CN112557523A - Impurities of isoshu pang blue injection, preparation method and detection method thereof - Google Patents
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Abstract
The invention provides impurities of an isosulindachene injection, and a preparation method and a detection method thereof, and belongs to the technical field of pharmacy. The structural formula of the impurity shown in the formula I is shown as follows. The impurities shown in the formula I are prepared when the isoshu panoxan injection is subjected to high-temperature damage and alkali damage. The invention can separate and prepare the impurities shown in the formula I, provides a new reference substance for impurity detection of the isosulubicin injection, and is more beneficial to development of a method for detecting related substances in the isosulubicin injection, thereby controlling the product quality.
Description
Technical Field
The invention belongs to the technical field of pharmacy, and particularly relates to impurities of an isoshu panoxan injection, and a preparation method and a detection method thereof.
Background
The ISHUFULAN injection is available under the trade name LYMPHAZURIN, developed by Hirsch Industries in 1981, and approved by FDA at 29 days 07/1981 for marketing in the United states. The isoshu blue injection belongs to a lymphatic vessel developer, has been used in the United states for nearly 40 years, and is safe and effective. It is an assisted lymphatic contrast agent: primary and secondary lymphedema of the extremities; chyluria, chylomicronemia ascites or chylomicron; lymph node invasion of primary or secondary tumors; and lymph node response to treatment modalities. At present, no original grinding product of the isoshu pang blue injection is on the market in China, and no similar product is on the market.
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.
The isoshu bluing injection can be gradually degraded to generate impurities during the stability period, and if the structure of the impurities is not determined, the impurities bring a greater risk to the quality control of the isoshu bluing injection; only by determining the chemical structure of the impurities, the generation mechanism of the impurities is known, and then the reaction operation in the step is effectively controlled, so that the quality requirement of the isoshu bluing injection can be met.
Disclosure of Invention
The invention aims to provide impurities of isosulubine injection on the basis of the prior art.
The invention also aims to provide a preparation method of the impurities of the isosulubine injection.
The third purpose of the invention is to provide a method for detecting the impurities in the isosulubine injection.
A fourth object of the invention is the use of the impurity for the quality testing of an injection solution of isosulubine.
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:
the isoshu bluing 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 finds the impurity shown in the formula I in the isoshu blue injection, and the content of the impurity exceeds the identification threshold value by 0.2% along with the prolonging of the stable standing time, so that no literature reports that the isoshu blue injection contains the unknown impurity at present. According to the ICH guiding principle, the generation mechanism of the unknown impurity is necessary to be known, 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 product quality of the ISHUFULAN injection is effectively controlled.
Based on the structural analysis of the isosulubine, the inventor of the invention respectively adopts the destruction conditions of high temperature, acid, alkali, oxidation and the like to carry out destruction research on the isosulubine injection and the aqueous solution of the isosulubine bulk drug and investigate the degradation path of the unknown impurities. 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.
When the isoshupeng blue injection and the aqueous solution of the isoshupeng blue raw material medicine are damaged at high temperature, the high temperature generally refers to 50-100 ℃.
In a preferred embodiment, when the inventor of the present invention destroys an isosulubine injection at high temperature, it comprises the following steps: the isosulubicin injection is subjected to chemical reaction at 50-70 ℃ to prepare the impurity shown in the formula I.
In a more preferable scheme, when the isoshu blue injection is damaged at high temperature, the reaction temperature is 60 ℃, and the reaction time is 36-60 hours, preferably 48 hours.
In one embodiment, the method for performing alkali destruction on the isosulubine injection comprises the following steps: the isosulubicin blue injection and alkali solution are subjected to chemical reaction at the temperature of 10-65 ℃ to prepare the impurity shown in the formula I.
For the present invention, when the alkali destruction is performed on the isoshu pan blue injection, the used alkali is an inorganic alkali, and may be, but is not limited to, one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate or ammonia water, and for example, sodium hydroxide may be selected.
