CN118108644A - Separation and structure identification method of unknown impurities in acetylcysteine - Google Patents
Separation and structure identification method of unknown impurities in acetylcysteine Download PDFInfo
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- CN118108644A CN118108644A CN202311793172.4A CN202311793172A CN118108644A CN 118108644 A CN118108644 A CN 118108644A CN 202311793172 A CN202311793172 A CN 202311793172A CN 118108644 A CN118108644 A CN 118108644A
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- 239000012535 impurity Substances 0.000 title claims abstract description 116
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 title claims abstract description 59
- 229960004308 acetylcysteine Drugs 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000000926 separation method Methods 0.000 title claims abstract description 13
- 239000000243 solution Substances 0.000 claims abstract description 44
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims abstract description 20
- 230000015556 catabolic process Effects 0.000 claims abstract description 18
- 238000006731 degradation reaction Methods 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 238000004458 analytical method Methods 0.000 claims abstract description 13
- 239000007864 aqueous solution Substances 0.000 claims abstract description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 7
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 6
- 230000000593 degrading effect Effects 0.000 claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 3
- 238000012494 forced degradation Methods 0.000 claims description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- 239000000377 silicon dioxide Substances 0.000 claims description 3
- 239000003814 drug Substances 0.000 abstract description 8
- 229940079593 drug Drugs 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 6
- 230000014759 maintenance of location Effects 0.000 abstract description 3
- HSPYGHDTVQJUDE-LURJTMIESA-N dacisteine Chemical compound CC(=O)N[C@H](C(O)=O)CSC(C)=O HSPYGHDTVQJUDE-LURJTMIESA-N 0.000 description 4
- 238000002514 liquid chromatography mass spectrum Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- YTPQSLLEROSACP-YUMQZZPRSA-N (2R)-2-acetamido-3-[[(2R)-2-acetamido-2-carboxyethyl]disulfanyl]propanoic acid Chemical compound CC(=O)N[C@H](C(O)=O)CSSC[C@@H](C(O)=O)NC(C)=O YTPQSLLEROSACP-YUMQZZPRSA-N 0.000 description 3
- 230000006652 catabolic pathway Effects 0.000 description 3
- 150000001793 charged compounds Chemical class 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 206010067484 Adverse reaction Diseases 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000010525 oxidative degradation reaction Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- PWKSKIMOESPYIA-UHFFFAOYSA-N 2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)NC(CS)C(O)=O PWKSKIMOESPYIA-UHFFFAOYSA-N 0.000 description 1
- -1 3,3'-Disulfanediylbis [ (2R) -2-aminopropanoicacid ] (L-cystine) 3,3' -disulfanediylbis [ (2R) -2-aminopropionic acid ] (L-cystine) Chemical compound 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 206010006458 Bronchitis chronic Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- KKQBDKHQSHCYFS-BBSPRICRSA-N N[C@@H](CS)C(O)=O.N[C@@H](CS)C(O)=O.N[C@@H](CS)C(O)=O.N[C@@H](CS)C(O)=O Chemical compound N[C@@H](CS)C(O)=O.N[C@@H](CS)C(O)=O.N[C@@H](CS)C(O)=O.N[C@@H](CS)C(O)=O KKQBDKHQSHCYFS-BBSPRICRSA-N 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000007451 chronic bronchitis Diseases 0.000 description 1
- 230000008094 contradictory effect Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 238000010579 first pass effect Methods 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
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- 210000003097 mucus Anatomy 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention provides a separation method of unknown impurities in acetylcysteine, which comprises the following steps: s1, forcibly degrading an acetylcysteine aqueous solution to obtain a degradation solution; s2, separating the degradation solution by a liquid chromatograph, detecting the wavelength of 205-220 nm, and collecting fractions of the unknown impurities to obtain an unknown impurity solution; in the step S2, the mobile phase is an ammonium sulfate buffer solution-methanol system, and the unknown impurity solution is obtained by taking components with RT of 27-28 min. In the technical scheme, the high performance liquid chromatograph is adopted to determine that the impurity is the acetylcysteine degradation impurity according to the retention time, the LC-MS is used for further confirming the chemical structure of the unknown impurity and analyzing the generation reason, the method is simple, the method can be used for impurity analysis control of the acetylcysteine, and the quality and medication safety are ensured.
Description
Technical Field
The invention belongs to the technical analysis field of medicines, and particularly relates to a separation and structure identification method of unknown impurities of acetylcysteine.
