CN112782327B - Method for separating and determining carbocisteine and impurities thereof by liquid chromatography - Google Patents
Method for separating and determining carbocisteine and impurities thereof by liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method for separating and determining carbocisteine and impurities thereof by high performance liquid chromatography, which adopts a chromatographic column with octadecylsilane chemically bonded silica as a filler, and has the following detection conditions: chromatographic conditions are as follows: the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, the mobile phase is phosphate-ion pair buffer solution, the pH value of the mobile phase is 1.6-2.0, and the detection wavelength is 215 nm. The method has high precision, good repeatability and high recovery rate, and can be widely used for quality detection of carbocisteine raw material medicines with different sources and corresponding preparations thereof.
Description
Technical Field
The invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for separating and determining carbocisteine and impurities thereof by high performance liquid chromatography.
Background
Carbocisteine (S-Carboxymethyl-L-cysteine) with molecular formula C is used for treating cough and expectoration caused by chronic bronchitis and bronchial asthma, and can be used for treating infantile non-suppurative otitis media to prevent deafness5H9NO4And S. The molecular weight is 179.19, and the carbocisteine structural formula is:
during the production and storage of carbocisteine, the purity of the drug may be affected by incomplete removal of starting materials and intermediates and by degradation impurities generated during storage, and these substances affecting the purity of the drug are referred to as related substances. The above related substances have no therapeutic effect, and may affect the stability and curative effect of the medicine, even harm human health.
The impurities A to G can be generated during the production and storage of carbocisteine, and the information of the structural formula, the chemical formula and the like is shown in the following table:
chinese patent CN-201611113881.3 discloses a method for separating and detecting carbocisteine bulk drugs and related substances containing carbocisteine preparations by liquid chromatography, which is characterized in that octadecyl silane bonded silica gel is used as a chromatographic column of a filler, and a buffer solution-organic phase with a certain proportion is used as a mobile phase to realize the separation and the determination of carbocisteine and the following three related substances.
Yujoehe et al published 'HPLC assay of Carbocisteine and its preparation related substances' and reported a method for testing Carbocisteine and its preparation related substances by HPLC method, using DiamonsilTMC18 column (4.6 mm. times.150 mm,5 μm) chromatography column; mobile phase: taking 6.8g of monopotassium phosphate and 0.5g of sodium heptanesulfonate, adding water to dissolve and dilute to 1000mL (adjusting the pH to 2.5 by using phosphoric acid); the detection wavelength is 215 nm; the flow rate is 1.0 mL/min; the column temperature is 35 ℃; the amount of the sample was 20. mu.L. The methodCan separate out thermal decomposition products, acid hydrolysis products, alkali hydrolysis products and oxidation products of carbocisteine.
The content determination of carbocisteine in carbocisteine sugar-free oral solution and related substances by an HPLC method is published in Shanghai Yun, and the like in Tankang, and the document reports a method for determining the content of carbocisteine and detecting the related substances. By HPLC using DiamonsilTMC18 column (4.6 mm. times.150 mm,5 μm) was used as chromatographic column, 0.68% potassium dihydrogen phosphate and 0.05% sodium hexane sulfonate (pH2.5) were used as mobile phase, and the detection wavelength was 215 nm. The method can separate acid hydrolysis product, alkaline hydrolysis product, thermal decomposition product, strong light damage product, and oxidation degradation product.
Impurity A, impurity C, impurity D, impurity E and impurity F, wherein impurity G is an impurity which is easy to introduce in the synthesis process of carbocisteine, and impurity B is an initial raw material for synthesizing carbocisteine. The applicant finds that the above patent documents can only partially separate and measure carbocisteine and impurities thereof, but cannot realize simultaneous separation and measurement of carbocisteine and the seven impurities, and no method for simultaneous separation and measurement of carbocisteine and the seven impurities is available at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for separating and determining carbocisteine and impurities thereof by high performance liquid chromatography, which has universality compared with the method disclosed by the prior art and is represented as follows: the method can realize the simultaneous separation of various impurities, is favorable for realizing more accurate quantitative analysis of related substances in the bulk drugs or the preparations, and is favorable for realizing effective quality control of unknown impurities which are potentially generated in the processes of preparation and storage of the bulk drugs or the preparations.
