CN112979531A - Preparation method of carbazochrome sodium sulfonate degradation impurities - Google Patents
Preparation method of carbazochrome sodium sulfonate degradation impurities Download PDFInfo
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- 229960004353 carbazochrome sodium sulfonate Drugs 0.000 title claims abstract description 135
- HLFCZZKCHVSOAP-WXIWBVQFSA-M sodium;(5e)-5-(carbamoylhydrazinylidene)-1-methyl-6-oxo-2,3-dihydroindole-2-sulfonate Chemical compound [Na+].NC(=O)N\N=C/1C(=O)C=C2N(C)C(S([O-])(=O)=O)CC2=C\1 HLFCZZKCHVSOAP-WXIWBVQFSA-M 0.000 title claims abstract description 135
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- 229960001763 zinc sulfate Drugs 0.000 claims abstract description 12
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- RAYLUPYCGGKXQO-UHFFFAOYSA-N n,n-dimethylacetamide;hydrate Chemical compound O.CN(C)C(C)=O RAYLUPYCGGKXQO-UHFFFAOYSA-N 0.000 claims description 3
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- 238000010438 heat treatment Methods 0.000 description 18
- 239000000523 sample Substances 0.000 description 18
- 238000005191 phase separation Methods 0.000 description 13
- 238000002347 injection Methods 0.000 description 10
- 239000007924 injection Substances 0.000 description 10
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 9
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- XSXCZNVKFKNLPR-SDQBBNPISA-N carbazochrome Chemical class NC(=O)N/N=C/1C(=O)C=C2N(C)CC(O)C2=C\1 XSXCZNVKFKNLPR-SDQBBNPISA-N 0.000 description 2
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- IAZDPXIOMUYVGZ-WFGJKAKNSA-N DMSO-d6 Substances [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a preparation method of carbazochrome sodium sulfonate degradation impurities, which comprises the following steps: taking a carbazochrome sodium sulfonate raw material medicine, adding a solvent for dissolving, and then carrying out stirring reaction at the temperature of 80-120 ℃ to obtain a crude product containing carbazochrome sodium sulfonate target degradation impurities; the preparation method comprises the steps of adopting a preparation liquid phase, taking an acidic buffer solution-acetonitrile system as a mobile phase, taking a C8 or C18 filler as a preparation column, carrying out elution separation on the obtained crude product containing the target carbazochrome sodium sulfonate degradation impurity, collecting the peak flow fraction of the target carbazochrome sodium sulfonate degradation impurity, and carrying out drying after adding zinc sulfate in the flow fraction after treatment. According to the technical scheme, the carbazochrome sodium sulfonate bulk drug is converted into target degradation impurities under the action of high temperature, the required crude product is obtained through one-step reaction, the step aggregation is simple and convenient, the high-efficiency and low-cost impurity crude product enrichment can be realized, the zinc sulfate is further added to obtain a drug impurity reference substance meeting the drug quality research requirement, and the subsequent quality research and analysis of carbazochrome sodium sulfonate are facilitated.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a preparation method of carbazochrome sodium sulfonate degradation impurities.
Background
Carbazochrome sodium sulfonate is a derivative of carbazochrome sodium. Carbazochrome sodium is also called as Anluxue, because there is no salicylic acid group in the molecular structure, it is difficult to dissolve in water. The sodium sulfonate group is a molecular structure of the newly introduced carbazochrome sodium sulfonate, and can effectively solve the problem of low solubility of the Anluo blood, so that the solubility is increased, and an obvious hemostatic effect is generated. The carbazochrome sodium sulfonate is firstly developed successfully by Nihonda pharmaceutical corporation, is mainly used for bleeding of diseases of urinary system, upper digestive tract, respiratory tract and obstetrics and gynecology in clinic, has obvious curative effect on bleeding of urinary system, and can also be used for bleeding of trauma and operation. The carbazochrome sodium sulfonate injection has obvious curative effect on hemostasis, is particularly widely applied to various diseases such as digestion, respiration, operation and the like, and recent researches show that the carbazochrome sodium sulfonate injection also provides wide application space for the carbazochrome sodium sulfonate in endoscopic surgery, kidney biopsy and certain infectious diseases. The carbazochrome sodium sulfonate has the advantages of quick effect, long-lasting effect, less adverse reaction and the like, is widely applied clinically, and is worthy of further clinical popularization.