Background
The acetylcysteine preparation is suitable for patients with cough, such as chronic bronchitis, with sticky phlegm and difficult expectoration. Studies in humans with labeled N-acetylcysteine showed rapid and complete drug absorption following oral administration, reaching plasma peaks at hours 2-3. Measurements of lung tissue levels taken 5 hours after administration indicated the presence of significant concentrations of N-acetylcysteine. Due to the pronounced pre-systemic metabolism, the bioavailability of free acetylcysteine is only around 10%. In vivo, acetylcysteine moieties are in unaltered form, moieties in the form of oxidized metabolites in free form, and bind to plasma proteins in a reversible manner via disulfide bonds. Acetylcysteine diffuses mainly into the aqueous medium of the extracellular space. It is located in particular in liver, kidney, lung and bronchial mucus. Metabolism begins immediately after taking the product: acetylcysteine is deacetylated at the intestinal wall level and during its first liver entry into L-cysteine, also active, and then metabolized to inactive bonds. About 30% of the administered dose is directly expelled through the kidneys. The major metabolites are cystine and cysteine. In addition, small amounts of taurine and sulfate are also discharged.
The synthesis scheme of acetylcysteine is as follows:
。
According to the structural characteristics of the acetylcysteine raw material and the synthesis process, analyzing impurities generated in the process, impurities generated in the raw material stability process and impurities possibly generated in the preparation production and stability process, and combining the requirements of related guiding principles, giving chemical structures to known impurities and analyzing impurity sources, wherein the chemical structures and the impurity sources are shown in the following table:
| Impurity name | Impurity attribution/generation path |
| 3,3'-Disulfanediylbis [ (2R) -2-aminopropanoicacid ] (L-cystine) 3,3' -disulfanediylbis [ (2R) -2-aminopropionic acid ] (L-cystine) | Oxidative degradation of SH-oxidation to disulfide bonds in impurities/YXBB |
| (2R) -2-amino-3-sulfanylpropanoicacid (L-cysteine) (2R) -2-amino-3-sulfanyl propionic acid (L-cysteine) | Degradation impurity/amide bond hydrolysis impurity |
| (2R, 2 'R) -3,3' -disulfanediylbis [2- (acetylamino) propanoicacid ] (N, N '-diacetyl-L-cystine) (2R, 2' R) -3,3'-disulfanediylbis [2- (acetamido) propionic acid ] (N, N' -diacetyl-L-cystine) | Oxidative degradation of SH-oxidation in impurities/acetylcysteines to disulfide bonds |
| (2R) -2- (acetylamino) -3- (acetylsulfanyl) propanoicacid (N, S-diacetyl-L-cysteine) (2R) -2- (acetylamino) -3- (acetylthio) propionic acid (N, S-diacetyl-L-cysteine) | Degradation impurity/SH-acetyl acylation |
The structural formula of the impurities is as follows:
、、、。
The daily maximum dose (MDD) of the product is 0.6g, and according to ICH guidelines and technical guidelines for research on chemical drug impurities, the three limits of impurities are respectively:
| Project | Reporting limits | Authentication limit | Quality control limit |
| Limit of | 0.05% | 0.10% | 0.15% |
| Limit basis | MDD=0.6g.ltoreq.2g, reporting limit is 0.05% | MDD=0.6 g.ltoreq.2 g, identification limit 0.10% | MDD=0.6 g.ltoreq.2 g, identification limit 0.15% |
Calculated, the identification limit is 0.10%, and the quality control limit is 0.15%.
Wherein, the unknown single impurity in the reference preparation and the self-grinding product is more than 0.15%, and the unknown impurities of the reference preparation and the sample detected by the applicant are shown in the following table.
| Sample of | Unknown impurity content (%) |
| Self-grinding product | 0.23 |
| Reference formulation | 0.45 |
Existing methods and literature attribute some of the impurities, but still the larger impurities are unknown impurities, which are not controlled by the prior art. The acetylcysteine contains unknown impurities, so that the use safety of the product is directly affected, adverse reactions are uncontrollable, and the impurities in the sample are thoroughly researched to further ensure the quality of the medicine, so that the reasons of the impurities are analyzed, and the impurity content is strictly limited, so that the technical problem to be solved is urgent.
Disclosure of Invention
The invention provides a separation and structure identification method of unknown impurities in acetylcysteine, and aims to solve the technical problem of limiting the content of the unknown impurities in the acetylcysteine.
