CN108169362B - Method for separating carbamazepine and related substances by liquid chromatography - Google Patents

Abstract

The invention relates to a method for separating carbamazepine and related substances by using liquid chromatography, which is high performance liquid chromatography. The invention can improve the sensitivity of impurity detection, especially the sensitivity of impurity E detection, by changing the mobile phase system and the sample injection amount, thereby effectively controlling the quality of carbamazepine.

Description

Method for separating carbamazepine and related substances by liquid chromatography

Technical Field

The invention belongs to the technical field of medicine detection, and particularly relates to a method for separating carbamazepine and related substances by using liquid chromatography.

Background

Carbamazepine is a common psychotropic drug. The clinical application is mainly as follows: epilepsy: partial seizures: complex partial seizures, simple partial seizures and secondary generalized seizures. Systemic attack: tonic, clonic, tonic clonic attack. The onset of trigeminal neuralgia and glossopharyngeal neuralgia, and is also used as a long-term preventive drug after the remission of trigeminal neuralgia. Can also be used for tuberculosis of spinal cord and multiple sclerosis, and the carbamazepine has the following names: carbamoylbenzene, carbamoylazepine, atropine, furazolidone, delidol, delavirdol, fenlepsin, carbamoylbenzene, carbaimizine, carbamazepine, chemical name 5H-dibenzo [ b, f ] azepine-5-carboxamide, CAS number 298-46-4.

During the preparation and storage of the drug, it is necessary to monitor impurities that may be generated and degraded during the synthesis process. Such impurities are also referred to as related substances because their chemical structures are generally similar or have a source relationship with the active ingredient. Therefore, the separation of the carbamazepine and the related substances thereof is realized, and the method has important practical significance in the aspects of synthesis of the carbamazepine and quality control of a preparation process.

The standard of carbamazepine medicine is recorded on page 182 of the second part of the Chinese pharmacopoeia 2015 edition, and an inspection method of related substances of carbamazepine is described in the standard, specifically, the product is taken about 50mg and placed in a50m L measuring flask, methanol 25m L is added to dissolve the product, the product is diluted to scales by water and shaken up to be used as a test solution, the test solution lm L is precisely measured and placed in a50m L measuring flask, the test solution lm 1 is diluted to scales by methanol-water (1:1) and shaken up to scales, the test solution and the test solution are precisely measured to be 20 mu L respectively according to chromatographic conditions and methods under content items, a chromatogram is precisely measured and injected into a liquid chromatograph, the chromatogram is recorded until the chromatogram is 6 times of the retention time of a main component peak, if an impurity peak exists in the test solution, the peak area of a single impurity cannot be larger than that of the main component of the test solution (the area of the single impurity peak area is not larger than 0.2%) and the area of the main component of the impurity solution is not larger than 2.5 times of the main component of the reference solution (5% of the impurity peak area).

The specific liquid chromatographic conditions are that nitrile propyl silane bonded silica gel is used as a filling agent in chromatographic condition and system applicability tests, methanol-tetrahydrofuran-water (120:30:850) is used as a mobile phase (0.2 m L formic acid and 0.5m L triethylamine are added into 1000m L solution), the detection wavelength is 230nm, about 25mg of carbamazepine reference substance is taken, the carbamazepine reference substance is placed into a 100m L measuring flask, the methanol-water (1:1) is used for dissolving and diluting to a scale, the mixture is shaken up, 20 mu L is taken and injected into a liquid chromatograph, the number of theoretical plates is not less than 5000 according to the carbamazepine, and the separation degree of the carbamazepine peak and the adjacent impurity peak meets the requirement.

The European pharmacopoeia, the United states pharmacopoeia and the Japanese pharmacopoeia also specify a method for examining substances related to carbamazepine, and the liquid phase conditions are the same as those in the Chinese pharmacopoeia except that the European pharmacopoeia, the United states pharmacopoeia and the Japanese pharmacopoeia list specific impurity structural formulas of carbamazepine and the content requirements of part of impurities. The impurity structures for carbamazepine are specified in the following table:

as is clear from the above table, the japanese pharmacopoeia is relatively simple in the regulation of impurities of carbamazepine, and therefore, in the case where the detection conditions are the same, the standards in the united states pharmacopoeia and european pharmacopoeia are preferable in the regulation of the impurity type and the impurity amount of carbamazepine. The liquid phase and impurity content specifications in the chinese pharmacopoeia, the united states pharmacopoeia, and the european pharmacopoeia for carbamazepine are shown in the following table:

from the above-mentioned specifications of the chinese pharmacopoeia, the united states pharmacopoeia and the european pharmacopoeia on the amount of the particular impurities of carbamazepine, the most important impurities of carbamazepine are impurity a, impurity B and impurity E, and no specific specification and requirement on the amount of other impurities are made.

Impurities of carbamazepine are associated with specific synthetic routes. The common synthetic route of carbamazepine (research on clean production process of carbamazepine) is as follows:

route 1:

route 2:

the synthesis route of the carbamazepine raw material medicine of the company is as follows:

referring to the european pharmacopoeia, the united states pharmacopoeia and the japanese pharmacopoeia, it is found that carbamazepine contains impurity a, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G, and it is possible to determine potential process impurities and degradation impurities into impurity a, impurity B, impurity C, impurity D and impurity E according to the synthetic route provided by the company, wherein impurity F and impurity G are not generated by the synthetic route. In the synthetic route of the company, the specific degradation routes of the impurity A, the impurity B, the impurity C, the impurity D and the impurity E are as follows:

impurity A

Impurity a results from the synthesis process and degradation pathway:

the process approach is as follows:

degradation route:

impurities B and D

Impurity B, D API hydrolysate and isomerate of hydrolysate, possible reaction pathway (verifiable by destructive testing and with L CMS):

in addition impurity D is also the starting material for route 2;

impurity C

The impurity C is a byproduct of a reaction product of the intermediate 3 and isocyanic acid and a degradation impurity;

degradation route: degrading carbamazepine into impurity A and formamide under an acidic condition; under acidic conditions, formamide reacts with carbamazepine to form impurity C.

