CN114563495B - Detection method of acetylcysteine and related substances thereof - Google Patents
Detection method of acetylcysteine and related substances thereof Download PDFInfo
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Abstract
The application provides a detection method of acetylcysteine and related substances thereof, and relates to the technical field of medicine analysis. Related substances include impurities and/or degradation products introduced during the production and storage of acetylcysteine. The detection method is characterized in that acetylcysteine and related substances thereof are separated and detected through high performance liquid chromatography, wherein the high performance liquid chromatography adopts a chromatographic column taking anion-cation intercalation modified alkyl bonding silica gel as a stationary phase, and adopts phosphoric acid aqueous solution as a mobile phase A and phosphoric acid acetonitrile solution as a mobile phase B for gradient elution, so that the acetylcysteine and related substances thereof are completely separated. The detection method can completely separate the acetylcysteine and related substances thereof, thereby improving the sensitivity and accuracy of detection.
Description
Technical Field
The application relates to the technical field of medicine analysis, in particular to a detection method of acetylcysteine and related substances thereof.
Background
Acetylcysteine is an essential amino acid for synthesizing glutathione, is an antioxidant containing sulfhydryl, has the functions of resisting oxidation, resisting inflammation, protecting liver, lung, heart, nerve cells and the like, and is used for treating lung diseases caused by the combined occurrence of phlegm adhesion caused by acute and chronic bronchitis and other reasons. Acetylcysteine is also useful in the treatment of HIV virus infected individuals to improve the physiological and immune functions of patients suffering from viral-induced cysteine deficiency.
At present, the impurity and the content of the acetylcysteine in the pharmacopoeia of each country are strictly regulated, wherein the content of the acetylcysteine is measured by adopting an iodometric method in the Chinese pharmacopoeia, but related substances are not regulated; the content of acetylcysteine was measured using HPLC (High Performance Liquid Chromatography ) and ODS (Octadecylsilyl-Silica Gel) columns using phenylalanine as an internal standard, and the related substances were not specified; the HPLC and ODS column are used for analyzing acetylcysteine and related substances, the mobile phase system used by the method is simpler, but the organic phase ratio of the mobile phase in the analysis method is lower (only 3 percent) than the bearing lower limit of the common ODS column (the lowest organic phase ratio is 5 percent), the pH of the mobile phase is lower, and the service life of the chromatographic column is influenced after long-term use.
The acetylcysteine bulk drug is easy to introduce various related substances such as L-cystine, L-cysteine, N' -diacetylcysteine and the like in the production process and the storage process, and the acetylcysteine can be degraded under a certain factor to generate degradation impurities. The existing analysis method can not separate related substances and degradation impurities of the acetylcysteine bulk drug, so that the detection is inaccurate. Therefore, there is a need to develop a more suitable detection method for acetylcysteine and related substances.
Disclosure of Invention
The present application has been made in view of the above problems, and has as its object to provide a method for detecting acetylcysteine and related substances which overcomes or at least partially solves the above problems.
An object of the present application is to provide a method for detecting acetylcysteine and its related substances, which can completely separate acetylcysteine and its related substances, thereby improving the sensitivity and accuracy of detection.
A further object of the present application is to extend the useful life of a chromatographic column.
According to an aspect of the embodiment of the application, a detection method of acetylcysteine and related substances thereof is provided, wherein the related substances comprise impurities and/or degradation products introduced in the production and storage processes of the acetylcysteine, the acetylcysteine and related substances thereof are separated and detected through high performance liquid chromatography, the high performance liquid chromatography adopts a chromatographic column taking anion-cation intercalation modified alkyl bonded silica gel as a stationary phase, and phosphoric acid aqueous solution is taken as a mobile phase A and phosphoric acid acetonitrile solution is taken as a mobile phase B for gradient elution, so that the acetylcysteine and related substances thereof are completely separated.
Optionally, the related substance includes at least one of:
l-cystine, L-cysteine, N-diacetyl-L-cystine, N, S-diacetyl-L-cysteine, (2R) -2-amino-3-sulfinylpropionic acid monohydrate, (R) -2-acetamido-3- (((R) -2-amino-2-carboxyethyl) disulfanyl) propionic acid, N- (2-mercaptoethyl) acetamide.
Optionally, the chromatography column is a ACE Comixsil ACRP chromatography column.