The above object of the present invention is achieved by the following technical solutions:
the invention discloses a method for separating and determining carbocisteine and impurities thereof by high performance liquid chromatography, which adopts a chromatographic column with octadecylsilane chemically bonded silica as a filler, and has the following detection conditions:
chromatographic conditions are as follows: the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, elution is carried out by taking phosphate-ion pair buffer solution as a mobile phase, the detection wavelength is 215nm, the column temperature is 20-30 ℃, the speed of the mobile phase is 0.8-1.20 mL/min, and the pH value of the phosphate-ion pair buffer solution is adjusted to 1.6-2.0 by using phosphoric acid;
preparing a sample solution: respectively preparing solutions containing 1-2.5 mg/mL of carbocisteine and impurities thereof by using a phosphate solution;
and (3) determination: and (3) injecting the solution into a high performance liquid chromatograph with the sample amount of 20-30 mu L, recording a chromatogram and analyzing.
The ion pair reagent is sodium octane sulfonate, and the concentration of the ion pair reagent is 8.0-12.5 mmol/L.
In the method for separating and determining carbocisteine and impurities thereof by the high performance liquid chromatography, the adopted chromatographic column, the phosphate buffer solution in the mobile phase, the concentration and the type of the ion pair reagent, the pH value and the speed of the mobile phase and the column temperature are important factors influencing the detection effect.
Specifically, the phosphate-ion pair buffer solution in the mobile phase adopted by the high performance liquid phase method consists of a phosphate buffer solution and an ion pair reagent. Carbocisteine is not substantially retained in C18 column, and has structure with 2 carboxyl groups and 1 amino group, and according to this feature, proper amount of phosphate, preferably dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and most preferably potassium dihydrogen phosphate, is added into mobile phase to control dissociation of these groups. In addition, the concentration and pH value of the phosphate buffer can affect the detection effect; for the concentration of the phosphate buffer solution in the mobile phase, a detection sample is a test solution prepared from a raw material medicine or a preparation, when the pH value is kept unchanged, the low-concentration phosphate buffer solution mobile phase cannot realize baseline separation of all impurities, and although the high-concentration phosphate buffer solution mobile phase can realize better impurity separation degree, the peak time is longer, so that the detection efficiency is influenced. Specifically, in the method for separating and determining carbocisteine and impurities thereof by high performance liquid chromatography, the concentration of the phosphate buffer solution is preferably 15-25 mmol/L, less preferably 18-23 mmol/L, and most preferably 20 mmol/L.
The retention time of anions in carbocisteine in a sample on a chromatographic column can be increased by adding a proper amount of ion pair reagent into the mobile phase. The ion pair reagent is sodium octane sulfonate. The concentration of the ion pair reagent is preferably 8.0-12.5 mmol/L, and most preferably 9.2 mmol/L. The change of ion to reagent kind and concentration is different to the retention time influence of each impurity, and too high and too low concentration all can influence the separation effect, and too low concentration can make impurity E and impurity C go out the peak and make an advance to cause the phenomenon that the peak overlaps, and too high concentration not only fails to detect impurity B and impurity C, still can appear the interference of unknown peak, consequently. Only at the appropriate species and concentrations described above, the impurities can achieve effective separation from the baseline.
For the pH value of the mobile phase, a detection sample is a test solution prepared from a raw material medicine or a preparation, when the concentration of a phosphate buffer solution is kept unchanged, the mobile phase with a low pH value is compatible with the mobile phase and is easy to damage an analytical column, the service life of the analytical column is shortened, the mobile phase with a high pH value cannot realize baseline separation of all impurities, when the detection sample is used for destroying the test solution of an experiment, the pH value is increased to influence the separation degree of unknown impurities and a main peak, so that the detection effect is influenced, and when the pH value of the mobile phase is 1.7, the best separation of the impurities in each test solution by detection is favorably realized.