In a preferred embodiment, when the alkali destruction is performed on the isosulubine injection, the concentration of the alkali solution is 1-3 mol/L, preferably 1-2 mol/L, and more preferably 2 mol/L.
The concentration of the isosulbactam blue in the isosulbactam blue injection is 6-10 mg/mL, and preferably 10 mg/mL.
In a preferred scheme, when alkali destruction is carried out on the isosulindac blue injection, the volume ratio of the isosulindac blue injection to the alkali solution is 1: 1-3; preferably 1: 1-2; for example, 1: 2.
In a preferred embodiment, when the alkali destruction is performed on the ISHUFULAN injection, the reaction temperature is 30 ℃ to 50 ℃, for example, but not limited to, 30 ℃, 35 ℃, 40 ℃, 45 ℃ or 50 ℃.
Further, the reaction time is 1 to 3 hours, preferably 2 hours.
For the impurities represented by formula I prepared when the isoshu pan blue injection is subjected to alkali destruction, the inventors tried to separate the impurities by using high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography comprise: 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 an ammonium acetate solution, and the mobile phase B1 is acetonitrile; 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 90:10 for 0-2 minutes; (2) the volume ratio of the mobile phase A1 to the mobile phase B1 is uniformly graded from 90:10 to 50:50 within 2-12 minutes; (2) the volume ratio of the mobile phase A1 to the mobile phase B1 is gradually changed from 50:50 to 5:95 at a constant speed within 12-12.2 minutes; (4) the volume ratio of the mobile phase A1 to the mobile phase B1 is kept constant at 5:95 within 12.2-15 minutes; (5) the volume ratio of the mobile phase A1 to the mobile phase B1 is gradually changed from 5:95 to 90:10 at a constant speed within 15-15.2 minutes; (6) the volume ratio of mobile phase A1 to mobile phase B1 remained constant at 90:10 for 15.2-17 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 x 250mm, 5 μm, pore size 100A.
In a preferable scheme, the mobile phase A1 is a 15-25 mmol/L ammonium acetate solution, and the pH value is adjusted to 4 by acetic acid. In a more preferred embodiment, mobile phase a1 is a 20mmol/L ammonium acetate solution adjusted to pH 4 with acetic acid.
Furthermore, the detection wavelength is 350-370 nm, preferably 360 nm.
Further, the flow rate is 30-40 mL/min, preferably 35mL/min
Furthermore, the column temperature is 20-40 ℃, and preferably 30 ℃.
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 a detection method of the impurity shown in the formula I in the isoshu pan-blue injection, the detection method adopts a high performance liquid chromatography to carry out detection, and carries out qualitative or quantitative detection on the impurity, 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 an ammonium acetate solution, and the mobile phase B2 is a mixed solution of acetonitrile and ammonia water; 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 90:10 for 0-5 minutes; (2) the volume ratio of the mobile phase A2 to the mobile phase B2 is uniformly graded from 90:10 to 20:80 within 5-25 minutes; (3) the volume ratio of the mobile phase A2 to the mobile phase B2 is kept constant at 20:80 within 25-45 minutes; (4) the volume ratio of the mobile phase A2 to the mobile phase B2 is uniformly graded from 20:80 to 90:10 within 45-50 minutes; (5) the volume ratio of mobile phase a2 to mobile phase B2 remained constant at 90:10 for 50-70 minutes. The detailed elution procedure is shown in table 2 below.
TABLE 2 gradient elution procedure
In the detection method, an octadecylsilane bonded silica gel column is used as a chromatographic column, and the model of the chromatographic column is 250mm multiplied by 4.6mm, 5.0 μm, for example: zorbax Eclipse XDB-C18 column or Luna C18.
For the purposes of the present invention, mobile phase a2 is a solution of ammonium acetate, and in a preferred embodiment, the concentration of ammonium acetate in mobile phase a2 is from 1 wt% to 3 wt%, e.g., 1 wt%.