Optionally, the gradient elution conditions are:
the volume ratio of the mobile phase A gradually changes from 95-100% to 15-25% from 0min to 20 min, and the volume ratio of the mobile phase B gradually changes from 0-5% to 75-85%;
the volume ratio of the mobile phase A gradually changes from 15-25% to 95-100% from 20 minutes to 22 minutes, and the volume ratio of the mobile phase B gradually changes from 75-85% to 0-5%;
maintaining the volume ratio of the mobile phase A to be 95-100% from 22 minutes, and the volume ratio of the mobile phase B to be 0-5%;
the volume ratio of the mobile phase A is kept to be 95-100% from 32 minutes, and the volume ratio of the mobile phase B is kept to be 0-5% until the detection is finished.
Optionally, the gradient elution conditions are:
the volume ratio of the mobile phase A gradually changes from 100% to 20% from 0 minutes to 20 minutes, and the volume ratio of the mobile phase B gradually changes from 0% to 80%;
the volume ratio of the mobile phase A gradually changes from 20% to 100% from 20 minutes to 22 minutes, and the volume ratio of the mobile phase B gradually changes from 80% to 0%;
maintaining the volume ratio of mobile phase a at 100% from 22 minutes;
the volume ratio of mobile phase a was kept at 100% from 32 minutes until the end of the test.
Optionally, the volume ratio of water to phosphoric acid in the mobile phase a is 100:0.03, and the volume ratio of acetonitrile to phosphoric acid in the mobile phase B is 100:0.03.
Optionally, the flow rate of the high performance liquid chromatography is 0.8-1.2ml/min, the column temperature is 25-35 ℃, the detection wavelength is 220nm, and the sample injection temperature is 5 ℃.
Optionally, the column temperature of the high performance liquid chromatography is 30 ℃.
Optionally, the detection method specifically includes the following steps:
dissolving an acetylcysteine sample to be detected in hydrochloric acid and diluting with a diluent to prepare a sample solution with a first concentration;
dissolving acetylcysteine reference substance in the diluent to prepare a reference substance solution with a second concentration;
precisely weighing acetaminophen and proper amounts of each related substance, and dissolving the acetaminophen and the proper amounts of each related substance in the diluent together to prepare a system applicability solution containing the third concentration of acetylcysteine and the fourth concentration of each related substance;
taking the system applicability solution sample for high performance liquid chromatography analysis to determine the retention time of acetylcysteine and each related substance;
and respectively taking the sample solution to be tested and the reference solution for sample injection to perform high performance liquid chromatography analysis, and calculating the contents of acetylcysteine and related substances in the sample solution by a main component external standard method of adding correction factors.
Optionally, the ratio of the third concentration to the fourth concentration is in the range of 150:1 to 250:1;
the first concentration is within + -10% of the third concentration;
the second concentration is within + -10% of the fourth concentration.
According to the detection method of the acetylcysteine and related substances, the related substances comprise impurities and/or degradation products introduced in the production and storage processes of the acetylcysteine, the acetylcysteine and related substances are separated and detected through high performance liquid chromatography, wherein the high performance liquid chromatography adopts a chromatographic column taking anion-cation intercalation modified alkyl bonding silica gel as a stationary phase, and a gradient elution mode is adopted, so that the acetylcysteine and the impurities and degradation products thereof can be completely separated, and the detection sensitivity and detection accuracy of the acetylcysteine and related substances are improved. The method has the advantages of strong specificity, good accuracy, high precision and good repeatability, meets the technical requirements of drug quality research standards, and has stable and reliable results.
Further, in the detection method of acetylcysteine and related substances thereof provided by the application, the stationary phase of the chromatographic column is high in water resistance, and the loss of the adopted flow relative to the chromatographic column is small, so that the service life of the chromatographic column can be prolonged, and the detection cost is reduced.
The foregoing description is only an overview of the present application, and is intended to be implemented in accordance with the teachings of the present application in order that the same may be more clearly understood and to make the same and other objects, features and advantages of the present application more readily apparent.