Impurity E

The impurity E provides process impurities and degradation impurities caused by starting materials of a synthetic route for the route 1 and a manufacturer;

degradation route:

impurity F

Impurity F is the third intermediate product of scheme 1.

Impurity G

The synthetic route provided by this company shows that the impurity G is not an impurity generated by this synthetic route, and the explanation thereof is omitted.

The potential process and degradation impurities can be identified according to the company providing a synthetic route to impurities a, B, C, D and E, wherein impurities F and G are not produced by the synthetic route. Therefore, only the impurity a, the impurity B, the impurity C, the impurity D, and the impurity E need to be detected based on the principle applicable to the specific situation of the present company.

The inventor finds in a specific test that detection of impurities of carbamazepine according to the liquid phase conditions of the Chinese pharmacopoeia has the defects that the signal-to-noise ratio of each impurity of carbamazepine is low at 9-10 mug, the risk is high when the method is carried out, and particularly the sensitivity of the impurity E is low, when the impurity E of carbamazepine is 0.5 mug/m L, the integration fails, when the impurity E is 2.0 mug/m L, the integration can be carried out, but the signal-to-noise ratio is too low, the concentrations of solutions of ChP, USP and EP test samples are 1.0-1.5mg, the impurity limit is about 0.1%, the limit concentration is 1.0-1.5 mug/m L, the signal-to-noise ratio of the impurity E2 mug/m L is only 1.67, the sensitivity of the method is not sufficient, when Waters E2695-2489 is used, the signal-to-noise ratio of the impurity peak is not greatly changed, when the impurity concentration is 1 mug/m L, the signal-to-signal-to-noise ratio of the impurity E, and therefore, the new liquid.

Disclosure of Invention

The invention aims to provide a method for separating carbamazepine and related substances thereof.

The invention also aims to provide a purity identification method of the carbamazepine raw material drug or the preparation thereof so as to realize the quality control of the carbamazepine raw material drug or the preparation thereof.

The invention provides a method for separating carbamazepine and related substances by using liquid chromatography, which is high performance liquid chromatography.

The high performance liquid chromatography comprises the following chromatographic conditions that a chromatographic column filler is nitrile propyl silane bonded silica gel, a mobile phase is ammonium dihydrogen phosphate solution-acetonitrile, the elution mode of the mobile phase is linear gradient elution, a detector is an ultraviolet detector, and the sample injection amount is 20 mu L-50 mu L.

The concentration of the ammonium dihydrogen phosphate solution is 30-60 mmol/L, preferably, the concentration of the ammonium dihydrogen phosphate solution is 50 mmol/L.

The mobile phase linear gradient elution conditions are as follows: the elution time is 50-70min, the initial volume ratio of the ammonium dihydrogen phosphate solution to the acetonitrile is 90:10-80:20, the final volume ratio of the ammonium dihydrogen phosphate solution to the acetonitrile is 80:20-60:40, and the flow rate of the mobile phase is 0.8-1.2 ml/min.

Preferably, the mobile phase linear gradient elution conditions are as follows: the initial volume of the ammonium dihydrogen phosphate solution and the acetonitrile is preferably 89:11, the termination ratio is 70:30, and the flow rate of the mobile phase is preferably 1.0 ml/min; the elution time is preferably 65 min.

The sample size was 50 μ L.

The detection wavelength of the ultraviolet detector is 215 nm.

The carbamazepine is carbamazepine, a carbamazepine eutectic or a carbamazepine intermediate; the related substances are impurities A to E.

The invention relates to a method for separating carbamazepine and related substances by using liquid chromatography, which comprises the following steps:

(1) chromatographic conditions are as follows:

performing linear gradient elution according to the following table by using nitrile propyl silane bonded silica gel as a filling agent, a mobile phase A of 50 mmol/L ammonium dihydrogen phosphate solution and a mobile phase B of acetonitrile, wherein a detector is an ultraviolet detector, and the detection wavelength is 215 nm;

(2) inspection method

Preparing a test solution, namely precisely weighing 50mg of carbamazepine, putting the carbamazepine into a50m L measuring flask, adding 25m L of methanol to dissolve the carbamazepine, diluting the carbamazepine with water to a scale, and shaking up the solution to be used as the test solution;

preparation of control solution A sample solution lm L was precisely measured, placed in a50m L measuring flask, diluted to the scale with methanol-water (1:1), shaken up, 5m L was precisely measured, placed in a50m L measuring flask, diluted to the scale with methanol-water (1:1), shaken up, and used as a control solution;

precisely measuring the reference solution and the sample solution at 50 μ L, respectively, injecting into a liquid chromatograph, and recording chromatogram.

The above method for separating carbamazepine and related substances by liquid chromatography is suitable for separating and detecting carbamazepine, carbamazepine preparations and impurities A-E of the carbamazepine related substances.

