CN104020238B - The detection method of related substance in compound Anlin Babituo injection - Google Patents
The detection method of related substance in compound Anlin Babituo injection Download PDFInfo
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Abstract
The invention discloses the detection method of related substance in a kind of compound Anlin Babituo injection, it measures with high performance liquid chromatography.Detection method of the present invention, can be separated each for compound Anlin Babituo injection effective constituent completely with impurity peaks simultaneously, provide more efficiently related substance detection method; Meanwhile, the impurity produced in product after the method effectively can also detect long storage periods or degraded, the scope of application is comparatively wide, for the drug safety of this parenteral solution provides guarantee.
Description
Technical field
The present invention relates to the detection method of related substance in compound Anlin Babituo injection.
Background technology
Compound Anlin Babituo injection is the compound preparation be made up of aminopyrine 100mg, antipyrine 40mg, barbital 18mg, the analgesic-antipyretic of promptly bringing down a fever when being clinical conventional acute high fever.Aminopyrine and antipyrine belong to pyrazolone analgesic-antipyretic, can suppress synthesis and the release of hypothalamus prostaglandin, recover the orthocrasia of heat-regulating centers receptive neuron and play antipyretic effect; Also play analgesic activity by suppressing the synthesis of prostaglandin etc. simultaneously.In aminopyrine energy inflammation-inhibiting local organization, the synthesis of prostaglandin and release, stablize lysosome membrane, affect cytophagous phagocytosis and play antiinflammatory action.Share barbital, can Postoperation effect.
2003, compound Anlin Babituo injection went on the market at home, and current operative norm mostly is the national drug standards of National Drug Administration WS-10001-(HD-1327)-2003.The content assaying method of the national drug standards is that neutralisation measures aminopyrine content, colorimetric method for determining antipyrine content, and silver nitrate titration method measures barbital content.Also there is bibliographical information to carry out method for measuring to three components content at present simultaneously, but need to use ion-pairing agent and pH value is higher, easily cause chromatographic column to be consumed fast, and, said preparation is the compound preparation of three kinds of compositions, and related substance detection method is set up more difficult.At present also not about the bibliographical information of compound Anlin Babituo injection related substance detection method.
Summary of the invention
The object of the present invention is to provide the detection method of related substance in compound Anlin Babituo injection.
The invention provides the detection method of related substance in compound Anlin Babituo injection, it measures with high performance liquid chromatography, and its operation steps is as follows:
(1) get compound Anlin Babituo injection, after dilution, obtain need testing solution;
(2) need testing solution is injected high performance liquid chromatograph, adopts following chromatographic condition to detect:
Determined wavelength: 220 ± 2nm;
Stationary liquid: the chromatographic column taking octadecyl silane as filler;
Mobile phase: gradient elution: mobile phase A 50% → 100%v/v, Mobile phase B 50% → 0%v/v, wherein, mobile phase A is damping fluid: acetonitrile=(90:10) ~ (80:20) v/v or damping fluid: methyl alcohol=(70:30) ~ (80:20) V/V, and Mobile phase B is acetonitrile;
The pH value of described damping fluid is 5.5-6.0.
Wherein, mobile phase A is damping fluid: acetonitrile=(90:10) ~ (80:20) v/v, and its condition of gradient elution is as follows:
| Time/minute | Mobile phase A/% | Acetonitrile/% |
| 0 | 100 | 0 |
| 20～30 | 100 | 0 |
| 40～50 | 50 | 50 |
| 45～55 | 50 | 50 1 --> |
| 46～56 | 100 | 0 |
| 55～65 | 100 | 0 |
Further, mobile phase A is damping fluid: acetonitrile=80:20v/v, and its condition of gradient elution is as follows:
| Time/minute | Mobile phase A/% | Acetonitrile/% |
| 0 | 100 | 0 |
| 25 | 100 | 0 |
| 45 | 50 | 50 |
| 50 | 50 | 50 |
| 51 | 100 | 0 |
| 60 | 100 | 0 |
；
Or mobile phase A is damping fluid: acetonitrile=83:17v/v, and its condition of gradient elution is as follows:
| Time/minute | Mobile phase A/% | Acetonitrile/% |
| 0 | 100 | 0 |
| 20 | 100 | 0 |
| 40 | 50 | 50 |
| 45 | 50 | 50 |
| 46 | 100 | 0 |
| 55 | 100 | 0 |
；
Or mobile phase A is damping fluid: acetonitrile=85:15v/v, and its condition of gradient elution is as follows:
| Time/minute | Mobile phase A/% | Acetonitrile/% |
| 0 | 100 | 0 |
| 30 | 100 | 0 |
| 50 | 50 | 50 |
| 55 | 50 | 50 |
| 56 | 100 | 0 |
| 65 | 100 | 0 |
Further, in step (1), using acetonitrile solution or damping fluid: acetonitrile=(90:10) ~ (80:20) v/v dilutes as solvent.
