CN112684022A

CN112684022A - Vitamin B1Injection impurity and preparation and detection method thereof

Info

Publication number: CN112684022A
Application number: CN202011385343.6A
Authority: CN
Inventors: 唐咏群; 黄锡伟; 郑俊丽; 胡铮; 田欣欣
Original assignee: NANJING JIANYOU BIOCHEMICAL PHARMACEUTICAL CO Ltd
Current assignee: NANJING JIANYOU BIOCHEMICAL PHARMACEUTICAL CO Ltd
Priority date: 2020-12-01
Filing date: 2020-12-01
Publication date: 2021-04-20
Anticipated expiration: 2040-12-01
Also published as: CN112684022B

Abstract

The invention provides vitamin B₁Injection impurities and a preparation method and a detection method thereof, belonging to the technical field of pharmacy. The structural formula of the impurity shown in the formula I is shown as follows. Vitamin B of the invention₁When the injection is subjected to high-temperature destruction and alkali destruction, impurities shown in formula I are prepared. The invention can separate and prepare the impurity shown in the formula I, namely vitamin B₁The impurity detection of the injection provides a new reference substance, and is more beneficial to vitamin B₁The development of a method for detecting related substances in the injection can control the quality of the product.

Description

Vitamin B1Injection impurity and preparation and detection method thereof

Technical Field

The invention belongs to the technical field of pharmacy, and particularly relates to vitamin B₁Injection impurity, and its preparation method and detection method are provided.

Background

Vitamin B₁Injection commodityIs named LYMPHAZURIN, developed by FRESENIUS KABI USA LLC, and is approved by the FDA for marketing in the United states in 1982. Vitamin B₁The injection is suitable for vitamin B₁Lack of treatment for the resulting beriberi or Wernicke's encephalopathy. Can also be used for vitamin B₁The adjuvant treatment of peripheral neuritis and dyspepsia caused by deficiency. Has been used in the united states for nearly 40 years, safe and effective.

On 14 days 05 and 2020, the national drug administration issued "a notice on the development of evaluation work on the quality and efficacy consistency of imitation drugs for chemical injections (62: 2020), and required the consistency of the quality and efficacy of imitation drugs for injections and the original drugs, wherein an important aspect is the consistency study of the impurity spectrum.

Vitamin B₁The injection can be gradually degraded during the stability period to generate impurities, and if the impurities are not determined by structure, vitamin B can be given₁The quality control of the injection brings about a great risk; vitamin B can be ensured only by determining the chemical structure of the impurity, knowing the mechanism of impurity generation and then effectively controlling the reaction operation in the step₁The injection meets the quality requirement.

ChP contains crude drug and injection, EP and USP contain crude drug. Comparison of vitamin B in ChP and USP methods₁The time to peak is too early and the number of impurities is less than that of the EP method; vitamin B in EP Process₁The time of peak emergence was too late.

Disclosure of Invention

The invention aims to provide vitamin B based on the prior art₁Impurities of the injection.

Another object of the present invention is to provide the above-mentioned vitamin B₁A preparation method of impurities of injection.

It is a third object of the present invention to provide vitamin B₁A method for detecting the impurities in the injection.

The technical scheme of the invention is as follows:

an impurity of formula (I) having the formula:

the invention also provides a preparation method of the impurity shown in the formula I, which comprises the following steps:

vitamin B₁The injection can be gradually degraded to generate impurities in the stability period, and the degradation amount of the impurities is continuously increased along with the extension of the stability lofting time. The inventor of the invention uses vitamin B₁The impurity shown in the formula I is found in the injection, the content of the impurity exceeds the identification threshold value by 0.2 percent along with the prolonging of the stable standing time, and no vitamin B is reported in the literature at present₁The injection contains the unknown impurities. According to the ICH guiding principle, the generation mechanism of the unknown impurity is necessary to be understood, the unknown impurity is further prepared and separated, and the structure of the impurity is determined by characterization means such as LC-MS and NMR, so that the vitamin B can be effectively controlled₁Product quality of the injection.