The above, as well as additional objectives, advantages, and features of the present application will become apparent to those skilled in the art from the following detailed description of a specific embodiment of the present application when read in conjunction with the accompanying drawings.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the application. Also, like reference numerals are used to designate like parts throughout the figures. In the drawings:
FIG. 1 is a schematic flow chart of a detection method of acetylcysteine and related substances according to an embodiment of the application;
FIG. 2 shows a chromatogram obtained by high performance liquid chromatography with a system-applicable solution according to example 1 of the present application;
FIG. 3 is a chromatogram obtained by high performance liquid chromatography with a system-applicable solution in comparative example 1;
FIG. 4 is a chromatogram obtained by high performance liquid chromatography with a system-applicable solution in comparative example 2;
FIG. 5 is a chromatogram obtained by high performance liquid chromatography with a system-applicable solution in comparative example 3;
FIG. 6 is a chromatogram obtained by high performance liquid chromatography with a system-applicable solution in comparative example 4.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
Through intensive researches, the inventor discovers that the acetylcysteine bulk drug is easy to introduce various related substances such as L-cystine, L-cysteine, N' -diacetylcysteine and the like in the production process and the storage process. In addition, the inventors have studied the experiments of influencing factors such as illumination, high temperature and oxidation, forced degradation experiments such as acid-base damage and the like according to the requirements of drug application and registration, and found various degradation impurities such as (2R) -2-amino-3-sulfinyl propionic acid monohydrate, (R) -2-acetamido-3- (((R) -2-amino-2-carboxyethyl) disulfanyl) propionic acid, N- (2-mercaptoethyl) acetamide and the like. The related substances and degradation impurities of the acetylcysteine bulk drug cannot be separated in the prior art, so that the detection is inaccurate.
In order to solve or at least partially solve the above problems, the present application provides a detection method of acetylcysteine and related substances thereof. The substances mentioned herein may include impurities (e.g., byproducts) and/or degradation products introduced during the production and storage of acetylcysteine.
The detection method of the acetylcysteine and the related substances thereof provided by the application separates and detects the acetylcysteine and the related substances thereof through High Performance Liquid Chromatography (HPLC). Specifically, the high performance liquid chromatography adopts a chromatographic column with anion-cation intercalation modified alkyl bonded silica gel as a stationary phase, and adopts phosphoric acid aqueous solution as mobile phase A and phosphoric acid acetonitrile solution as mobile phase B for gradient elution so as to completely separate acetylcysteine and related substances thereof. The anion-cation intercalation modified alkyl bonding silica gel is bonding silica gel with anions and cations simultaneously intercalated with alkyl for modification, and can provide stable anion-cation exchange effect (cation-anion exclusion effect) so as to improve the separation capability.
The detection method of the acetylcysteine and related substances provided by the embodiment of the application can realize the complete separation of the acetylcysteine and impurities and degradation products thereof, thereby improving the detection sensitivity and detection accuracy of the acetylcysteine and related substances.
Specifically, the acetylcysteine-related substance may include at least one of L-cystine, L-cysteine, N-diacetyl-L-cystine, N, S-diacetyl-L-cysteine, (2R) -2-amino-3-sulfinylpropionic acid monohydrate, (R) -2-acetamido-3- (((R) -2-amino-2-carboxyethyl) disulfanyl) propionic acid, N- (2-mercaptoethyl) acetamide, and the like. For convenience of description, the chemical structural formulas, chemical names and designations herein of the various related substances are listed in table 1 below.
TABLE 1 list of acetylcysteines and related substances
In some embodiments, a ACE Comixsil ACRP column may be used.
Further, the specification of the column may be 250mm×4.6mm, with a diameter of 5 μm.
In some specific embodiments, the volume ratio of water to phosphoric acid in the aqueous phosphoric acid solution (i.e., water-phosphoric acid mixed solution) as mobile phase A is preferably 100 (0.01-0.05), e.g., 100:0.02, 100:0.03, 100:0.04. More preferably, the volume ratio of water to phosphoric acid in mobile phase a is 100:0.03.
In addition, in some specific embodiments, the volume ratio of acetonitrile to phosphoric acid in the acetonitrile phosphate solution (i.e., acetonitrile-phosphoric acid mixed solution) as mobile phase B is preferably 100 (0.01-0.05), e.g., 100:0.02, 100:0.03, 100:0.04. More preferably, the volume ratio of acetonitrile to phosphoric acid in mobile phase B is 100:0.03.
In the detection method of acetylcysteine and related substances, the stationary phase of the chromatographic column is water-resistant anion-cation intercalation modified alkyl bonding silica gel, the organic phase of the adopted mobile phase is high in proportion, the loss on the chromatographic column is small, the service life of the chromatographic column can be prolonged, and therefore the detection cost is reduced.