The method for determining the separation effect of the carbamazepine and related substances comprises the steps of precisely weighing 25mg of the carbamazepine, placing the weighed carbamazepine into a 100m L measuring flask, diluting the carbamazepine with methanol-water (1:1) until the calibration is uniform, taking 20 mu L of a control solution, injecting the control solution into a liquid chromatograph, calculating the theoretical plate number by using the peak of the carbamazepine to be not less than 5000, and determining the separation degree of the peak of the carbamazepine and the adjacent impurity peak to be more than 1.7.

If an impurity peak exists in a chromatogram of the test solution, the contents of impurities A and E: no more than half (0.10%) of the peak area of carbamazepine in the chromatogram of control solution (a) for each impurity; unspecified impurities: no more than half (0.10%) of the peak area of carbamazepine in the chromatogram of control solution (a); total amount: not more than 2.5 times (0.5%) of the peak area of carbamazepine in the chromatogram of control solution (a).

The separation method of carbamazepine and related substances thereof provided by the invention has the following advantages:

1. compared with the method for separating the carbamazepine and related substances by using the liquid chromatography recorded in pharmacopoeia, the invention can improve the sensitivity of detecting impurities, particularly the sensitivity of detecting the impurity E by changing a mobile phase system and a sample amount, thereby effectively controlling the quality of the carbamazepine.

2. The method provided by the invention can be applied to the detection of the individual purity of each relevant substance of carbamazepine. Because each related substance of the carbamazepine has a mother nucleus with the same chemical structure as the carbamazepine, the structure is very similar, and the detection method of each related substance is the same as that of the carbamazepine.

3. The method provided by the invention can effectively separate the carbamazepine and 5 related substances thereof, can comprehensively detect and control the related substance impurities possibly existing in the carbamazepine, and can also be used for accurately quantifying the carbamazepine and the related substances thereof.

4. The separation method adopts a universal chromatographic column, and is simple, accurate and convenient to operate.

Drawings

Fig. 1 is a liquid chromatogram of a systematic adaptability experiment performed on carbamazepine according to chromatographic conditions specified in the chinese pharmacopoeia.

FIG. 2 is a liquid chromatogram of a linear range determination experiment on carbamazepine according to chromatographic conditions specified in the Chinese pharmacopoeia, with an impurity concentration of carbamazepine of 0.5 μ g/m L.

FIG. 3 is a liquid chromatogram of a linear range determination experiment on carbamazepine according to chromatographic conditions specified in the Chinese pharmacopoeia, with an impurity concentration of 2.0 μ g/m L.

FIG. 4 is a linear range determination experimental chromatogram of carbamazepine with an impurity concentration of 1.0 μ g/m L using a different instrument (Waterse2695-2489) according to the chromatographic conditions specified in the Chinese pharmacopoeia.

FIG. 5 is a graph of the separation of impurities from carbamazepine using chromatographic conditions of an acetonitrile-water gradient elution regime, with a carbamazepine impurity concentration of 9 μ g/m L.

FIG. 6 shows a mobile phase A of 0.4% formic acid and 0.10% triethylamine solution, and a mobile phase B of acetonitrile gradient elution mode for testing the separation degree of impurities of carbamazepine, wherein the impurity concentration of carbamazepine is 9 mug/m L.

FIG. 7 is a chromatogram of optimized mobile gradient phases of carbamazepine and its impurities (mobile phase A0.4% formic acid and 0.10% triethylamine solution; mobile phase B acetonitrile).

FIG. 8 is a chromatogram 3D of carbamazepine and its impurities after further optimization of the mobile gradient phase (mobile phase A0.4% formic acid and 0.10% triethylamine solution; mobile phase B acetonitrile).

FIG. 9 is a chromatogram of carbamazepine and its impurities after further optimization of mobile gradient phase (mobile phase A0.4% formic acid and 0.10% triethylamine solution; mobile phase B acetonitrile).

FIG. 10 is a chromatogram of carbamazepine and its impurities (mobile phase A0.4% formic acid and 6 g/L sodium dihydrogen phosphate solution; mobile phase B acetonitrile).

FIG. 11 is a chromatogram of carbamazepine and its impurities in an isocratic mobile phase (mobile phase A0.4% formic acid and 6 g/L sodium dihydrogen phosphate solution; mobile phase B acetonitrile).

FIG. 12 is a chromatogram of carbamazepine and various impurities under an optimized mobile phase gradient (mobile phase A0.4% formic acid and 0.10% triethylamine solution; mobile phase B is acetonitrile, detection wavelength 215nm, and impurity sample concentration is about 9 μ g/ml).

FIG. 13 is a chromatogram of carbamazepine and its impurities under an optimized mobile phase gradient (mobile phase A0.4% formic acid and 0.10% triethylamine solution; mobile phase B is acetonitrile, detection wavelength 215nm, and impurity sample concentration is about 0.5. mu.g/ml).

FIG. 14 is a chromatogram of carbamazepine and various impurities under an optimized mobile phase gradient (mobile phase A is a50mmol ammonium dihydrogen phosphate solution; mobile phase B is acetonitrile, the detection wavelength is 215nm, and the concentration of the impurity sample is about 5 mu g/ml).

FIG. 15 is a chromatogram of carbamazepine and various impurities under the condition of further optimizing the gradient of the mobile phase and increasing the sample volume (mobile phase A is 50mmol ammonium dihydrogen phosphate solution; mobile phase B is acetonitrile, the detection wavelength is 215nm, and the concentration of the impurity sample is about 5 mu g/ml).