In order to achieve the purpose of the invention, the separation method of unknown impurities in acetylcysteine provided by the invention comprises the following steps:
s1, forcibly degrading an acetylcysteine aqueous solution to obtain a degradation solution;
S2, separating the degradation solution by a liquid chromatograph, detecting the wavelength of 205-220 nm, and collecting fractions of the unknown impurities to obtain an unknown impurity solution;
In the step S2, the mobile phase is an ammonium sulfate buffer solution-methanol system, and the unknown impurity solution is obtained by taking components with RT of 27-28 min.
Optionally, in the step S1, the acetylcysteine aqueous solution is subjected to forced degradation by being placed at a high temperature of 60 ℃ for 24 hours.
Optionally, in the step S1, the content of the unknown impurities in the degradation solution is 1.0wt% to 10.0wt%.
Optionally, the mobile phase further comprises sodium heptanesulfonate.
Optionally, the concentration of the sodium heptanesulfonate is 1.0g/L to 5.0g/L.
Optionally, in the step S2, the flow rate in the chromatographic conditions of the liquid chromatograph separation is 0.5-1.5 ml/min, the sample injection volume is 100 μl, and the column temperature is 30-40 ℃.
Alternatively, in the step S2, the column separated by the liquid chromatograph uses octadecylsilane chemically bonded silica as a filler.
Optionally, in the step S2, the content of the unknown impurities in the unknown impurity solution is more than or equal to 70wt%.
The invention also provides an unknown impurity in the acetylcysteine separated by the separation method, and the structure formula of the acetylcysteine is as follows through LC-MS analysis:
。
optionally, the limit of the unknown impurity is set to not more than 0.3%.
The invention also provides a structure identification method of unknown impurities in acetylcysteine, which comprises the following steps:
analyzing the separated impurity solution by LC-MS;
The analysis conditions of the LC-MS were:
mobile phase A is 0.15% acetic acid solution, mobile phase B is acetonitrile;
the gradient elution conditions were: 0-3min,90% A to 90% A;3-10min,90% A to 20% A;10-12min,20% A;12-12.01min,20% A to 90% A;12.01-15min,90% A;
the column temperature is 28-32 ℃;
the wavelength of the ultraviolet detector is 205-220nm;
the flow rate of the column is 0.3-1mL/min;
the LC-MS scan type: fullmass/dd-MS2 (topN);
Sheath air flow rate (Arb): 35;
auxiliary gas flow rate (Arb): 20, a step of;
Auxiliary gas heater temperature: 350 ℃;
Capillary temperature: 320 ℃;
resolution ratio: 70000, collision energy 10, 20, 40.
In the technical scheme of the invention, the impurity is determined to be acetylcysteine degradation impurity according to the retention time by adopting a high performance liquid chromatograph, and then the molecular weight of the unknown impurity is determined by LC-MS; the accurate molecular ion peak m/z is obtained by LC-MS spectrum, the molecular weight of the unknown impurity is: 163.19, deducing the molecular formula according to degradation pathways: c 5H9NO3 S. The scheme adopts a liquid chromatograph to realize the separation and enrichment of unknown impurities, further confirms the chemical structure of the unknown impurities by using an LC-MS, analyzes the generation reasons, has simple method, can be used for the impurity analysis control of acetylcysteine, and ensures the quality and medication safety.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic flow chart of an embodiment of a method for separating unknown impurities from acetylcysteine according to the present invention;
FIG. 2 is a liquid chromatogram of an embodiment of unknown impurities in acetylcysteine provided by the present invention;
FIG. 3 is a LC-MS spectrum of an embodiment of unknown impurities in acetylcysteine provided by the present invention.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention.
The specific conditions were not specified in the examples, and the examples were conducted under the conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Some impurities in finished acetylcysteine products are attributed to existing methods and literature, but the larger impurities still belong to unknown impurities, and the prior art does not control the impurities. The acetylcysteine contains unknown impurities, so that the use safety of the product is directly affected, adverse reactions are uncontrollable, and the impurities in the sample are thoroughly researched to further ensure the quality of the medicine, so that the reasons of the impurities are analyzed, and the impurity content is strictly limited, so that the technical problem to be solved is urgent.
In view of the above, the present invention provides a method for separating an unknown impurity from acetylcysteine, and in combination with a schematic flow chart of an embodiment of the method for separating an unknown impurity from acetylcysteine provided in fig. 1, the method for separating an unknown impurity from acetylcysteine includes the following steps.
And S1, forcibly degrading the acetylcysteine aqueous solution to obtain a degradation solution.