However, in long-term stability tests of the carbazochrome sodium sulfonate injection, the carbazochrome sodium sulfonate injection is found to degrade an unknown impurity (the content reaches 0.95 percent), which is far beyond the quality control limit specified by ICH, and has important influence on the safety and the effectiveness of the medicine, so that the preparation and the structure confirmation of the degraded impurity are necessary. Meanwhile, in recent years, each national drug administration progressively strengthens the supervision work of drug application, particularly imitation drug application, and especially puts forward higher requirements on the quality of imitation drugs, pharmaceutical enterprises are required to carry out intensive research on various impurities influencing the safety and the effectiveness of the drugs, and various pharmaceutical researches can be carried out only by obtaining a certain amount of impurity reference substances in the research process, so that the preparation of the impurity reference substances is very important work.
Disclosure of Invention
Aiming at the technical problems, the invention discloses a preparation method of carbazochrome sodium sulfonate degradation impurities, which realizes the high-efficiency and low-cost impurity crude product enrichment and can better research the impurities.
In contrast, the technical scheme adopted by the invention is as follows:
a preparation method of carbazochrome sodium sulfonate degradation impurities comprises the following steps:
step S1, adding a solvent into a carbazochrome sodium sulfonate raw material medicine for dissolving, and then carrying out stirring reaction at the temperature of 80-120 ℃ to obtain a crude product containing carbazochrome sodium sulfonate target degradation impurities; in this step, the chemical conversion reaction route is as follows:
the structural formula of the carbazochrome sodium sulfonate degradation impurity is shown as the structural formula on the right side of the formula.
And S2, adopting a preparation liquid phase, taking an acidic buffer solution-acetonitrile system as a mobile phase, taking C8 or C18 filler as a preparation column, eluting and separating the obtained crude product containing the target carbazochrome sodium sulfonate degradation impurity, and collecting the peak flow of the target carbazochrome sodium sulfonate degradation impurity. Further, C18 packing was used as a preparation column.
By adopting the technical scheme, the carbazochrome sodium sulfonate degradation impurities can be obtained, the step aggregation is simple and convenient, and the impurity crude product enrichment with high efficiency and low cost can be realized.
As a further improvement of the invention, the preparation method of the carbazochrome sodium sulfonate degradation impurity further comprises a step S3, zinc sulfate is added into the collected peak flow of the target carbazochrome sodium sulfonate degradation impurity, and then a rotary evaporator is adopted to carry out reduced pressure evaporation to dryness, so as to obtain the carbazochrome sodium sulfonate degradation impurity with the purity of more than 95%.
The carbazochrome sodium sulfonate degradation impurities are particularly unstable in the post-treatment process and are also difficult in impurity storage. By adopting the technical scheme, the zinc sulfate is added as the stabilizer, so that a stable solid impurity reference substance can be obtained, and the carbazochrome sodium sulfonate degradation impurity can be conveniently stored for a long time and studied.
As a further improvement of the present invention, in step S1, the solvent is water-propylene glycol, water-dimethyl sulfoxide or water-dimethyl acetamide.
As a further improvement of the invention, the pH value of the solvent is 3-6. Further preferably, the pH of the solvent is 4.5. The research shows that the pH value mainly influences the yield of the carbazochrome sodium sulfonate degradation impurities. When the pH value exceeds 6, the carbazochrome sodium sulfonate degradation impurities cannot be obtained, the pH value is less than 3, and the yield of the carbazochrome sodium sulfonate degradation impurities is low.
