CN106706787B - The detection method and application of lysine content in a kind of aspirin-Al-lysine for injection - Google Patents
The detection method and application of lysine content in a kind of aspirin-Al-lysine for injection Download PDFInfo
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- 239000004472 Lysine Substances 0.000 title claims abstract description 74
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 title claims abstract description 72
- 238000001514 detection method Methods 0.000 title claims abstract description 33
- 238000002347 injection Methods 0.000 title claims abstract description 21
- 239000007924 injection Substances 0.000 title claims abstract description 21
- 238000012360 testing method Methods 0.000 claims abstract description 89
- 238000000034 method Methods 0.000 claims abstract description 30
- 239000000523 sample Substances 0.000 claims description 80
- 229960003646 lysine Drugs 0.000 claims description 69
- 239000000243 solution Substances 0.000 claims description 50
- 238000005303 weighing Methods 0.000 claims description 23
- 229960001138 acetylsalicylic acid Drugs 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 20
- JJBCTCGUOQYZHK-ZSCHJXSPSA-N 2-acetyloxybenzoic acid;(2s)-2,6-diaminohexanoic acid Chemical compound NCCCC[C@H](N)C(O)=O.CC(=O)OC1=CC=CC=C1C(O)=O JJBCTCGUOQYZHK-ZSCHJXSPSA-N 0.000 claims description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 13
- 238000007865 diluting Methods 0.000 claims description 13
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 13
- QGPGUZIKJKOKRF-UHFFFAOYSA-M potassium;acetonitrile;dihydrogen phosphate Chemical compound [K+].CC#N.OP(O)([O-])=O QGPGUZIKJKOKRF-UHFFFAOYSA-M 0.000 claims description 11
- 239000013558 reference substance Substances 0.000 claims description 11
- 239000012085 test solution Substances 0.000 claims description 11
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 claims description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 238000005341 cation exchange Methods 0.000 claims description 10
- 239000000945 filler Substances 0.000 claims description 10
- 229960005337 lysine hydrochloride Drugs 0.000 claims description 10
- 239000012488 sample solution Substances 0.000 claims description 10
- 239000000741 silica gel Substances 0.000 claims description 10
- 229910002027 silica gel Inorganic materials 0.000 claims description 10
- 238000010812 external standard method Methods 0.000 claims description 8
- 239000012088 reference solution Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000004364 calculation method Methods 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 12
- 150000001413 amino acids Chemical class 0.000 abstract description 6
- 238000001212 derivatisation Methods 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 3
- 238000004811 liquid chromatography Methods 0.000 abstract 1
- 239000003085 diluting agent Substances 0.000 description 21
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000011084 recovery Methods 0.000 description 9
- 239000012071 phase Substances 0.000 description 8
- 238000013112 stability test Methods 0.000 description 7
- 239000011550 stock solution Substances 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical group CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 6
- 238000005259 measurement Methods 0.000 description 5
- 230000006378 damage Effects 0.000 description 4
- QKFJKGMPGYROCL-UHFFFAOYSA-N phenyl isothiocyanate Chemical compound S=C=NC1=CC=CC=C1 QKFJKGMPGYROCL-UHFFFAOYSA-N 0.000 description 4
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- LOTKRQAVGJMPNV-UHFFFAOYSA-N 1-fluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C([N+]([O-])=O)=C1 LOTKRQAVGJMPNV-UHFFFAOYSA-N 0.000 description 2
- VYZAHLCBVHPDDF-UHFFFAOYSA-N Dinitrochlorobenzene Chemical compound [O-][N+](=O)C1=CC=C(Cl)C([N+]([O-])=O)=C1 VYZAHLCBVHPDDF-UHFFFAOYSA-N 0.000 description 2
- 238000005903 acid hydrolysis reaction Methods 0.000 description 2
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 229940054441 o-phthalaldehyde Drugs 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 2
- 229940117953 phenylisothiocyanate Drugs 0.000 description 2
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229960004889 salicylic acid Drugs 0.000 description 2
- 238000011003 system suitability test Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001754 anti-pyretic effect Effects 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- 239000003907 antipyretic analgesic agent Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- ULJWXUZHULWYTB-UHFFFAOYSA-M sodium;acetonitrile;dihydrogen phosphate Chemical compound [Na+].CC#N.OP(O)([O-])=O ULJWXUZHULWYTB-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 125000000542 sulfonic acid group Chemical group 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a kind of high-efficiency liquid chromatography method for detecting of lysine, lysine content in drug " aspirin-Al-lysine for injection " can accurately and reliably be measured, the range of linearity is wide, system suitability is good, accuracy is high, and stability, precision, durability and specificity are good, and operation is simple, compared with derivatization method and automatic amino acid analyzer detection method, testing cost is greatly reduced.