For the invention, the mobile phase B2 is a mixed solution of acetonitrile and ammonia water, and in a preferable scheme, the volume concentration of the ammonia water in the mobile phase B2 is 3-10%. In a more preferred embodiment, the concentration of ammonia in mobile phase B2 is 5% by volume.
When detecting impurities shown in formula I in the isoshuVanilla injection, the method comprises the following steps of:
control solution: the impurity represented by formula I, which was prepared when alkali destruction was performed on the isoshuwuron blue injection, was precisely weighed as a control, and was dissolved and diluted with a diluent (the volume ratio of mobile phase A2 to acetonitrile was 9: 1) to prepare a control containing 0.01mg per 1mL as a control solution.
Test solution: taking the isosulubine injection destroyed by the alkali solution, diluting with a diluent (the volume ratio of the mobile phase A2 to the acetonitrile is 9: 1) to prepare the solution containing 2mg of isosulubine per 1mL as a test solution.
The invention also provides application of the impurity shown in the formula I in detecting the quality of the isosulubicin injection, for example, the impurity is used as a reference substance, and the high performance liquid chromatography is used for detecting the impurity in the isosulubicin injection, so that the quality of the isosulubicin injection is controlled.
The qualitative detection in the present invention can be performed by using conventional methods, such as corresponding analysis by external standard method with reference substance, or qualitative analysis by conventional identification means, such as mass spectrometry, thin layer, ultraviolet, etc. after separating each component by HPLC.
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, 225 nm.
Further, the flow rate is 0.6 to 1.2mL/min, preferably 1.0 mL/min.
Further, the column temperature is 20-40 ℃, and preferably 30 ℃.
Furthermore, the sample amount is 12 to 30 μ L, preferably 20 μ L.
By adopting the technical scheme of the invention, the advantages are as follows:
(1) the invention provides a new impurity generated in the isosulindblue injection, defines the degradation path of the impurity and is beneficial to the quality control of the isosulindblue product.
(2) The invention provides a method for separating and preparing impurities shown in the formula I from an isosulubine injection damaged by alkali, which does not need to use excessive chemical reagents and post-treatment, has high reaction conversion rate and is easy to separate and prepare.
(3) According to the invention, the impurities shown in the formula I are separated and prepared, so that a new reference substance is provided for impurity detection of the isoshu blue injection, and the development of a method for detecting related substances in the isoshu blue injection is facilitated, so that the product quality is controlled.
Drawings
FIG. 1 is a graph of impurities in the isosulubicin injection prepared in example 41H NMR chart;
FIG. 2 shows the impurities in the isosulubine injection prepared in example 413C NMR chart;
FIG. 3 is a DEPT90 plot of the impurities of the isosulubine injection prepared in example 4;
FIG. 4 is a DEPT135 plot of the impurities of the isosulubine injection prepared in example 4;
FIG. 5 is a COSY plot of the impurities of the isosulubine injection prepared in example 4;
FIG. 6 is a graph of HSQC of the impurities of the isosulubine injection prepared in example 4;
FIG. 7 is a HMBC plot of the impurities of the isosulubine injection prepared in example 4;
FIG. 8 is a LC-MS (positive ion) plot of the impurities of the isosulubine blue injection prepared in example 4;
FIG. 9 is a LC-MS (negative ion) profile of the impurities of the isosulubine injection prepared in example 4;
FIG. 10 is an HPLC chart of the control solution in example 5;
FIG. 11 is a HPLC chart of the test solution in example 5.
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
10mL of 10mg/mL of isosulubine injection is taken, HPLC analysis shows that the impurity shown in the formula I is not detected, 20mL of 1mol/L hydrochloric acid solution is added into the injection, reaction is carried out at 30 ℃ for 24 hours, and HPLC analysis shows that the impurity shown in the formula I is still not detected.