And placing the acetylcysteine aqueous solution at a high temperature of 60 ℃ for 24 hours to perform forced degradation, wherein the content of unknown impurities in the degradation solution is 1.0-10.0 wt%. The acetylcysteine aqueous solution is prepared from acetylcysteine raw material and distilled water.
And S2, separating the degradation solution by a liquid chromatograph, detecting the wavelength of 205-220 nm, and collecting fractions of the unknown impurities to obtain an unknown impurity solution.
In the step S2, the mobile phase is an ammonium sulfate buffer solution-methanol system, and the unknown impurity solution is obtained by taking components with RT of 27-28 min. The mobile phase also comprises sodium heptanesulfonate, the concentration of the sodium heptanesulfonate is 1.0 g/L-5.0 g/L, and the concentration is in chromatographic conditions of liquid chromatograph separation: the flow rate is 0.5-1.5 mL/min, the sample injection volume is 100 mu L, the column temperature is 30-40 ℃, and octadecylsilane chemically bonded silica is used as a filler for a chromatographic column.
The content of the unknown impurities in the unknown impurity solution is more than or equal to 70wt percent after separation.
In the technical scheme of the invention, the impurity is determined to be the degrading impurity of acetylcysteine according to the retention time by adopting a high performance liquid chromatograph, and can be confirmed by a liquid chromatogram thereof.
The invention also provides a structure identification method of unknown impurities in acetylcysteine, which comprises the following steps:
analyzing the separated impurity solution by LC-MS;
The analysis conditions of the LC-MS were:
mobile phase A is 0.15% acetic acid solution, mobile phase B is acetonitrile;
the gradient elution conditions were: 0-3min,90% A to 90% A;3-10min,90% A to 20% A;10-12min,20% A;12-12.01min,20% A to 90% A;12.01-15min,90% A;
the column temperature is 28-32 ℃;
the wavelength of the ultraviolet detector is 205-220nm;
the flow rate of the column is 0.3-1mL/min;
the LC-MS scan type: fullmass/dd-MS2 (topN);
Sheath air flow rate (Arb): 35;
auxiliary gas flow rate (Arb): 20, a step of;
Auxiliary gas heater temperature: 350 ℃;
Capillary temperature: 320 ℃;
resolution ratio: 70000, collision energy 10, 20, 40.
The structural formula of the unknown impurity in the acetylcysteine is obtained by LC-MS analysis:
。
preferably, in this embodiment, the limit of the unknown impurities in the acetylcysteine is set to not more than 0.3%.
In the technical scheme of the invention, the molecular weight of the unknown impurity is determined by LC-MS; the accurate molecular ion peak m/z is obtained by LC-MS spectrum, the molecular weight of the unknown impurity is: 163.19, deducing the molecular formula according to degradation pathways: c 5H9NO3 S. The scheme adopts LC-MS to confirm the chemical structure of the unknown impurity, analyzes the generation reason, has simple method, can be used for the impurity analysis control of acetylcysteine, and ensures the quality and medication safety.
The following description of the embodiments of the present invention is further provided in detail with reference to specific examples, which should be understood as merely illustrative of the present invention and not limiting thereof.
Example 1
The unknown impurities in the acetylcysteine are separated and purified by the following method:
60mL of acetylcysteine aqueous solution is placed at 60 ℃ for 24 hours to obtain a degradation solution of the unknown impurity with the content of about 5 percent. Separating the degradation solution by a liquid chromatograph, detecting the wavelength of 205-220 nm, and collecting fractions of the impurities to obtain an unknown impurity solution; referring to fig. 2, it can be confirmed from the liquid chromatogram that the unknown impurity in the solution is an acetylcysteine degradation impurity.
Wherein the mobile phase is an ammonium sulfate buffer solution-methanol system, and the components with RT of 27-28 min are taken to obtain an unknown impurity solution;
Mobile phase: ammonium sulfate buffer (2.25 g of ammonium sulfate, 1.82g of sodium heptanesulfonate, diluted with water to 450mL, and adjusted to pH 1.4 with 7mol/L hydrochloric acid solution) -methanol (90:10); the detection wavelength is 210nm; the column temperature is 30 ℃; the flow rate is 1.0mL per minute; sample injection volume 25. Mu.L;
The column was WelchLP-C184.6X105 mm,5 μm.
Example 2
The structure analysis is carried out on the obtained unknown impurities by the following method:
molecular weight measurement: the unknown impurity solution in example 1 was taken and subjected to structural analysis by LC-MS.