Description
Technical Field
The invention relates to a detection method and application of lysine, in particular to a detection method and application for detecting lysine in a medicine through HPLC.
Background
The medicine lysinpirine is aspirin and lysine double salt, is a nonsteroidal anti-inflammatory antipyretic analgesic, and has antipyretic, analgesic and anti-inflammatory effects. The clinically applied preparation is aspirin-lysine for injection.
The 'lysine-aspirin for injection' is newly added in the 'Chinese pharmacopoeia' 2015 edition, wherein the contents of lysine and aspirin are respectively determined under the 'content determination' item. However, the pharmacopoeia only describes "separation and measurement using an appropriate amino acid analyzer or high performance liquid chromatograph" for the measurement of the content of lysine, and does not describe specific detection conditions.
Currently, High Performance Liquid Chromatography (HPLC) is mainly used to detect the content of amino acids by derivatization, for example, o-phthalaldehyde (OPA), 2, 4-dinitrochlorobenzene (CDNB), Phenylisothiocyanate (PITC), and 2, 4-Dinitrofluorobenzene (DNFB) are used as derivatization reagents to measure the content of amino acids. However, the derivatization reaction and chromatographic conditions in the prior art known at present are not suitable for determining the content of lysine in aspirin-lysine for injection, and more than one derivatization compound is detected by the prior art, so that the specificity is poor.
High Performance Liquid Chromatography (HPLC) is a common detection and analysis method, but requires a large number of experiments for a specific analyte to screen out the optimal selection of various parameters in chromatographic conditions. Particularly, in the detection of the medicine of aspirin-lysine, the aspirin and the lysine exist, and the aspirin and the lysine have fast peak appearance under the conventional reverse phase chromatographic condition and interfere with each other, so that the aspirin-lysine and the lysine are difficult to directly separate and measure.
Therefore, it is very necessary to develop a lysine detection method, which can accurately and reliably detect the lysine content in aspirin-lysine for injection, and is used as a medicine quality standard.
Disclosure of Invention
The invention provides a high performance liquid chromatography detection method of lysine, which comprises the following chromatographic conditions: sulfonic cation exchange bonded silica gel is used as a filling agent, a mixed solution of phosphate solution and acetonitrile is used as a mobile phase, wherein the concentration of the phosphate solution is 10-60mmol/L, the volume percentage of the acetonitrile is 5-25%, the detection wavelength is 200-210nm, and the detection wavelength is the terminal wavelength capable of detecting the ultraviolet absorption of amino acid.
Preferably, the invention provides a high performance liquid chromatography detection method of lysine, the chromatographic conditions of which are as follows: sulfonic acid group cation exchange bonded silica gel is used as a filling agent, 20mmol/L potassium dihydrogen phosphate-acetonitrile (85:15) is used as a mobile phase, and the detection wavelength is 205 nm.
Further, the high performance liquid chromatography detection method of lysine of the invention has the following chromatographic conditions: sulfonic cation exchange bonded silica gel is used as a filling agent, 20mmol/L potassium dihydrogen phosphate-acetonitrile (85:15) is used as a mobile phase, the detection wavelength is 205nm, the flow rate is 1.0ml/min, and the column temperature is 35 ℃.
Furthermore, the theoretical plate number of the high performance liquid chromatography detection method of lysine is not less than 3000 according to lysine peaks.
