CN108732279B - Method for analyzing and determining genotoxic impurities in valsartan by using HPLC (high performance liquid chromatography) method - Google Patents

Abstract

The invention provides a method for analyzing and determining genotoxic impurities in valsartan by using an HPLC (high performance liquid chromatography) method, belonging to the technical field of pharmaceutical analysis. The invention adopts phenyl silane bonded silica gel as a stationary phase, trifluoroacetic acid aqueous solution and trifluoroacetic acid acetonitrile solution as mobile phases, and adopts a gradient elution mode, and the established high performance liquid analysis method can accurately determine 2 potential genotoxic impurities in valsartan, namely 4 ' -bromomethyl-2-cyanobiphenyl (impurity I) and 4 ', 4 ' -dibromomethyl-2-cyanobiphenyl (impurity II). The HPLC-UV method is simple to operate, short in analysis time, and high in sensitivity up to 0.5ppm, and provides a reliable basis for improving the quality standard of valsartan and researching other sartan genotoxic impurities.

Description

Method for analyzing and determining genotoxic impurities in valsartan by using HPLC (high performance liquid chromatography) method

Technical Field

The invention belongs to the technical field of pharmaceutical analysis, and particularly discloses a method for determining two potential genotoxic impurities in valsartan by using an HPLC method: a method for analyzing a 4 ' -bromomethyl-2-cyanobiphenyl (impurity I) and a 4 ', 4 ' -dibromomethyl-2-cyanobiphenyl (impurity II).

Background

Valsartan of chemical name N-valeryl-N- [ [2'- (1H-tetrazol-5-yl) [1,1' -biphenyl]-4-yl]Methyl radical]-L-valine of formula C₂₄H₂₉N₅O₃Molecular weight of 435.52, and the structural formula is as follows:

valsartan is an angiotensin II receptor Antagonist (ARB) drug, and is the only ARB product which has three indications of hypertension, heart failure and myocardial infarction approved by the FDA in the United states at the same time. Since its introduction into the united states 1997, the potent antihypertensive therapeutic and cardiovascular protective properties of valsartan have been demonstrated by abundant clinical use. Research shows that the blood pressure reducing effect of valsartan is similar to that of other ARBs including olmesartan, and the valsartan is more prominent in the aspect of stable blood pressure reduction. More rarely, valsartan has wider indications than other ARBs, and is the only ARB to select for patients with heart failure and myocardial infarction.

In view of good curative effect and wide demand in clinical aspect, enterprises have continuously and deeply studied raw materials and preparation processes of valsartan, and the synthesis methods of valsartan are reported in various documents such as US005399578A, CN102417486B, WO05049586A and the like. The disclosures of the above patents, although different in terms of process parameters and specific routes, all use 4' -bromomethyl-2-cyanobiphenyl (impurity i) as the starting material. In patents CN1628094A and CN101597243A, 4 ' -methyl-2-cyanobiphenyl is used as a raw material to synthesize the impurity i through a free radical reaction, a byproduct 4 ', 4 ' -dibromomethyl-2-cyanobiphenyl (impurity ii) is also generated during the reaction, the reaction process is shown in the following formula, and the impurity i and the impurity ii are both possibly transferred to a final product during the preparation of valsartan.

The impurity I and the impurity II have halogenated alkane structures, the compounds with warning structures are defined as potential genotoxic impurities by ICH (International harmonization for registration technical requirements of human drugs), FDA (food and drug administration in the United states) and EMEA (European drug evaluation agency), and the risk that most drugs can be accepted is recognized by taking 1.5 mu g of genotoxic impurities at most every day according to the ICH guiding principles of genetic toxicity test and result analysis of human drugs. The maximum daily intake of valsartan is 320mg, so the corresponding limits for impurity i and impurity ii should be (1.5 μ g/day)/(320mg/day) 4.7 ppm.

In recent years, with the gradual soundness of relevant regulations of domestic and foreign medical administration departments, research on genotoxic impurities has become one of the keys for ensuring the product quality. At present, no report of toxic impurities of valsartan genes is found in USP, EP, JP, Chinese pharmacopoeia and related documents and patents.

Disclosure of Invention

The invention provides a method for analyzing and determining two potential genotoxic impurities, namely 4 ' -bromomethyl-2-cyanobiphenyl (impurity I) and 4 ', 4 ' -dibromomethyl-2-cyanobiphenyl (impurity II) in valsartan by using a high performance liquid chromatography.