Based on para-vitamin B₁The inventors of the present invention used destructive conditions such as high temperature, acid, alkali, oxidation, etc. to treat vitamin B₁Injection and vitamin B₁The aqueous solution of the drug substance was subjected to a destruction study to investigate the degradation pathway of the unknown impurity. The inventors detected this unknown impurity in both the high temperature and base-destruct samples, and the impurity degraded significantly under base-destruct conditions. Therefore, the degradation pathways of the unknown impurities are mainly high temperature destruction and alkali destruction.

The invention converts vitamin B into vitamin B₁Injection and vitamin B₁When the aqueous solution of the raw material medicine is damaged at high temperature, the high temperature generally refers to 75-100 ℃.

In a preferred embodiment, the present inventors compound vitamin B₁When the injection is damaged at high temperature, the method comprises the following steps: vitamin B₁And carrying out chemical reaction on the injection at 75-85 ℃ to prepare the impurity shown in the formula I.

In a more preferred embodiment, vitamin B₁When the injection is damaged at high temperature, the reaction temperature is 80 ℃, the reaction time is 18-30 hours, and preferably 24 hours.

In one embodiment, the present invention employs vitamin B₁When the injection is subjected to alkali destruction, the method comprises the following steps: mixing vitamin B₁The injection and the alkali solution are subjected to chemical reaction at the temperature of 25-60 ℃ to prepare the impurity shown in the formula I.

For the purposes of the present invention, vitamin B₁When the injection is subjected to alkali destruction, the alkali used is an inorganic alkali, and can be, but is not limited to, one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate or ammonia water, and for example, sodium hydroxide can be selected.

In a preferred embodiment, vitamin B₁When the injection is subjected to alkali destruction, the concentration of the alkali solution is 1-3 mol/L, preferably 1-2 mol/L, and more preferably 2 mol/L.

Vitamin B as mentioned in the invention₁Vitamin B in injection₁The concentration of (B) is 100mg/mL to 200mg/mL, preferably 200 mg/mL.

In a preferred embodiment, vitamin B₁When the injection is subjected to alkali destruction, vitamin B₁The volume ratio of the injection to the alkali solution is 1: 1-3; preferably 1: 1-2; for example, 1: 2.

In a preferred embodiment, vitamin B₁When the injection is subjected to alkali destruction, the reaction temperature is 30-40 ℃.

Further, the reaction time is 0.5 to 2 hours, preferably 1 hour.

For vitamin B₁The inventors tried to separate the impurities represented by formula I prepared when the injection solution was subjected to alkali destruction by using high performance liquid chromatography under conditions including: the chromatographic column is Welch Ultimate AQ, and gradient elution is carried out by adopting a mixed mobile phase of a mobile phase A1 and a mobile phase B1, wherein the mobile phase A1 is 0.05-0.5% trifluoroacetic acid solution, and the mobile phase B1 is methanol; the ladderThe elution comprises the following steps: (1) the volume ratio of the mobile phase A1 to the mobile phase B1 was kept constant at 95:5 for 0-16 minutes; (2) the volume ratio of the mobile phase A1 to the mobile phase B1 is uniformly graded from 95:5 to 5:95 within 16-16.2 minutes; (3) the volume ratio of the mobile phase A1 to the mobile phase B1 is kept constant at 5:95 within 16.2-18 minutes; (4) the volume ratio of the mobile phase A1 to the mobile phase B1 is gradually changed from 5:95 to 95:5 at a constant speed within 18-18.2 minutes; (5) the volume ratio of mobile phase a1 to mobile phase B1 remained constant at 95:5 for 18.2-20 minutes. The detailed elution procedure is shown in table 1 below.

TABLE 1 gradient elution procedure

In a preferred embodiment, the impurities are separated by HPLC, using a column of Welch Ultimate AQ 30mm X250 mm, 5 μm.

In a preferred embodiment, mobile phase a1 is a 0.1% trifluoroacetic acid solution.

Furthermore, the detection wavelength is 245-250 nm, preferably 248 nm.

