Preparative liquid chromatography for separating nifedipine photodegradation impurities

Abstract

The invention provides a preparative liquid chromatography method of nifedipine photodegradation impurities. The method comprises the following steps: placing nifedipine under an illumination condition, carrying out isocratic elution on impurities generated by a sample by adopting preparative liquid chromatography, taking octadecylsilane chemically bonded silica as a stationary phase and taking acetonitrile-water as a mobile phase, collecting effluent at the wavelength of 265 nm, and enriching to obtain the nifedipine compound. The method is high in separation degree, and the purity of impurity monomers can reach 90% or above; acetonitrile-water is used as a mobile phase for elution, so that the method is simple and efficient; the method is suitable for separation of difficult-to-separate components. An impurity reference substance is more sufficiently provided for nifedipine impurity research, so that the quality of nifedipine is effectively controlled.

Description

Technical Field

[0001] The invention relates to the technical field of chemical pharmacy, and in particular to a preparation method for separating photodegradable impurities of nifedipine. Background Art

[0002] Nifedipine, chemical name is 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydro-3,5-pyridinedicarboxylic acid dimethyl ester, molecular formula is C 17 H 18 N2O6, the structural formula is as follows.

[0003]

[0004] Nifedipine is a yellow crystalline powder, odorless, and unstable in the presence of light. It is a dihydropyridine calcium antagonist used to prevent and treat angina pectoris caused by coronary heart disease, especially variant angina pectoris and angina pectoris caused by coronary artery spasm. It has no adverse effects on respiratory function, so it is suitable for patients with angina pectoris suffering from obstructive respiratory diseases, and its efficacy is better than that of beta-receptor antagonists. It is also suitable for various types of hypertension, and has good effects on refractory and severe hypertension. Because it can reduce afterload, it also has good effects on refractory congestive heart failure and is suitable for long-term use.

[0005] The study of the main degradation products of dihydropyridine calcium ion antagonists (Master's thesis of Tianjin University) has reported that nifedipine impurity I can degrade at a high rate under light conditions. This impurity has a warning group-nitroso. At the same time, the disappearance of the N-H bond in the parent nucleus of the nifedipine molecule-phenyl-1,4-dihydropyridine ring and the planarization of the parent nucleus will lead to the weakening of the activity of the drug. Therefore, the content of this impurity needs to be controlled. Referring to the requirements for impurity content in the 2020 edition of the Chinese Pharmacopoeia, this impurity needs to be controlled below 0.1%.

[0006] Nifedipine impurity II is obtained by oxidative dehydrogenation of nifedipine. Nifedipine impurity II can be obtained under both light and oxidation conditions. The active group in this impurity, the phenyl-1,4-dihydropyridine ring, undergoes oxidation, and its activity is weakened, so its content needs to be controlled. Referring to the requirements for impurity content in the 2020 edition of the Chinese Pharmacopoeia, this impurity needs to be controlled below 0.1%.

[0007] Nifedipine impurities III and IV are impurities obtained by deep oxidation of nifedipine molecules to form N-oxidized pyridine derivatives. These two impurities are mainly obtained under oxidative conditions. These two impurities are not included in the Chinese Pharmacopoeia, and there are no literature reports on their related pharmacological and toxicological properties, but both impurities contain a warning group - N-oxidized pyridine ring.

[0008] The impurity I (2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester) and impurity II (2,6-dimethyl-4-(2-nitrosophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester) of nifedipine have been included in the Chinese Pharmacopoeia, and their content in nifedipine raw materials is controlled to be no more than 0.1%; impurity I is controlled to be no more than 1.0% and impurity II is controlled to be no more than 0.5% in nifedipine tablets, as important testing items for drug inspection. However, in the actual study of the nifedipine photodegradation test of this project, in addition to the two main degradation products of impurity I and impurity II, other degradation products were also produced. Under light conditions, impurity A accounts for about 0.5% of nifedipine raw materials. Under light conditions, impurity A in nifedipine tablets increases to about 0.5% with the extension of light exposure time (as shown in the table below). Considering the structural characteristics of nifedipine, it is very likely to produce a warning structure. The impurity is unknown and its content is relatively large, exceeding 0.1%. According to the drug application requirements, the impurity needs to be separated and prepared, and its structure needs to be confirmed. The impurity is similar to nifedipine impurity I and nifedipine impurity II in terms of spatial structure, and the active groups are inactivated, so its content needs to be controlled.

