CN104655786A - Method for measuring substances related to formoterol intermediate by separation of liquid chromatography - Google Patents
Method for measuring substances related to formoterol intermediate by separation of liquid chromatography Download PDFInfo
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- CN104655786A CN104655786A CN201510092202.8A CN201510092202A CN104655786A CN 104655786 A CN104655786 A CN 104655786A CN 201510092202 A CN201510092202 A CN 201510092202A CN 104655786 A CN104655786 A CN 104655786A
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Abstract
The invention belongs to the field of analytical chemistry, and discloses a method for measuring the chemical purities of substances related to formoterol intermediate (R)-1-(3-amino-4-(benzyloxy) phenyl)-2-(benzyl (R)-1-(4-methoxyphenyl) propan-2-yl) amino) ethanol by separation of liquid chromatography. According to the method, phenyl silane bonded silica gel is taken as a chromatographic column of filler and a buffer salt solution-organic phase with a certain proportion is taken as a mobile phase, and the contents of the formoterol intermediate and the substances related to the formoterol intermediate can be measured, so that the mass of the formoterol intermediate can be effectively controlled, and the mass of the final product of formoterol is controllable. The method is high in specificity, high in accuracy and simple and convenient to operate.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical field, in particular to a kind of method of quality control of Formoterol, particularly relate to a kind of detection method of Formoterol intermediate related substance.
Background technology
Formoterol is a kind of long-acting beta-2-adrenoreceptor agonists, for the bronchoconstriction disease that long term maintenance therapy chronic obstructive pulmonary disease (COPD) causes, comprises chronic bronchitis and pulmonary emphysema etc.Formoterol intermediated chemistry is called (R)-1-(3-amino-4-(benzyloxy) phenyl)-2-(benzyl (R)-1-(4-methoxyphenyl) propan-2-yl) amino) ethanol, molecular formula is C
32h
36n
2o
3.Formoterol intermediate structure formula is:
In the process of this compound of synthesis, there is the important intermediate of a few step may affect purity and the quality of Formoterol bulk drug owing to removing not exclusively, related substance for the synthesis major control of Formoterol intermediate has 5, related substance 1:2-Bromo-1-[3-nitro-4-(phenylmethoxy) phenyl]-ethanone respectively, related substance 2:(R)-1-(4-Benzyloxy-3-nitrophenyl)-2-bromoethanol, related substance 3:(R)-2-(4-(benzyloxy)-3-nitrophenyl) oxirane, related substance 4:(R)-1-(4-Methoxyphenyl)-2-benzylaminopropane, related substance 5:(R)-1-(4-(benzyloxy)-3-nitrophenyl)-2-benzyl (R)-1-(4-methoxyphenyl) propan-2-yl) amino) ethanol.Structural formula is respectively:
Impurity removal in Formoterol intermediate is incomplete, will introduce bulk drug finished product, thus affect pharmaceutical purity and quality.Therefore, realize the separation of Formoterol intermediate and related substance thereof, have important practical significance in the production and quality control thereof of Formoterol.
Summary of the invention
In view of the deficiencies in the prior art, the present inventor has groped a kind of separation Formoterol intermediate and related substance thereof by great many of experiments, the method of its chemical purity can be measured simultaneously, thus realize the separated island form of Formoterol intermediate and its related substance, thus ensure that Formoterol is quality controllable.
A kind of method measuring Formoterol intermediate related substance of the present invention adopts phenyl silane bonded silica gel to be the chromatographic column of filler, with a certain proportion of buffer salt solution-organic phase for mobile phase;
Above-mentioned said chromatographic column is with phenyl silane bonded silica gel for filling agent, and chromatographic column is selected from as brands such as Kromasil, Ultimate and Agilent.
Above-mentioned said organic phase is acetonitrile or methyl alcohol.
Above-mentioned said buffer salt can be phosphate or acetate, preferably phosphoric acid potassium dihydrogen.