Preferably, the lysine detecting method by high performance liquid chromatography of the invention, wherein the concentration of the lysine to be detected is in the range of 0.2-2.0058 mg/ml.
Preferably, the lysine high performance liquid chromatography detection method is used for detecting the lysine, wherein the to-be-detected product is aspirin-lysine for medicine injection.
Preferably, the method for detecting lysine by high performance liquid chromatography comprises the following steps:
chromatographic conditions and system applicability test: sulfonic cation exchange bonded silica gel is used as a filling agent, and 20mmol/L potassium dihydrogen phosphate-acetonitrile (85:15) is used as a mobile phase; the detection wavelength is 205nm, and the number of theoretical plates is not less than 3000 according to lysine peak.
The determination method comprises the following steps: taking a proper amount of an aspirin-lysine test sample for injection, precisely weighing, adding 0.02mmol/L hydrochloric acid solution for dissolving, quantitatively diluting to obtain a solution containing about 0.8mg of lysine in each 1ml as a test sample solution, precisely weighing 20 mu L, injecting into a liquid chromatograph, and recording a chromatogram; and taking a proper amount of lysine hydrochloride reference substances, and measuring by the same method. And calculating the content of lysine by peak area according to an external standard method to obtain the lysine.
The invention also provides application of the high performance liquid chromatography detection method for lysine, which is used for detecting the content of lysine in aspirin-lysine for medicine injection.
The high performance liquid chromatography detection method for lysine can accurately and reliably detect the content of lysine in the medicine lysine aspirin for injection, has good linear relation in the concentration range of 0.2-2.0mg/ml, has good system applicability, high accuracy, stability, precision, durability and specificity, is simple and easy to operate, and greatly reduces the detection cost compared with a derivatization method and an amino acid automatic analyzer detection method.
Drawings
FIG. 1: the concentration versus peak area linear regression curve for the lysine hydrochloride control in example 2.
FIG. 2: chromatogram results of example 10.
FIG. 3: chromatogram results of example 11.
Detailed Description
The present invention is further described in detail by the following examples, and it will be appreciated by those skilled in the art that modifications may be made without departing from the scope and spirit of the invention.
Example 1: chromatographic conditions and samples
High performance liquid chromatograph: shimadzu LC-20A liquid chromatograph.
Chromatographic conditions are as follows: sulfonic cation exchange bonded silica gel is used as a filling agent, and 20mmol/L potassium dihydrogen phosphate-acetonitrile (85:15) is used as a mobile phase; the flow rate is 1.0ml/min, the column temperature is 35 ℃, the detection wavelength is 205nm, and the number of theoretical plates is not less than 3000 according to lysine peaks.
Lysine hydrochloride control: from the China food and drug testing institute, 140673-: 99.9 percent.
And (3) testing the sample: national medicine group, national rui pharmaceutical industry limited company, lysine aspirin for injection, specification: 0.9g, batch number: 1510101
Diluent agent: 0.02mol/L hydrochloric acid solution.
Example 2: linear test
A lysine hydrochloride control (0.10029 g) was precisely weighed, placed in a 50ml measuring flask, dissolved and diluted to the scale with a diluent, and shaken up to serve as a stock solution for a linear test (100% concentration). Precisely measuring 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml and 8ml of linear test stock solutions, respectively placing the stock solutions into a 10ml measuring flask, diluting the stock solutions to a scale by using a diluent, shaking the stock solutions uniformly to obtain linear test series concentration solutions which are respectively a 10% concentration solution, a 20% concentration solution, a 30% concentration solution, a 40% concentration solution, a 50% concentration solution, a 60% concentration solution, a 70% concentration solution and an 80% concentration solution.
And respectively and precisely measuring 20 mu l of each concentration solution, injecting the solution into a liquid chromatograph, carrying out sample introduction for 2 times on each sample, and recording a chromatogram. The results of the experiments show that lysine is well correlated linearly over a concentration range of about 0.20058mg/ml to 2.0058 mg/ml. The peak areas were subjected to linear regression with concentration, the test results are shown in Table 1 below, and the linear regression curve is shown in FIG. 1.