[0009] Summary of the invention

[0010] Purpose of the invention: The present invention aims to separate the photodegradation impurities of nifedipine with higher purity for stricter control of drug quality.

[0011] Technical solution: The enrichment and analysis of nifedipine photodegradation impurities of the present invention comprises the following steps:

[0012]

[0013] The technical solution provided by the present invention comprises the following steps:

[0014] (1) Samples after nifedipine light test;

[0015] (2) Separation: The sample obtained in step (1) is subjected to preparative high pressure liquid separation. The specific preparation method is as follows:

[0016] Instrument: preparative liquid chromatograph; chromatographic column: chromatographic column with octadecylsilane bonded silica gel filler as stationary phase; acetonitrile-water (40:60v / v~60:40v / v) as mobile phase for isocratic elution; flow rate 10ml / min~25ml / min; wavelength 200nm~400nm; column temperature 25℃~35℃; injection volume 0.5ml~2.0ml;

[0017] (3) The prepared solution in step (2) is taken and enriched to finally obtain impurities.

[0018] In the present invention, the sample used is a sample of nifedipine placed in a light box for 10 days.

[0019] In the present invention, the preparation instrument used is a conventional preparation liquid chromatography instrument in the art, such as AgelaHP100, and its parameters and operations are conventional operations in the art.

[0020] In the present invention, the chromatographic column used preferably uses octadecylsilane bonded silica gel filler as the stationary phase, such as Innova! ODS-2, and the chromatographic column specification is 30 mm×250 mm, 5 μm.

[0021] In the present invention, preferably, the volume ratio of acetonitrile to water in the mobile phase is 50:50 (v / v).

[0022] In the present invention, preferably, the flow rate is 20 ml per minute, the detection wavelength is 265 nm, the column temperature is 30° C., an automatic sampler is used, and the injection volume is 1.0 ml.

[0023] In the present invention, the high-purity nifedipine photodegradation impurity A prepared can be used as a reference substance, the impurity can be qualitatively judged according to the peak time, and its content can be controlled by using relevant methods according to the specific structure.

[0024] The present invention mainly provides a method for preparing nifedipine photodegradation impurities, which is simple to operate and highly efficient, and the purity of the obtained compound monomer can reach more than 90%. Using the separation and preparation method of the present application, nifedipine photodegradation impurities can be effectively prepared, and the specificity is good, and the target substance can be effectively separated. The obtained product can be used as an impurity reference substance for subsequent quality research of nifedipine raw materials and preparations, providing a guarantee for the safe use of nifedipine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Preparative liquid chromatogram of nifedipine photodegradation impurities in Example 1

[0026] Figure 2 Example 1: Photodegradation of nifedipine impurities 1 HNMR spectrum

[0027] Figure 3 Example 1: Photodegradation of nifedipine impurities 13 CNMR spectrum

[0028] Figure 4 Mass spectrum of nifedipine photodegradation impurities in Example 1 (positive ion mode)

[0029] Figure 5 Mass spectrum of nifedipine photodegradation impurities in Example 1 (negative ion mode)

[0030] Figure 6 HPLC chromatogram of nifedipine photodegradation impurities in Example 1

[0031] Figure 7 Preparative liquid chromatogram of nifedipine photodegradation impurities in Example 2

[0032] Figure 8 HPLC chromatogram of nifedipine photodegradation impurities in Example 2

[0033] Fig. 9 Preparative liquid chromatogram of nifedipine photodegradation impurities in Example 3

[0034] Fig.10 HPLC chromatogram of nifedipine photodegradation impurities in Example 3

[0035] Fig.11 Preparative liquid chromatogram of nifedipine photodegradation impurities in Example 4

[0036] Fig.12 HPLC chromatogram of nifedipine photodegradation impurities in Example 4

[0037] Fig.13 Preparative liquid chromatogram of nifedipine photodegradation impurities in Example 5

[0038] Fig.14 HPLC chromatogram of nifedipine photodegradation impurities in Example 5

[0039] Fig.15 HPLC chromatogram of nifedipine API after 1h illumination

[0040] Fig.16 HPLC chromatogram of nifedipine API after 2h illumination

[0041] Fig.17 HPLC chromatogram of nifedipine API after 4 hours of illumination

[0042] Fig.18 HPLC chromatogram of nifedipine API after 6 days of illumination

[0043] Fig.19 HPLC chromatogram of nifedipine tablets after 3 hours of illumination