The column temperature is also one of the important factors for determining the separation effect, specifically, for a test solution prepared from the bulk drug or the preparation, the separation degree of the impurity peak and the main peak is gradually increased along with the reduction of the column temperature, but when the detection sample is a destructive test solution, the separation degree of the unknown impurity and the main peak is gradually decreased along with the reduction of the column temperature until the separation degree is overlapped; the inventor comprehensively obtains that the column temperature is preferably 25 ℃, and the separation effect is the best.
The octadecylsilane bonded silica gel chromatographic column adopted by the high performance liquid chromatography can be understood by those skilled in the art that even if the chromatographic column is filled with the same material, the performance of the chromatographic column is different, and the detection effect is influenced; specifically, the octadecylsilane bonded silica column used in the present invention may be a ZORBAX SB-Aq column (4.6 mm. times.250 mm,5 μm), a Shim-pack GIST C18 column (4.6 mm. times.250 mm,5 μm) or a CAPCELL PAK C18 AQ column (4.6 mm. times.250 mm,5 μm), preferably a ZORBAX SB-Aq column (4.6 mm. times.250 mm,5 μm).
According to the method for separating and determining carbocisteine and impurities thereof by high performance liquid chromatography, the flow phase speed is preferably 1mL/min, the concentration of the sample preparation solution is preferably 2mg/mL, and the sample injection volume is preferably 25 mu L, so that a good separation effect can be obtained, and the analysis efficiency is ensured.
In the method for separating and determining carbocisteine and impurities thereof by using the high performance liquid chromatography, the impurities are impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G, and the structural formula of the impurities is as follows:
in a preferred embodiment of the present invention, in the method for separating and determining carbocisteine and impurities thereof by high performance liquid chromatography according to the present invention, the impurities are impurity a, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G, and the structural formula of the impurities is:
the method comprises the following steps:
chromatographic conditions are as follows: the chromatographic column is a ZORBAX SB-Aq chromatographic column (4.6mm multiplied by 250mm,5 mu m), a phosphate-ion pair buffer solution consisting of 20mmol/L potassium dihydrogen phosphate and 9.2mmol/L sodium octane sulfonate reagent is used as a mobile phase for elution, the detection wavelength is 215nm, the column temperature is 25 ℃, the speed of the mobile phase is 1.0mL/min, and the pH value of the phosphate-ion pair buffer solution is adjusted to 1.7 by phosphoric acid;
preparing a sample solution: respectively preparing a solution containing 2mg/mL of carbocisteine and impurities thereof by adopting a phosphate solution;
and (3) determination: the sample amount was 25. mu.L, and the solution was injected into a high performance liquid chromatograph, and a chromatogram was recorded and analyzed.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for separating and determining carbocisteine and impurities thereof by high performance liquid chromatography, which can control the content of impurities in carbocisteine, can realize the simultaneous and complete separation of the carbocisteine and the impurities, has the separation degree with the impurities larger than 2.0, has the theoretical plate number reaching 21012 calculated according to carbocisteine peak, has the detection limit of about 0.02 mu g/mL and the sample injection concentration of about 0.0002-0.008 mg/mL in the linear range when the sample injection volume is 25 mu L and the same signal-to-noise ratio is 4:1, and has the advantages of high precision, good repeatability and high recovery rate. The method can accurately carry out quantitative analysis on related substances of the bulk drug carbocisteine and the preparation thereof, thereby ensuring the quality controllability of the carbocisteine and the preparation thereof.
Drawings
FIG. 1 is the HPLC chromatogram obtained in example 1.
FIG. 2 is the HPLC chromatogram obtained in example 2.
FIG. 3 is the HPLC chromatogram obtained in example 3.
FIG. 4 is a HPLC chromatogram obtained in comparative example 1.
FIG. 5 is a HPLC chart obtained in comparative example 2.
FIG. 6 is a HPLC chart obtained in comparative example 3.
FIG. 7 is a HPLC chart obtained in comparative example 4.
FIG. 8 is a HPLC chart obtained in comparative example 5.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the invention are not limited thereto.