FIG. 16 is a chromatogram of carbamazepine and various impurities under the condition of further optimizing the gradient of the mobile phase and increasing the sample volume (mobile phase A is 50mmol ammonium dihydrogen phosphate solution; mobile phase B is acetonitrile, the detection wavelength is 215nm, and the concentration of the impurity sample is about 0.5 mu g/ml).

FIG. 17 is a chromatogram of undamaged carbamazepine in an acid destruction test (mobile phase A50mmol ammonium dihydrogen phosphate solution; mobile phase B acetonitrile, detection wavelength 215 nm).

FIG. 18 is a chromatogram of carbamazepine subjected to acid destruction in the acid destruction test (mobile phase A50mmol ammonium dihydrogen phosphate solution; mobile phase B acetonitrile, detection wavelength 215 nm).

FIG. 19 is a chromatogram of undamaged carbamazepine in a base destruction test (mobile phase A50mmol ammonium dihydrogen phosphate solution; mobile phase B acetonitrile, detection wavelength 215 nm).

FIG. 20 is a chromatogram of carbamazepine subjected to base destruction in the base destruction test (mobile phase A50mmol ammonium dihydrogen phosphate solution; mobile phase B acetonitrile, detection wavelength 215 nm).

FIG. 21 is a chromatogram of undamaged carbamazepine in a high temperature destruction test (mobile phase A50mmol ammonium dihydrogen phosphate solution; mobile phase B acetonitrile, detection wavelength 215 nm).

FIG. 22 is a chromatogram of carbamazepine destroyed at high temperature in the high temperature destruction test (mobile phase A50mmol ammonium dihydrogen phosphate solution; mobile phase B acetonitrile, detection wavelength 215 nm).

FIG. 23 is a chromatogram of undamaged carbamazepine in a light irradiation damage test (mobile phase A50mmol ammonium dihydrogen phosphate solution; mobile phase B acetonitrile, detection wavelength 215 nm).

FIG. 24 is a chromatogram of a radiation-damaged carbamazepine in a radiation damage test (mobile phase A50mmol ammonium dihydrogen phosphate solution; mobile phase B acetonitrile, detection wavelength 215 nm).

FIG. 25 shows a liquid chromatogram of a carbamazepine drug (lot 2017120501) (mobile phase A50mmol ammonium dihydrogen phosphate solution; mobile phase B acetonitrile, detection wavelength 215 nm).

FIG. 26 shows a liquid chromatogram of a commercial sample, 1 carbamazepine, as a crude drug (mobile phase A50mmol ammonium dihydrogen phosphate solution; mobile phase B acetonitrile, detection wavelength 215 nm).

FIG. 27 is a chromatogram of the bulk drug liquid of a commercial sample, 2 carbamazepine (mobile phase A50mmol ammonium dihydrogen phosphate solution; mobile phase B acetonitrile, detection wavelength 215 nm).

In the above figures, the unit of the abscissa is min, and the unit of the ordinate is AU.

Detailed Description

The invention will be further described with reference to the accompanying drawings and specific embodiments so that those skilled in the art may better understand the invention, but the invention is not limited thereto.

Unless otherwise specified, concentrations generally refer to the weight to volume ratio of solute to solution, g/ml. If the solute is a liquid, the concentration is by volume, e.g., 0.1% aqueous triethylamine solution means 0.1ml triethylamine dissolved in 100ml water.

The purity of the standard substance of the carbamazepine and related substances thereof is more than 99 percent, and the standard substance is provided by Shandong Zhengyao medicine science and technology limited company;

the purity of the chemical reagents is of chromatographic purity grade and is respectively purchased from Beijing limited chemical reagents of national drug group.

The sample preparation methods are as follows: carbamazepine and impurity A, B, C, D, E were weighed out precisely and diluted with methanol-water (1:1) to the concentrations in the examples and experiments.

Experimental example 1 verification of chromatographic conditions in pharmacopoeia

A systematic adaptation experiment was performed on carbamazepine according to the chromatographic conditions specified in the chinese pharmacopoeia, using a sample of carbamazepine produced by this company under batch number 2017021303. The specific chromatographic conditions were as follows:

(1) chromatographic conditions

(2) Sample preparation

(3) The results are shown in FIG. 1. Separation of carbamazepine and impurities a, B, C, D and E was as follows.

Name (R)	Retention time	Area of	Number of theoretical plate	Degree of separation	S/N (Signal-to-noise ratio)
						Impurity B	9.153	439898	3861		58.06
Impurity A	12.379	162994	3594	4.70	15.93
						Carbamazepine	14.193	8501116	3802	2.13	725.00
Impurity C	21.205	482508	3563	6.03	26.41
						Impurity D	41.771	536433	4750	10.87	17.94
Impurity E	54.148	149517	5535	4.65	4.27

(4) Conclusion

As can be seen from fig. 1 and the above results, the signal to noise ratio of each impurity of carbamazepine is low at 9-10 μ g, and the risk is high when the method is confirmed, especially the sensitivity of impurity E is low.

A linear range determination experiment was performed on carbamazepine according to the chromatographic conditions specified in the chinese pharmacopoeia, using a sample of carbamazepine produced by this company, batch number 2017021601. The specific chromatographic conditions were as follows:

(1) chromatographic conditions

(2) When the impurity concentration of carbamazepine is 0.5 mu g/m L, the chromatogram is shown in figure 2. the separation of carbamazepine and impurities A, B, C, D and E is as follows.