Method of separating and assaying of the present invention, can realize in accordance with the following methods:
1) get Formoterol intermediate and related substance sample thereof appropriate, use mobile phase sample dissolution respectively, be mixed with the sample solution of every 1 mL containing Formoterol intermediate and related substance 0.1 ~ 1.5 mg thereof;
2) arranging flow rate of mobile phase is 0.5 ~ 1.5 mL/min, determined wavelength 205 ~ 280 nm;
3) mobile phase A is 15 ~ 25 mmol/L potassium dihydrogen phosphates, adjusts pH to 5.0 ~ 7.0 with potassium hydroxide; Mobile phase B is acetonitrile or methyl alcohol;
4) get 1) sample solution 10 ~ 50 μ L injection liquid chromatography, complete the separation determination of Formoterol intermediate and related substance thereof.Wherein:
The model of high performance liquid chromatograph, has no special requirements, and the chromatograph that the present invention adopts is Shimadzu high performance liquid chromatograph:
LC-20AT，CBM-20A，SIL-20AC，SPD-M20A，CTO-10ASvp
Chromatographic column: Phenyl(Kromasil, 250 × 4.6 mm, 5 μm)
Mobile phase: A:20 mmol/L potassium dihydrogen phosphate (pH 6.0)
B: acetonitrile
Flow velocity: 1.0 mL/min
Determined wavelength: 215 nm
Sampling volume: 10 μ L
Gradient:
| Time (min) | Mobile phase A | Mobile phase B |
| 0 | 48 | 52 |
| 27 | 48 | 52 |
| 30 | 42 | 58 |
| 65 | 42 | 58 |
| 80 | 30 | 70 |
| 81 | 48 | 52 |
| 95 | 48 | 52 |
The present invention adopts Phenyl(Kromasil, 250 × 4.6 mm, 5 μm) chromatographic column, can effectively be separated Formoterol intermediate and related substance thereof.The invention solves the separation determination problem of Formoterol and related substance thereof, thus ensure that quality controllable (the results are shown in accompanying drawing 1 ~ 7) of Formoterol intermediate and bulk drug.
Accompanying drawing explanation
Formoterol intermediate when Fig. 1 is embodiment 1 and related substance HPLC thereof scheme;
When Fig. 2 is embodiment 1, Formoterol intermediate HPLC schemes;
Formoterol intermediate when Fig. 3 is embodiment 2 and related substance HPLC thereof scheme;
The HPLC figure of Formoterol intermediate when Fig. 4 is embodiment 2;
Solvent HPLC when Fig. 5 is embodiment 3 schemes;
Formoterol intermediate when Fig. 6 is embodiment 3 and related substance HPLC thereof scheme;
The HPLC figure of Formoterol intermediate when Fig. 7 is embodiment 3.
embodiment:
Following examples are used for understanding the present invention further, but are not limited to the scope of this experiment.
Embodiment 1
Instrument and condition
High performance liquid chromatograph: Shimadzu: LC-20AT, CBM-20A, SIL-20AC, SPD-M20A, CTO-10ASvp;
Chromatographic column: C
8(Apollo, 250 × 4.6 mm, 5 μm);
Mobile phase: 20 mmol/L sodium hydrogen phosphates (pH 3.0)-acetonitrile (55:45)
Flow velocity: 1.0 mL/min
Determined wavelength: 215 nm
Sampling volume: 10 μ L.
Experimental procedure
Get Formoterol intermediate and related substance thereof appropriate, use mobile phase sample dissolution respectively, be mixed with the sample solution containing Formoterol intermediate and related substance about 0.5 mg/mL thereof.Efficient liquid phase chromatographic analysis is carried out, record chromatogram by above-mentioned condition.To the results are shown in retention time in accompanying drawing 1 ~ 2, Fig. 1 be the chromatographic peak of 15.090 min is Formoterol intermediate, and all the other chromatographic peaks are the chromatographic peak of each related substance of Formoterol intermediate; In Fig. 2, retention time is the chromatographic peak of 14.601 min is Formoterol intermediate.
Embodiment 2
Instrument and condition
High performance liquid chromatograph: Shimadzu: LC-20AT, CBM-20A, SIL-20AC, SPD-M20A, CTO-10ASvp;
Chromatographic column: C
8(Apollo, 250 × 4.6 mm, 5 μm);
Mobile phase: 20 mmol/L potassium dihydrogen phosphates (pH 6.0)-acetonitrile (30:70)
Flow velocity: 1.0 mL/min
Determined wavelength: 215 nm
Sampling volume: 10 μ L.