TABLE 1 lysine content determination method Linear test results
Example 3: system suitability test
Taking a 40% concentration solution under a linear test item as a system applicability solution, precisely measuring 20 mu l of the solution, injecting the solution into a liquid chromatograph, continuously injecting samples for 6 times, and recording a chromatogram. The test result shows that the method has good system applicability, and the test result is shown in the following table 2.
TABLE 2 lysine content determination method System suitability test results
| Number of measurements | 1 | 2 | 3 | 4 | 5 | 6 | Average | RSD％ |
| Retention time (min) | 25.944 | 25.941 | 25.935 | 25.936 | 25.935 | 25.934 | 25.938 | 0.02 |
| Peak area | 489875 | 489813 | 490356 | 490332 | 490151 | 489632 | 490026.5 | 0.07 |
| Number of theoretical plate | 14735.8 | 14770.2 | 14748.5 | 14745.0 | 14685 | 14762.5 | 14741.2 | 0.20 |
Example 4: accuracy test (sample recovery test)
Recovery determination stock preparation: 0.38106g of aspirin lysine for injection (the content of lysine is accurately determined to be 44.70%, namely the total content of 0.1703g of lysine) is precisely weighed, placed in a 250ml measuring flask, dissolved and diluted to the scale by a diluent, shaken up and used as a stock solution for determining the recovery rate.
Preparation of recovery determination solution: precisely weighing 9 parts of lysine hydrochloride reference substances (the actual input amount of lysine in each 1 part is shown in table 3), respectively placing the reference substances into 9 50ml measuring flasks, precisely adding 25ml of the recovery rate measuring stock solution into each measuring flask (namely, the amount of lysine in the sample contained in each measuring flask is 0.01703g), respectively diluting the solution to a constant volume with a diluent, and shaking the solution uniformly to obtain the recovery rate measuring solution.
Injecting 20 mul of each of the 9 recovery rate determination solutions into a liquid chromatograph, injecting each solution for 2 times, and recording a chromatogram; and (3) taking a 40% concentration solution under a linear test item as a reference solution, calculating the measured quantity by a peak area according to an external standard method, and calculating the sample-adding recovery rate according to the following formula. Test results show that the method is high in accuracy and capable of meeting detection requirements, and the test results are shown in the following table 3.
TABLE 3 lysine content determination method sample application recovery test results
Example 5: stability test of solution
Taking a 40% concentration solution under a linear test item as a reference substance stability test solution; precisely weighing 0.07545g of a test sample, placing the test sample in a 50ml measuring flask, dissolving the test sample by using a diluent, and diluting the test sample to a scale to obtain a test sample stability test solution; the two solutions are placed in a sample holder of a liquid chromatograph, and the temperature of the sample holder is room temperature. Injecting 20 μ l of the control stability test solution into a liquid chromatograph at 0, 2, 5, 9, 14, 19, 24, 29 and 35 hours, and recording chromatogram; the test solution for stability test of the test article was taken 20. mu.l at 0, 3, 6, 9, 14, 21, 26 and 32 hours, respectively, and injected into a liquid chromatograph, and the chromatogram was recorded. Test results show that the reference solution is stable after being placed at room temperature for 35 hours; the test solution was stable at room temperature for 32 hours. The results of the tests are given in Table 4 below.
TABLE 4 lysine content determination of solution stability test results
Example 6: precision test
Preparing 5 parts of test solution for stability test by the same method with the test solution as sample 1, and 6 parts of sample, injecting 20 μ l of the solution into a liquid chromatograph, introducing sample 2 times for each sample, and recording chromatogram; and (3) taking a 40% concentration solution under a linear test item as a reference solution, and calculating the content of lysine in the test sample by peak area according to an external standard method. Test results show that the method has good precision. The results of the tests are shown in Table 5 below.