Further, the flow rate is 10 to 50ml/min, preferably 30 ml/min.

Further, the column temperature is 15-35 ℃, and preferably 25 ℃.

Furthermore, the sample injection amount is 1000-5000 μ L, preferably 4000 μ L.

Collecting the target peak preparation solution, pre-freezing with dry ice and ethanol, and freeze-drying in the dark to obtain the target impurity solid shown in the formula I.

On the basis of providing an impurity separation preparation method, the invention also provides vitamin B₁The detection method of the impurities shown in the formula I in the injection adopts high performance liquid chromatography for detection, carries out qualitative or quantitative detection on the impurities, and the conditions of the high performance liquid chromatography comprise: performing gradient elution by using an octadecylsilane bonded silica gel column as a chromatographic column and adopting a mobile phase A2 and a mobile phase B2 as mixed mobile phases, wherein the mobile phase A2 is an ammonium hexanesulfonate solution, and the method is characterized in thatMobile phase B2 was methanol; the gradient elution comprises the following steps: (1) the volume ratio of the mobile phase A2 to the mobile phase B2 was kept constant at 95:5 for 0-5 minutes; (2) the volume ratio of the mobile phase A2 to the mobile phase B2 is uniformly graded from 95:5 to 50:50 within 5-15 minutes; (3) the volume ratio of the mobile phase A2 to the mobile phase B2 is kept constant at 50:50 within 15-35 minutes; (4) the volume ratio of the mobile phase A2 to the mobile phase B2 is uniformly graded from 50:50 to 95:5 within 35-36 minutes; (5) the volume ratio of mobile phase a2 to mobile phase B2 remained constant at 95:5 for 36-45 minutes. The detailed elution procedure is shown in table 2 below.

TABLE 2 gradient elution procedure

In the detection method, an octadecylsilane chemically bonded silica gel column is used as a chromatographic column, and the model of the chromatographic column is Shimadzu Inertsil ODS-3, 250mm multiplied by 4.6mm, and 5.0 μm.

For the purposes of the present invention, mobile phase a2 is a solution of ammonium hexane sulfonate, and in a preferred embodiment, the concentration of ammonium hexane sulfonate in mobile phase a2 is from 0.5 wt% to 2 wt%, e.g., 1 wt%.

In the detection of vitamin B₁When the impurities shown in the formula I in the injection are impurities, the following solutions are prepared:

control solution: with vitamin B₁The impurities represented by formula I prepared by alkali destruction of injection are used as reference substance, precisely weighed, and diluted with [ 0.75% glacial acetic acid ]]Dissolved and diluted to make a control solution containing 0.01mg of the total amount of the drug per 1 mL.

Test solution: taking vitamin B destroyed by alkali solution₁Injection, with diluent [ 0.75% glacial acetic acid ]]Diluting to 1mL containing 1mg vitamin B₁As a test solution.

In the quantitative detection of the invention, the content can be calculated by using the conventional methods such as an external standard method, an area normalization method and the like.

During quantitative analysis, if an external standard method is used, a standard curve is manufactured by a conventional method for calculation; however, in the qualitative analysis, a standard curve is not required to be prepared, and the determination can be made by the retention time.

In the invention, the parameters such as column temperature, flow rate, sample injection amount and the like can be selected in a common range.

In a preferred embodiment, the detection wavelength is 210-400 nm, preferably 210-300 nm, for example 248 nm.

Further, the flow rate is 0.6 to 1.2mL/min, preferably 0.8 mL/min.

Further, the column temperature is 20-40 ℃, and preferably 30 ℃.

Furthermore, the sample amount is 20 to 50 μ L, preferably 25 μ L.

By adopting the technical scheme of the invention, the advantages are as follows:

(1) the present invention provides vitamin B₁The new impurities generated in the injection define the degradation path of the impurities and are beneficial to vitamin B₁And (5) controlling the quality of the product.

(2) The invention provides a method for synthesizing impurities shown in a formula I.