Experimental procedure
Get Formoterol intermediate and related substance thereof appropriate, use mobile phase sample dissolution respectively, be mixed with the sample solution containing Formoterol intermediate and related substance about 0.5 mg/mL thereof.Efficient liquid phase chromatographic analysis is carried out, record chromatogram by above-mentioned condition.To the results are shown in retention time in accompanying drawing 3 ~ 4, Fig. 3 be the chromatographic peak of 10.323 min is Formoterol intermediate, and all the other chromatographic peaks are the chromatographic peak of each related substance of Formoterol intermediate; In Fig. 4, retention time is the chromatographic peak of 10.315 min is Formoterol intermediate.
Embodiment 3
Instrument and condition
High performance liquid chromatograph: Shimadzu: LC-20AT, CBM-20A, SIL-20AC, SPD-M20A, CTO-10ASvp;
Chromatographic column: Phenyl(Kromasil, 250 × 4.6 mm, 5 μm);
Mobile phase: A:20 mmol/L potassium dihydrogen phosphate (pH 6.0)
B: acetonitrile
Flow velocity: 1.0 mL/min
Determined wavelength: 215 nm
Sampling volume: 10 μ L
Gradient:
| Time (min) | Mobile phase A | Mobile phase B |
| 0 | 48 | 52 |
| 27 | 48 | 52 |
| 30 | 42 | 58 |
| 65 | 42 | 58 |
| 80 | 30 | 70 |
| 81 | 48 | 52 |
| 95 | 48 | 52 |
The following items of above-mentioned Formoterol intermediate and Related substance method thereof is verified:
1, system suitability
Under the above-mentioned chromatographic condition determined, get Formoterol intermediate and related substance thereof appropriate, use mobile phase sample dissolution respectively, be mixed with the sample solution containing Formoterol intermediate and related substance about 0.5 mg/mL thereof; Separately get mobile phase in right amount as blank solvent.Efficient liquid phase chromatographic analysis is carried out, record chromatogram by above-mentioned condition.The results are shown in accompanying drawing 5 ~ 7, Fig. 5 is solvent chromatogram; In Fig. 6, retention time is the chromatographic peak of 53.574 min is Formoterol intermediate, all the other chromatographic peaks are the chromatographic peak of each related substance of Formoterol intermediate, as seen from the figure, Formoterol intermediate and its related substance can reach baseline separation, meet the requirement of Chinese Pharmacopoeia; In Fig. 7, retention time is the chromatographic peak Formoterol intermediate of 53.750 min, and can find out that between Formoterol intermediate and its related substance, degree of separation meets the requirements under this condition, peak purity and single-point threshold value all meet the requirements.
2, stability of solution
Get the test liquid of Formoterol intermediate and related substance thereof, respectively at 0,2,4,6,8,10,12,24 hour sample introduction, investigate the stability of solution when sample size measures, from result, this solution is stable in 24 hours.
3, durability
Because the above-mentioned chromatographic condition determined is gradient elution, and determine corresponding flow velocity, column temperature, mobile phase pH and chromatographic column model, therefore these conditions are done corresponding fine setting, investigate the durability of chromatographic condition.
Change in flow is within the scope of ± 0.1 mL/min, and the peak shape of Formoterol intermediate and related substance thereof does not change, but retention time have corresponding reach and after move; Column temperature change is within the scope of ± 5 DEG C, and the peak shape of each material and retention time are all without larger change; The pH change of mobile phase is in ± 0.2 scope, and the peak shape of each material and retention time are also without larger change; In the durability investigation of chromatographic column, go out peak during Ulitmate Phenyl chromatogram column analysis very fast, but do not affect peak purity and the degree of separation of each material, during Agilent Phenyl chromatogram column analysis, each material retention time and degree of separation are without significant change.