Example 1
The instrument comprises the following steps: agilent 1260Infinity II;
a chromatographic column: ZORBAX SB-Aq column (4.6 mm. times.250 mm,5 μm);
mobile phase: 20mmol/L potassium dihydrogen phosphate-9.2 mmol/L sodium octane sulfonate ion pair buffer solution;
column temperature: 25 ℃;
flow rate: 1 mL/min;
detection wavelength: 215 nm;
preparing a mixture containing carbocisteine and impurity concentration thereof: 2 mg/mL;
sample introduction amount: 25 mu L of the solution;
preparation of a mobile phase: taking 2.72g of monopotassium phosphate and 2g of octane sodium sulfonate, adding water to dissolve and dilute the monopotassium phosphate and the octane sodium sulfonate to 1000mL, and adjusting the pH value to 1.7 by using phosphoric acid;
preparing a positioning solution: accurately weighing 5mg of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G respectively, placing the impurities in a 50mL measuring flask respectively, adding a diluent (0.02mol/L dipotassium hydrogen phosphate solution) to dissolve and dilute the impurities to a scale, and shaking the solution uniformly to obtain stock solutions of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G. Taking appropriate amount of stock solutions of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G as positioning solutions.
Preparation of a carbocisteine system applicability solution: precisely weighing 20mg of carbocisteine working reference substance, placing the carbocisteine working reference substance into a 10mL measuring flask, adding 1mL of each of stock solutions of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G, adding a diluent (0.02mol/L dipotassium hydrogen phosphate solution), dissolving and diluting to scale, and shaking uniformly to obtain the carbocisteine compound.
And (3) determination: injecting 25 μ L of the positioning solution into a high performance liquid chromatograph, and detecting impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G with retention time of 9.249min, 13.281min, 48.205min, 8.072min, 4.362min (4.574min), 17.562min and 3.206min respectively. Injecting 25 μ L of mixed solution of carbocisteine and impurities into a high performance liquid chromatograph, and recording chromatogram, wherein the retention time of carbocisteine peak and impurities A, B, C, D, E, F and G is 14.779min, 9.238min, 13.312min, 48.512min, 8.111min, 4.368min (4.582min), 17.640min and 3.209min respectively. The chromatographic condition of the embodiment can effectively distinguish the peaks of carbocisteine and the impurities A-G, the carbocisteine and the impurities A-G achieve baseline separation, and the method has strong specificity and high sensitivity. In experiments, the fact that the mobile phase is added with a potassium dihydrogen phosphate buffer solution of an ion pair reagent sodium octane sulfonate with a proper concentration can inhibit the dissociation of carboxyl and amino on carbocisteine, and meanwhile, the retention time of anions on a chromatographic column is prolonged, so that peaks of carbocisteine and impurities A-G are good, and qualitative detection and quantitative detection of the impurities A-G can be achieved by adopting the method. By adopting the chromatographic conditions of the embodiment, peaks of carbocisteine and impurities A-G can be effectively distinguished, the carbocisteine and the impurities A-G are separated from each other by a base line, the base line is stable, the condition of peak cracking does not exist, the separation degree of adjacent chromatographic peaks is greater than 2.0, and identification and positioning are facilitated.
Example 2
The instrument comprises the following steps: agilent 1260 Infinity;
a chromatographic column: shim-pack GIST C18 column (4.6 mm. times.250 mm,5 μm);
mobile phase: 15mmol/L sodium dihydrogen phosphate-8.0 mmol/L sodium octane sulfonate ion pair buffer solution;
column temperature: 23 ℃;
flow rate: 0.8 mL/min;
detection wavelength: 215 nm;
preparing a mixture containing carbocisteine and impurity concentration thereof: 1 mg/mL;
sample introduction amount: 20 mu L of the solution;
preparation of a mobile phase: 1.8g of sodium dihydrogen phosphate and 1.73g of octane sodium sulfonate are taken, water is added for dissolution and dilution to 1000mL, and the pH value is adjusted to 1.6 by phosphoric acid;
the solution preparation method and the determination method are carried out according to example 1, and the spectrum of the carbocisteine tablet solution is shown in figure 2. The chromatographic condition of the embodiment can effectively distinguish the peaks of carbocisteine and impurities A-G, the retention time of the carbocisteine peak, the impurities A, B, C, D, E, F and G is respectively 13.600min, 9.460min, 12.240min, 43.387min, 7.873min, 4.500min (4.673min), 16.627min and 3.240min, the carbocisteine and the impurities A-G reach baseline separation, and the method has strong specificity and high sensitivity. In experiments, the mobile phase is added with a sodium dihydrogen phosphate buffer solution of an ion pair reagent sodium octane sulfonate with a proper concentration, so that the dissociation of carboxyl and amino on carbocisteine can be inhibited, and the retention time of anions on a chromatographic column is prolonged, therefore, the peaks of carbocisteine and impurities A-G are good, and the qualitative detection and the quantitative detection of the impurities A-G can be realized by adopting the method.