In some embodiments, when performing the gradient elution procedure, it may be performed according to the conditions shown in table 2 below.
TABLE 2 gradient elution conditions
That is, in the gradient elution, the volume ratio of the mobile phase A gradually changes from 95 to 100% to 15 to 25% and the volume ratio of the mobile phase B gradually changes from 0 to 5% to 75 to 85% from 0 to 20 minutes; the volume ratio of the mobile phase A gradually changes from 15-25% to 95-100% from 20 minutes to 22 minutes, and the volume ratio of the mobile phase B gradually changes from 75-85% to 0-5%; maintaining the volume ratio of the mobile phase A to be 95-100% from 22 minutes, and the volume ratio of the mobile phase B to be 0-5%; the volume ratio of the mobile phase A is kept to be 95-100% from 32 minutes, and the volume ratio of the mobile phase B is kept to be 0-5% until the detection is finished.
In some preferred embodiments, the gradient elution procedure may be performed according to the conditions shown in table 3 below.
TABLE 3 gradient elution conditions
That is, in the gradient elution, the volume ratio of the mobile phase A gradually changes from 100% to 20% and the volume ratio of the mobile phase B gradually changes from 0% to 80% from 0 minutes to 20 minutes; the volume ratio of the mobile phase A gradually changes from 20% to 100% from 20 minutes to 22 minutes, and the volume ratio of the mobile phase B gradually changes from 80% to 0%; maintaining the volume ratio of mobile phase a at 100% from 22 minutes; the volume ratio of mobile phase a was kept at 100% from 32 minutes until the end of the test.
By setting proper gradient elution conditions, the separation effect of acetylcysteine and related substances can be further ensured, so that the sensitivity and accuracy of detection are further improved.
In some specific embodiments, the flow rate of the high performance liquid chromatography may be set to 0.8-1.2ml/min, such as 0.9ml/min, 1.0ml/min, 1.1ml/min, etc. Preferably, the flow rate may be set to 1.0ml/min.
The column temperature of the chromatographic column can be set to 25-35deg.C, such as 26deg.C, 27deg.C, 28deg.C, 29 deg.C, 30deg.C, 31deg.C, 32deg.C, 33deg.C, and 34 deg.C. Preferably, the column temperature may be set at 30 ℃.
The high performance liquid chromatography can adopt ultraviolet detection, and the detection wavelength can be set to 220nm.
To ensure the stability of the sample, the sample introduction temperature may be set at 4-8deg.C, such as 5deg.C, 6deg.C, 7deg.C, preferably 5deg.C.
The sample volume may be set to 5-15. Mu.l, for example 6. Mu.l, 7. Mu.l, 8. Mu.l, 9. Mu.l, 10. Mu.l, 11. Mu.l, 12. Mu.l, 13. Mu.l, 14. Mu.l, preferably 10. Mu.l.
The conditions and parameters of HPLC analysis in the detection method of acetylcysteine and related substances of the present application are described above, and the following steps of the detection method are specifically described.
Fig. 1 shows a flow chart of a detection method of acetylcysteine and related substances according to an embodiment of the application. As shown in fig. 1, the detection method may include the following steps S1 to S5.
Step S1: and dissolving the acetylcysteine sample to be detected in hydrochloric acid and diluting with a diluent to prepare a first-concentration sample solution.
Step S2: dissolving acetylcysteine reference substance in diluent to obtain reference substance solution with second concentration.
Step S3: accurately weighing acetaminophen and appropriate amounts of each related substance, and dissolving the acetaminophen and the related substances in a diluent together to prepare a system applicability solution containing the third concentration of acetylcysteine and the fourth concentration of each related substance.
Step S4: and taking a system applicability solution sample for carrying out high performance liquid chromatography analysis to determine the retention time of acetylcysteine and each related substance.
Step S5: taking sample solutions of the sample and the reference solution respectively, performing high performance liquid chromatography analysis, and calculating the contents of acetylcysteine and related substances in the sample solution by adding correction factors.
In this example, the hplc analysis in steps S4 and S5 can be performed according to the conditions and parameters of the hplc analysis described above.
In addition, steps S1, S2 and S3 are not in the order defined before and after, and their order may be interchanged or performed simultaneously.