TABLE 5 lysine content determination method precision test results
| Sample 1 | Sample 2 | Sample 3 | Sample No. 4 | Sample No. 5 | Sample No. 6 | RSD(％) | |
| Peak area of sample No. 1 | 512097 | 511470 | 509518 | 515252 | 516587 | 516639 | --- |
| Peak area of 2 nd sample injection | 512588 | 512155 | 509325 | 515145 | 516240 | 516138 | --- |
| Lysine content (%) | 44.76 | 44.64 | 44.69 | 44.77 | 44.64 | 44.67 | 0.13 |
Example 7: intermediate precision test
Another analyst performed experiments at different times using a different model high performance liquid chromatograph (shimadzu LC-2010CHT) than example 6 under the same chromatographic conditions.
Weighing about 76mg of a test sample, placing the test sample in a 50ml measuring flask, dissolving the test sample by using a diluent, and diluting the test sample to a scale to obtain a test sample solution; preparing 5 parts of sample solution by the same method, wherein 6 parts of sample are prepared, respectively taking 20 mu l of the solution to inject into a liquid chromatograph, introducing the sample for 2 times, and recording a chromatogram; precisely weighing 0.02023g of reference substance, placing in a 25ml measuring flask, dissolving with diluent and diluting to scale, using as reference substance solution, and calculating the lysine content in the sample by peak area according to external standard method. The test results show that the RSD of the lysine content in 12 samples of two analysts in example 6 and example 7 is 0.59%, which indicates that the method has good intermediate precision. The test results are shown in tables 6 and 7 below.
TABLE 6 determination of lysine content intermediate precision test results
| Sample 1 | Sample 2 | Sample 3 | Sample No. 4 | Sample No. 5 | Sample No. 6 | RSD(％) | |
| Peak area of sample No. 1 | 536976 | 530281 | 535771 | 530206 | 538252 | 532216 | --- |
| Peak area of 2 nd sample injection | 536467 | 529918 | 535569 | 530544 | 536990 | 534081 | --- |
| Lysine content (%) | 45.11 | 45.08 | 45.10 | 44.87 | 45.50 | 45.00 | 0.48 |
TABLE 7 lysine content precision test results
Example 8: durability test
Taking a proper amount of lysine hydrochloride reference substance, and adding a diluent to dissolve. The column temperature was 30 ℃ and 40 ℃, the flow rate was 0.8ml/min and 1.2ml/min, the phosphate concentration was 18mmol and 22mmol, the organic phase (acetonitrile) ratio was 13% and 17%, respectively, and the other corresponding chromatographic conditions were unchanged. Precisely measuring 20 μ l, injecting into liquid chromatograph, and recording chromatogram. The test result shows that the method has good durability. The test results are shown in Table 8 below.
TABLE 8 lysine content determination method durability test results
Example 9: specificity test
(1) Acid hydrolysis (5mol/L HCl room temperature 72 h): weighing 76mg of a test sample, placing the test sample in a 50ml measuring flask, adding 5ml of 5mol/L HCl, standing at room temperature for 72h, neutralizing with NaOH, adding a diluent to dilute to a scale, shaking up, taking the solution, and filtering to obtain the test sample.
(2) Alkaline hydrolysis (5mol/L NaOH room temperature 72 h): weighing 76mg of a test sample, placing the test sample in a 50ml measuring flask, adding 5ml of 5mol/L NaOH, standing at room temperature for 72h, neutralizing with HCl, adding a diluent to dilute to a scale, shaking up, taking the solution, and filtering to obtain the test sample.
(3) High temperature failure (6 h at 85 ℃): weighing 152mg of the test sample, placing the test sample in a 25 x 40mm weighing bottle, spreading, heating at 85 ℃ for 6 hours, cooling to room temperature, dissolving with a diluent, transferring to a 100ml weighing bottle, fixing the volume to the scale with the diluent, and shaking uniformly to obtain the test sample.
(4) Oxidative destruction (30% H)2O2Standing at room temperature for 72 h): weighing 76mg of the test sample, placing the test sample in a 50ml measuring flask, adding 5ml of 30% H2O2, standing at room temperature for 72H, adding a diluent to dilute to a scale, shaking up, and filtering the solution to obtain the test sample.
