CN103512967A - High performance liquid chromatography (HPLC) method for measuring residual quantity of acetic acid in aspisol - Google Patents
High performance liquid chromatography (HPLC) method for measuring residual quantity of acetic acid in aspisol Download PDFInfo
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- lysine
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 title claims abstract description 182
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000004128 high performance liquid chromatography Methods 0.000 title abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 156
- 238000012360 testing method Methods 0.000 claims abstract description 50
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 42
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 21
- 235000019253 formic acid Nutrition 0.000 claims abstract description 21
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000001514 detection method Methods 0.000 claims abstract description 7
- 238000010828 elution Methods 0.000 claims abstract description 5
- 239000000945 filler Substances 0.000 claims abstract description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 8
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 238000004811 liquid chromatography Methods 0.000 claims description 4
- 238000013016 damping Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 abstract description 15
- 239000006184 cosolvent Substances 0.000 abstract description 3
- 150000002500 ions Chemical class 0.000 abstract description 2
- JJBCTCGUOQYZHK-UHFFFAOYSA-N 2-acetyloxybenzoate;(5-amino-1-carboxypentyl)azanium Chemical compound OC(=O)C(N)CCCC[NH3+].CC(=O)OC1=CC=CC=C1C([O-])=O JJBCTCGUOQYZHK-UHFFFAOYSA-N 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 abstract 1
- 230000015556 catabolic process Effects 0.000 abstract 1
- 238000006731 degradation reaction Methods 0.000 abstract 1
- 239000008055 phosphate buffer solution Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 67
- 229960000583 acetic acid Drugs 0.000 description 56
- 239000000523 sample Substances 0.000 description 29
- 239000013558 reference substance Substances 0.000 description 18
- 239000012071 phase Substances 0.000 description 15
- 238000011084 recovery Methods 0.000 description 12
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 8
- 229960001138 acetylsalicylic acid Drugs 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 7
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 6
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 6
- 229960004889 salicylic acid Drugs 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000011550 stock solution Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 238000010812 external standard method Methods 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000013459 approach Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
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- 150000003839 salts Chemical class 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 206010019133 Hangover Diseases 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000003822 preparative gas chromatography Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
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- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a high performance liquid chromatography (HPLC) method for measuring the residual quantity of acetic acid in aspisol. The method comprises the steps of (1) taking formic acid as a cosolvent for dissolving a test article; (2) carrying out gradient elution by taking octadecylsilane chemically bonded silica as a filling agent, a phosphate buffer solution and methanol as mobile phases A and methanol as a mobile phase B, wherein the volume ratio of the buffer solution to the methanol is 90:10 to 95:5, the detection wavelength is 210+/-5nm, and the flow rate is 1.0-1.5ml/min. By using the method, acetate ions can be effectively separated from other similar ions, and a false positive result caused by acetic acid generated by the degradation of aspirin-dl-lysine can be avoided, so that the accuracy of a measured result can be ensured. The method is good in specificity, high in precision and accuracy and capable of rapidly and accurately measuring the residual quantity of the acetic acid.
Description
Technical field
The present invention relates to the detection method of residual solvent in medicine, relate to specifically a kind of HPLC analytical method of measuring acetic acid residual quantity.
Background technology
Di-lysine-aspirin is the double salt of aspirin and lysine, is nonsteroidal analgesic-antipyretic, has analgesic, analgesia, antiinflammatory action, is applicable to heating and light, moderate pain.Owing to having used acetic acid as organic solvent, be therefore necessary to set up the assay method of controlling acetic acid residual quantity in production technology.The conventional detection method of Determination of Residual Organic Solvents is vapor-phase chromatography at present.
At present to be embodied in (1) acetic acid polarity large for the deficiency of method, and boiling point is high, and gas chromatography no better than one ought to is unfavorable for the residual quantity of Accurate Determining acetic acid; (2) di-lysine-aspirin is very easily degraded and is produced salicylic acid and acetic acid in aqueous solution and under hot conditions, makes to record result higher than actual value, obtains false positive results, can not truly reflect product quality.(3) di-lysine-aspirin is not soluble in organic solvent, and in need testing solution, acetate concentration is low, method insufficient sensitivity.Therefore, need to find a kind of new analytical approach and measure the acetic acid residual quantity in di-lysine-aspirin.It is high that high performance liquid chromatography has separation efficiency, and selectivity is good, and detection sensitivity is high, operation automation, and the advantages such as applied range, adopt high performance liquid chromatography can avoid di-lysine-aspirin at high temperature to decompose generation acetic acid; But need to select suitable solvent to prepare di-lysine-aspirin sample solution, can suppress its degraded and can increase again sample concentration to meet detection needs.Through retrieval, in the acetous sample of easily degrading, the assay method of acetic acid residual quantity has no open report at present, therefore need to set up a kind of can this type of sample of rapid and accurate determination in the new method of acetic acid residual quantity.