According to the structural analysis of the impurity I and the impurity II, aiming at the conditions that a compound has a benzene ring structure, bromine substitution difference and the like, the inventor firstly selects phenyl silane bonded silica gel which has steric hindrance characteristics and has unique retention effect on an aromatic compound as a stationary phase, so that the separation effect among the impurity I, the impurity II and adjacent unknown impurities is ensured; according to the dissociation characteristics of the valsartan functional group, trifluoroacetic acid aqueous solution with extremely low pH value and trifluoroacetic acid acetonitrile solution are adopted as mobile phases, so that the peak shape of a main peak is improved; a gradient elution mode is adopted, so that the response value of the impurities under the condition of the same concentration is improved; the inventor simultaneously considers the characteristics of no dissociative group and small polarity of the impurity I and the impurity II, and adopts high-concentration acetonitrile water solution as an extraction solvent to improve the extraction recovery rate of the impurity.

The invention provides a method for analyzing and determining genotoxic impurities in valsartan by using an HPLC method, which comprises the following steps:

(1) chromatographic conditions are as follows: taking phenyl silane bonded silica gel as a stationary phase, trifluoroacetic acid aqueous solution as a mobile phase A, and trifluoroacetic acid acetonitrile solution as a mobile phase B, and performing gradient elution;

(2) preparation of sample solution: preparing a sample to be detected into a sample solution by using an acetonitrile aqueous solution as a solvent;

(3) separation and analysis: injecting the sample solution into a high performance liquid chromatograph to complete the determination of the toxic impurities of the valsartan gene,

wherein the genotoxic impurities are 4 ' -bromomethyl-2-cyanobiphenyl (impurity I) and 4 ', 4 ' -dibromomethyl-2-cyanobiphenyl (impurity II).

In the above method of the present invention, a preferable technical solution is that the stationary phase phenylsilane bonded silica chromatographic column has a particle size of 5 μm, a length of 250mm, and an inner diameter of 4.6 mm.

Preferably, the concentration of acetonitrile in the aqueous acetonitrile solution used as the solvent in step (2) is from 85% to 95%, preferably 90%.

In the above method of the present invention, another preferable technical means is that, in the aqueous trifluoroacetic acid solution as the mobile phase a, the concentration of trifluoroacetic acid is 0.05% to 0.15%, preferably 0.08% to 0.12%, and most preferably 0.1%; the concentration of trifluoroacetic acid in acetonitrile as mobile phase B is 0.05% to 0.15%, preferably 0.08% to 0.12%, most preferably 0.1%.

According to the technical scheme of the invention, the flow rate of the mobile phase is preferably 0.9-1.1 ml/min, and preferably 1.0 ml/min.

In the above method of the present invention, a further preferred embodiment is that the column temperature of the chromatographic column is 35 to 45 ℃, preferably 38 to 42 ℃, and more preferably 40 ℃.

Preferably, the detection wavelength is 254 nm; the injection volume was 20. mu.l.

More preferably, in one embodiment of the present invention, the elution gradient is:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	60	40
3	60	40
			12	20	80
22	20	80
			23	60	40
30	60	40

The method for analyzing and determining the genotoxic impurities in the valsartan has the sensitivity of 0.5ppm, plays an important role in establishing and improving the quality standard of valsartan raw material medicaments and preparations, and provides a reference basis for researching the genotoxic impurities of other ARB medicaments adopting similar starting raw materials and synthetic routes.

Drawings

FIG. 1A schematic representation of a localized solution of valsartan in example 1;

FIG. 2 is a schematic view of a solution for localizing impurity I in example 1;

FIG. 3 is a schematic view of a positioning solution of impurity II in example 1;

FIG. 4 is an enlarged view of a part of a white solvent in example 1;

FIG. 5 is an enlarged view of a portion of the proprietary solution of example 1;

FIG. 6 is an enlarged view of a part of the quantitative limiting solution in example 2;

FIG. 7 is an enlarged view of a portion of the linear solution of example 3;

FIG. 8 is a linear operating curve of impurity I in example 3;

FIG. 9 is a linear operating graph of impurity II in example 3.

Detailed Description

The invention is further illustrated by the following specific examples. It should be understood that: the examples of the present invention are provided for illustration only and not for limitation of the present invention. The technical scheme obtained by simply improving the invention or equivalently replacing the conventional means or components on the basis of the technical scheme of the invention belongs to the protection scope of the invention.

Example 1 specificity and impurity localization test

(1) Preparation of sample solution

Solvent: 90% acetonitrile in water

Positioning solution: taking appropriate amount of valsartan, impurity I and impurity II, precisely weighing, respectively placing in different measuring bottles, adding a solvent (90% acetonitrile water solution) for dissolving and diluting to prepare solutions containing valsartan, impurity I and impurity II, and 0.1mg/ml of each solution, and using the solutions as compound positioning solutions.