(5) Photo disruption (4500Lux exposure 72 h): weighing 152mg of the test sample, placing the test sample in a 25 x 40mm weighing bottle, spreading, irradiating for 72 hours under the condition of 4500Lux, dissolving the test sample by using a diluent, transferring the test sample into a 100ml weighing bottle, fixing the volume to the scale by using the diluent, and shaking uniformly to obtain the test sample.
Precisely measuring 20 mu l of each solution, injecting the solution into a liquid chromatograph, detecting by using a DAD detector, recording a chromatogram, wherein the purity of the lysine main peak meets the requirement (is more than 0.999) under each condition, which shows that the method has good specificity and the separation degree between the degradation product and the main peak is more than 1.5.
In addition, an appropriate amount of aspirin reference (Chinese food and drug inspection research institute, 100113 + 201405, content: 99.8%), salicylic acid reference (Chinese food and drug inspection research institute, 100106 + 201104, content: 99.9%), acetic acid (chemical reagent Co., Ltd., Chinese medicine group, 20131016, content: not less than 99.5%) and the reference are taken, a diluent is used for preparing a solution with an appropriate concentration, 20 mul of the solution and the solvent are respectively injected into a liquid chromatograph, and the chromatogram is recorded. Test results show that known components aspirin and main degradation products salicylic acid and acetic acid thereof in the aspirin-lysine for injection and other unknown degradation impurities are effectively separated, the determination of lysine is not interfered, and the method has good specificity.
The results of the tests are shown in Table 9 below:
TABLE 9 lysine content determination method specificity test separation and Peak purity
| Destructive condition | Degree of separation of main peak from adjacent impurities | Index of peak purity |
| Acid hydrolysis | 34.147 | 0.999951 |
| Alkaline hydrolysis | 32.868 | 0.999899 |
| Oxidative destruction | 17.580 | 0.999955 |
| High temperature destruction | 7.190 | 0.999583 |
| Damage of strong light | 36.847 | 0.999868 |
Example 10: measurement example 1
Chromatographic conditions are as follows: sulfonic cation exchange bonded silica gel is used as a filling agent, 20mmol/L potassium dihydrogen phosphate-acetonitrile (85:15) is used as a mobile phase, the detection wavelength is 205nm, the flow rate is 1.0ml/min, and the column temperature is 35 ℃.
The specification of the test article is as follows: 0.9 g/count. 5 samples were taken, and the average loading of the samples was determined to be 0.9102 g/sample.
Precisely weighing 82.60mg of test sample, placing in a 50ml measuring flask, dissolving with diluent, diluting to scale, shaking to obtain test sample solution (C)Sample (A)82.60/50-1.6520 mg/ml); precisely measuring 20 mul, injecting into a liquid chromatograph, recording a chromatogram, and injecting each sample solution for 2 times; precisely weighing 50.44mg lysine hydrochloride reference substance, placing in 50ml measuring flask, dissolving with diluent, and diluting to scale as pairPhoto solution (C)To pair50.44 × 0.999 × 0.8004/50 ═ 0.80664mg/ml), determined in the same manner. The lysine content in the test sample is 44.61% by peak area calculation according to an external standard method. The chromatograms of the test and control are shown in FIG. 2.
ASample (A): the mean value of the areas of lysine peaks in the test solution; a. theTo pair: mean lysine peak area in control solution.
Example 11: measurement example 2
Chromatographic conditions are as follows: sulfonic cation exchange bonded silica gel is used as a filling agent, 25mmol/L sodium dihydrogen phosphate-acetonitrile (80: 20) is used as a mobile phase, the detection wavelength is 208nm, the flow rate is 0.8ml/min, and the column temperature is 30 ℃.
The specification of the test article is as follows: 0.9 g/count. 5 samples were taken, and the average loading of the samples was determined to be 0.9098 g/sample.
