -2- (3-hydroxypropyl) -6,7-dimethoxy-2-methyl-1- (3,4,5-trimethoxybenzyl) -1,2,3, 4-tetrahydroisoquinoline-2-ammonium chloride, the English name is (1R) -2- (3-hydroxypropyl) -6,7-dimethoxy-2-methyl-1- (3,4, 5-trimethoxybenzzyl) -1,2,3, 4-tetrahydroquinonyl-2-i μm chloride, the molecular weight is 482.1, and the structural formulas of the intermediate (code MIV-G) and the enantiomer thereof are shown in Table 1. The generation of the intermediate (code MIV-G) is a step of chiral center generation, belongs to a key step of quality control, and for mikul ammonium chloride intermediates, detection methods of enantiomers of the mikul ammonium chloride intermediates are not collected in pharmacopoeias of various countries at present, so that in the synthesis process, a method with specificity is developed to detect the purity of four isomer configurations of the intermediate (code MIV-G), a reasonable and feasible diastereoisomer proportion range and enantiomer impurity limit are formulated, and the method is an important part for quality control of the intermediate (code MIV-G) and also an important part for ensuring the product quality of the mikul ammonium chloride and preparations thereof.

TABLE 1 intermediate MIV-G and its enantiomeric formula

The applicant consults a large amount of Chinese and foreign patent and non-patent documents, and at present, no document report for detecting the purity of the four isomers of the intermediate MIV-G by adopting a chiral chromatographic column high performance liquid chromatography method exists, which is not beneficial to the control of an enterprise on the product quality.

Disclosure of Invention

In order to solve the problems in the prior art, the inventor provides a method for separating and analyzing four chiral configurations of mikuammonium chloride intermediate MIV-G through a large amount of theoretical research and experimental exploration, so that the quality of mikuammonium chloride products is better controlled.

In order to realize the purpose of the invention, the technical scheme provided by the invention is as follows:

an HPLC detection method of miV-G isomer of mikui ammonium chloride intermediate comprises the following steps: adopts a polysaccharide derivative coating type chiral chromatographic column (normal phase) as a filling agent, and takes a mixed solution of normal hexane and an alcohol compound as a flowing relative mikui ammonium chloride intermediate MIV-G with four chiral configurations

A separation assay was performed using the following chromatographic conditions for the HPLC assay:

the flow rate of the mobile phase is 0.8-1.2 mL/min;

the temperature of the chromatographic column is 20-40 ℃;

the detector adopts an ultraviolet detector, and the detection wavelength is 200-240 nm;

preferably, the chromatographic column is

OX-H (4.6X 250mm), filler particle size 5 μm;

preferably, the filler is cellulose-tris (4-chloro-3-methylphenyl carbamate) coated silica gel;

preferably, in the mobile phase, the volume of the alcohol compound accounts for 20-40% of the total volume of the mobile phase, and is optimally 28%;

preferably, the alcohol compound is isopropanol;

preferably, the chromatographic column temperature is 25 ℃;

preferably, the detection wavelength is 210 nm;

preferably, the flow rate of the mobile phase is 1.0 mL/min;

preferably, the sample size is 10. mu.L.

Further, the detection method comprises the following steps:

1.1, preparing a solution of a to-be-detected product by using a diluent;

1.2, injecting the solution of the to-be-detected product obtained in the step 1.1 into a chromatographic column of a high performance liquid chromatograph for detection; the diluent is a mobile phase with the volume of the alcohol compound accounting for 28 percent of the total volume of the mobile phase.

Compared with the prior art, the method of the invention has the following advantages and beneficial effects:

1. the method realizes effective separation and detection of the four isomers of the mikul ammonium chloride intermediate MIV-G, so that the detection method can better realize quality control of the intermediate MIV-G and the finished mikul ammonium chloride, improve the quality of the final product, and ensure the safety and effectiveness of clinical medication.

2. The detection method is simple and convenient to operate, low in cost and capable of effectively separating isomers with four configurations, the separation degrees of the isomers with the four configurations are all larger than 1.5, the peak patterns of main peaks are symmetrical, specific data refer to fig. 11, and the simple, stable and reliable analysis detection method is provided for research and development and production of products.