As a further improvement of the invention, in step S1, the temperature of the stirring reaction is 90-110 ℃. Further preferably, the temperature of the stirring reaction is 100 ℃. Researches show that the reaction temperature can influence the yield of the target impurity, namely the carbazochrome sodium sulfonate degradation impurity, and the carbazochrome sodium sulfonate degradation impurity cannot be obtained when the reaction temperature is too low or too high. The technical proposal adopts the stirring reaction temperature of 90-110 ℃, and can obtain higher yield.
As a further improvement of the present invention, in step S2, the acidic buffer-acetonitrile system is 0.1% aqueous formic acid solution-acetonitrile, 0.1% aqueous trifluoroacetic acid solution-acetonitrile or 0.1% aqueous acetic acid solution-acetonitrile. Wherein 0.1% of the 0.1% formic acid aqueous solution-acetonitrile, 0.1% trifluoroacetic acid aqueous solution-acetonitrile or 0.1% acetic acid solution-acetonitrile is a volume ratio.
As a further improvement of the present invention, in step S2, the acidic buffer-acetonitrile system is 0.1% trifluoroacetic acid in water-acetonitrile.
Compared with the prior art, the invention has the beneficial effects that:
firstly, the technical scheme of the invention adopts a high-temperature destructive chemical conversion method to convert the carbazochrome sodium sulfonate bulk drug into target degradation impurities under the action of high temperature, and the required crude product can be obtained by one-step reaction, so that the step aggregation is simple and convenient, and the high-efficiency and low-cost impurity crude product enrichment can be realized. The method realizes the separation and preparation of the largest impurity generated in the carbazochrome sodium sulfonate stability test for the first time, and realizes the structural identification of the impurity through HRMS and NMR for the first time to identify the impurity as a new compound. Furthermore, because the degraded impurities are unstable in a solid state, the technical scheme of the invention skillfully adds zinc sulfate as a stabilizer to obtain a drug impurity reference substance meeting the drug quality research requirements, which is beneficial to the subsequent quality research and analysis of carbazochrome sodium sulfonate.
Secondly, the preparation liquid is adopted to carry out one-step separation preparation relative to the carbazochrome sodium sulfonate destruction crude product, the adopted mobile phase is organic acid solution and acetonitrile, the prepared fraction can be directly decompressed and evaporated to dryness after the stabilizer is added, and the post-treatment is simple, low in cost and low in pollution.
Drawings
Figure 1 is an HPLC purity profile of carbazochrome sodium sulfonate degradation impurity prepared in example 1 of the present invention.
FIG. 2 is a high-resolution mass spectrum of carbazochrome sodium sulfonate degradation impurity prepared in example 1 of the present invention.
FIG. 3 shows the carbazochrome sodium sulfonate degradation impurity prepared in example 1 of the present invention1And (4) H spectrum atlas.
FIG. 4 shows the carbazochrome sodium sulfonate degradation impurity prepared in example 1 of the present invention 13And (C) spectrum atlas.
FIG. 5 is a COSY spectrum of carbazochrome sodium sulfonate degradation impurity prepared in example 1 of the present invention.
FIG. 6 is an HSQC spectrum of carbazochrome sodium sulfonate degradation impurities prepared according to example 1 of the present invention.
FIG. 7 is an HMBC spectrum of the carbazochrome sodium sulfonate degradation impurity prepared in example 1 of the present invention.
Detailed Description
Preferred embodiments of the present invention are described in further detail below.
A preparation method of a carbazochrome sodium sulfonate degradation impurity reference substance comprises the following steps:
(1) and (3) enriching a crude product through chemical conversion: taking a proper amount of carbazochrome sodium sulfonate raw material medicine, selecting a proper amount of solvent system with a proper pH value range for dissolving, stirring and reacting under a heating condition, and preferably reacting to the end point to obtain a crude product containing carbazochrome sodium sulfonate target degradation impurities. Wherein, the pH value can be 3-6, and is preferably 4.5; the solvent system is water-propylene glycol, water-dimethyl sulfoxide or water-dimethyl acetamide. The heating temperature is 90-110 ℃, and preferably 100 ℃.