Example 3
The instrument comprises the following steps: agilent 1260 Infinity;
a chromatographic column: CAPCELL PAK C18 AQ chromatography column (4.6mm X250 mm,5 μm);
mobile phase: 25mmol/L dipotassium hydrogen phosphate-12.5 mmol/L octane sodium sulfonate ion pair buffer solution;
column temperature: 30 ℃;
flow rate: 1.2 mL/min;
detection wavelength: 215 nm;
preparing a mixture containing carbocisteine and impurity concentration thereof: 2.5 mg/mL;
sample introduction amount: 30 mu L of the solution;
preparation of a mobile phase: dissolving 4.3g of dipotassium phosphate and 2.70g of octane sodium sulfonate in water, diluting to 1000mL, and adjusting the pH value to 2.0 by using phosphoric acid;
the solution preparation method and the determination method are carried out according to example 1, and the spectrum of the carbocisteine tablet solution is shown in figure 3. The results show that the method can realize qualitative detection and quantitative detection of the impurities A to G. The chromatographic condition of the embodiment can effectively distinguish the peaks of carbocisteine and impurities A-G, the retention time of the carbocisteine peak, the retention time of the impurities A, the retention time of the impurities B, the retention time of the impurities C, the retention time of the impurities D, the retention time of the impurities E, the retention time of the impurities F and the retention time of the impurities G are 14.430min, 9.49min, 12.980min, 46.907min, 8.040min, 4.560min (4.760min), 16.957min and 3.293min, the carbocisteine and the impurities A-G are separated from each other by a base line, and the carbocisteine and the impurities A-G are high in specificity and sensitivity. In experiments, the mobile phase is added with a proper concentration of dipotassium hydrogen phosphate buffer solution of ion pair reagent sodium octane sulfonate, which can inhibit the dissociation of carboxyl and amino on carbocisteine and increase the retention time of anions on a chromatographic column, so that the peaks of carbocisteine and impurities A-G are good, and the qualitative detection and the quantitative detection of the impurities A-G can be realized by adopting the method.
Comparative example 1
The instrument comprises the following steps: agilent 1260 Infinity;
a chromatographic column: ZORBAX SB-Aq column (4.6 mm. times.250 mm,5 μm);
mobile phase: 10mmol/L potassium dihydrogen phosphate-5.7 mmol/L sodium pentane sulfonate ion pair buffer solution;
column temperature: 15 ℃;
flow rate: 0.5 mL/min;
detection wavelength: 215 nm;
preparing a mixture containing carbocisteine and impurity concentration thereof: 0.5mg/mL
Sample introduction amount: 15 mu L of the solution;
preparation of a mobile phase: taking 13.6g of monopotassium phosphate and 1g of sodium pentanesulfonate, adding water to dissolve and dilute the monopotassium phosphate and the sodium pentanesulfonate to 1000mL, and adjusting the pH value to 1.5 by using phosphoric acid;
the solution preparation method and the determination method are carried out according to example 1, and the spectrum of the carbocisteine tablet solution is shown in figure 4. The results show that the chromatographic conditions of the comparative example cannot effectively distinguish peaks of carbocisteine and impurities A-G, the impurity G overlaps with the impurity E in peak type, the impurity B overlaps with the impurity C in peak type, and the impurity F overlaps with the impurity D in peak type, so that the impurities cannot be distinguished. Analysis may be related to the selection of ion pair reagents and the like, and when sodium pentane sulfonate is selected, the relative retention time of some impurities is changed, so that the relative retention time is overlapped with other peak types. Therefore, the method cannot completely realize qualitative detection and quantitative detection of the impurities A to G.