Further the, in step (1), the concentration of described acetonitrile solution is 5-15%v/v.
Further, determined wavelength is 220nm.
Wherein, described damping fluid is phosphate buffer or acetate buffer.
Further, described phosphate buffer is sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate or ammonium dihydrogen phosphate (ADP) damping fluid; Described acetate buffer is ammonium acetate, sodium acetate or potassium acetate damping fluid.
Further, the concentration of described damping fluid is 0.01mol/L-0.05mol/L.
Wherein, in step (2), chromatogram column temperature is 30 ± 5 DEG C.
Wherein, in described high performance liquid chromatography, area normalization method or major component Self-control method is adopted to calculate its related substances.
Detection method of the present invention, can be separated each for compound Anlin Babituo injection effective constituent completely with impurity peaks simultaneously, provide more efficiently related substance detection method; Meanwhile, the impurity produced in product after the method effectively can also detect long storage periods or degraded, the scope of application is comparatively wide, for the drug safety of this parenteral solution provides guarantee.
Accompanying drawing explanation
Fig. 1 reference substance HPLC collection of illustrative plates
The HPLC collection of illustrative plates of Fig. 2 embodiment 1, pH of buffer=5.5; Wherein, A is solvent blank contrast, and B is test sample collection of illustrative plates
The HPLC collection of illustrative plates of Fig. 3 embodiment 2, pH of buffer=6.0; Wherein, A is solvent blank contrast, and B is test sample collection of illustrative plates
HPLC collection of illustrative plates under the different eluent gradient of Fig. 4
HPLC collection of illustrative plates under the different eluent gradient of Fig. 5
The HPLC collection of illustrative plates of Fig. 6 embodiment 3; Buffer salinity is 0.01mol/L
The HPLC collection of illustrative plates of Fig. 7 embodiment 4; Buffer salinity is 0.05mol/L
36 months sample HPLC collection of illustrative plates in Fig. 8 compound Anlin Babituo injection peak
Fig. 9 aminopyrine solution illumination HPLC collection of illustrative plates of 2 months
Figure 10 antipyrine solution illumination HPLC collection of illustrative plates of 2 months
Figure 11 barbital solution illumination HPLC collection of illustrative plates of 2 months
Figure 12 ammonia woods barbital parenteral solution acid degradation HPLC collection of illustrative plates
Figure 13 aminopyrine acid degradation HPLC collection of illustrative plates
Figure 14 antipyrine acid degradation HPLC collection of illustrative plates
Figure 15 barbiturates degraded HPLC collection of illustrative plates
Figure 16 ammonia woods barbital parenteral solution high temperature degradation HPLC collection of illustrative plates
Figure 17 aminopyrine high temperature degradation HPLC collection of illustrative plates
Figure 18 antipyrine high temperature degradation HPLC collection of illustrative plates
Figure 19 barbital high temperature degradation HPLC collection of illustrative plates
Figure 20 ammonia woods barbital parenteral solution oxidative degradation HPLC collection of illustrative plates (room temperature 2 hours)
Figure 21 ammonia woods barbital parenteral solution oxidative degradation HPLC collection of illustrative plates (water-bath 2.5 hours)
Figure 22 aminopyrine oxidative degradation HPLC collection of illustrative plates
Figure 23 antipyrine oxidative degradation HPLC collection of illustrative plates
Figure 24 barbital oxidative degradation HPLC collection of illustrative plates
Figure 25 ammonia woods barbital parenteral solution alkaline degradation (60 degrees Celsius 1 hour) HPLC collection of illustrative plates
Figure 26 ammonia woods barbital parenteral solution alkaline degradation (water-bath 2.5 hours) HPLC collection of illustrative plates
Figure 27 aminopyrine alkaline degradation HPLC collection of illustrative plates
Figure 28 antipyrine alkaline degradation HPLC collection of illustrative plates
Figure 29 barbital alkaline degradation HPLC collection of illustrative plates
Figure 30 uses HPLC collection of illustrative plates during acetate buffer
Figure 31 uses HPLC collection of illustrative plates during ammonium dihydrogen phosphate (ADP) damping fluid
Figure 32 uses HPLC collection of illustrative plates during phosphate sodium dihydrogen buffer solution
Embodiment
The detection of related substance of the present invention, all can adopt the conventional method such as area normalization method, major component Self-control method to calculate impurity content.