Name (R)	Retention time	Area of	Theoretical towerNumber of plates	S/N
					Impurity B	8.806	27501	3624	5.00
Impurity A	12.441	10907	1535	1.40
					Impurity C	21.333	23036	3920	2.14
Impurity D	41.443	13035	11516	1.00
					Impurity E	N/A	N/A

(3) When the impurity of carbamazepine is 2.0 mu g/m L, the chromatogram is shown in figure 3. the separation of carbamazepine and impurities A, B, C, D and E is as follows.

(4) Cause analysis that the mobile phase used in the ChP, USP and EP methods is methanol to tetrahydrofuran to water 12:3:85, 0.2m L anhydrous formic acid and 0.5m L triethylamine were added to 1.0L of the solution, and the tetrahydrofuran, triethylamine and formic acid have uv absorbance at 230nm, masking a portion of the uv absorbance of each impurity of carbamazepine, causing the response of each impurity of carbamazepine to be lower, resulting in a lower signal to noise ratio.

The concentrations of the ChP, USP and EP test solution are 1.0-1.5mg, the impurity limit is about 0.1%, the limit concentration is 1.0-1.5 mu g/m L, the signal-to-noise ratio of the impurity E2 mu g/m L is only 1.67, and the method has insufficient sensitivity.

Linear range determination experiments were performed on carbamazepine using different instruments (Waters e2695-2489) according to the chromatographic conditions specified in the chinese pharmacopoeia. The specific chromatographic conditions were as follows:

(1) chromatographic conditions

(2) When the impurity concentration of carbamazepine is 1. mu.g/m L, the chromatogram is shown in FIG. 4.

(3) Conclusion

When Waters E2695-2489 was used, the signal-to-noise ratio of the impurity peak did not change much, and when the impurity concentration was 1. mu.g/m L, the signal-to-noise ratio of the impurity E could not satisfy the conditions for verification.

The inventor finds in a specific test that detection of impurities of carbamazepine according to the liquid phase conditions of the Chinese pharmacopoeia has the defects that the signal-to-noise ratio of each impurity of carbamazepine is low at 9-10 mug, the risk is high when the method is carried out, and particularly the sensitivity of the impurity E is low, when the impurity E of carbamazepine is 0.5 mug/m L, the integration fails, when the impurity E is 2.0 mug/m L, the integration can be carried out, but the signal-to-noise ratio is too low, the concentrations of solutions of ChP, USP and EP test samples are 1.0-1.5mg, the impurity limit is about 0.1%, the limit concentration is 1.0-1.5 mug/m L, the signal-to-noise ratio of the impurity E2 mug/m L is only 1.67, the sensitivity of the method is not sufficient, when Waters E2695-2489 is used, the signal-to-noise ratio of the impurity peak is not greatly changed, when the impurity concentration is 1 mug/m L, the signal-to-signal-to-noise ratio of the impurity E, and therefore, the new liquid.

Example 1 investigation of conditions for detection of carbamazepine-related substance

1. Firstly, a mobile phase system is changed, tetrahydrofuran is contained in the mobile phase system, and the tetrahydrofuran is absorbed at a low wavelength position, possibly covering impurity peaks, and an acetonitrile-water gradient elution mode is adopted. The new chromatographic conditions were as follows:

(1) chromatographic conditions

(2) The chromatogram obtained is shown in FIG. 5. The separation of carbamazepine and various impurities was as follows:

(3) conclusion

The concentration of each impurity of carbamazepine is about 9 mu g/m L, one impurity in the carbamazepine has no peak, and other impurities are well separated.

2. Changing the mobile phase A into 0.4% formic acid aqueous solution and 0.10% triethylamine aqueous solution, wherein the specific chromatographic conditions are as follows:

(1) chromatographic conditions

(2) And (6) obtaining the result. The liquid chromatogram is shown in figure 6.

Name (R)	Retention time
		Impurity B	12.971
Impurity A	17.479
		Impurity C	24.903
Impurity D	33.285
		Impurity E	35.439

(3) Conclusion

Using 0.4% formic acid and 0.10% triethylamine solution and acetonitrile as mobile phases, the impurities of carbamazepine were completely separated at this gradient.

3. The flowing gradient phase is optimized, and the specific chromatographic conditions are as follows:

(1) chromatographic conditions

The results are shown in FIG. 7. As can be seen from FIG. 7, impurity C appears on the gradient peak

4. The mobile phase gradient is further optimized, and the specific chromatographic conditions are as follows:

(1) chromatographic conditions

(2) The results are shown in FIGS. 8 and 9.

(3) And (4) conclusion: the peak of impurity C appears on the gradient peak. The optimal absorption wavelength for all impurities is 215nm as seen by the 3D spectrum. 5. The salt in the mobile phase is changed into sodium dihydrogen phosphate, and the specific chromatographic conditions are as follows:

(1) chromatographic conditions

(2) The results are shown in FIG. 10.

(3) And (4) conclusion: after formic acid and triethylamine in the mobile phase are changed into disodium hydrogen phosphate, the peak of the carbamazepine impurity is reduced by one.

6. Using isocratic mobile phases

(1) Chromatographic conditions

(2) The results are shown in FIG. 11.

(3) And (4) conclusion: impurity E does not peak.

7. Optimizing mobile phase gradients

(1) Chromatographic conditions

(2) The results are shown in FIG. 12. The respective impurities and the specific separation are as follows.

Name (R)	Retention time	Area of	Number of theoretical plate	S/N
					Impurity B	12.660	760298	5482	224.03
Impurity A	18.440	782884	5526	163.65
					Carbamazepine	20.613	23979200	5551	4406.03
Impurity C	29.316	1169718	5167	150.88
					Impurity D	38.942	915085	92716	358.86
Impurity E	41.932	1235700	60399	355.03

(3) Conclusion

The triethylamine and formic acid are used as salts in the mobile phase, so that a larger gradient is generated, the noise is larger, and the improvement of the signal-to-noise ratio is not ideal.