The sodium hexanesulfonate and the sodium heptanesulfonate with the same concentration are selected as the ion pair reagent, the obtained spectrogram pattern is poor, and the peaks of the impurities are overlapped, so that the other ion pair reagents except the sodium octanesulfonate cannot completely separate the carbocisteine and the seven impurities thereof.
Comparative example 2
The instrument comprises the following steps: agilent 1260 Infinity;
a chromatographic column: ZORBAX SB-Aq column (4.6 mm. times.250 mm,5 μm);
mobile phase: 30mmol/L potassium dihydrogen phosphate-17.2 mmol/L sodium pentane sulfonate ion pair buffer solution;
column temperature: 35 ℃;
flow rate: 1.5 mL/min;
detection wavelength: 215 nm;
preparing a mixture containing carbocisteine and impurity concentration thereof: 3 mg/mL;
sample introduction amount: 35 mu L of the solution;
preparation of a mobile phase: taking 4.08g of monopotassium phosphate and 3g of sodium pentanesulfonate, adding water to dissolve and dilute the monopotassium phosphate and the sodium pentanesulfonate to 1000mL, and adjusting the pH value to 2.5 by using phosphoric acid;
the preparation method and the determination method of the solution are carried out according to example 1, and the spectrum of the carbocisteine tablet solution is shown in figure 5. The result shows that the chromatographic conditions of the comparative example can not effectively distinguish the peaks of carbocisteine and the impurities A to G, the peak types of the impurity G and the impurity E are overlapped and cannot be distinguished, and the impurity B and the impurity C cannot be detected. Analysis of the method may relate to selection of ion pair reagents and the like, when sodium pentane sulfonate is selected, relative retention time of certain impurities is changed, so that the impurities are overlapped with other peak types, and excessive pH enables retention time to be reduced and peak emergence to be accelerated. Therefore, the method cannot completely realize qualitative detection and quantitative detection of the impurities A to G.
Comparative example 3
Isolation determination was carried out by reference to the method reported in patent CN201611113881.3
The instrument comprises the following steps: agilent 1260 Infinity;
a chromatographic column: an Altima C18 chromatography column (4.6 mm. times.250 mm,5 μm);
mobile phase: 20mmol/L potassium dihydrogen phosphate-20 mmol/L sodium octane sulfonate ion pair buffer solution-methanol;
column temperature: 30 ℃;
flow rate: 1.0 mL/min;
detection wavelength: 210 nm;
preparation of a mobile phase: taking 2.72g of monopotassium phosphate and 4.33g of octane sodium sulfonate, adding water to dissolve and dilute the monopotassium phosphate and the octane sodium sulfonate to 960mL, then adding 40mL of methanol, and adjusting the pH value to 2.5 by using phosphoric acid;
taking proper amount of carbocisteine or a preparation containing carbocisteine, dissolving a sample by using a mobile phase, and preparing a solution containing carbocisteine of about 2 mg/mL; respectively taking appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G, dissolving with a mobile phase to prepare each impurity solution with the concentration of about 0.5 mg/mL; taking appropriate amount of each impurity solution and carbocisteine solution, diluting with mobile phase, and preparing into system applicability solution; performing high performance liquid chromatography analysis according to the conditions, and recording chromatogram 6. The result shows that the chromatographic conditions of the comparative example can not effectively distinguish the peaks of carbocisteine and the impurities A to G, and the impurity B and the carbocisteine main peak have overlapped peak types and can not be distinguished. The analysis is probably related to the concentration of the ion-pair reagent, when the concentration of the ion-pair reagent is more than 13mmol, the phenomenon of peak cracking is easy to generate, and the bearing capacity of the chromatographic column reaches the limit. Therefore, the method cannot completely realize qualitative detection and quantitative detection of the impurities A to G.