Embodiment 1 detection method
Instrument and condition: Agilent1200 liquid chromatograph, DAD detecting device, chromatographic column: AgilentTC-C18 (250mm*4.6mm, 5um); Determined wavelength: 220nm; Column temperature: 30 DEG C; With 0.02mol/L potassium phosphate buffer (pH is adjusted to 5.5)-acetonitrile=85:15 be mobile phase A, with acetonitrile for Mobile phase B, according to the form below carries out linear gradient elution.
| Time/minute | Mobile phase A/% | Acetonitrile/% |
| 0 | 100 | 0 |
| 30 | 100 | 0 |
| 50 | 50 | 50 |
| 55 | 50 | 50 |
| 56 | 100 | 0 |
| 65 | 100 | 0 |
Main peak is located
The reference substance getting aminopyrine, antipyrine and barbital is respectively appropriate, adds the solution that mobile phase A is mixed with concentration 0.05mg/ml, 0.02mg/ml, 0.009mg/ml.Separately get compound Anlin Babituo injection 1ml and be placed in 100ml measuring bottle, add mobile phase A constant volume, measure in accordance with the law, Fig. 1 is shown in by HPLC collection of illustrative plates.
As seen from Figure 1: barbital, antipyrine, aminopyrine peak retention time be followed successively by 7.801 minutes, 11.143 minutes, 22.453 minutes.Be separated good between each main peak.
Parenteral solution detection method and result:
The compound Anlin Babituo injection 1ml getting different batches is placed in 100ml measuring bottle, and after 10% acetonitrile solution dilution constant volume, detect as stated above, Fig. 2 is shown in by HPLC collection of illustrative plates, and testing result sees table (major component Self-control method):
Table 1
Embodiment 2 detection method
Instrument and condition: Agilent1200 liquid chromatograph, DAD detecting device, chromatographic column: AgilentTC-C18 (250mm*4.6mm, 5um); Determined wavelength: 220nm; Column temperature: 30 DEG C; With 0.02mol/L potassium phosphate buffer (pH is adjusted to 6.0)-acetonitrile=85:15 be mobile phase A, with acetonitrile for Mobile phase B, according to the form below carries out linear gradient elution.
| Time/minute | Mobile phase A/% | Acetonitrile/% 4--> |
| 0 | 100 | 0 |
| 30 | 100 | 0 |
| 50 | 50 | 50 |
| 55 | 50 | 50 |
| 56 | 100 | 0 |
| 65 | 100 | 0 |
Get compound Anlin Babituo injection 1ml and be placed in 100ml measuring bottle, after mobile phase A dilution constant volume, detect as stated above, Fig. 3 is shown in by HPLC collection of illustrative plates.