8. Optimizing mobile phase gradients

(1) Chromatographic conditions

(2) The results are shown in FIG. 13.

Name (R)	Retention time	Area of	Number of theoretical plate	S/N
					Impurity B	15.343	36948	7333	27.10
Impurity A	18.577	40236	7535	24.27
					Impurity C	29.580	48880	8428	19.86
Impurity D	40.448	39787	40952	27.06
					Impurity E	46.050	53088	33839	28.96

(3) Conclusion

The effect of changing the height of the gradient peak is not obvious by making the gradient of the mobile phase become gentle.

9. Changing the salt in the mobile phase to ammonium phosphate salt

Due to the terminal absorption of triethylamine, the absorption is obvious at 215nm, a large gradient peak is generated during gradient change, and meanwhile, impurity signals are buried to a certain extent. Therefore, ammonium dihydrogen phosphate having a small terminal absorption was selected for the test.

(1) Chromatographic conditions

(2) The results are shown in FIG. 14

Name (R)	Retention time	Area of	Number of theoretical plate	Degree of separation	S/N
						Impurity B	14.254	450486	4949		260.16
Impurity A	18.752	483671	5657	5.07	234.26
						Carbamazepine	20.814	168040465	4338	1.66	52889.68
Impurity C	29.740	598529	5960	5.91	190.53
						Impurity D	40.792	584927	37155	9.36	320.95
Impurity E	46.697	730909	26678	6.04	298.09

(3) Conclusion

After the salt in the mobile phase A is changed into ammonium dihydrogen phosphate, the signal-to-noise ratio of impurities in the carbamazepine is improved, the gradient peak is very slow, and the risk that the integral of unknown impurities on the gradient peak cannot be obtained is reduced.

10. The mobile phase gradient is optimized, the sample size is changed to 50 mu L, and the aim of the operation is to continuously improve the signal-to-noise ratio

(1) Chromatographic conditions

(2) The results are shown in FIGS. 15 and 16.

Name (R)	Retention time	Area of	Number of theoretical plate	Degree of separation	S/N
						Impurity B	15.358	1130860	4868		627.19
Impurity A	20.674	1242623	5028	5.28	538.16
						Carbamazepine	23.268	265594201	2787	1.50	58267.54
Impurity C	34.244	1771282	9588	5.96	624.54
						Impurity D	43.881	1470892	52146	9.03	927.80
Impurity E	48.881	1896889	33710	5.57	852.55

Name (R)	Retention time	Area of	Number of theoretical plate	Degree of separation	S/N
						Impurity B	15.495	100094	4490		57.02
Impurity A	20.712	123726	4621	5.09	55.53
						Impurity C	34.283	156252	9738	10.98	62.03
Impurity D	43.878	111748	50582	9.24	74.81
						Impurity E	48.866	171804	32749	5.52	81.98

(3) Conclusion

After the sample injection amount is increased from 20 mu L to 50 mu L, the signal-to-noise ratio is obviously increased, but the separation degree of the impurity A and the main peak is reduced to 1.50, the conditions are met, the gradient is used for slowing down the gradient peak, the integral of the impurity peak is not greatly influenced, the benefit and disadvantage factors are comprehensively considered, and finally the liquid phase condition of the method is used.

The chromatographic conditions and the method of the finally drawn up carbamazepine related substances are as follows:

example 2 destructive testing

1 acid, base and oxidative destruction (sample set: 2017032801)

1.1 undamaged raw material medicine

Taking 75mg of carbamazepine raw material, precisely weighing, placing in a50 ml measuring flask, adding 25ml of methanol for dissolving, performing ultrasonic treatment to dissolve, adding water for diluting to a scale, and shaking up to obtain the carbamazepine. Weighing 5.00ml of solution into a50 ml volumetric flask, adding 22ml of methanol, adding water to dilute to the scale, and shaking up to obtain the product.

The results are shown in FIG. 17:

name (R)	Retention time	Peak name	Angle of purity	Threshold of purity	Area of	Area%	Degree of separation	s/n
									1	21.338	Main peak	0.232	0.345	52876503	100		12862.92

1.2 acid breakdown

Acid-base blank, transferring 1 mol/L hydrochloric acid solution and 5.00ml sodium hydroxide solution into a50 ml volumetric flask, adding 25ml methanol solution, adding water to dilute to scale, and shaking up to obtain the final product.

Taking 75mg of carbamazepine raw material, precisely weighing, placing in a50 ml measuring flask, adding 5ml of methanol for dissolving, then adding 5ml of 1 mol/L hydrochloric acid solution, heating for 3 hours under the condition of 80 ℃ water bath, cooling, adding 5ml of 1 mol/L sodium hydroxide solution for neutralizing, adding 20ml of methanol, ultrasonically dissolving, adding water for diluting to scale, shaking uniformly to obtain the carbamazepine water soluble tablet, measuring 5.00ml of solution in a50 ml measuring flask, adding 22ml of methanol, adding water for diluting to scale, and shaking uniformly to obtain the carbamazepine water soluble tablet.