Drawings

FIG. 1 is an HPLC chromatogram of a mixed control solution of R-MIV-G and S-MIV-G controls obtained from experiment (one) of example 1 using a chiral column packed with cellulose-tris (3, 5-dichlorophenyl carbamate) bonded silica gel;

FIG. 2 is an HPLC chromatogram of a mixed control solution of R-MIV-G and S-MIV-G controls obtained from experiment (II) of example 1 using a chiral chromatographic column packed with cellulose-tris (4-chloro-3-methylphenyl carbamate) -coated silica gel;

FIG. 3 is a chromatogram obtained by detection in example 2 when the mobile phase is in the chromatographic condition (1);

FIG. 4 is a chromatogram obtained by detection in example 2 when the mobile phase is in the chromatographic condition (2);

FIG. 5 is a chromatogram obtained by detection in example 2 when the mobile phase is in the chromatographic condition (3);

FIG. 6 is a chromatogram obtained by detection in example 2 when the mobile phase is in the chromatographic condition (4);

FIG. 7 is a chromatogram obtained by detection in example 2 when the mobile phase is in the chromatographic condition (5);

FIG. 8 shows the column temperatures in example 3 (1): HPLC profile at 25 ℃;

FIG. 9 shows the column temperature in example 3 (2): HPLC profile at 30 ℃;

FIG. 10 shows the column temperature in example 3 (3): HPLC profile at 35 ℃;

FIG. 11 is a chromatogram of the mixed control solution of example 4;

FIG. 12 is an HPLC chromatogram of 146-37-12 batches of mickey ammonium chloride intermediate R-MIV-G from example 4;

FIG. 13 is an HPLC chromatogram of intermediate R-MIV-G from mikammonium chloride batches 146-56-19 of example 4;

FIG. 14 is an HPLC chromatogram of intermediate R-MIV-G from micstore ammonium chloride batches 146-72-05 of example 4;

FIG. 15 is an HPLC chromatogram of the S-MIV-G sample in example 4.

Detailed Description

The technical solutions of the present invention are further described below with reference to specific examples in order to facilitate the understanding of the present invention by those skilled in the art, but the following should not limit the scope of the present invention as claimed in the claims in any way.

In the following examples, the instruments and reagents used were as follows:

the instrument comprises the following steps: thermoelectric U3000 high performance liquid chromatograph.

Reagent: absolute ethyl alcohol, isopropanol and normal hexane are chromatographic grade;

trifluoroacetic acid and triethylamine are analytical grade;

the R-MIV-G reference and the S-MIV-G reference were made by the Applicant in accordance with the prior art EP0181055A1 (see pages P16-17).

Continuing to react the R-MIV-G to finally generate a final product of micaceous ammonium chloride;

and (4) continuing to react the S-MIV-G to finally generate the mikul ammonium chloride isomer.

Chromatography column Chiralpak IC (4.6X 250mm,5 μm),

OX-H (4.6X 250mm,5 μm) were purchased from Dailn pharmaceutical chiral technologies (Shanghai) Co., Ltd.

Example 1 separation and analysis of four chiral configurations of mikui ammonium chloride intermediate MIV-G using different chiral columns, the procedure was as follows:

(one) stationary phase: cellulose-tris (3, 5-dichlorophenyl carbamate) bonded silica gel

A chromatographic column: chiralpak IC 4.6X 250mm,5 μm

Chromatography apparatus: thermo Fisher U3000 high performance liquid chromatograph;

detector and wavelength: UV-210 nm;

mobile phase: n-hexane-absolute ethanol (n-hexane: absolute ethanol 75:25, v/v);

flow rate: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

column temperature: 25 ℃;

preparing a test solution:

R-MIV-G control solution: accurately weighing about 25mg of R-MIV-G reference substance, placing in a 25mL measuring flask, adding mobile phase for dissolving, diluting to scale, and shaking.

S-MIV-G control solution: accurately weighing about 25mg of S-MIV-G reference substance, placing in a 25mL measuring flask, adding mobile phase for dissolving, diluting to scale, and shaking.

R-MIV-G and S-MIV-G mixed control solution: precisely measuring 5mL of the prepared R-MIV-G reference substance solution and 5mL of the prepared S-MIV-G reference substance solution, placing the solutions in the same 10mL measuring flask, and shaking up to obtain the final product.

10. mu.L of the mixed control solution was taken and injected into a liquid chromatograph, and a chromatogram was recorded, and the result is shown in FIG. 1.

Only two peaks were separated, and 2 peaks were not separated, wherein the peak-off time of the two peaks was 20.508min and 25.832min, respectively.

The separation effect of the chromatographic column is poor.

(II) stationary phase: cellulose-tris (4-chloro-3-methylphenyl carbamate) coated silica gel chromatography instrument: thermo Fisher U3000 high performance liquid chromatograph;

a chromatographic column:

OX-H 4.6×250mm,5μm

detector and wavelength: UV-210 nm;

mobile phase: n-hexane-absolute ethanol (n-hexane: absolute ethanol ═ 50:50, v/v);

flow rate: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

column temperature: 25 ℃;

diluent agent: n-hexane-absolute ethanol (n-hexane: absolute ethanol ═ 50:50, v/v);

preparing a test solution:

10. mu.L of the mixed control solution was injected into a liquid chromatograph, and a chromatogram was recorded, and the result is shown in FIG. 2.