Embodiment 3 compound Anlin Babituo injection determination of related substances
Instrument and condition: Agilent1200 liquid chromatograph, DAD detecting device, chromatographic column: AgilentTC-C18 (250mm*4.6mm, 5um); Determined wavelength: 220nm; Column temperature: 30 DEG C; With 0.02mol/L potassium phosphate buffer (pH is adjusted to 5.5)-acetonitrile=83:17 be mobile phase A, with acetonitrile for Mobile phase B, according to the form below carries out linear gradient elution.
| Time/minute | Mobile phase A/% | Acetonitrile/% |
| 0 | 100 | 0 |
| 20 | 100 | 0 |
| 40 | 50 | 50 |
| 45 | 50 | 50 |
| 46 | 100 | 0 |
| 55 | 100 | 0 |
Experimental procedure: measure compound Anlin Babituo injection preparation long-term 36 months sample 1ml and be placed in 100ml measuring bottle, add mobile phase A constant volume and shake up, measuring in accordance with the law, Fig. 4 is shown in by HPLC collection of illustrative plates.
Embodiment 4 compound Anlin Babituo injection determination of related substances
Instrument and condition: Agilent1200 liquid chromatograph, DAD detecting device, chromatographic column: AgilentTC-C18 (250mm*4.6mm, 5um); Determined wavelength: 220nm; Column temperature: 30 DEG C; With 0.02mol/L potassium phosphate buffer (pH is adjusted to 5.5)-acetonitrile=80:20 be mobile phase A, with acetonitrile for Mobile phase B, according to the form below carries out linear gradient elution.
| Time/minute | Mobile phase A/% | Acetonitrile/% |
| 0 | 100 | 0 |
| 25 | 100 | 0 |
| 45 | 50 | 50 |
| 50 | 50 | 50 |
| 51 | 100 | 0 |
| 60 | 100 | 0 |
Experimental procedure: measure compound Anlin Babituo injection preparation long-term 36 months sample 1ml and be placed in 100ml measuring bottle, add mobile phase A constant volume and shake up, measuring in accordance with the law, Fig. 5 is shown in by HPLC collection of illustrative plates.
Embodiment 5 compound Anlin Babituo injection determination of related substances
Instrument and condition: Agilent1200 liquid chromatograph, DAD detecting device, chromatographic column: AgilentTC-C18 (250mm*4.6mm, 5um); Determined wavelength: 220nm; Column temperature: 30 DEG C; With 0.01mol/L potassium phosphate buffer (pH is for 5.5)-acetonitrile=85:15 be mobile phase A, with acetonitrile for Mobile phase B, according to the form below carries out linear gradient elution.
| Time/minute | Mobile phase A/% | Acetonitrile/% |
| 0 | 100 | 0 |
| 30 | 100 | 0 |
| 50 | 50 | 50 |
| 55 | 50 | 50 |
| 56 | 100 | 0 |
| 65 | 100 | 0 |
Get compound Anlin Babituo injection 1ml and be placed in 100ml measuring bottle, after damping fluid dilution constant volume, detect as stated above, result is see Fig. 6.
Embodiment 6 compound Anlin Babituo injection determination of related substances
Instrument and condition: Agilent1200 liquid chromatograph, DAD detecting device, chromatographic column: AgilentTC-C18 (250mm*4.6mm, 5um); Determined wavelength: 220nm; Column temperature: 30 DEG C; With 0.05mol/L potassium phosphate buffer (pH is for 5.5)-acetonitrile=85:15 be mobile phase A, with acetonitrile for Mobile phase B, according to the form below carries out linear gradient elution.
| Time/minute | Mobile phase A/% | Acetonitrile/% |
| 0 | 100 | 0 |
| 30 | 100 | 0 |
| 50 | 50 | 50 |
| 55 | 50 | 50 |
| 56 | 100 | 0 |
| 65 | 100 | 0 |
Get compound Anlin Babituo injection 1ml and be placed in 100ml measuring bottle, after mobile phase A dilution constant volume, detect as stated above, result is see Fig. 7.