The results are shown in FIG. 18:

name (R)	Retention time	Peak name	Angle of purity	Threshold of purity	Area of	Area%	Degree of separation	s/n
									1	15.105	Impurity B	1.155	1.292	53190	0.11		23.69
2	18.721	Impurity A	1.738	1.768	32505	0.06	3.58	15.93
									3	21.237	Main peak	0.136	0.320	45952506	91.68	2.33	16864.60
4	42.030	Impurity D	0.186	0.247	4042905	8.07	20.99	2155.77
									5	52.271	Unknown impurity	8.080	7.669	44332	0.09	8.21	13.4

And (4) conclusion: the carbamazepine is degraded in an acidic solution of methanol, and the separation degree of each degradation impurity from a main peak and known impurities is more than 1.5; the purity angle of the main peak is smaller than the purity threshold value, and the product is qualified.

1.3 base disruption

Taking 75mg of carbamazepine raw material, precisely weighing, placing in a50 ml measuring flask, adding 10ml of methanol for dissolving, then adding 5ml of 1 mol/L sodium hydroxide solution, heating for 3h under the condition of 80 ℃ water bath, cooling, adding 5ml of 1 mol/L hydrochloric acid solution for neutralizing, adding 15ml of methanol, ultrasonically dissolving, adding water for diluting to scale, shaking up to obtain the carbamazepine water soluble tablet, measuring 5.00ml of solution in a50 ml measuring flask, adding 22ml of methanol, adding water for diluting to scale, and shaking up to obtain the carbamazepine water soluble tablet.

The results are shown in FIG. 19:

name (R)	Retention time	Peak name	Angle of purity	Threshold of purity	Area of	Area%	Degree of separation	s/n
									1	21.126	Main peak	0.173	0.351	51256559	98.58		12913.80
2	41.940	Impurity D	0.455	0.369	738151	1.42	20.6	271.79

And (4) conclusion: the carbamazepine is degraded in an alkaline solution of methanol, and the separation degree of degraded impurities and a main peak is more than 1.5; the main peak purity angle is less than the purity threshold.

1.4 oxidative destruction

Oxidation blank: transferring 5.00ml of 30% hydrogen peroxide solution into a50 ml volumetric flask, adding 25ml of methanol solution, adding water to dilute to a scale, and shaking up to obtain the product.

Taking 75mg of carbamazepine as a raw material, precisely weighing, placing in a50 ml volumetric flask, adding 5ml of methanol for dissolution, then adding 5ml of 30% hydrogen peroxide solution, and heating for 3 hours under the condition of 80 ℃ water bath; cooling, adding 20ml of methanol, dissolving by ultrasonic treatment, diluting with water to scale, and shaking. Weighing 5.00ml of solution into a50 ml volumetric flask, adding 22ml of methanol, adding water to dilute to the scale, and shaking up to obtain the product.

The results are shown in FIG. 20.

Name (R)	Retention time	Peak name	Angle of purity	Threshold of purity	Area of	Area%	Degree of separation	s/n
									1	5.645	Unknown impurity	13.66	14.436	7402	0.01		7.62
2	8.222	Unknown impurity	4.889	5.097	28848	0.06	6.11	19.41
									3	9.299	Unknown impurity	3.986	3.903	57949	0.11	1.73	26.09
4	10.23	Unknown impurity	47.07	15.776	42193	0.08	1.28	6.53
									5	13.384	Unknown impurity	18.779	14.806	19929	0.04	4.08	8.18
6	18.724	Impurity A	2.46	2.288	20359	0.04	5.74	6.91
									7	21.048	Main peak	0.155	0.353	50750657	99.65	2.11	13661.60

And (4) conclusion: the carbamazepine is degraded in a hydrogen peroxide solution of methanol, and the separation degree of each degradation impurity from a main peak and known impurities is more than 1.5; the purity angle of the main peak is smaller than the purity threshold value, and the product is qualified.

1.5 conservation of Material

Calculating the formula:

A₁peak area of the raw material medicine;

A₂sample peak area sum;

m₁the mass of the raw material medicine;

m₂sample mass;

2 high temperature Damage (sample set: 2017031702)

2.1 undamaged raw material medicine

Taking 75mg of carbamazepine raw material, precisely weighing, placing in a50 ml measuring flask, adding 25ml of methanol for dissolving, performing ultrasonic treatment to dissolve, adding water for diluting to a scale, and shaking up to obtain the carbamazepine. Weighing 5.00ml of solution into a50 ml volumetric flask, adding 22ml of methanol, adding water to dilute to the scale, and shaking up to obtain the product.

The results are shown in FIG. 21:

name (R)	Retention time	Angle of purity	Threshold of purity	Area of	Area%	Degree of separation
							1	22.215	0.21	0.309	52103843	100

2.2 high temperature destruction

Taking 75mg of carbamazepine raw material heated for 4 hours in a 130 ℃ oven, precisely weighing, placing in a50 ml volumetric flask, adding 25ml of methanol, adding water to dilute to a scale, and shaking up to obtain the carbamazepine. Weighing 5.00ml of solution into a50 ml volumetric flask, adding 22ml of methanol, adding water to dilute to the scale, and shaking up to obtain the product.

The results are shown in FIG. 22:

name (R)	Retention time	Angle of purity	Threshold of purity	Area of	Area%	Degree of separation	s/n
								1	22.175	0.201	0.302	52664229	100		12932.07

And (4) conclusion: can exist stably at 130 ℃; the purity angle of the main peak is less than the purity threshold.