(2) Preparation and liquid phase separation: and (3) adopting a preparation type liquid phase, taking a pH value as an acidic buffer solution-acetonitrile system as a mobile phase, taking a C18 filler as a preparation column, eluting and separating the target carbazochrome sodium sulfonate degradation impurity from other components, and collecting a target carbazochrome sodium sulfonate degradation impurity peak flow component. The pH value is acidic buffer solution-acetonitrile system of 0.1% formic acid water solution-acetonitrile, 0.1% trifluoroacetic acid water solution-acetonitrile, 0.1% acetic acid solution-acetonitrile, preferably 0.1% trifluoroacetic acid water solution-acetonitrile)
(3) And adding zinc sulfate as a stabilizer into the collected target carbazochrome sodium sulfonate degradation impurity peak flow, and then performing reduced pressure evaporation by using a rotary evaporator to obtain a carbazochrome sodium sulfonate degradation impurity reference substance with the purity of more than 95%.
The following description will be given with reference to specific examples.
Example 1
The instrument comprises the following steps: preparing a liquid phase (Jiangsu Hanbang science and technology Co., Ltd.); rotary evaporator (IKA corporation); heat collection type constant temperature heating magnetic stirrer (Ongyi Zhihua instrument Co., Ltd.)
Preparing a carbazochrome sodium sulfonate degradation impurity crude product: dissolving 2g of carbazochrome sodium sulfonate raw material medicine in 50ml of water-propylene glycol (9:1, pH is adjusted to be 4.5 by phosphoric acid), and reacting for 10 hours at the reaction temperature of 100 ℃ in a heat collection type constant-temperature heating magnetic stirrer.
Preparing a column: the filler is Kromasil EternitytXT-10-C18, specification 50 x 250 mm.
Preparation of liquid phase separation chromatographic conditions: performing isocratic elution by using 0.1% trifluoroacetic acid water solution-acetonitrile (70: 30, V/V) as a mobile phase; the flow rate is 100 mL/min; the detection wavelength is 230 nm; the column temperature is room temperature; the sample is manually injected, and the sample injection amount is 2 ml.
Preparing a sample solution: concentrating the prepared carbazochrome sodium sulfonate degradation impurity crude product to 20ml, and filtering with 0.45um organic filter membrane.
Collecting carbazochrome sodium sulfonate degradation fractions: and (3) after 8 minutes of preparing a liquid phase separation chromatographic condition balance system, manually injecting a sample for elution separation, starting to collect fractions when the carbazochrome sodium sulfonate degradation impurities just generate peaks, ending at the tail part of the peaks, and storing the carbazochrome sodium sulfonate degradation impurity fractions at normal temperature. The instrument was repeated several times and all fractions of carbazochrome sodium sulfonate degradation impurities were pooled.
Carrying out flow separation post-treatment on the carbazochrome sodium sulfonate degradation impurities: adding 100mg of zinc sulfate into all fractions obtained by separation, and then evaporating to dryness at 30 ℃ under reduced pressure by using a rotary evaporator to obtain 500mg of carbazochrome sodium sulfonate degraded impurities with the purity of 97.47%, wherein the yield is 24.37%.
The carbazochrome sodium sulfonate degradation impurities obtained by the steps are analyzed and tested as follows:
(1) LC-MS test:
the instrument comprises the following steps: thermo Vanquish-Q active liquid chromatograph-mass spectrometer
The test method comprises the following steps: shear gas flow rate:50, S-lens RF level:55, Aux gas flow rate:15, Spray voltage:3.5 kV; polar: Negative; Scan type: Full MS; Capillary temp.: 350 ℃.
And (3) testing results: the observed M/z299.04460 is that the prepared carbazochrome sodium sulfonate degrades heteroleptic [ M-H]-Ion, whose molecule is calculated to be C10H12N4O5S, the HPLC purity map of the prepared carbazochrome sodium sulfonate degradation impurity is shown in figure 1.
(2) NMR measurement:
the instrument comprises the following steps: agilent 400 NMR superconducting pulse Fourier transform nuclear magnetic resonance spectrometer
The test method comprises the following steps: the samples were dissolved in DMSO-d6Testing of1H spectrum,13C-NMR spectra, COSY spectra, HSQC spectra and HMBC spectra.