Embodiment 7 compound Anlin Babituo injection determination of related substances
Instrument and condition: Agilent1200 liquid chromatograph, DAD detecting device, chromatographic column: AgilentTC-C18 (250mm*4.6mm, 5um); Determined wavelength: 220nm; Column temperature: 30 DEG C; With 0.02mol/L potassium phosphate buffer (pH is adjusted to 5.5)-acetonitrile=85:15 be mobile phase A, with acetonitrile for Mobile phase B, according to the form below carries out linear gradient elution.
| Time/minute | Mobile phase A/% | Acetonitrile/% |
| 0 | 100 | 0 |
| 30 | 100 | 0 |
| 50 | 50 | 50 |
| 55 | 50 | 50 |
| 56 | 100 | 0 |
| 65 | 100 | 0 |
1, the determination of related substances after parenteral solution preservation for a long time in 36 months
Experimental procedure: measure compound Anlin Babituo injection preparation long-term 36 months sample 1ml and be placed in 100ml measuring bottle, add mobile phase A constant volume and shake up, measuring in accordance with the law, Fig. 8 is shown in by HPLC collection of illustrative plates.
As seen from Figure 8: the degree of separation of barbital and antipyrine is 12.08, antipyrine is 5.00 with the degree of separation of impurity peaks thereafter, the degree of separation of aminopyrine impurity peaks front with it is 3.25, and each main peak and impurity peaks are all separated very well, and main peak peak purity all conforms with the regulations.
2, the related substance after compound Anlin Babituo injection illumination degrading detects
Get aminopyrine, antipyrine, barbital respectively respectively 25mg, 10mg be placed in 50ml measuring bottle, barbital 9mg is placed in 100ml measuring bottle, adds mobile phase A constant volume, in 4500Lux illumination box place 60 days.Measure, Fig. 9 ~ 11 are shown in by HPLC collection of illustrative plates in accordance with the law.
As seen from the figure: aminopyrine is poor at illumination condition stability inferior, the impurity peaks being about 0.20 and 0.85 relative to aminopyrine main peak retention time obviously increases, and three main peaks all can be separated with impurity peaks very well, and each main peak peak purity all conforms with the regulations simultaneously.
3, the related substance after compound Anlin Babituo injection acid destruction detects
Get this product 1ml and be placed in 100ml measuring bottle, add 1mol/l hydrochloric acid solution 3ml, 100 DEG C of heating water baths 2.5 hours, adjust PH to neutral with 1mol/l sodium hydroxide solution, add mobile phase A and be diluted to scale, shake up, filter, measure, Figure 12 ~ 15 are shown in by HPLC collection of illustrative plates in accordance with the law.
As seen from the figure: this product is very stable in acid condition, each main peak peak purity all conforms with the regulations.
4, the related substance after compound Anlin Babituo injection high temperature detects
Get this product 100 DEG C of heating water baths 5 hours, measure 1ml and be placed in 100ml measuring bottle, add mobile phase A and be diluted to scale, shake up, filter, measure, Figure 16 ~ 19 are shown in by HPLC collection of illustrative plates in accordance with the law.
As seen from the figure: aminopyrine is less stable at high temperature, the contrast parenteral solution collection of illustrative plates of 36 months finds, the degradation impurity that the impurity peaks being about 0.58,0.79,0.85 relative to aminopyrine main peak retention time in long-term setting-out may produce when high-temperature sterilization for aminopyrine.Each main peak peak purity all conforms with the regulations.
5, the related substance after compound Anlin Babituo injection Oxidative demage detects
Get compound Anlin Babituo injection 1ml and be placed in 100ml measuring bottle, add 3% hydrogen peroxide 3ml, 100 DEG C of heating water baths mobile phase A constant volume after 2.5 hours, separately get aminopyrine, antipyrine, barbital respectively respectively 25mg, 10mg be placed in 50ml measuring bottle, barbital 9mg is placed in 100ml measuring bottle, add 3% hydrogen peroxide 3ml, aminopyrine room temperature places 2 hours, antipyrine and barbital 100 DEG C of heating water baths 2 hours, mobile phase A constant volume, measure, Figure 20 ~ 24 are shown in by HPLC collection of illustrative plates in accordance with the law.
As seen from the figure: three components is oxidized equal instability under the high temperature conditions, wherein aminopyrine stability is the poorest, the impurity peaks being about 0.14,0.18 and 0.85 relative to aminopyrine main peak retention time obviously increases, test in conjunction with high temperature degradation, infer that sample mainly produces impurity and derives from sterilizing and oxidative degradation under high temperature, three main peaks all can be separated with impurity peaks very well, and each main peak peak purity all conforms with the regulations simultaneously.