Precisely weighing 98.51mg of test sample, placing in a 50ml measuring flask, dissolving with diluent, diluting to scale, shaking to obtain test sample solution (C)Sample (A)98.51/50-1.9702 mg/ml); precisely measuring 20 mul, injecting into a liquid chromatograph, recording a chromatogram, and injecting each sample solution for 2 times; precisely weighing lysine hydrochloride reference substance 50.12mg, placing in 50ml measuring flask, dissolving with diluent, and diluting to scale to obtain reference solution (C)To pair50.12 × 0.999 × 0.8004/50 — 0.80152mg/ml), and the same method. The lysine content in the sample was 44.70% calculated by peak area according to the external standard method. The chromatograms of the test and control are shown in FIG. 3.
ASample (A): the mean value of the areas of lysine peaks in the test solution; a. theTo pair: mean lysine peak area in control solution.
Claims (6)
1. A high performance liquid chromatography detection method for lysine in aspirin lysine for injection comprises the following chromatographic conditions: sulfonic cation exchange bonded silica gel is used as a filling agent, 20mmol/L potassium dihydrogen phosphate-acetonitrile is used as a mobile phase, and the detection wavelength is 200-210 nm; wherein the volume ratio of 20mmol/L potassium dihydrogen phosphate-acetonitrile is 85:15, and the concentration range of lysine to be detected is 0.2-2.0058 mg/ml.
2. The detection method according to claim 1, wherein the chromatographic conditions are as follows: the detection wavelength was 205 nm.
3. The detection method according to claim 2, wherein the chromatographic conditions are as follows: the flow rate was 1.0ml/min and the column temperature was 35 ℃.
4. The detection method according to any one of claims 1 to 3, wherein the number of theoretical plates is not less than 3000 in terms of lysine peak.
5. A method for detecting the content of lysine in aspirin-lysine for injection comprises the following steps:
(1) chromatographic conditions and system applicability test: sulfonic cation exchange bonded silica gel is used as a filling agent, and 20mmol/L potassium dihydrogen phosphate-acetonitrile is used as a mobile phase; the detection wavelength is 205nm, and the number of theoretical plates is not less than 3000 according to lysine peaks; wherein the volume ratio of 20mmol/L potassium dihydrogen phosphate-acetonitrile is 85: 15;
(2) the determination method comprises the following steps: taking a proper amount of an aspirin-lysine test sample for injection, precisely weighing, adding 0.02mmol/L hydrochloric acid solution for dissolving, quantitatively diluting to obtain a solution containing about 0.8mg of lysine in each 1ml as a test sample solution, precisely weighing 20 mu L, injecting into a liquid chromatograph, and recording a chromatogram; taking a proper amount of lysine hydrochloride reference substance, and measuring by the same method; the lysine content was calculated as peak area by external standard method.
6. A method for detecting the content of lysine in aspirin-lysine for injection comprises the following steps:
chromatographic conditions are as follows: sulfonic cation exchange bonded silica gel is used as a filling agent, 20mmol/L potassium dihydrogen phosphate-acetonitrile is used as a mobile phase, the detection wavelength is 205nm, the flow rate is 1.0ml/min, and the column temperature is 35 ℃; wherein the volume ratio of 20mmol/L potassium dihydrogen phosphate-acetonitrile is 85: 15;
the determination method comprises the following steps: precisely weighing a test sample, placing the test sample in a 50ml measuring flask, dissolving the test sample by using 0.02mmol/L hydrochloric acid solution, diluting the test sample to a scale, and shaking up to obtain a test sample solution; precisely measuring 20 mu l, injecting into a liquid chromatograph, recording a chromatogram, and carrying out sample injection for each sample solution for 2 times; precisely weighing lysine hydrochloride reference substance, placing in 50ml measuring flask, dissolving with 0.02mmol/L hydrochloric acid solution, diluting to scale, and measuring by the same method; calculating the content of lysine in the test sample by peak area according to an external standard method, wherein the calculation formula is as follows:
wherein,
Asample (A)Is the mean value of the lysine peak area in the test solution; a. theTo pairIs the mean value of the lysine peak area in the reference solution; cTo pairConcentration of control solution for accurate preparation, CSample (A)Is the nominal concentration of the accurately prepared test solution; the average filling amount is the average filling amount of the contents of each test sample container; the specification is the marked amount of each sample.
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