Comparative example 4
The separation and the determination are carried out according to the method reported in the literature, namely the HPLC determination of the carbocisteine and the related substances of the preparation thereof
The instrument comprises the following steps: agilent 1260 Infinity;
a chromatographic column: diamondTMC18 column (4.6 mm. times.150 mm,5 μm);
mobile phase: 50mmol/L potassium dihydrogen phosphate-2.5 mmol/L sodium heptanesulfonate ion pair buffer solution;
column temperature: 35 ℃;
flow rate: 1.0 mL/min;
detection wavelength: 215 nm;
preparation of a mobile phase: taking 6.80g of monopotassium phosphate and 0.5g of sodium heptanesulfonate, adding water to dissolve and dilute the monopotassium phosphate and the sodium heptanesulfonate to 1000mL, and adjusting the pH value to 2.5 by using phosphoric acid;
preparing a positioning solution: accurately weighing 5mg of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G respectively, placing in a 50mL measuring flask respectively, adding a diluent (mobile phase) for dissolving and diluting to a scale, and shaking uniformly to obtain stock solutions of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G. Taking proper amounts of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G as positioning solutions.
Taking proper amount of carbocisteine or a preparation containing carbocisteine, dissolving a sample by using a mobile phase, and preparing a solution containing carbocisteine of about 2 mg/mL;
1mL of each impurity stock solution and 20mg of carbocisteine working reference substance are taken and placed in a 10mL measuring flask, and are dissolved and diluted to a scale by using a mobile phase to prepare a system applicability solution; performing high performance liquid chromatography analysis according to the conditions, and recording chromatogram 7. The result shows that the chromatographic conditions of the comparative example can not effectively distinguish peaks of carbocisteine and impurities A-G, and the peaks of the impurities A and C are overlapped with the peaks of the impurities G and cannot be distinguished. Analysis of the pH may be related to pH, and increasing the pH affects the separation of impurities from the main peak, so that the peak emergence time of some impurities is advanced, and the peak patterns are overlapped. Therefore, the method cannot completely realize qualitative detection and quantitative detection of the impurities A to G.
Comparative example 5
The content of the carbocisteine in the carbocisteine sugar-free oral solution and related substances are separated and determined according to the method reported in the literature
The instrument comprises the following steps: agilent 1260 Infinity;
a chromatographic column: diamondTMC18 column (4.6mm X150 mm,5 μm)
Mobile phase: 50mmol/L potassium dihydrogen phosphate-2.7 mmol/L sodium hexanesulfonate ion pair buffer solution;
column temperature: 25 ℃;
flow rate: 1.0 mL/min;
detection wavelength: 215 nm;
preparation of a mobile phase: taking 6.80g of monopotassium phosphate and 0.5g of sodium hexanesulfonate, adding water to dissolve and dilute the monopotassium phosphate and the sodium hexanesulfonate to 1000mL, and adjusting the pH value to 2.5 by using phosphoric acid;
preparing a positioning solution: accurately weighing 5mg of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G respectively, placing in a 50mL measuring flask respectively, adding a diluent (water) to dissolve and dilute to a scale, and shaking uniformly to obtain stock solutions of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G. Taking proper amounts of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G as positioning solutions.
Taking a proper amount of carbocisteine or a preparation containing carbocisteine, dissolving a sample by using a diluent, and preparing a solution containing carbocisteine of about 2 mg/mL;
1mL of each impurity stock solution and 20mg of carbocisteine working reference substance are taken and put into a 10mL measuring flask and diluted by a diluent to prepare a system applicability solution; performing high performance liquid chromatography analysis according to the conditions, and recording chromatogram 8. The result shows that the chromatographic conditions of the comparative example can not effectively distinguish peaks of carbocisteine and impurities A-G, the peaks of the impurities B and E are overlapped with the peaks of the impurities G and cannot be distinguished, and the impurity A cannot be detected. Analysis of the pH may be related to pH, and increasing the pH affects the separation of impurities from the main peak, so that the peak emergence time of some impurities is advanced, and the peak patterns are overlapped. Therefore, the method cannot completely realize qualitative detection and quantitative detection of the impurities A to G.
In the subsequent analysis methodology verification, the methods of example 1, example 2 and example 3 are adopted, the separation degree of each impurity is more than 2.0, the theoretical plate number reaches 21012 calculated according to carbocisteine peak, the detection limit is about 0.02 mu g/mL when the same signal-to-noise ratio is 4:1 and the sample injection volume is 25 mu L, the sample injection concentration in a linear range is about 0.0002-0.008 mg/mL, the precision is high, the repeatability is good, the recovery rate is high, and the requirements of the analysis method in the drug quality control process are met.