Further, to ensure separation and detection precision, the ratio of the third concentration to the fourth concentration may be set in the range of 150:1 to 250:1, for example 160:1, 170:1, 180:1, 190:1, 200:1, 210:1220:1, 230:1, 240:1. Preferably, the ratio of the third concentration to the fourth concentration may be 200:1.
In a specific embodiment, the third concentration and the fourth concentration may be specifically set to 8mg/ml and 40 μg/ml, respectively.
Further, the first concentration may be set within ±10% of the third concentration. The second concentration may be set within + -10% of the fourth concentration. For example, the first concentration may be set to the same 8mg/ml as the third concentration. The second concentration may be set to the same 40 μg/ml as the fourth concentration.
The following embodiments of the present application will be described in detail with reference to specific examples, and those skilled in the art will readily appreciate that the present application is further advantageous and advantageous for carrying out the present application. The application may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present application.
Among the acetylcysteine and related substance controls used in the following examples, impurities other than impurity O are commercially available. The impurity O was prepared by the inventors themselves.
The synthetic route of impurity O ((R) -2-acetamido-3- (((R) -2-amino-2-carboxyethyl) disulfanyl) propionic acid) is shown in the following formula (1).
The preparation process of the impurity O is as follows:
acetylcysteine, naOH solution, L-cysteine, mnO 2 Added into a 50ml three-necked flask and stirred into black turbid liquid. After 2 hours of reaction, the system became a gray turbid liquid. Filtering to obtain pale yellow solution. The filtrate was lyophilized to give 2.48g of yellow solid. The mixture was separated by chromatography to give 500mg of a white solid. The white solid was detected by nuclear magnetism, LCMS (Liquid Chromatography-Mass Spectrometry ), infrared, ultraviolet, etc., to confirm that its structure was consistent with the impurity O profile, wherein LCMS purity was 98.95%, M/z= 283.34 (m+h) + )， 1 H-NMR(500MHz,DMSO-d6)δ8.20(s，1H)，4.55(s，1H)，3.92(s，1H)，3.35(m，2H)，2.847-3.01(m，2H)，1.86(s，3H)。
Example 1
The chromatographic conditions in this example 1 are as follows:
chromatographic column: ACE Comixsil ACRP,250 mm. Times.4.6 mm,5 μm; mobile phase a: water-phosphoric acid solution (volume ratio 100:0.03), mobile phase B: acetonitrile-phosphoric acid solution (volume ratio 100:0.03); gradient elution was performed and the gradient elution settings are shown in table 4 below:
TABLE 4 gradient elution conditions
Flow rate: 1ml/min; column temperature: 30 ℃; detection wavelength: 220nm; sample introduction temperature: sample injection volume at 5 ℃): 10 μl.
The detection method and the detection result are as follows:
(1) Solution preparation
Taking an appropriate amount of acetylcysteine sample (acetylcysteine reference substance in the embodiment), precisely weighing, adding 0.01mol/L hydrochloric acid for dissolution and quantitatively diluting to prepare a solution containing about 8mg of acetylcysteine in 1ml as a sample solution; taking a proper amount of acetylcysteine reference substance, precisely weighing, adding a diluent for dissolving and quantitatively diluting to prepare a solution with the concentration of 40 mug per 1ml serving as the reference substance solution.
(2) Specialization of
Taking proper amounts of acetylcysteine, impurities A, B, C, D, E, O and R, precisely weighing, placing into a same measuring flask, adding a diluent for dissolving and diluting to prepare a solution containing 8mg of acetylcysteine and about 40 mug of each impurity in each 1ml, and taking the solution as a system applicability solution. Precisely measuring 10 μl of system applicability solution, injecting into high performance liquid chromatograph, recording chromatogram, and determining retention time of acetylcysteine and each impurity. The separation detection result of the system applicability solution is shown in fig. 2, and the peak sequence is as follows: retention time rt5.115 is impurity R, rt5.856 is impurity B, rt8.003 is impurity O, rt9.724 is acetylcysteine, rt10.949 is impurity D, rt11.743 is impurity a, rt13.689 is impurity E, and rt19.39 is impurity C.