(3) The invention provides vitamin B after alkali destruction₁The method for separating and preparing the impurities shown in the formula I in the injection does not need to use excessive chemical reagents and post-treatment, has high reaction conversion rate and is easy to separate and prepare.

(4) The invention prepares the impurity shown in the formula I, namely vitamin B through separation₁The impurity detection of the injection provides a new reference substance, and is more beneficial to vitamin B₁The development of a method for detecting related substances in the injection can control the quality of the product.

Drawings

FIG. 1 is vitamin B prepared in example 6₁Of impurities in the injection¹H NMR chart;

FIG. 2 is an enlarged view of a portion of FIG. 1;

FIG. 3 is vitamin B prepared in example 6₁Of impurities in the injection¹³C NMR chart;

FIG. 4 is vitamin B prepared in example 6₁DEPT90 plot of injection impurities;

FIG. 5 is a block diagramVitamin B prepared in example 6₁DEPT135 plot of injection impurities;

FIG. 6 is vitamin B prepared in example 6₁COSY plot of injection impurities;

FIG. 7 is vitamin B prepared in example 6₁HSQC plot of injection impurities;

FIG. 8 is vitamin B prepared in example 6₁HMBC profile-1/3 of injection impurities;

FIG. 9 is vitamin B prepared in example 6₁HMBC profile-2/3 of injection impurities;

FIG. 10 is vitamin B prepared in example 6₁HMBC profile-3/3 of injection impurities;

FIG. 11 is vitamin B prepared in example 6₁LC-MS (positive ion) profile of injection impurities;

FIG. 12 is an HPLC chart of the control solution in example 7;

FIG. 13 is a HPLC chart of the test solution in example 7.

Detailed Description

Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

The present invention will be described in further detail with reference to specific examples. It will be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

Comparative example 1

5mL of 200mg/mL vitamin B₁The injection was analyzed by HPLC, and no impurity represented by formula I was detected, and 5mL of a 2mol/L hydrochloric acid solution was added thereto, reacted at 25 ℃ for 24 hours, and analyzed by HPLC, and no impurity represented by formula I was detected.

Comparative example 2

5mL of 200mg/mL vitamin B₁The injection, after HPLC analysis, has no impurity detected as shown in formula I, 5mL of 3% hydrogen peroxide solution is added, and the mixture reacts at 25 deg.C for 24h, and after HPLC analysis, the mixture still remainsThe impurities shown in formula I were not detected.

Example 1

5mL of 200mg/mL vitamin B₁The injection is analyzed by HPLC, the impurities shown in the formula I are not detected, the injection is placed in an oven at the temperature of 80 ℃ for reaction for 24 hours, and the content of the impurities shown in the formula I is about 0.5% by HPLC analysis.

Example 2

5mL of 200mg/mL vitamin B₁The injection is analyzed by HPLC, the impurities shown in the formula I are not detected, 5mL of 1mol/L sodium hydroxide solution is added into the injection, the reaction is carried out at 25 ℃ for 1h, and the content of the impurities shown in the formula I is about 0.5% by HPLC analysis.

Example 3

5mL of 200mg/mL vitamin B₁The injection is analyzed by HPLC, the impurities shown in the formula I are not detected, 5mL of 2mol/L sodium hydroxide solution is added into the injection, the reaction is carried out at 25 ℃ for 1h, and the content of the impurities shown in the formula I is about 1.0 percent by HPLC analysis.

Example 4

5mL of 200mg/mL vitamin B₁The injection is analyzed by HPLC, the impurities shown in the formula I are not detected, 5mL of 3mol/L sodium hydroxide solution is added into the injection to react for 1h at 25 ℃, and the impurities shown in the formula I are not effectively separated from the surrounding peaks by the analysis of HPLC.

Example 5

5mL of 200mg/mL vitamin B₁The injection is analyzed by HPLC, the impurities shown in the formula I are not detected, 10mL of 2mol/L sodium hydroxide solution is added into the injection, the reaction is carried out at 25 ℃ for 1h, and the content of the impurities shown in the formula I is about 5.0% by HPLC analysis.