Summary of the invention
The object of the invention is to set up a kind of HPLC analytical method that can measure fast and accurately acetic acid residual quantity, while particularly containing the acetous material of degradable in sample, measure the method for acetic acid residual quantity, the present invention adopts methyl alcohol as solvent, the formic acid of usining is prepared need testing solution as cosolvent, when suppressing major component degraded, increase sample solution concentration, to make up the deficiencies in the prior art.
For achieving the above object, technical scheme of the present invention is as follows:
Measure a HPLC analytical method for acetic acid residual quantity, its concrete chromatographic condition is as follows:
Chromatographic column: the chromatographic column that the octadecylsilane chemically bonded silica of take is filling agent, column packing particle diameter is 3.5 μ m～5 μ m, column length is 150mm～250mm.
Mobile phase: mobile phase A (being called for short A phase): methyl alcohol-phosphate buffer (containing 1.0～1.5ml phosphoric acid, adjust pH with sodium hydroxide solution in 1000ml damping fluid, pH is 2.5～3.5), the volume ratio of methyl alcohol-phosphate buffer is 10: 90～5: 95; Mobile phase B: methyl alcohol.According to following table, carry out gradient elution:
| Time | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 5 | 100 | 0 |
| 6 | 40 | 60 |
| 12 | 40 | 60 |
| 13 | 100 | 0 |
| 20 | 100 | 0 |
Flow velocity: 1.0ml/min～1.5ml/min.
Column temperature: 20 ℃～30 ℃.
Detect wavelength: 210 ± 5nm.
Sample size: 5 μ l～20 μ l.
Preferred color of choice spectral condition: the chromatographic column that the octadecylsilane chemically bonded silica of take is filling agent; Methyl alcohol-the phosphate buffer (pH3.0) of take is mobile phase A, and wherein the volume ratio of methyl alcohol-phosphate buffer is 8: 92; Take methyl alcohol as Mobile phase B, carry out gradient elution; Flow velocity is 1.2ml/min; Detection wavelength is 210nm; Sample size is 10 μ l.
The preparation of need testing solution: get di-lysine-aspirin 0.2g, accurately weighed, put in 10ml measuring bottle, add appropriate methyl alcohol, then add 100 μ l formic acid, ultrasonic make to dissolve completely after, with methyl alcohol, be diluted to scale, shake up, as need testing solution.Separately get acetic acid 0.5g, accurately weighed, put in 100ml measuring bottle, with methyl alcohol, be diluted to scale, shake up, then precision measures 1ml in 50ml measuring bottle, with methyl alcohol, be diluted to scale, shake up, in contrast product solution.
In aqueous solution, di-lysine-aspirin is very easily hydrolyzed generation acetic acid, for avoiding hydrolysis, adopts methyl alcohol to prepare need testing solution as solvent.But di-lysine-aspirin is not soluble in methyl alcohol, extend ultrasonic time, can not obviously increase di-lysine-aspirin solubleness, and ultrasonic time extends, solution temperature raises, and the acetic acid that its degraded produces obviously increases on the contrary.Find after deliberation, in sample, add a small amount of formic acid, can increase the solubleness of di-lysine-aspirin in methyl alcohol, can reach and measure the residual required concentration of acetic acid, and can shorten sample dissolution required time, increase the stability of sample.
Under general condition, a little less than acetate ion retains in C18 chromatographic column, cannot separate the phosphate-buffered salt that is 2.5～3.5 by pH value with solvent peak and other ions, and using low-proportion methanol as mobile phase, can improve the separation efficiency of acetate ion in C18 chromatographic column.Find after deliberation, when A mutually in methyl alcohol ratio lower than 5% time, the last chromatographic peak hangover of acetate, interference measurement, and acetate chromatogram peak-to-peak type is asymmetric.When A mutually in methyl alcohol ratio higher than 10% time, acetate can not be completely separated with adjacent peak.A mutually in methyl alcohol ratio 5%～10% time, can meet separated requirement, analysis time is also reasonable.At acetic acid, go out behind peak, B phase ratio is risen to 60%, with other the strong compositions that retain in quick wash-out test sample, to avoid it to measure to produce to subsequent sample, disturb, and shorten analysis time.