In conclusion, the analysis method can simultaneously detect the impurities A to G, realize the one-time effective separation of the related impurities, further realize the qualitative detection and the quantitative detection, and is beneficial to the quality control of the carbocisteine bulk drug and the preparation. The method disclosed by the prior art can only partially separate and measure carbocisteine and impurities thereof, and cannot realize the simultaneous separation and measurement of carbocisteine and the seven impurities. Therefore, the detection method has higher universality compared with the detection method disclosed by the prior art.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. A method for separating and measuring carbocisteine and impurities thereof by high performance liquid chromatography is characterized in that a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted in the measuring method, and the detection conditions are as follows: chromatographic conditions are as follows: the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, elution is carried out by taking phosphate-ion pair buffer solution as a mobile phase, the detection wavelength is 215nm, the column temperature is 23-30 ℃, the speed of the mobile phase is 0.8-1.20 mL/min, and the pH value of the phosphate-ion pair buffer solution is adjusted to 1.6-2.0 by using phosphoric acid;
preparing a sample solution: respectively preparing solutions containing 1-2.5 mg/mL of carbocisteine and impurities thereof by using a phosphate solution;
and (3) determination: injecting the solution into a high performance liquid chromatograph with the sample amount of 20-30 mu L, recording a chromatogram and analyzing;
the ion pair reagent is sodium octane sulfonate, and the concentration of the ion pair reagent is 9.2 mmol/L;
the chromatographic column is a ZORBAX SB-Aq chromatographic column with the diameter of 4.6mm multiplied by 250mm and the diameter of 5 mu m;
the phosphate is potassium dihydrogen phosphate;
the concentration of the phosphate buffer solution is 18-23 mmol/L.
2. The method for separating and determining carbocisteine and impurities thereof according to claim 1, wherein the concentration of the phosphate buffer is 20 mmol/L.
3. The method for separating and determining carbocisteine and impurities thereof according to claim 1, wherein the pH is 1.7 and the column temperature is 25 ℃.
4. The method for separating and determining carbocisteine and impurities thereof according to claim 1, wherein the mobile phase velocity is 1mL/min, and the concentration of carbocisteine and impurities thereof is 2 mg/mL.
5. The method for separating and determining carbocisteine and impurities thereof according to claim 1, wherein the sample volume is 25 μ L.
6. The method for separating and determining carbocisteine and impurities thereof according to any one of claims 1 to 5, wherein the impurities are impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G, and the structural formula of the impurities is as follows:
7. a method for separating and determining carbocisteine and impurities thereof by high performance liquid chromatography, wherein the impurities comprise an impurity A, an impurity B, an impurity C, an impurity D, an impurity E, an impurity F and an impurity G, and the structural formula of the impurities is as follows:
the method is characterized by comprising the following steps:
chromatographic conditions are as follows: the chromatographic column is a ZORBAX SB-Aq chromatographic column with the thickness of 4.6mm multiplied by 250mm and the thickness of 5 mu m, a phosphate-ion pair buffer solution consisting of 20mmol/L potassium dihydrogen phosphate and 9.2mmol/L sodium octane sulfonate reagent is used as a mobile phase for elution, the detection wavelength is 215nm, the column temperature is 25 ℃, the speed of the mobile phase is 1.0mL/min, and the pH value of the phosphate-ion pair buffer solution is adjusted to 1.7 by phosphoric acid;
preparing a sample solution: respectively preparing a solution containing 2mg/mL of carbocisteine and impurities thereof by adopting a phosphate solution;
and (3) determination: the sample amount was 25. mu.L, and the solution was injected into a high performance liquid chromatograph, and a chromatogram was recorded and analyzed.
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| CN106680386A (en) * | 2016-12-07 | 2017-05-17 | 万全万特制药（厦门）有限公司 | Method for separating and testing related substance of carbocisteine raw material medicine and preparation by using liquid chromatography |
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