(3) Precision of
And precisely measuring the sample solution of the sample solution and the reference solution, performing high performance liquid chromatography analysis, and recording chromatograms. Calculating the content of each related substance in 6 parts of test sample solution according to the external standard method of the main component added with the correction factors. The content of detected impurity C in 6 parts of sample solution is 0.02%, and RSD is 0% and less than 3.0%. Therefore, the detection method meets the requirements of high performance liquid chromatography on detection of related substances.
(4) Linearity and range
Taking a proper amount of acetylcysteine and an impurity reference substance thereof, adding a diluent for dissolving and diluting to prepare a solution with the concentration of 0.4mg/ml, and taking the solution as a stock solution. 1ml of stock solution was taken and placed in 20ml, 2ml was placed in 25ml, 2ml was placed in 20ml, 4ml was placed in 20ml, 3ml was placed in a 10ml measuring flask, and diluted to scale with a diluent to prepare a linear test solution. Linear regression was performed on the concentration with peak area to obtain a linear equation (listed in table 5 below). The results show that acetylcysteine and its impurities have good linear relationship in the linear range.
TABLE 5 Linear measurement results
| Name of the name | Concentration range (μg/ml) | Linear equation | Correlation coefficient (r) |
| Acetylcysteine | 2.3669～136.0474 | y＝12.4316x+0.5385 | 1.0000 |
| Impurity A | 4.0469～124.0637 | y＝9.9467x+4.3313 | 0.9996 |
| Impurity B | 3.4260～121.1071 | y＝5.2925x-1.5300 | 0.9998 |
| Impurity C | 1.7886～113.0090 | y＝17.9722x+6.8492 | 1.0000 |
| Impurity D | 0.9779～123.9312 | y＝33.2357x+0.5627 | 0.9999 |
| Impurity E | 0.6679～114.8666 | y＝56.2419x-5.2636 | 1.0000 |
| Impurity O | 1.5801～103.7548 | y＝11.8090x+3.3607 | 1.0000 |
| Impurity R | 1.3371～116.2986 | y＝12.2550x-2.3095 | 0.9999 |
(5) Quantitative limit and detection limit
Taking a proper amount of acetylcysteine and an impurity reference substance thereof to prepare a series of solutions, wherein the solutions are used as quantitative limiting solutions when S/N is more than or equal to 10; when S/N is more than or equal to 3, the solution is used as a detection limit solution. The quantitative limit and the detection limit of acetylcysteine and its impurities are shown in Table 6.
TABLE 6 quantitative limit and detection limit results
| Name of the name | Detection limit (mug/ml) | Signal to noise ratio (S/N) | Limit of quantification (μg/ml) | Signal to noise ratio (S/N) |
| Acetylcysteine | 0.7101 | 5.6 | 2.3669 | 15.0 |
| Impurity A | 1.2141 | 4.9 | 4.0469 | 13.7 |
| Impurity B | 1.0278 | 4.7 | 3.4260 | 13.5 |
| Impurity C | 0.5366 | 6.1 | 1.7886 | 14.4 |
| Impurity D | 0.3258 | 4.7 | 0.9779 | 12.5 |
| Impurity E | 0.2004 | 3.4 | 0.6679 | 12.2 |
| Impurity O | 0.4740 | 4.9 | 1.5801 | 14.2 |
| Impurity R | 0.4011 | 4.3 | 1.3371 | 13.2 |
From the above results, it can be seen that acetylcysteine and its impurities and degradation products can be completely separated under the chromatographic conditions of this example. The method has the advantages of strong specificity, good accuracy, high precision and good repeatability, meets the technical requirements of drug quality research standards, and has stable and reliable results.
Comparative example 1
The test method of European pharmacopoeia was used in comparative example 1, and the chromatographic conditions were as follows:
chromatographic column: hypersil ODS,250 mm. Times.4.6 mm,5 μm; mobile phase: water-acetonitrile-phosphoric acid (97:3:0.1 by volume); isocratic elution was performed for 30min.
Flow rate: 1ml/min; column temperature: 30 ℃; detection wavelength: 220nm; sample introduction temperature: sample injection volume at 5 ℃): 10 μl.
Taking proper amounts of acetylcysteine, impurities A, B, C, D, E, O and R, precisely weighing, placing into a same measuring flask, adding a diluent to dissolve and dilute to prepare a solution containing 50 mug of acetylcysteine and about 10 mug of each impurity in each 1ml, and taking the solution as a system applicability solution. And (3) feeding the system applicability solution, and performing high performance liquid chromatography analysis according to the chromatographic conditions. The chromatogram of the system applicability solution is shown in fig. 3.