Example 6

5mL of 200mg/mL vitamin B₁The injection, through HPLC analysis, does not detect the impurity shown in formula I, to which 10mL 2mol/L sodium hydroxide solution is added, reaction at 40 ℃ for 1h, through HPLC analysis, the content of degradation impurity is about 10.0%.

For vitamin B after alkali destruction of this example₁The impurities shown in formula I generated by the injection can be separated by adopting high performance liquid chromatography, and the high performance liquid chromatography conditions comprise that:

a chromatographic column: welch Ultimate AQ, 30mm × 250mm, 5 μm; detection wavelength: 248 nm; the mobile phases comprise a mobile phase A1, a mobile phase B1, a mobile phase A1: 0.1% trifluoroacetic acid solution; mobile phase B1: methanol; flow rate: 30 ml/min; column temperature: 25 ℃; sample introduction amount: 4000 l; the mixed mobile phase is eluted by a gradient elution method, and the elution procedure is as follows in the following table 1:

TABLE 1 gradient elution procedure

Collecting the preparation liquid of the target peak, pre-freezing the preparation liquid by using dry ice and ethanol, and then freeze-drying the preparation liquid in a dark place to obtain 35mg of target impurity solid shown in the formula I, wherein the yield is 35%. LC-MS (M + H +): ESI m/z 230.09592, HPLC purity: 93.4 percent.

TABLE 3¹H NMR (DMSO-d6) data

The chemical shifts δ (ppm) of the hydrogen spectra with peaks m, dp were averaged.

TABLE 4¹³C NMR (DMSO-d6) data

Example 7

Vitamin B₁The detection method of the impurities shown in the formula I in the injection comprises the following steps:

a chromatographic column: octadecylsilane chemically bonded silica gel column, the model of chromatographic column is Shimadzu Inertsil ODS-3, 250mm is multiplied by 4.6mm, and 5.0 μm; the mobile phases comprise a mobile phase A2, a mobile phase B2, a mobile phase A2: 1 wt% ammonium hexane sulfonate solution; mobile phase B: methanol; the mobile phase was eluted by gradient elution, according to the following procedure in Table 2:

TABLE 2 gradient elution procedure

The column temperature is 30 ℃; the flow rate was 0.8mL per minute; the sample volume is 25 mu L; the detection wavelength was 248 nm.

The impurity of formula I prepared in example 6 was used as a control, precisely weighed, and diluted with [ 0.75% glacial acetic acid ]]Dissolving and diluting to obtain a reference substance containing 0.01mg per 1mL as a reference solution; vitamin B after destruction of the aqueous alkali solution of example 6₁Injection, with diluent [ 0.75% glacial acetic acid ]]Diluting to 1mL containing 1mg vitamin B₁As a test solution.

Precisely measuring 25 μ L of each of the test solution and the reference solution, injecting into a liquid chromatograph, and recording chromatogram; according to the detection result, the absolute retention time of the impurity in the reference solution is 13.020min, and the content of the impurity is 95.4% by an area normalization method (see fig. 12); the absolute retention time of the impurity of the present invention in the test solution was 13.025min, and the content thereof was about 9% by area normalization (see FIG. 13).

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An impurity of formula I, having the formula:

2. a process for the preparation of the impurity of claim 1, comprising the steps of:

3. a process for the preparation of impurities according to claim 2, characterized in that it comprises the following steps: mixing vitamin B₁Carrying out chemical reaction on the injection at 75-85 ℃ to prepare impurities shown in the formula I; preferably, the reaction temperature is 80 ℃, and the reaction time is 18-30 hours, preferably 24 hours.

4. The process for producing impurities according to claim 2, wherein vitamin B is added₁The injection and the alkali solution are subjected to chemical reaction at the temperature of 25-40 ℃ to prepare the impurities shown in the formula I.

5. The method for the production of impurities according to claim 4, wherein the base is an inorganic base, preferably sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate or aqueous ammonia, more preferably sodium hydroxide; the concentration of the alkali solution is 1-3 mol/L, preferably 1-2 mol/L, and more preferably 2 mol/L.