4, quantitative limit and detectability
Get Formoterol intermediate and related substance in right amount each, accurately weighed, add mobile phase and dissolve the test liquid that each sample is made into response respectively, then precision to measure test liquid appropriate, stepwise dilution, sample introduction is investigated.Each material quantitative limit and detectability data as shown in the table:
| Project | Main peak | Impurity 1 | Impurity 2 | Impurity 3 | Impurity 4 | Impurity 5 |
| Quantitative limit (ng/mL) | 1.96 | 2.45 | 1.64 | 1.85 | 1.56 | 1.34 |
| Detectability (ng/mL) | 0.58 | 1.56 | 0.56 | 0.46 | 0.62 | 0.49 |
Claims (9)
1. measure a method for Formoterol intermediate related substance, it is characterized in that: adopt reversed-phased high performace liquid chromatographic, phenyl silane bonded silica gel is the chromatographic column of filler, with a certain proportion of buffer salt solution-organic phase for mobile phase.
2. method of separating and assaying according to claim 1, chromatographic column is selected from Kromasil, Ultimate and Agilent brand.
3. method of separating and assaying according to claim 1, said organic phase is methyl alcohol or acetonitrile.
4. method of separating and assaying according to claim 1, said buffer salt can be phosphate or acetate.
5. method of separating and assaying according to claim 1, said buffer salt solution pH is 5.0 ~ 7.0.
6. method of separating and assaying according to claim 4, said buffer salt preferably phosphoric acid potassium dihydrogen.
7. method of separating and assaying according to claim 1, is characterized in that:
1) get Formoterol intermediate and related substance sample thereof appropriate, use mobile phase sample dissolution respectively, be mixed with the sample solution of every 1 mL containing Formoterol intermediate and related substance 0.1 ~ 1.5 mg thereof;
2) arranging flow rate of mobile phase is 0.5 ~ 1.5 mL/min, determined wavelength 205 ~ 280 nm;
3) mobile phase A is 15 ~ 25 mmol/L potassium dihydrogen phosphates, adjusts pH to 5.0 ~ 7.0 with potassium hydroxide; Mobile phase B is acetonitrile or methyl alcohol; The gradient of mobile phase arranges as follows:
4) 1 is got) sample solution 10 ~ 50 μ L injecting chromatograph, complete Formoterol intermediate and related substance separation determination thereof.
8. the new method of mensuration Formoterol intermediated chemistry purity according to claim 7, is characterized in that: mobile phase A is 20 mmol/L potassium dihydrogen phosphates, adjusts pH to 6.0 with potassium hydroxide; Mobile phase B is acetonitrile.
9. the new method of mensuration Formoterol intermediated chemistry purity according to claim 7, is characterized in that: chromatographic column is Phenyl, 5 μm, 250 × 4.6 mm(I.D.), flow velocity is 1.0 mL/min, and determined wavelength is 215 nm, can detect under room temperature condition.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108362813A (en) * | 2017-12-29 | 2018-08-03 | 江苏悦兴医药技术有限公司 | A kind of method for detecting purity of principal component |
| CN110376313A (en) * | 2019-08-20 | 2019-10-25 | 广州健康元呼吸药物工程技术有限公司 | A method of impurity in detection formoterol fumarate or its related preparations |
| CN114213285A (en) * | 2021-12-29 | 2022-03-22 | 斯坦德标准技术研究(湖北)有限公司 | Formoterol related substance, preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108362813A (en) * | 2017-12-29 | 2018-08-03 | 江苏悦兴医药技术有限公司 | A kind of method for detecting purity of principal component |
| CN110376313A (en) * | 2019-08-20 | 2019-10-25 | 广州健康元呼吸药物工程技术有限公司 | A method of impurity in detection formoterol fumarate or its related preparations |
| CN110376313B (en) * | 2019-08-20 | 2022-06-07 | 广州健康元呼吸药物工程技术有限公司 | Method for detecting impurities in formoterol fumarate or related preparation thereof |
| CN114213285A (en) * | 2021-12-29 | 2022-03-22 | 斯坦德标准技术研究(湖北)有限公司 | Formoterol related substance, preparation method and application thereof |
| CN114213285B (en) * | 2021-12-29 | 2022-07-29 | 斯坦德标准技术研究(湖北)有限公司 | Formoterol related substance, preparation method and application thereof |
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