2.3 conservation of Material

Calculating the formula:

A₁peak area of the raw material medicine;

A₂sample peak area sum;

m₁the mass of the raw material medicine;

m₂sample mass;

3 photo disruption (sample set: 2017032701)

3.1 undamaged raw material medicine

Taking 75mg of carbamazepine raw material, precisely weighing, placing in a50 ml measuring flask, adding 25ml of methanol for dissolving, performing ultrasonic treatment to dissolve, adding water for diluting to a scale, and shaking up to obtain the carbamazepine. Weighing 5.00ml of solution into a50 ml volumetric flask, adding 22ml of methanol, adding water to dilute to the scale, and shaking up to obtain the product.

The results are shown in FIG. 23:

name (R)	Retention time	Angle of purity	Threshold of purity	Area of	Area%	Degree of separation
							1	21.682	0.145	0.371	52878243	100

3.2 photo disruption

The longitude illumination is 4500lx, and the near ultraviolet energy is more than or equal to 200 w.hr/m²Irradiating for 13 days to obtain 75mg of carbamazepine raw material, precisely weighing, placing in a50 ml volumetric flask, adding 25 methanol, performing ultrasonic treatment to dissolve, adding water to dilute to a scale, and shaking up to obtain the carbamazepine. Weighing 5.00ml of solution into a50 ml volumetric flask, adding 22ml of methanol, adding water to dilute to the scale, and shaking up to obtain the product.

The results are shown in FIG. 24:

name (R)	Retention time	Angle of purity	Threshold of purity	Area of	Area%	Degree of separation	s/n
								1	21.393	0.152	0.362	53162134	100		16061.61

And (4) conclusion: the carbamazepine can stably exist under the irradiation of white light and ultraviolet; the purity angle of the main peak is less than the purity threshold.

3.3 conservation of Material

Calculating the formula:

A₁peak area of the raw material medicine;

A₂sample peak area sum;

m₁the mass of the raw material medicine;

m₂sample mass;

example 3 substance testing of carbamazepine drug substance

The samples to be tested are two batches of carbamazepine bulk drug (batch number 2017120501) produced by Shandong Zhengji pharmaceutical technology Co., Ltd and commercially available samples, namely a commercially available sample 1 and a commercially available sample 2.

The detection steps are as follows:

preparing a test solution, namely precisely weighing 50mg of carbamazepine, putting the carbamazepine into a50m L measuring flask, adding 25m L of methanol to dissolve the carbamazepine, diluting the carbamazepine with water to a scale, and shaking up the solution to be used as the test solution;

preparation of control solution A sample solution lm L was precisely measured, placed in a50m L measuring flask, diluted to the scale with methanol-water (1:1), shaken up, 5m L was precisely measured, placed in a50m L measuring flask, diluted to the scale with methanol-water (1:1), shaken up, and used as a control solution;

precisely measuring the reference solution and the sample solution at 50 μ L, respectively, injecting into a liquid chromatograph,

the results of the tests are shown in FIG. 25, FIG. 26 and FIG. 27, and the results are shown in the following table.

Content of related substances

Batch number	2017120501	Commercial sample 1	Commercial sample 2	Standard provisions
					Impurity A	0.02	0.05	0.05	＜0.10％
Impurity B	Not detected out	Not detected out	Not detected out	＜0.10％
					Impurity C	0.07	0.11	Not detected out	＜0.10％
Impurity D	Not detected out	Not detected out	Not detected out	＜0.10％
					Impurity E	Not detected out	Not detected out	Not detected out	＜0.10％
Total impurities	0.09	0.82	0.81	＜0.50％

The results show that: the purity of the equine Western sample produced by the company is 99.91%, and the detection result meets the quality standard requirement. The purities of the samples sold in the market are 99.18% and 99.19%, respectively, and the detection result does not meet the quality standard requirement. The method can be effectively applied to the quality control of the carbamazepine raw material medicine.

Claims (1)

1. Separation of carbamazepine by liquid chromatography and its related methodA method of producing a substance, characterized in that the substance of interest is impurity A

Impurity B

Impurity C

Impurity D

Impurity E

The liquid chromatography method comprises the following steps:

(1) chromatographic conditions are as follows:

nitrile propyl silane bonded silica gel is used as a filling agent, a mobile phase A is a50 mmol/L ammonium dihydrogen phosphate solution, a mobile phase B is acetonitrile, a detector is an ultraviolet detector, and the detection wavelength is 215 nm;

the linear gradient elution conditions were:

the gradient adopted in 0-28 minutes is that the mobile phase A is 89%, and the mobile phase B is 11%;

the gradient adopted in 28-38 minutes is that the mobile phase A is from 89% to 70%, and the mobile phase B is from 11% to 30%;

the gradient adopted in 38-55 minutes is that the mobile phase A is 70 percent, and the mobile phase B is 30 percent;

the gradient adopted in 55-56 minutes is that the mobile phase A is 70-89%, and the mobile phase B is 30-11%;

the gradient adopted for 56-65 minutes is that the mobile phase A is 89 percent and the mobile phase B is 11 percent;

(2) inspection method

Preparing a test solution, namely precisely weighing 50mg of carbamazepine, putting the carbamazepine into a50m L measuring flask, adding 25m L of methanol to dissolve the carbamazepine, diluting the carbamazepine with water to a scale, and shaking up the solution to be used as the test solution;

preparation of control solution A sample solution lm L was precisely measured, placed in a50m L measuring flask, diluted to scale with methanol-water 1:1, shaken up, 5m L was precisely measured, placed in a50m L measuring flask, diluted to scale with methanol-water 1:1, shaken up, and used as a control solution;

precisely measuring the reference solution and the sample solution at 50 μ L, respectively, injecting into a liquid chromatograph, and recording chromatogram.

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