As can be seen from fig. 2, the initial separation of 4 compounds, further optimization of the process, resulted in the desired results.

And (4) conclusion: the chiral chromatographic column adopting the cellulose-tri (4-chloro-3-methylphenyl carbamate) coated silica gel as the filler can preliminarily separate out four-configuration compounds, and the method is further optimized, so that four-configuration isomers can be completely separated.

Example 2 separation and analysis of four chiral configurations of mikui ammonium chloride intermediate MIV-G with different mobile phases and ratios comprises the following steps: chromatographic conditions are as follows:

(1) mobile phase: n-hexane: absolute ethanol (50:50, v/v);

(2) mobile phase: n-hexane: absolute ethanol (60:40, v/v);

(3) mobile phase: n-hexane: isopropanol (75:25, v/v);

(4) mobile phase: n-hexane: isopropanol (0.2% TFA, 0.1% TEA, v/v) (75:25, v/v);

(5) mobile phase: n-hexane: isopropanol (0.3% TFA, 0.2% TEA, v/v) (72:28, v/v);

note: TFA: trifluoroacetic acid, TEA: triethylamine.

a chromatographic column:

OX-H 4.6×250mm，5μm

detector and wavelength: UV-210 nm;

flow rate: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

column temperature: 25 ℃;

preparing a test solution: the control solution was mixed as in example 1.

Diluent agent: mobile phase corresponding to chromatographic conditions.

10. mu.L of the mixed control solution was taken and injected into a liquid chromatograph, and detected by different mobile chromatography phases in (1) to (5) of this example, and chromatograms were recorded, and the results are shown in FIGS. 3,4,5, 6, and 7.

When the mobile phase of (1) is used, 4 isomers are initially separated as shown in FIG. 3, but the baseline is not completely separated, and optimization is continued.

When the mobile phase in the step (2) is adopted, the peak emergence time of the four configurations is 13.910min, 15.170min, 21.413min and 24.188min in sequence, the minimum separation degree between the four configuration isomers of the intermediate MIV-G is 1.87, but the tailing of 21.413min is serious, and a symmetric factor cannot be obtained.

When the mobile phase in the step (3) is adopted, the peak emergence time of the four configurations is 25.183min, 27.765min, 42.428min and 49.207min in sequence, the minimum separation degree between the four configuration isomers of the intermediate MIV-G is 2.08, but the peak type tailing of 42.428min is serious, and the peak emergence time is long, so that the condition is optimized.

When the mobile phase in the step (4) is adopted, the peak emergence time of the four configurations is 24.902min, 27.022min, 40.065min and 48.500min in sequence, the minimum separation degree between the four configuration isomers of the intermediate MIV-G is 1.76, but the peak form of 40.065min is improved, the asymmetry degree is 1.80, but the peak emergence time is longer, and the condition is optimized.

When the mobile phase in the step (5) is adopted, the peak emergence time of the four configurations is 22.978min, 24.757min, 35.208min and 43.558min in sequence, the minimum separation degree between the four configuration isomers of the intermediate MIV-G is 1.63, the peak type symmetry of 35.208min is improved, the asymmetry degree is 1.630, and the peak emergence time is proper, so that the method is adopted to detect the MIV-G isomer.

And (4) conclusion: the four chiral configurations can be successfully separated by adopting the mobile phase, and the preferred mobile phase is n-hexane: isopropanol (0.3% TFA, 0.2% TEA) (72:28, v/v).

Example 3 separation and analysis of four chiral configurations of mikui ammonium chloride intermediate MIV-G at different column temperatures, the steps are as follows:

(1) column temperature: 25 ℃;

(2) column temperature: 30 ℃;

(3) column temperature: 35 ℃;

(II) stationary phase: cellulose-tris (4-chloro-3-methylphenyl carbamate) -coated silica gel

a chromatographic column:

OX-H 4.6×250mm，5μm

detector and wavelength: UV-210 nm;

mobile phase: n-hexane: isopropanol (0.3% TFA, 0.2% TEA) (72:28, v/v);

flow rate: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

preparing a test solution: the control solution was mixed as in example 1.

Diluent agent: a mobile phase.

10. mu.L of the mixed control solution was taken and injected into a liquid chromatograph, and detection was performed at different column temperatures in (1), (2), and (3) of the present example (the chromatographic conditions other than the column temperature were the same as in (5) of example 2), and chromatograms were recorded, and the results are shown in FIGS. 8, 9, and 10.