Comparative example 2
10mL of 10mg/mL of isoshuwuron blue injection is taken, HPLC analysis shows that the impurity shown in the formula I is not detected, 20mL of 10% hydrogen peroxide solution is added into the injection, the reaction is carried out at 30 ℃ for 24h, and HPLC analysis shows that the impurity shown in the formula I is still not detected.
Example 1
10mL of 10mg/mL isosulubine injection is taken, and analyzed by HPLC, the impurity shown in the formula I is not detected, placed in a 60 ℃ oven for reaction for 48 hours, and analyzed by HPLC, the content of the impurity shown in the formula I is 0.5%.
Example 2
10mL of 10mg/mL of isoshuwulan injection is taken, HPLC analysis shows that the impurity shown in the formula I is not detected, 30mL of 1mol/L sodium hydroxide aqueous solution is added into the injection, the reaction is carried out at 40 ℃ for 2h, and HPLC analysis shows that the content of the impurity shown in the formula I is 8%.
Example 3
10mL of 10mg/mL of isoshuwulan injection is taken, HPLC analysis shows that the impurity shown in the formula I is not detected, 10mL of 3mol/L sodium hydroxide aqueous solution is added into the injection, the reaction is carried out at 30 ℃ for 2h, and HPLC analysis shows that the content of the impurity shown in the formula I is 15%.
Example 4
10mL of 10mg/mL of isoshuwulan injection is taken, HPLC analysis shows that the impurity shown in the formula I is not detected, 20mL of 2mol/L sodium hydroxide aqueous solution is added into the injection, the reaction is carried out at 50 ℃ for 2h, and HPLC analysis shows that the content of the impurity shown in the formula I is 40%.
For the impurities shown in formula I produced by the alkali-destroyed isoshu blue injection of the embodiment, the impurities can be separated by high performance liquid chromatography, and the conditions of the high performance liquid chromatography include:
a chromatographic column: welch Ultimate AQ, 30mm × 250mm, 5 μm, pore size 100A; detection wavelength: 360 nm; flow rate: 35 mL/min; column temperature: 30 ℃; the mobile phases comprise a mobile phase A1, a mobile phase B1, a mobile phase A1: 20mmol/L ammonium acetate solution (pH 4 adjusted with acetic acid); mobile phase B1: acetonitrile; the mobile phase was eluted by gradient elution, according to the following procedure in table 1:
TABLE 1 gradient elution procedure
The target peak preparation was collected, prefreezed with dry ice and ethanol, and lyophilized in the dark to obtain 38mg of the target impurity solid with a yield of 38%. LC-MS (M + H)+)：ESI m/z＝414.06761。LC-MS(M+H-): ESI m/z 412.05231. HPLC purity: 98.4 percent.
TABLE 31H NMR (DMSO-d6) data
The chemical shifts δ (ppm) of the peaks d, t, q, m in nuclear magnetic hydrogen spectra were averaged.
TABLE 413C NMR (DMSO-d6) data
Example 5
The method for detecting the impurities shown in the formula I in the isosulubine injection comprises the following steps:
a chromatographic column: zorbax Eclipse XDB C18, 250mm × 4.6mm, 5.0 μm; the mobile phases comprise a mobile phase A2, a mobile phase B2, a mobile phase A2: 1 wt% ammonium acetate solution; mobile phase B: a mixed solution of ammonia water and acetonitrile (the volume concentration of the ammonia water is 5%); 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 1.0mL per minute; the sample volume is 20 mu L; the detection wavelength was 225 nm.
Taking the impurity shown in the formula I prepared in the example 4 as a reference substance, precisely weighing, dissolving and diluting by using a diluent (the volume ratio of the mobile phase A2 to the acetonitrile is 9: 1) to prepare a reference substance containing 0.01mg in each 1mL, and using the reference substance as a reference substance solution; the isosumatriptan injection destroyed by the alkali solution in example 2 was diluted with a diluent (the volume ratio of mobile phase A2 to acetonitrile was 9: 1) to prepare an isosumatriptan containing 2mg per 1mL as a test solution.