As can be seen from fig. 3, under this chromatographic condition, the peak-exiting sequence is in turn: retention times rt1.961 for impurities a and E, rt2.177 for impurity B, rt2.503 for impurity O, rt4.243 for acetylcysteine, rt8.009 for impurity C, rt9.204 for impurity D, and rt18.894 for impurity R. It can be seen that the impurities A, E, B and O peak very early with little retention and the impurities a and E are not separated, which affects quantitative results and qualitative decisions of the impurities.
Comparative example 2
The chromatographic conditions in comparative example 2 are as follows:
chromatographic column: supersil C18, 250mm 4.6mm,5 μm; mobile phase: (10 mmol/L potassium dihydrogen phosphate, 30mmol/L sodium heptanesulfonate, pH 2.0 adjusted by phosphoric acid) -methanol (volume ratio 90:10); isocratic elution was performed for 30min.
Flow rate: 1ml/min; column temperature: 40 ℃; detection wavelength: 220nm; sample introduction temperature: sample injection volume at 5 ℃): 20 μl.
The system applicability solution was prepared as in comparative example 1. And (3) feeding the system applicability solution, and performing high performance liquid chromatography analysis according to the chromatographic conditions. The chromatogram of the system applicability solution is shown in fig. 4.
As can be seen from fig. 4, under this chromatographic condition, the peak-exiting sequence is in turn: retention time rt2.267 is impurity E, rt4.769 is acetylcysteine, rt5.534 is impurity R, rt6.556 is impurity C, rt8.706 is impurity D, rt11.938 is impurities B and O, and rt23.656 is impurity a. It can be seen that the impurities B and O are not separated, and the quantitative result and qualitative judgment of the impurities are affected.
Comparative example 3
The chromatographic conditions for comparative example 3 are as follows:
chromatographic column: supersil C18, 250mm 4.6mm,5 μm; mobile phase: (10 mmol/L potassium dihydrogen phosphate, 20mmol/L sodium heptanesulfonate, phosphoric acid adjusted pH 2.0) -methanol (volume ratio 90:10); isocratic elution was performed for 30min.
Flow rate: 1ml/min; column temperature: 40 ℃; detection wavelength: 220nm; sample introduction temperature: sample injection volume at 5 ℃): 20 μl.
The system applicability solution was prepared as in comparative example 1. And (3) feeding the system applicability solution, and performing high performance liquid chromatography analysis according to the chromatographic conditions. The chromatogram of the system applicability solution is shown in fig. 5.
As can be seen from fig. 5, under the chromatographic conditions, the peak-exiting sequence is: retention time rt2.261 is impurity E, rt5.286 is acetylcysteine, rt6.057 is impurity R, rt8.219 is impurity C, rt8.929 is impurity B, rt10.319 is impurities D and O, and rt14.434 is impurity a. It can be seen that the impurities D and O are not separated, and the quantitative result and qualitative judgment of the impurities are affected.
Comparative example 4
The chromatographic conditions for comparative example 4 are as follows:
chromatographic column: ACE Comixsil ACRP,250 mm. Times.4.6 mm,5 μm; mobile phase a: water-formic acid (volume ratio 100:0.03), mobile phase B: acetonitrile-formic acid (volume ratio 100:0.03); gradient elution was performed and set up as in table 7 below:
TABLE 7 gradient elution conditions
Flow rate: 1ml/min; column temperature: 30 ℃; detection wavelength: 220nm; sample introduction temperature: sample injection volume at 5 ℃): 10 μl.
The system applicability solution was prepared as in example 1. And (3) feeding the system applicability solution, and performing high performance liquid chromatography analysis according to the chromatographic conditions. The chromatogram of the system applicability solution is shown in fig. 6.
As can be seen from fig. 6, under the chromatographic conditions, the peak-exiting sequence is: retention time rt3.721 is impurity a and rt18.327 is acetylcysteine. Only acetylcysteine and impurity A show peaks, and the rest impurities do not respond, so that quantitative results and qualitative judgment of the impurities are affected.
In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the application may be practiced without these specific details. In some instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.