[0044] Fig. 20 HPLC chromatogram of nifedipine tablets after 6 hours of illumination

[0045] Fig.21 HPLC chromatogram of nifedipine tablets after 12 hours of illumination

[0046] Fig. 22 HPLC chromatogram of nifedipine tablets after 24 hours of illumination DETAILED DESCRIPTION

[0047] Example 1

[0048] (1) Experimental instruments and conditions

[0049] Instrument: Preparative high pressure liquid chromatography Agela HP100;

[0050] Chromatographic column: Innova! ODS-2, 30 mm × 250 mm, 5 μm;

[0051] Detector: UV detector;

[0052] Elution mode: isocratic elution;

[0053] Mobile phase: acetonitrile-water (50:50 v / v);

[0054] Column temperature: 30°C;

[0055] Flow rate: 20ml per minute;

[0056] Injection volume: 1.0ml.

[0057] (2) Experimental procedures

[0058] Solution preparation: Take a certain amount of nifedipine sample after 10 days of light exposure test, add acetonitrile to dissolve and dilute to a solution containing about 0.01g per 1ml.

[0059] Preparation of impurities: Collect the effluent at a wavelength of 265 nm according to the method under (1), and obtain the impurities after enrichment and rotary evaporation.

[0060] like Figure 1-6 As shown, the structure of the nifedipine photodegradation impurities obtained in the above example was analyzed by NMR and mass spectrometry data: 1 HNMR (400MHZ, CDCl3), δ7.70 (dd, 1H), 7.58 (dd, 1H), 7.47 (td, 1H), 7.30-7.26 (m, 1H), 5.75 (s, 1H), 5.69 (s, 1H), 3.61 (s, 6H), 2.37 (s, 6H). It can be seen that the separation and preparation method of the present application can effectively prepare nifedipine photodegradation impurity A, with a molecular weight of 347.12. The method has a high degree of separation, and the purity of the impurity A monomer can reach 96.24%.

[0061] The structural formula is as follows:

[0062]

[0063] Example 2

[0064] (2) Experimental instruments and conditions

[0065] Instrument: Preparative high pressure liquid chromatography Agela HP100;

[0066] Chromatographic column: Innova! ODS-2, 30 mm × 250 mm, 5 μm;

[0067] Detector: UV detector;

[0068] Elution mode: isocratic elution;

[0069] Mobile phase: acetonitrile-water (60:40 v / v);

[0070] Column temperature: 30°C;

[0071] Flow rate: 20ml per minute;

[0072] Injection volume: 1.0ml.

[0073] (2) Experimental procedures

[0074] Solution preparation: Take a certain amount of nifedipine sample after 10 days of light exposure test, add acetonitrile to dissolve and dilute to a solution containing about 0.01g per 1ml.

[0075] Preparation of impurities: Collect the effluent at a wavelength of 265 nm according to the method under (1), enrich it by rotary evaporation, and then freeze-dry it to obtain the impurities.

[0076] like Figure 7 , 8 As shown, it is the preparative liquid chromatogram of the nifedipine photodegradation impurity A obtained in the above example. Using the separation and preparation method of the present application, nifedipine photodegradation impurity A can be effectively prepared, and the monomer purity is 90.71%.

[0077] Example 3

[0078] (1) Experimental instruments and conditions

[0079] Instrument: Preparative high pressure liquid chromatography Agela HP100;

[0080] Chromatographic column: Innova! ODS-2, 30 mm × 250 mm, 5 μm;

[0081] Detector: UV detector;

[0082] Elution mode: isocratic elution;

[0083] Mobile phase: acetonitrile-water (40:60 v / v);

[0084] Column temperature: 30°C;

[0085] Flow rate: 20ml per minute;

[0086] Injection volume: 1.0ml.

[0087] (2) Experimental procedures

[0088] Solution preparation: Take a certain amount of nifedipine sample after 10 days of light exposure test, add acetonitrile to dissolve and dilute to a solution containing about 0.01g per 1ml.

[0089] Preparation of impurities: Collect the effluent at a wavelength of 265 nm according to the method under (1), enrich it by rotary evaporation, and then freeze-dry it to obtain the impurities.

[0090] like Fig. 9 , 10 As shown, it is the preparative liquid chromatogram of the nifedipine photodegradation impurity A obtained in the above example. Using the separation and preparation method of the present application, nifedipine photodegradation impurity A can be effectively prepared, and the monomer purity is 92.46%.