6, the related substance after compound Anlin Babituo injection alkali destruction detects
Get this product 1ml and be placed in 100ml measuring bottle, add 1mol/l hydrochloric acid solution 3ml, 100 DEG C of heating water baths 2.5 hours, adjust PH to neutral with 1mol/l sodium hydroxide solution, add mobile phase A and be diluted to scale, shake up, filter, measure, Figure 25 ~ 29 are shown in by HPLC collection of illustrative plates in accordance with the law.
As seen from the figure: barbital is unstable in the basic conditions, aminopyrine and antipyrine are stable under high-temperature alkaline condition, and three main peaks and impurity peaks degree of separation are greater than 1.5 simultaneously, and each main peak peak purity all conforms with the regulations.
Brief summary:
Above-mentioned experiment shows, the inventive method also can effectively be separated the product after compound Anlin Babituo injection degraded, is conducive to each the implementation quality control in period to product.
The screening of embodiment 8 testing conditions of the present invention
The determination of 1 determined wavelength
The bulk drug getting aminopyrine, antipyrine and barbital is respectively appropriate, adds the solution that mobile phase A is mixed with concentration 0.05mg/ml, 0.02mg/ml, 0.009mg/ml.Respectively at the interscan of 200-400nm wavelength coverage, aminopyrine has absorption maximum at 233nm and 268nm place, antipyrine has absorption maximum at 243nm and 261nm place, barbital has absorption and successively decreases along with the increase of wavelength at 200-240nm place, consider that in three components, the content of barbital is lower, simultaneously in order to avoid the interference at nearly 200nm wavelength place mobile phase, 220nm is selected to be the determined wavelength tested.
The screening of 2 mobile phase pH
Instrument and condition: Agilent1200 liquid chromatograph, DAD detecting device, chromatographic column: AgilentTC-C18 (250mm*4.6mm, 5um); Determined wavelength: 220nm; Column temperature: 30 DEG C; With 0.02mol/L potassium phosphate buffer (pH sees table)-acetonitrile=85:15 be mobile phase A, with acetonitrile for Mobile phase B, according to the form below carries out linear gradient elution.
| Time/minute | Mobile phase A/% | Acetonitrile/% |
| 0 | 100 | 0 |
| 30 | 100 | 0 |
| 50 | 50 | 50 |
| 55 | 50 | 50 |
| 56 | 100 | 0 |
| 65 | 100 | 0 |
The experimental result that phosphate buffer pH screens:
| Numbering | PH value | Peak shape | Impurity detects |
| 1 | 3.5 | Forward position, aminopyrine serious peak | Impurity is not separated with main peak |
| 2 | 4.5 | Forward position, aminopyrine serious peak | Impurity is not separated with main peak |
| 3 | 5.0 | Symmetrical | Impurity does not detect completely |
| 4 | 5.5 | Symmetrical | Impurity and main peak good separating effect |
| 5 | 6.0 | Symmetrical | Impurity and main peak good separating effect |
| 6 | 6.5 | Symmetrical | Impurity does not detect completely |
From above-mentioned experiment, only have when pH5.5 ~ 6.0 of phosphate buffer, the good separation of impurity and main peak.
The investigation of 3 damping fluids
The selection of 3.1 buffer solution kinds
Adopt the elution requirement of embodiment 1, Dichlorodiphenyl Acetate salt buffer solution and phosphate buffer are investigated, and confirm the kind of damping fluid.In the present embodiment, for ammonium acetate buffer, mobile phase A is 0.02mol/L ammonium acetate buffer: acetonitrile=85:15, and Mobile phase B is with embodiment 1.
Wherein, the testing result of phosphate buffer is with embodiment 1; The testing result of acetate buffer is shown in Figure 30.Contrast known, both all effectively can be separated effective constituent in parenteral solution and impurity, and wherein, acetate buffer in later stage baseline wander, but does not affect the mensuration to related substance.