Adopt high-efficient liquid phase chromatogram condition of the present invention and test sample compound method, having realized the residual liquid phase of acetic acid in di-lysine-aspirin detects, can avoid sample degraded to produce false positive results, can truly reflect acetic acid residual condition in test sample, effective control for product quality.
Adopt the present invention to measure the residual HPLC analytical method of acetic acid, the retention time of acetate is about 3.5 minutes, and analysis time is short, and peak shape symmetry is good.The recovery of this method is high, and average recovery rate is 100.9%; Highly sensitive, quantitative limit reaches 51.6ng.Adopt the residual quantity of acetic acid in method energy rapid and accurate determination di-lysine-aspirin of the present invention.
The method is applicable to the mensuration with the middle acetic acid residual quantity of similar other samples of di-lysine-aspirin (easily degraded produces acetic acid, and poorly soluble in organic solvent) too.
Accompanying drawing explanation
Fig. 1 is acetic acid reference substance solution chromatogram.
Fig. 2 is the residual mensuration chromatogram of acetic acid in di-lysine-aspirin.
Fig. 3 is the methanol solution chromatogram of formic acid.
Fig. 4 is LYS chromatogram.
Fig. 5 is aspirin chromatogram.
Fig. 6 is salicylic methanol solution chromatogram.
Fig. 7 is typical curve.
Embodiment
Below by embodiment, the present invention is further illustrated, but embodiment does not limit the scope of the invention.
Key instrument and chromatographic condition:
Agilent 1200 series of high efficiency liquid chromatographs; Join VWD UV-detector, automatic sampler, Agilent chromatographic work station.
Chromatographic column: the chromatographic column that the octadecylsilane chemically bonded silica of take is filling agent.
Reagent and medicine:
Glacial acetic acid, NaOH, phosphoric acid, formic acid, methyl alcohol (HPLC level).
In experiment, water is homemade purified water.
the selection of embodiment 1 chromatographic condition
The selection of 1.1 wavelength
Get reference substance solution, take mobile phase A as blank correction liquid, according to ultraviolet-visible spectrophotometry (two appendix IV A of Chinese Pharmacopoeia version in 2010), in 200nm～400nm place, scan.Result demonstration, reference substance solution has absorption maximum at 210nm wavelength place, therefore using 210nm ± 5nm as detecting wavelength.
The shaker test of 1.2 methods
This test, by adjusting composition and ratio and flow velocity, the column temperature etc. of mobile phase, is screened the chromatographic process of this test.Concrete scheme is in Table 1.
The screening scheme of table 1 method
Phosphoric acid 1.0～1.5ml in the preparation 1000ml damping fluid of phosphate buffer, adjusts pH with 4mol/L sodium hydroxide solution, obtains.
Get the about 0.2g of di-lysine-aspirin, accurately weighed, put in 10ml measuring bottle, add appropriate methyl alcohol and 100 μ l formic acid, ultrasonic make to dissolve completely after, with methyl alcohol, be diluted to scale, shake up, as need testing solution; Get again the about 0.5g of acetic acid, accurately weighed, put in 100ml measuring bottle, with methyl alcohol, be diluted to scale, shake up, then precision measures 1ml in 50ml measuring bottle, with methyl alcohol, be diluted to scale, shake up, in contrast product solution.Accurate need testing solution and the reference substance solution that measures 10 μ l, in injection liquid chromatography, records chromatogram respectively.Investigate the variation of acetate ion and adjacent peak degree of separation.The results are shown in Table 2, the chromatogram of scheme 1 gained acetic acid reference substance and di-lysine-aspirin need testing solution is shown in respectively Fig. 1, Fig. 2.