The resulting mass spectrum is shown in figure 2,1the spectrum of the H spectrum is shown in figure 3,13the C spectrum is shown in figure 4, the COSY spectrum is shown in figure 5, the HSQC spectrum is shown in figure 6, and the HMBC spectrum is shown in figure 7.
The chemical structural formula of the carbazochrome sodium sulfonate degradation impurity is as follows:
TABLE 1 NMR data attribution of Carbazochrome sodium sulfonate degrading impurities (400 and 100 MHz, DMSO-d 6))
Example 2
The instrument comprises the following steps: preparing a liquid phase (Jiangsu Hanbang science and technology Co., Ltd.); rotary evaporator (IKA corporation); heat collection type constant temperature heating magnetic stirrer (Ongyi Zhihua instrument Co., Ltd.)
Preparing a carbazochrome sodium sulfonate degradation impurity crude product: dissolving 2g of carbazochrome sodium sulfonate raw material medicine in 50ml of water-dimethyl sulfoxide (8:2, pH is adjusted to 5.0 by phosphoric acid) solution, and reacting for 12 hours in a heat collection type constant temperature heating magnetic stirrer at the reaction temperature of 95 ℃.
Preparing a column: the filler is Kromasil EternitytXT-10-C18, specification 50 x 250 mm.
Preparation of liquid phase separation chromatographic conditions: performing isocratic elution with 0.1% acetic acid water solution-acetonitrile (70: 30, V/V) as mobile phase; the flow rate is 100 mL/min; the detection wavelength is 230 nm; the column temperature is room temperature; the sample is manually injected, and the sample injection amount is 2 ml.
Preparing a sample solution: concentrating the prepared carbazochrome sodium sulfonate degradation impurity crude product to 20ml, and filtering with 0.45um organic filter membrane.
Collecting carbazochrome sodium sulfonate degradation fractions: and (3) after 8 minutes of preparing a liquid phase separation chromatographic condition balance system, manually injecting a sample for elution separation, starting to collect fractions when the carbazochrome sodium sulfonate degradation impurities just generate peaks, ending at the tail part of the peaks, and storing the carbazochrome sodium sulfonate degradation impurity fractions at normal temperature. The instrument was repeated several times and all fractions of carbazochrome sodium sulfonate degradation impurities were pooled.
Carrying out flow separation post-treatment on the carbazochrome sodium sulfonate degradation impurities: adding 100mg of zinc sulfate into all fractions obtained by separation, and then evaporating to dryness at 30 ℃ under reduced pressure by using a rotary evaporator to obtain 460mg of carbazochrome sodium sulfonate degradation impurities with the purity of 96.36%, wherein the yield is 22.16%.
Example 3
The instrument comprises the following steps: preparing a liquid phase (Jiangsu Hanbang science and technology Co., Ltd.); rotary evaporator (IKA corporation); heat collection type constant temperature heating magnetic stirrer (Ongyi Zhihua instrument Co., Ltd.)
Preparing a carbazochrome sodium sulfonate degradation impurity crude product: dissolving 2g of carbazochrome sodium sulfonate raw material medicine in 50ml of water-propylene glycol (9:1, pH is adjusted to be 4.5 by phosphoric acid), and reacting for 8 hours at the reaction temperature of 110 ℃ in a heat collection type constant-temperature heating magnetic stirrer.
Preparing a column: the filler is Kromasil EternitytXT-10-C18, specification 50 x 250 mm.
Preparation of liquid phase separation chromatographic conditions: performing isocratic elution by using 0.1% aqueous trifluorosolution-acetonitrile (70: 30, V/V) as a mobile phase; the flow rate is 100 mL/min; the detection wavelength is 230 nm; the column temperature is room temperature; the sample is manually injected, and the sample injection amount is 2 ml.
Preparing a sample solution: concentrating the prepared carbazochrome sodium sulfonate degradation impurity crude product to 20ml, and filtering with 0.45um organic filter membrane.