The LC-MS analysis conditions preferably employ ACQUITUPLCBEHC column 1.7 μm,2.1 x 100mm, flow rate 0.4mL/min, sample volume 10. Mu.L, sample tray temperature 4 ℃, mobile phase A0.1% acetic acid solution, mobile phase B acetonitrile. The gradient ratios are shown in the following table:
| Time min | 0.1% Formic acid water | Acetonitrile |
| 0 | 90% | 10% |
| 3 | 90% | 10% |
| 10 | 20% | 80% |
| 12 | 20% | 80% |
| 12.01 | 90% | 10% |
| 15 | 90% | 10% |
Analysis of LC-MS employs Electrospray (ESI) positive ion detection, scan type: fullmass/dd-MS2 (topN); sheath air flow rate (Arb): 35; auxiliary gas flow rate (Arb): 20, a step of; auxiliary gas heater temperature: 350 ℃; capillary temperature: 320 ℃; resolution ratio: 70000, collision energy 10, 20, 40.
Determining the molecular weight of the unknown impurity by LC-MS, referring to FIG. 3, obtaining an accurate molecular ion peak m/z from the LC-MS spectrum of the unknown impurity solution; the molecular weight of the unknown impurity is: 163.19, deducing the molecular formula according to degradation pathways: c 5H9NO3 S, and deducing that the structural formula is as follows:
。
Analysis of the cause: the amide bond in acetylcysteine is hydrolyzed and then sulfhydryl-acetylated to form the unknown impurity, the production route is shown in the following formula:
。
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.
Claims (10)
1. A method for separating unknown impurities in acetylcysteine, comprising the steps of:
s1, forcibly degrading an acetylcysteine aqueous solution to obtain a degradation solution;
S2, separating the degradation solution by a liquid chromatograph, detecting the wavelength of 205-220 nm, and collecting fractions of the unknown impurities to obtain an unknown impurity solution;
In the step S2, the mobile phase is an ammonium sulfate buffer solution-methanol system, and the unknown impurity solution is obtained by taking components with RT of 27-28 min.
2. The method for separating unknown impurities from acetylcysteine according to claim 1, wherein in the step S1, the acetylcysteine aqueous solution is subjected to forced degradation by being left at a high temperature of 60 ℃ for 24 hours.
3. The method for separating unknown impurities from acetylcysteine according to claim 1, wherein the content of the unknown impurities in the degradation solution in the step S1 is 1.0wt% to 10.0wt%.
4. The method for separating unknown impurities in acetylcysteine according to claim 1, wherein the mobile phase further comprises sodium heptanesulfonate, and the concentration of the sodium heptanesulfonate is 1.0g/L to 5.0g/L.
5. The method for separating unknown impurities from acetylcysteine according to claim 1, wherein in the step S2, the flow rate is 0.5 to 1.5ml/min, the sample injection volume is 100 μl, and the column temperature is 30 ℃ to 40 ℃ under the chromatographic conditions of the liquid chromatograph separation.
6. The method according to claim 1, wherein in the step S2, the column separated by the liquid chromatograph is packed with octadecylsilane chemically bonded silica.
7. The method for separating unknown impurities from acetylcysteine according to claim 1, wherein the content of the unknown impurities in the unknown impurity solution in the step S2 is not less than 70wt%.
8. An acetylcysteine having an unknown impurity obtained by the separation method according to any one of claims 1 to 7, characterized by having the structural formula:
。
9. the acetylcysteine of claim 8, wherein the unknown impurities are limited to no more than 0.3%.
10. A method for identifying the structure of an unknown impurity in acetylcysteine according to claim 8, comprising the steps of:
analyzing the separated impurity solution by LC-MS;
The analysis conditions of the LC-MS were:
mobile phase A is 0.15% acetic acid solution, mobile phase B is acetonitrile;
the gradient elution conditions were: 0-3min,90% A to 90% A;3-10min,90% A to 20% A;10-12min,20% A;12-12.01min,20% A to 90% A;12.01-15min,90% A;
the column temperature is 28-32 ℃;
the wavelength of the ultraviolet detector is 205-220nm;
the flow rate of the column is 0.3-1mL/min;
the LC-MS scan type: fullmass/dd-MS2 (topN);
Sheath air flow rate (Arb): 35;
auxiliary gas flow rate (Arb): 20, a step of;
Auxiliary gas heater temperature: 350 ℃;
Capillary temperature: 320 ℃;
resolution ratio: 70000, collision energy 10, 20, 40.
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