The shaker test result of table 2 method
| Scheme | Degree of separation | Symmetrical factor |
| 1 | 4.15 | 0.88 |
| 2 | 4.11 | 0.83 |
| 3 | 4.08 | 0.79 |
| 4 | 5.15 | 0.98 |
| 5 | 3.22 | 0.73 |
| 6 | 2.84 | 0.72 |
| 7 | 2.63 | 0.70 |
| 8 | 3.92 | 0.79 |
Result shows, scheme 1 is to the chromatographic condition of scheme 8, and the degree of separation of acetate ion and adjacent peak is all greater than 1.5, has better peak type and suitable response, all can be used for the mensuration of acetic acid residual quantity.
Separately, at acetic acid, go out behind peak, adjust gradient, Mobile phase B ratio is increased to 60%, with the strong reservation composition in wash-out test sample, avoid its mensuration to subsequent sample to produce and disturb.
the compound method of embodiment 2 need testing solutions
Get di-lysine-aspirin appropriate, accurately weighed, put in 10ml measuring bottle, add appropriate methyl alcohol and different volumes formic acid, ultrasonic make to dissolve completely after, with methyl alcohol, be diluted to scale, shake up, precision measures 10 μ l injection liquid chromatographies respectively, records chromatogram, the impact of more different compound methods on measurement result, the results are shown in Table 3.
Table 3 need testing solution compound method is investigated result
| Scheme | Sample weighting amount (g) | Formic acid volume (μ l) | Ultrasonic time | Acetic acid residual quantity (%) |
| 1 | 0.1001 | 10 | 39min | 0.0182 |
| 2 | 0.1003 | 50 | About 50s | Do not detect |
| 3 | 0.1004 | 20 | 23min | 0.0126 |
| 4 | 0.1004 | 40 | 3min | 0.0047 |
| 5 | 0.2000 | 80 | 5min | 0.0053 |
| 6 | 0.2026 | 100 | About 1min | 0.0025 |
| 7 | 0.2006 | 200 | About 50s | 0.0020 |
| 8 | 0.2001 | 30 | About 5h | Can not dissolve completely |
Result shows, during ultrasonic dissolution di-lysine-aspirin, along with ultrasonic time increases, solution temperature raises, and degraded gained acetic acid increases.Add formic acid can promote this product to dissolve, shorten ultrasonic time.For acetic acid residual quantity in true reflection this product and guarantee that in test sample, acetate concentration meets testing requirement, final definite test sample compound method is: get this product 0.2g, accurately weighed, put in 10ml measuring bottle, add appropriate methyl alcohol, then add 50～200 μ l formic acid, ultrasonic make to dissolve completely after, with methyl alcohol, be diluted to scale, shake up, obtain need testing solution.
embodiment 3 specificity tests
Di-lysine-aspirin be take LYS and aspirin and is made as raw material, because aspirin facile hydrolysis produces salicylic acid, in di-lysine-aspirin, may contain LYS, aspirin and salicylic acid, therefore get respectively LYS, aspirin and salicylic acid appropriate, with methyl alcohol, be formulated as the solution of about 0.1mg/ml, chromatographic condition according to scheme 1 in embodiment 1 is tested, and the specificity of investigation method, records chromatogram.
Because using formic acid hydrotropy in test sample preparation, get formic acid 100 μ l in 10ml methyl alcohol, according to the chromatographic condition of scheme 1 in embodiment 1, test, record chromatogram.
Result shows, acetic acid retention time is about 3.3 minutes, formic acid was located and 4.4 minutes Chu Chu peaks about 2.5 minutes to 3 minutes, LYS retention time is about 2 minutes, aspirin retention time is about 10 minutes, and salicylic acid retention time is about 10.2 minutes, so di-lysine-aspirin raw material and other components that may exist can be completely separated with acetic acid, solvent peak does not disturb the mensuration of acetic acid residual quantity yet, and chromatogram is shown in Fig. 3～6.The specificity of this method is good.And employing gradient elution, makes to retain by force composition aspirin, salicylic acid and also at approximately 10 minutes, goes out peak, has shortened analysis time.
the stability of embodiment 4 solution
Need testing solution 1: get the about 0.2g of di-lysine-aspirin, accurately weighed, put in 10ml measuring bottle, add appropriate methyl alcohol and 100 μ l formic acid, ultrasonic make to dissolve completely after, with methyl alcohol, be diluted to scale, shake up, as need testing solution 1.
Need testing solution 2: get the about 0.2g of di-lysine-aspirin, accurately weighed, put in 10ml measuring bottle, with mobile phase, dissolve and be diluted to scale, as need testing solution 2.