Collecting carbazochrome sodium sulfonate degradation fractions: and (3) after 8 minutes of preparing a liquid phase separation chromatographic condition balance system, manually injecting a sample for elution separation, starting to collect fractions when the carbazochrome sodium sulfonate degradation impurities just generate peaks, ending at the tail part of the peaks, and storing the carbazochrome sodium sulfonate degradation impurity fractions at normal temperature. The instrument was repeated several times and all fractions of carbazochrome sodium sulfonate degradation impurities were pooled.
Carrying out flow separation post-treatment on the carbazochrome sodium sulfonate degradation impurities: adding 100mg of zinc sulfate into all fractions obtained by separation, and then evaporating to dryness at 30 ℃ under reduced pressure by using a rotary evaporator to obtain 400mg of carbazochrome sodium sulfonate degraded impurities with the purity of 95.69%, wherein the yield is 19.14%.
Example 4
The instrument comprises the following steps: preparing a liquid phase (Jiangsu Hanbang science and technology Co., Ltd.); rotary evaporator (IKA corporation); heat collection type constant temperature heating magnetic stirrer (Ongyi Zhihua instrument Co., Ltd.)
Preparing a carbazochrome sodium sulfonate degradation impurity crude product: dissolving 2g of carbazochrome sodium sulfonate raw material medicine in 50ml of water-propylene glycol (9:1, pH is adjusted to be 6.0 by phosphoric acid), and reacting for 10 hours at the reaction temperature of 100 ℃ in a heat collection type constant-temperature heating magnetic stirrer.
Preparing a column: the filler is Kromasil EternitytXT-10-C18, specification 50 x 250 mm.
Preparation of liquid phase separation chromatographic conditions: performing isocratic elution by using 0.1% trifluoroacetic acid water solution-acetonitrile (70: 30, V/V) as a mobile phase; the flow rate is 100 mL/min; the detection wavelength is 230 nm; the column temperature is room temperature; the sample is manually injected, and the sample injection amount is 2 ml.
Preparing a sample solution: concentrating the prepared carbazochrome sodium sulfonate degradation impurity crude product to 20ml, and filtering with 0.45um organic filter membrane.
Collecting carbazochrome sodium sulfonate degradation fractions: and (3) after 8 minutes of preparing a liquid phase separation chromatographic condition balance system, manually injecting a sample for elution separation, starting to collect fractions when the carbazochrome sodium sulfonate degradation impurities just generate peaks, ending at the tail part of the peaks, and storing the carbazochrome sodium sulfonate degradation impurity fractions at normal temperature. The instrument was repeated several times and all fractions of carbazochrome sodium sulfonate degradation impurities were pooled.
Carrying out flow separation post-treatment on the carbazochrome sodium sulfonate degradation impurities: adding 100mg of zinc sulfate into all fractions obtained by separation, and evaporating to dryness at 30 ℃ under reduced pressure by using a rotary evaporator to obtain 470mg of carbazochrome sodium sulfonate degradation impurities with the purity of 96.53%, wherein the yield is 22.68%.
Example 5
The instrument comprises the following steps: preparing a liquid phase (Jiangsu Hanbang science and technology Co., Ltd.); rotary evaporator (IKA corporation); heat collection type constant temperature heating magnetic stirrer (Ongyi Zhihua instrument Co., Ltd.)
Preparing a carbazochrome sodium sulfonate degradation impurity crude product: dissolving 2g of carbazochrome sodium sulfonate raw material medicine in 50ml of water-propylene glycol (9:1, pH is adjusted to 3.0 by phosphoric acid), and reacting for 10 hours at a reaction temperature of 90 ℃ in a heat collection type constant-temperature heating magnetic stirrer.
Preparing a column: the filler is Kromasil EternitytXT-10-C18, specification 50 x 250 mm.
Preparation of liquid phase separation chromatographic conditions: performing isocratic elution by using 0.1% trifluoroacetic acid water solution-acetonitrile (70: 30, V/V) as a mobile phase; the flow rate is 100 mL/min; the detection wavelength is 230 nm; the column temperature is room temperature; the sample is manually injected, and the sample injection amount is 2 ml.