The investigation of 3.2 phosphate buffers
Adopt the elution requirement of embodiment 1, for ammonium dihydrogen phosphate (ADP) damping fluid and phosphate sodium dihydrogen buffer solution, confirm the kind of phosphate buffer.In two groups of experiments, Mobile phase B is identical, and mobile phase A is 0.02mol/L ammonium dihydrogen phosphate (ADP) damping fluid: during acetonitrile=85:15, the results are shown in Figure 31; Mobile phase A is 0.02mol/L phosphate sodium dihydrogen buffer solution: during acetonitrile=85:15, the results are shown in Figure 32.According to result, sodium ascorbyl phosphate, ammonium phosphate salt all effectively can be separated effective constituent in parenteral solution and impurity, are applicable to the detection of related substance.
Conclusion:
From above-described embodiment:
(1) detection method of the present invention, can be separated each for compound Anlin Babituo injection effective constituent completely with impurity peaks simultaneously, provide more efficiently related substance detection method, for the drug safety of this parenteral solution provides guarantee.
(2) by gradient elution and isocratic elution more known, Gradient program can also detect 2 impurity that retention time is about 45 minutes, thus ensure impurity farthest detect and be separated, Gradient program is more excellent.
Claims (9)
1. the detection method of related substance in compound Anlin Babituo injection, it is characterized in that: it measures with high performance liquid chromatography, its operation steps is as follows:
(1) get compound Anlin Babituo injection, after dilution, obtain need testing solution;
(2) need testing solution is injected high performance liquid chromatograph, adopts following chromatographic condition to detect:
Determined wavelength: 220 ± 2nm;
Stationary liquid: the chromatographic column taking octadecyl silane as filler;
Mobile phase: Mobile phase B is acetonitrile;
Mobile phase A is damping fluid: acetonitrile=80:20v/v, and its condition of gradient elution is as follows:
0 minute, mobile phase A was 100v%; 25 minutes, mobile phase A was 100v%; 45 minutes, mobile phase A was 50v%, and acetonitrile is 50v%; 50 minutes, mobile phase A was 50v%, and acetonitrile is 50v%; 51 minutes, mobile phase A was 100v%; 60 minutes, mobile phase A was 100v%;
Or mobile phase A is damping fluid: acetonitrile=83:17v/v, and its condition of gradient elution is as follows:
0 minute, mobile phase A was 100v%; 20 minutes, mobile phase A was 100v%; 40 minutes, mobile phase A was 50v%, and acetonitrile is 50v%; 45 minutes, mobile phase A was 50v%, and acetonitrile is 50v%; 46 minutes, mobile phase A was 100v%; 55 minutes, mobile phase A was 100v%;
Or mobile phase A is damping fluid: acetonitrile=85:15v/v, and its condition of gradient elution is as follows: 0 minute, mobile phase A is 100v%; 30 minutes, mobile phase A was 100v%; 50 minutes, mobile phase A was 50v%, and acetonitrile is 50v%; 55 minutes, mobile phase A was 50v%, and acetonitrile is 50v%; 56 minutes, mobile phase A was 100v%; 65 minutes, mobile phase A was 100v%;
The pH value of described damping fluid is 5.5-6.0.
2. detection method according to claim 1, is characterized in that: in step (1), using acetonitrile solution or damping fluid: acetonitrile=(90:10) ~ (80:20) v/v dilutes as solvent.
3. detection method according to claim 2, is characterized in that: in step (1), and the concentration of described acetonitrile solution is 5-15%v/v.
4. detection method according to claim 1, is characterized in that: determined wavelength is 220nm.
5. the detection method according to claim 1-4 any one, is characterized in that: described damping fluid is phosphate buffer or acetate buffer.
6. detection method according to claim 5, is characterized in that: described phosphate buffer is sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate or ammonium dihydrogen phosphate (ADP) damping fluid; Described acetate buffer is ammonium acetate, sodium acetate or potassium acetate damping fluid.
7. detection method according to claim 5, is characterized in that: the concentration of described damping fluid is 0.01mol/L-0.05mol/L.
8. detection method according to claim 1, is characterized in that: in step (2), and chromatogram column temperature is 30 ± 5 DEG C.
9. the detection method according to claim 1-4,6 or 8 any one, is characterized in that: in described high performance liquid chromatography, adopts area normalization method or major component Self-control method to calculate its related substances.
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