At 0 minute, 30 minutes, within 60 minutes, precision measured need testing solution 10 μ l, according to the chromatographic condition of scheme 1 in embodiment 1, tests, and records chromatogram respectively.The results are shown in Table 4.
Table 4 stability of solution result
Result shows, adopts the mobile phase preparation gained need testing solution acetate ion that in the time of 2,30 minutes, sample hydrolysis produces to approach limit requirement, disturbs the residual mensuration of acetic acid; Take methyl alcohol as solvent, and formic acid is the need testing solution 1 of cosolvent preparation, more stable in 60 minutes, does not substantially affect the mensuration of acetic acid residual quantity, can be used for the mensuration of acetic acid residual quantity.
the making of embodiment 5 typical curves
Chromatographic condition: with the chromatographic condition described in scheme 1 in embodiment 1.
Precision takes acetic acid 0.5156g in 100ml measuring bottle, with methyl alcohol, is diluted to scale, shakes up, and must contrast stock solution 1; Precision measures 1ml contrast stock solution 1, puts respectively in 100ml, 50ml, 25ml measuring bottle, with methyl alcohol, is diluted to scale, shakes up, and obtains linear solution 3, linear solution 4, linear solution 5; Precision measures 2ml contrast stock solution 1 in 100ml measuring bottle, with methyl alcohol, is diluted to scale, shakes up, must contrast stock solution 2, precision measures 1ml contrast stock solution 2, puts respectively in 20ml, 10ml measuring bottle, with methyl alcohol, be diluted to scale, shake up, obtain linear solution 1, linear solution 2.
Line taking solution 1～5 sample introduction, with peak area and concentration mapping (see figure 7).It is linear that acetum is in the scope of 0.516 μ g/ml～20.64 μ g/ml, and equation of linear regression is:
Y=2.8322X-0.1895, coefficient R
2=1.0000.
Y: acetate ion peak area; X: acetate ion concentration, μ g/ml;
The residual limit of acetic acid is two appendix VIII P of 0.5%(Chinese Pharmacopoeia version in 2010), be 1000ng, when signal to noise ratio (S/N ratio) is 10:1, what record acetate ion is quantitatively limited to 51.56ng, far below requiring limit, show that in need testing solution, sample concentration can meet mensuration requirement.
Result shows, the linearity of this method is good, and quantitative limit can meet the requirement of acetic acid residual quantity in Accurate Determining di-lysine-aspirin completely.
embodiment 6 application of sample recovery tests
Chromatographic condition: with the chromatographic condition described in scheme 1 in embodiment 1.
In di-lysine-aspirin solution, add a certain amount of acetic acid, then measure the amount of acetic acid in solution, calculate recovery rate.The concentration of investigating is respectively 5.156 μ g/ml, 103.1 μ g/ml, 206.2 μ g/ml.
The preparation of reference substance solution: precision takes acetic acid 0.5156g in 100ml measuring bottle, is diluted to scale with methyl alcohol, shakes up, and obtains reference substance solution 1; Precision measures 2ml reference substance solution 1 in 100ml measuring bottle, with methyl alcohol, is diluted to scale, shakes up, and obtains reference substance solution 2; Precision measures 5ml reference substance solution 1 in 25ml measuring bottle, with methyl alcohol, is diluted to scale, shakes up, and obtains reference substance solution 3.
Need testing solution: get the about 0.2g of di-lysine-aspirin, accurately weighed, put in 10ml measuring bottle, add appropriate methyl alcohol and 100 μ l formic acid, ultrasonic make to dissolve completely after, with methyl alcohol, be diluted to scale, shake up, as need testing solution; With 6 parts of method preparations.
Recovery solution: take the about 0.2g of di-lysine-aspirin, accurately weighed, put in 10ml measuring bottle totally 9 parts; In 1-3 measuring bottle, add respectively 0.5ml reference substance solution 2; In 4-6 measuring bottle, add respectively 1ml reference substance solution 3; In 7-9 measuring bottle, add respectively 2ml reference substance solution 3; Add respectively more appropriate methyl alcohol, 100 μ l formic acid, ultrasonic making, after dissolving, is diluted to scale with methyl alcohol, shakes up, and obtains recovery solution 1-9.