Preparing a sample solution: concentrating the prepared carbazochrome sodium sulfonate degradation impurity crude product to 20ml, and filtering with 0.45um organic filter membrane.
Collecting carbazochrome sodium sulfonate degradation fractions: and (3) after 8 minutes of preparing a liquid phase separation chromatographic condition balance system, manually injecting a sample for elution separation, starting to collect fractions when the carbazochrome sodium sulfonate degradation impurities just generate peaks, ending at the tail part of the peaks, and storing the carbazochrome sodium sulfonate degradation impurity fractions at normal temperature. The instrument was repeated several times and all fractions of carbazochrome sodium sulfonate degradation impurities were pooled.
Carrying out flow separation post-treatment on the carbazochrome sodium sulfonate degradation impurities: adding 100mg of zinc sulfate into all fractions obtained by separation, and evaporating to dryness at 30 ℃ under reduced pressure by using a rotary evaporator to obtain 420mg of carbazochrome sodium sulfonate degraded impurities with the purity of 96.35%, wherein the yield is 20.23%.
Comparative example 1
The instrument comprises the following steps: preparing a liquid phase (Jiangsu Hanbang science and technology Co., Ltd.); rotary evaporator (IKA corporation); heat collection type constant temperature heating magnetic stirrer (Ongyi Zhihua instrument Co., Ltd.)
Preparing a carbazochrome sodium sulfonate degradation impurity crude product: dissolving 2g of carbazochrome sodium sulfonate raw material medicine in 50ml of water-propylene glycol (9:1, pH is adjusted to 4.5 by phosphoric acid), and reacting for 10 hours in a heat collection type constant temperature heating magnetic stirrer at the reaction temperature of 100 ℃.
Preparing a column: the filler is Kromasil EternitytXT-10-C18, specification 50 x 250 mm.
Preparation of liquid phase separation chromatographic conditions: performing isocratic elution by using 0.1% trifluoroacetic acid water solution-acetonitrile (70: 30, V/V) as a mobile phase; the flow rate is 100 mL/min; the detection wavelength is 230 nm; the column temperature is room temperature; the sample is manually injected, and the sample injection amount is 2 ml.
Preparing a sample solution: concentrating the prepared carbazochrome sodium sulfonate degradation impurity crude product to 20ml, and filtering with 0.45um organic filter membrane.
Collecting carbazochrome sodium sulfonate degradation fractions: and (3) after 8 minutes of preparing a liquid phase separation chromatographic condition balance system, manually injecting a sample for elution separation, starting to collect fractions when the carbazochrome sodium sulfonate degradation impurities just generate peaks, ending at the tail part of the peaks, and storing the carbazochrome sodium sulfonate degradation impurity fractions at normal temperature. The instrument was repeated several times and all fractions of carbazochrome sodium sulfonate degradation impurities were pooled.
Carrying out flow separation post-treatment on the carbazochrome sodium sulfonate degradation impurities: and (3) directly evaporating all fractions obtained by separation at 30 ℃ by using a rotary evaporator under reduced pressure, and detecting the HPLC purity to find that the content of the target impurity is only 5.23%, so that an impurity reference substance meeting the requirement cannot be obtained.
Comparative example 2
The instrument comprises the following steps: analytical liquid phase (Agilent technologies, Inc.); heat collection type constant temperature heating magnetic stirrer (Ongyi Zhihua instrument Co., Ltd.)
Preparing a carbazochrome sodium sulfonate degradation impurity crude product: taking two 2g portions of carbazochrome sodium sulfonate bulk drug, respectively dissolving the two portions of carbazochrome sodium sulfonate bulk drug in 50ml of water-propylene glycol (9:1, the pH value is adjusted to 2.5 by phosphoric acid) solution and 50ml of water-propylene glycol (9:1, the pH value is adjusted to 6.5 by phosphoric acid), respectively sampling in a heat collection type constant temperature heating magnetic stirrer at the reaction temperature of 100 ℃ for 6h, 8h, 12h and 24h for analysis liquid phase monitoring, and finding that the target impurity content is not higher than 3%, so that the carbazochrome sodium sulfonate bulk drug is not suitable for separation and purification.