By now it should be appreciated by those skilled in the art that while a number of exemplary embodiments of the application have been shown and described herein in detail, many other variations or modifications of the application consistent with the principles of the application may be directly ascertained or inferred from the present disclosure without departing from the spirit and scope of the application. Accordingly, the scope of the present application should be understood and deemed to cover all such other variations or modifications.
Claims (7)
1. The detection method of acetylcysteine and related substances thereof, wherein the related substances comprise impurities and/or degradation products introduced in the production and storage processes of the acetylcysteine, and the detection method is characterized in that the acetylcysteine and related substances thereof are separated and detected through high performance liquid chromatography, wherein the high performance liquid chromatography adopts a chromatographic column taking anion-cation intercalation modified alkyl bonding silica gel as a stationary phase, and adopts phosphoric acid aqueous solution as a mobile phase A and phosphoric acid acetonitrile solution as a mobile phase B for gradient elution, so that the acetylcysteine and related substances thereof are completely separated;
wherein the related substances include:
l-cystine, L-cysteine, N-diacetyl-L-cystine, N, S-diacetyl-L-cysteine, (2R) -2-amino-3-sulfinylpropionic acid monohydrate, (R) -2-acetamido-3- (((R) -2-amino-2-carboxyethyl) disulfanyl) propionic acid, and N- (2-mercaptoethyl) acetamide;
the conditions of the gradient elution are as follows:
the volume ratio of the mobile phase A gradually changes from 95-100% to 15-25% from 0min to 20 min, and the volume ratio of the mobile phase B gradually changes from 0-5% to 75-85%;
the volume ratio of the mobile phase A gradually changes from 15-25% to 95-100% from 20 minutes to 22 minutes, and the volume ratio of the mobile phase B gradually changes from 75-85% to 0-5%;
maintaining the volume ratio of the mobile phase A to be 95-100% from 22 minutes, and the volume ratio of the mobile phase B to be 0-5%;
the volume ratio of the mobile phase A is kept to be 95-100% from 32 minutes, and the volume ratio of the mobile phase B is kept to be 0-5% until the detection is finished;
the flow rate of the high performance liquid chromatography is 0.8-1.2ml/min, the column temperature is 25-35 ℃, the detection wavelength is 220nm, and the sample injection temperature is 4-8 ℃.
2. The method of claim 1, wherein the chromatographic column is a ACE Comixsil ACRP chromatographic column.
3. The method according to claim 1, wherein the conditions of the gradient elution are:
the volume ratio of the mobile phase A gradually changes from 100% to 20% from 0 minutes to 20 minutes, and the volume ratio of the mobile phase B gradually changes from 0% to 80%;
the volume ratio of the mobile phase A gradually changes from 20% to 100% from 20 minutes to 22 minutes, and the volume ratio of the mobile phase B gradually changes from 80% to 0%;
maintaining the volume ratio of mobile phase a at 100% from 22 minutes;
the volume ratio of mobile phase a was kept at 100% from 32 minutes until the end of the test.
4. The method according to claim 1, wherein the volume ratio of water to phosphoric acid in mobile phase a is 100:0.03, and the volume ratio of acetonitrile to phosphoric acid in mobile phase B is 100:0.03.
5. The method according to claim 1, wherein the column temperature of the high performance liquid chromatography is 30 ℃.
6. The method according to any one of claims 1-5, characterized in that the method comprises in particular the following steps:
dissolving an acetylcysteine sample to be detected in hydrochloric acid and diluting with a diluent to prepare a sample solution with a first concentration;
dissolving acetylcysteine reference substance in the diluent to prepare a reference substance solution with a second concentration;
precisely weighing acetaminophen and proper amounts of each related substance, and dissolving the acetaminophen and the proper amounts of each related substance in the diluent together to prepare a system applicability solution containing the third concentration of acetylcysteine and the fourth concentration of each related substance;
taking the system applicability solution sample for high performance liquid chromatography analysis to determine the retention time of acetylcysteine and each related substance;
and respectively taking the sample solution to be tested and the reference solution for sample injection to perform high performance liquid chromatography analysis, and calculating the contents of acetylcysteine and related substances in the sample solution by a main component external standard method of adding correction factors.
7. The method of claim 6, wherein the ratio of the third concentration to the fourth concentration is in the range of 150:1 to 250:1;
the first concentration is within + -10% of the third concentration;
the second concentration is within + -10% of the fourth concentration.
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