Precisely measuring 20 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 12.752min, and the content of the impurity is 98.4% by an area normalization method (see figure 10); the absolute retention time of the impurity of the present invention in the test solution was 12.669min, and its content was 8% by area normalization (see FIG. 11).
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 (10)
1. An impurity of formula I, having the formula:
2. a process for producing the impurity of claim 1,
3. a process for the preparation of impurities according to claim 2, characterized in that it comprises the following steps: carrying out chemical reaction on the isoshu pan-blue injection at 50-75 ℃ to prepare impurities shown in formula I; preferably, the reaction temperature is 60 ℃; the reaction time is 36 to 60 hours, preferably 48 hours.
4. The method for preparing impurities as claimed in claim 2, wherein the impurities of formula I are prepared by chemical reaction of ISHUFU blue injection with alkali solution at 10-65 ℃.
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. The method for preparing impurities according to claim 4, wherein the concentration of isosulubine in the isosulubine injection is 6-10 mg/mL, preferably 10 mg/mL; the reaction temperature is 30-50 ℃; the reaction time is 1-3 hours; the volume ratio of the isopantoea injection to the aqueous alkali 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 an ammonium acetate solution, and the mobile phase B1 is acetonitrile; 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 90:10 for 0-2 minutes; (2) the volume ratio of the mobile phase A1 to the mobile phase B1 is uniformly graded from 90:10 to 50:50 within 2-12 minutes; (2) the volume ratio of the mobile phase A1 to the mobile phase B1 is gradually changed from 50:50 to 5:95 at a constant speed within 12-12.2 minutes; (4) the volume ratio of the mobile phase A1 to the mobile phase B1 is kept constant at 5:95 within 12.2-15 minutes; (5) the volume ratio of the mobile phase A1 to the mobile phase B1 is gradually changed from 5:95 to 90:10 at a constant speed within 15-15.2 minutes; (6) the volume ratio of mobile phase A1 to mobile phase B1 remained constant at 90:10 for 15.2-17 minutes.
8. The method for detecting the impurity according to claim 1, wherein the method for detecting the impurity in the isoshu pan-blue injection adopts high performance liquid chromatography, 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 acetate solution, and the mobile phase B2 is a mixed solution of acetonitrile and ammonia water; 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 90:10 for 0-5 minutes; (2) the volume ratio of the mobile phase A2 to the mobile phase B2 is uniformly graded from 90:10 to 20:80 within 5-25 minutes; (3) the volume ratio of the mobile phase A2 to the mobile phase B2 is kept constant at 20:80 within 25-45 minutes; (4) the volume ratio of the mobile phase A2 to the mobile phase B2 is uniformly graded from 20:80 to 90:10 within 45-50 minutes; (5) the volume ratio of mobile phase a2 to mobile phase B2 remained constant at 90:10 for 50-70 minutes.
9. Use of the impurity of claim 1 for testing the quality of an injection of isosulubine.
10. The use of claim 9, wherein the impurities in the isosuzuran injection are detected by high performance liquid chromatography, 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 acetate solution, and the mobile phase B2 is a mixed solution of acetonitrile and ammonia water; 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 90:10 for 0-5 minutes; (2) the volume ratio of the mobile phase A2 to the mobile phase B2 is uniformly graded from 90:10 to 20:80 within 5-25 minutes; (3) the volume ratio of the mobile phase A2 to the mobile phase B2 is kept constant at 20:80 within 25-45 minutes; (4) the volume ratio of the mobile phase A2 to the mobile phase B2 is uniformly graded from 20:80 to 90:10 within 45-50 minutes; (5) the volume ratio of mobile phase a2 to mobile phase B2 remained constant at 90:10 for 50-70 minutes.
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