Comparative example 3
The instrument comprises the following steps: analytical liquid phase (Agilent technologies, Inc.); heat collection type constant temperature heating magnetic stirrer (Ongyi Zhihua instrument Co., Ltd.)
Preparing a carbazochrome sodium sulfonate degradation impurity crude product: taking two 2g portions of carbazochrome sodium sulfonate raw material medicine, respectively dissolving the two portions in 50ml of water-propylene glycol (9:1, pH is adjusted to 4.5 by phosphoric acid), respectively heating a magnetic stirrer in a heat collection type constant temperature, respectively sampling for 6h, 8h, 12h and 24h at the reaction temperature of 75 ℃ and 125 ℃ in an analysis liquid phase monitoring, and finding that the content of target impurities is not higher than 2%, so that the carbazochrome sodium sulfonate raw material medicine is not suitable for separation and purification.
As can be seen from the comparison between the above examples and comparative examples, the technical scheme of the invention is adopted to realize the separation and preparation of the degraded impurities, and the obtained carbazochrome sodium sulfonate degraded impurities have high purity, simple method, low cost and little pollution, and can obtain impurity reference substances which can meet the quality research requirements.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (8)
1. A preparation method of carbazochrome sodium sulfonate degradation impurities is characterized in that: which comprises the following steps:
step S1, adding a solvent into a carbazochrome sodium sulfonate raw material medicine for dissolving, and then carrying out stirring reaction at the temperature of 80-120 ℃ to obtain a crude product containing carbazochrome sodium sulfonate target degradation impurities;
step S2, adopting a preparation liquid phase, taking an acidic buffer solution-acetonitrile system as a mobile phase, taking C8 or C18 filler as a preparation column, eluting and separating the obtained crude product containing the target carbazochrome sodium sulfonate degradation impurity, and collecting the peak flow of the target carbazochrome sodium sulfonate degradation impurity;
and step S3, adding zinc sulfate into the collected target carbazochrome sodium sulfonate degradation impurity peak flow, and then carrying out reduced pressure evaporation to dryness to obtain carbazochrome sodium sulfonate degradation impurities with the purity of more than 95%.
2. The method for preparing carbazochrome sodium sulfonate degradation impurities according to claim 1, wherein: in step S1, the solvent is water-propylene glycol, water-dimethyl sulfoxide, or water-dimethylacetamide.
3. The method for preparing carbazochrome sodium sulfonate degradation impurities according to claim 2, characterized in that: the pH value of the solvent is 3-6.
4. The method for preparing carbazochrome sodium sulfonate degradation impurities according to claim 3, wherein: the pH of the solvent was 4.5.
5. The method for preparing carbazochrome sodium sulfonate degradation impurities according to claim 1, wherein: in step S1, the temperature of the stirring reaction is 90-110 ℃.
6. The method for preparing carbazochrome sodium sulfonate degradation impurities according to claim 5, wherein: in step S1, the temperature of the stirring reaction was 100 ℃.
7. The method for preparing carbazochrome sodium sulfonate degradation impurities according to any one of claims 1 to 6, wherein: in step S2, the acidic buffer-acetonitrile system is 0.1% aqueous formic acid solution-acetonitrile, 0.1% aqueous trifluoroacetic acid solution-acetonitrile, or 0.1% aqueous acetic acid solution-acetonitrile.
8. The method of preparing carbazochrome sodium sulfonate degradation impurities according to claim 7, characterized in that: in step S2, the acidic buffer-acetonitrile system is 0.1% trifluoroacetic acid aqueous solution-acetonitrile.
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| CN116789585A (en) * | 2022-12-30 | 2023-09-22 | 四川汇宇制药股份有限公司 | A kind of carbosodium sulfonate degradation impurity and its preparation method |
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