(1) Column temperature: the peak emergence time of the four configurational isomers is 27.327min, 29.597min, 41.887min and 53.107min in sequence at 25 ℃, and the minimum separation degree between the four configurational isomers of the intermediate MIV-G is 1.80.

(2) Column temperature: the peak emergence time of the four configurational isomers is 24.743min, 26.683min, 39.332min and 48.123min in sequence at 30 ℃, and the minimum separation degree between the four configurational isomers of the intermediate MIV-G is 1.60.

(3) Column temperature: the peak emergence time of the four configurational isomers is 22.978min, 24.757min, 35.208min and 43.558min in sequence at 35 ℃, and the minimum separation degree between the four configurational isomers of the intermediate MIV-G is 1.63.

And (4) conclusion: the column temperature is increased to improve the peak time of four configurational isomers of the intermediate MIV-G, but the separation degree is reduced, and the column temperature is preferably 25 ℃ comprehensively.

EXAMPLE 4 Miroconium chloride intermediate MIV-G isomer purity assay

Chromatographic conditions are as follows:

a chromatographic column:

OX-H 4.6×250mm，5μm

detector and wavelength: UV-210 nm;

mobile phase: n-hexane: isopropanol (0.3% TFA, 0.2% TEA) (72:28, v/v);

flow rate: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

column temperature: 25 ℃;

diluent agent: a mobile phase;

test article mixed control solution: the same as example 1;

test solution: the applicant prepares micstore ammonium chloride intermediate R-MIV-G (self-made according to the prior art EP0181055A1 (see page P16-17)), samples of 3 batches in Table 2, precisely weighs 25mg of the samples respectively, places the samples in a 25mL measuring flask, dissolves and dilutes the samples to scales by using a diluent, and shakes the samples uniformly to obtain the micstore ammonium chloride intermediate. 10 mu L of the test solution and the mixed reference solution are respectively injected into a liquid chromatograph, chromatogram is recorded, and the ratio of the peak areas of the two R-MIV-G peaks (which are enantiomers) and the purity of diastereoisomers are calculated, and the results are shown in Table 2.

S-MIV-G sample: the applicant prepared the isomer impurity S-MIV-G of the micstore ammonium chloride intermediate (prepared according to the prior art EP0181055A1 (see page P16-17)), 10mg was taken and placed in a 10ml volumetric flask, dissolved and diluted to the scale with a diluent, and shaken up to obtain the product, the determination result is shown in figure 15, the purity is 98.97%, and the R-MIV-G configuration impurity content is 1.04%.

The chromatogram results of the mixed control solution are shown in FIG. 11.

The chromatogram of batches 146-37-12, such as the chromatogram of batches 12, 146-56-19, such as the chromatogram of batches 13, 146-72-05, is shown in FIG. 14.

The ratio of the peak areas of R-MIV-G (which are enantiomers of each other) in the chromatogram of the test solution is shown in the following table, if an S-MIV-G (diastereoisomer) peak exists in the chromatogram of the test solution, the peak area is not more than 2.0% according to the peak area normalization method, the results in the table 2 meet the requirements, and the results are not detected and are 0.

TABLE 2 determination of MIV-G isomer ratio and purity of intermediate

Test sample batch number	R-MIV-G	S-MIV-G
			146-37-12	77.77％：22.23％	0
146-56-19	77.11％：22.89％	0
			146-72-05	76.92％：23.08％	0

Claims

1. An HPLC detection method for mikuonium chloride intermediate MIV-G isomer adopts a chiral chromatographic column with cellulose-tris (4-chloro-3-methylphenyl carbamate) coated silica gel as a filler, and adopts a mixed solution with a volume ratio of n-hexane to an isopropanol solution of 72:28 as a flowing phase to carry out separation and determination on four chiral configurations of mikuonium chloride intermediate MIV-G, wherein the HPLC detection adopts the following chromatographic conditions:

the flow rate of the mobile phase is 0.8-1.2 mL/min;

the temperature of the chromatographic column is 20-40 ℃;

the isopropanol solution contained 0.3v/v% trifluoroacetic acid and 0.2v/v% triethylamine;

in the method, the preparation process of the mixed reference solution and the test solution is as follows:

(1) dissolving R-MIV-G and S-MIV-G reference substances respectively with diluent to obtain R-MIV-G and S-MIV-G reference substance solutions, mixing the two solutions, and shaking up to obtain mixed reference substance solution;

(2) preparing a test solution by using a diluent;

the diluent is a mobile phase.