6. Process for the preparation of the impurity according to claim 4, wherein said vitamin B₁Vitamin B in injection₁The concentration of (A) is 100 mg/mL-200 mg/mL, preferably 200 mg/mL; the reaction temperature is 30-40 ℃; the reaction time is 0.5-2 hours; the vitamin B₁The volume ratio of the injection to the alkali solution is 1: 1-3; preferably 1: 1-2; more preferably 1: 2.

7. The process of claim 4, wherein the impurities are separated by HPLC under conditions comprising: the chromatographic column is Welch Ultimate AQ, and gradient elution is carried out by adopting a mixed mobile phase of a mobile phase A1 and a mobile phase B1, wherein the mobile phase A1 is 0.05-0.5% trifluoroacetic acid solution, and the mobile phase B1 is methanol; the gradient elution comprises the following steps: (1) the volume ratio of the mobile phase A1 to the mobile phase B1 was kept constant at 95:5 for 0-16 minutes; (2) the volume ratio of the mobile phase A1 to the mobile phase B1 is uniformly graded from 95:5 to 5:95 within 16-16.2 minutes; (3) the volume ratio of the mobile phase A1 to the mobile phase B1 is kept constant at 5:95 within 16.2-18 minutes; (4) the volume ratio of the mobile phase A1 to the mobile phase B1 is gradually changed from 5:95 to 95:5 at a constant speed within 18-18.2 minutes; (5) the volume ratio of mobile phase a1 to mobile phase B1 remained constant at 95:5 for 18.2-20 minutes.

8. The method for detecting impurities according to claim 1, wherein the method for detecting impurities comprises subjecting vitamin B to high performance liquid chromatography₁The impurities in the injection are detected, and the high performance liquid chromatography conditions comprise: performing gradient elution by using an octadecylsilane bonded silica gel column as a chromatographic column and adopting a mobile phase A2 and a mobile phase B2 as mixed mobile phases, wherein the mobile phase A2 is a hexane ammonium sulfonate solution, and the mobile phase B2 is methanol; the gradient elution comprises the following steps: (1) the volume ratio of the mobile phase A2 to the mobile phase B2 was kept constant at 95:5 for 0-5 minutes; (2) the volume ratio of the mobile phase A2 to the mobile phase B2 is uniformly graded from 95:5 to 50:50 within 5-15 minutes; (3) the volume ratio of the mobile phase A2 to the mobile phase B2 is kept constant at 50:50 within 15-35 minutes; (4) the volume ratio of the mobile phase A2 to the mobile phase B2 is uniformly graded from 50:50 to 95:5 within 35-36 minutes; (5) the volume ratio of mobile phase a2 to mobile phase B2 remained constant at 95:5 for 36-45 minutes.

9. Use of the impurity of claim 1 in the detection of vitamin B₁The quality of the injection.

10. Use according to claim 9, wherein high performance liquid chromatography is used for vitamin B₁The impurities in the injection are detected,the high performance liquid chromatography conditions comprise: performing gradient elution by using an octadecylsilane bonded silica gel column as a chromatographic column and adopting a mobile phase A2 and a mobile phase B2 as mixed mobile phases, wherein the mobile phase A2 is a hexane ammonium sulfonate solution, and the mobile phase B2 is methanol; the gradient elution comprises the following steps: (1) the volume ratio of the mobile phase A2 to the mobile phase B2 was kept constant at 95:5 for 0-5 minutes; (2) the volume ratio of the mobile phase A2 to the mobile phase B2 is uniformly graded from 95:5 to 50:50 within 5-15 minutes; (3) the volume ratio of the mobile phase A2 to the mobile phase B2 is kept constant at 50:50 within 15-35 minutes; (4) the volume ratio of the mobile phase A2 to the mobile phase B2 is uniformly graded from 50:50 to 95:5 within 35-36 minutes; (5) the volume ratio of mobile phase a2 to mobile phase B2 remained constant at 95:5 for 36-45 minutes.