Precision measures above-mentioned 6 parts of need testing solutions and nine parts of each 10 μ l injection high performance liquid chromatographs of recovery solution respectively, records chromatogram, presses external standard method with the calculated by peak area recovery.Recording acetic acid residual quantity in sample is 5.012 μ g, and the recovery is between 99%～102%.The results are shown in Table 5.
Table 5 average recovery measurement result
Result demonstration, average recovery rate is 100.9%; RSD is 0.85%, shows that the method recovery is good, and accuracy is high.
embodiment 7 replica tests
Chromatographic condition: with the chromatographic condition described in scheme 1 in embodiment 1.
Need testing solution: precision takes 6 parts of di-lysine-aspirins, the compound method of pressing need testing solution 1 in embodiment 4 respectively.
Reference substance solution: with embodiment 5 neutral line solution 4.
Get need testing solution and reference substance solution according to chromatographic condition sample introduction of the present invention, record chromatogram.By acetic acid residual quantity in external standard method calculation sample.The results are shown in Table 6.
Table 6 sample repeatability testing result
| Measure number of times | 1 | 2 | 3 | 4 | 5 | 6 | RSD（%） |
| Acetic acid residual (%) | 0.0022 | 0.0023 | 0.0021 | 0.0021 | 0.0022 | 0.0022 | 3.4 |
Result shows, replica test RSD is 3.4%, is less than 5%, and the method has better repeatability.
the mensuration of embodiment 8 samples
Chromatographic condition: with the chromatographic condition described in scheme 1 in embodiment 1.
Get di-lysine-aspirin 0.2g, accurately weighed, put in 10ml measuring bottle, add appropriate methyl alcohol, then add 100 μ l formic acid, ultrasonic make to dissolve completely after, with methyl alcohol, be diluted to scale, shake up, obtain need testing solution.Separately get acetic acid 0.5g, accurately weighed, put in 100ml measuring bottle, with methyl alcohol, be diluted to scale, shake up, precision measures 1ml in 50ml measuring bottle again, with methyl alcohol, is diluted to scale, shakes up, obtain reference substance solution, precision measures need testing solution and each 10 μ l injection liquid chromatographies of reference substance solution respectively, records chromatogram, residual with acetic acid in calculated by peak area this product by external standard method.The results are shown in Table 7.
The measurement result of acetic acid residual quantity in table 7 sample
| Lot number | Acetic acid residual quantity (%) |
| 126000150 | 0.0022 |
| 126000160 | 0.0025 |
| 126000180 | 0.0027 |
| 126000190 | 0.0023 |
Result shows, the specificity of this method is strong, highly sensitive, precision is good, and the symmetry of acetic acid is good, can measure quickly and accurately the residual quantity of acetic acid in di-lysine-aspirin.
Claims (5)
1. a method that adopts acetic acid residual quantity in high effective liquid chromatography for measuring di-lysine-aspirin, is characterized in that: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol-phosphate buffer as mobile phase A, wherein said phosphate buffer be in 1000ml damping fluid containing 1.0～1.5ml phosphoric acid, pH value be 2.5～3.5, A mutually the volume ratio of middle methyl alcohol-phosphate buffer be 10: 90～5: 95; Take methyl alcohol as Mobile phase B, according to following table, carry out gradient elution:
Detection wavelength is 210 ± 5nm; Flow velocity is 1.0ml/min～1.5 ml/min; Sample size is 10 μ l;
Need testing solution compound method: get di-lysine-aspirin 0.2g, accurately weighed, put in 10ml measuring bottle, add appropriate methyl alcohol, then add 50～200 μ l formic acid, ultrasonic make to dissolve completely after, with methyl alcohol, be diluted to scale, shake up, obtain need testing solution, face with now joining.
2. method as claimed in claim 1, is characterized in that phosphate buffer pH is 3.0.
3. method as claimed in claim 1 or 2, is characterized in that during A mutually, the volume ratio of methyl alcohol and phosphate buffer is 8: 92.
4. the method for claim 1, is characterized in that column temperature is 20 ℃～30 ℃.
5. the method for claim 1, is characterized in that need testing solution compound method, and the amount that adds formic acid is 100 μ l.
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| CN115932122A (en) * | 2023-02-14 | 2023-04-07 | 苏州正济医药研究有限公司 | Analytical method for determining residual acetic acid in protected amino acid based on RP-HPLC internal standard method |
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