[0091] Example 4

[0092] (1) Experimental instruments and conditions

[0093] Instrument: Preparative high pressure liquid chromatography Agela HP100;

[0094] Chromatographic column: Innova! ODS-2, 30 mm × 250 mm, 5 μm;

[0095] Detector: UV detector;

[0096] Elution mode: isocratic elution;

[0097] Mobile phase: acetonitrile-water (50:50 v / v);

[0098] Column temperature: 25°C;

[0099] Flow rate: 20ml per minute;

[0100] Injection volume: 2.0ml.

[0101] (2) Experimental procedures

[0102] Solution preparation: Take a certain amount of nifedipine sample after 10 days of light exposure test, add acetonitrile to dissolve and dilute to a solution containing about 0.01g per 1ml.

[0103] Preparation of impurities: Collect the effluent at a wavelength of 265 nm according to the method under (1), enrich it by rotary evaporation, and then freeze-dry it to obtain the impurities.

[0104] like Fig.11 , 12As shown, it is the preparative liquid chromatogram of the nifedipine photodegradation impurity A obtained in the above example. Using the separation and preparation method of the present application, nifedipine photodegradation impurity A can be effectively prepared, and the monomer purity is 75.31%.

[0105] Example 5

[0106] (1) Experimental instruments and conditions

[0107] Instrument: Preparative high pressure liquid chromatography Agela HP100;

[0108] Chromatographic column: Innova! ODS-2, 30 mm × 250 mm, 5 μm;

[0109] Detector: UV detector;

[0110] Elution mode: isocratic elution;

[0111] Mobile phase: acetonitrile-water (45:55 v / v);

[0112] Column temperature: 30°C;

[0113] Flow rate: 25ml per minute;

[0114] Injection volume: 2.0ml.

[0115] (2) Experimental procedures

[0116] Solution preparation: Take a certain amount of nifedipine sample after 10 days of light exposure test, add acetonitrile to dissolve and dilute to a solution containing about 0.01g per 1ml.

[0117] Preparation of impurities: Collect the effluent at a wavelength of 265 nm according to the method under (1), enrich it by rotary evaporation, and then freeze-dry it to obtain the impurities.

[0118] like Fig.13 , 14 As shown, it is the preparative liquid chromatogram of the nifedipine photodegradation impurity A obtained in the above example. Using the separation and preparation method of the present application, nifedipine photodegradation impurity A can be effectively prepared, and the monomer purity is 91.39%.

Claims

1. A preparative liquid chromatography method for separating nifedipine photodegradation impurity A, characterized in that Proceed as follows: (1) performing a light test on nifedipine to obtain a sample; (2) Separation: The sample obtained in step (1) is subjected to preparative high pressure liquid separation. The specific preparation method is as follows: Instrument: preparative liquid chromatograph; chromatographic column: chromatographic column with octadecylsilane bonded silica gel filler as stationary phase; acetonitrile-water (40:60v / v~60:40v / v) as mobile phase for isocratic elution; flow rate 10ml / min~25ml / min; wavelength 200nm~400nm; column temperature 25℃~35℃; injection volume 0.5ml~2.0ml; (3) The prepared solution in step (2) is enriched and rotary evaporated to finally obtain impurities.

2. The preparation method according to claim 1, characterized in that: The obtained sample is a sample after nifedipine has been placed in a light box for 10 days.

3. The preparation method according to claim 1, characterized in that: The preparation instrument used is a conventional preparation liquid chromatography instrument in the art, such as Agela HP100, and its parameters and operations are conventional operations in the art.

4. The preparation method according to claim 1, characterized in that: The chromatographic column used is an octadecylsilane bonded silica gel chromatographic column, such as Innova! ODS-2, and the chromatographic column specification is 30 mm×250 mm, 5 μm.

5. The preparation method according to claim 1, characterized in that: The volume ratio of acetonitrile to water in the mobile phase was 50:50 (v / v).

6. The preparation method according to claim 1, characterized in that: The flow rate was 20 ml / min, the detection wavelength was 265 nm, and the column temperature was 30 °C.

7. The preparation method according to claim 1, characterized in that: An autosampler was used with an injection volume of 1.0 ml.

8. Application of the method according to claim 1 in preparing high-purity photodegradation impurity A of nifedipine.

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