A special solution: 1.0mg of each of the impurity I and the impurity II is precisely weighed, placed in a same 1000ml measuring flask, dissolved and diluted to 1000ml scale by adding a solvent (90% acetonitrile aqueous solution), and shaken up to be used as stock solutions of the impurity I and the impurity II; taking 200mg of valsartan, precisely weighing the valsartan, placing the valsartan into a 10ml measuring flask, precisely measuring 1ml of impurity I and impurity II stock solution, placing the impurity I and impurity II stock solution into the measuring flask, adding a solvent (90% acetonitrile aqueous solution) to dissolve the valsartan, diluting the valsartan to 10ml scales, shaking the mixture evenly, and preparing a solution containing 20mg/ml of valsartan, 0.1 mu g/ml of impurity I and 0.1 mu g/ml of impurity II to serve as a special solution.

Precisely measuring 20 mul of blank solvent, positioning solution and special solution respectively, injecting into a liquid chromatograph, and detecting the chromatographic condition according to the embodiment of the invention.

(2) Chromatographic conditions

The instrument comprises the following steps: waters2695-2489 high performance liquid chromatograph

A chromatographic column: xbridge Phenyl (4.6X 250mm, 5 μm)

Mobile phase A: 0.1% trifluoroacetic acid solution

Mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution

Gradient elution procedure

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	60	40
3	60	40
			12	20	80
22	20	80
			23	60	40
30	60	40

Column temperature: 40 ℃; detection wavelength: 254 nm; flow rate: 1.0 ml/min; sample introduction volume: 20 μ l

(3) Test results

And (3) positioning test results: the retention time of valsartan, impurity I and impurity II is 9.582min, 12.578min and 13.618min respectively, and the spectra are shown in figures 1-3.

Results of the specificity test: the blank solvent does not interfere with the detection of the impurity I and the impurity II; in the special solution, the separation degrees of the impurity I and the impurity II and adjacent unknown impurity peaks are both greater than 1.5; and the chromatographic peak purity angles of the impurity I and the impurity II are both smaller than the purity threshold value, which indicates that the chromatographic peak is a single component. The above shows that the method specificity meets the requirements. The results are shown in Table 1, and the spectra are shown in figure 4 and figure 5.

TABLE 1 method specificity test results

Chromatographic peak name	Retention time (minutes)	Degree of separation	Purity angle/purity threshold
				Blank solvent	－	－	－
Unknown impurity 1	11.893	－	－
				Impurity I	12.567	3.21	4.707/5.295
Unknown impurity 2	13.152	2.39	－
				Impurity II	13.605	1.92	4.555/6.094
Unknown impurity 3	14.169	3.28	－

EXAMPLE 2 quantitative limits

(1) Preparation of sample solution

Determination of baseline noise: precisely measuring 20 mu l of solvent, injecting the solvent into a liquid chromatograph, continuously injecting a sample with 3 needles, and detecting the chromatographic condition according to the embodiment of the invention. And recording blank baseline noise in the peak-off time ranges of the impurity I and the impurity II, and calculating an average value.

Preparation of a quantitative limiting solution: taking a proper amount of each of the impurity I and the impurity II, precisely weighing, placing the impurity I and the impurity II in the same measuring flask, dissolving by using a solvent and gradually diluting, detecting according to the chromatographic condition of the embodiment of the invention until the impurity I and the impurity II are diluted until the ratio of the peak height to the corresponding noise (namely, the signal-to-noise ratio, S/N) is about 10, wherein the ratio of the sample concentration to the measured concentration of a sample is the limit of quantitation, continuously injecting samples for 6 needles according to the concentration, and calculating the average value of the peak heights.

(2) Chromatographic conditions

The chromatographic conditions were the same as in example 1

(3) Test results

The test result shows that the quantitative limits of the impurity I and the impurity II under the analysis method of the invention are both 0.5ppm and are below 15% of the impurity control limit (4.7ppm), namely, the impurity I and the impurity II with the content of more than 0.5ppm in the sample can be detected and accurately quantified. Thus, the sensitivity of the method is satisfactory. The results of quantitation limit are shown in Table 2, and the quantitation limit solution map is shown in FIG. 6.

TABLE 2 quantitative limit results

Name (R)	Concentration (μ g/ml)	Average peak height (μ V)	Average noise (μ V)	Signal to noise ratio	Limit of quantitation (ppm)
						Impurity I	0.0102	95.0	8.0	11.9	0.5
Impurity II	0.0103	66.5	5.7	11.7	0.5

Example 3 linearity

(1) Sample solution preparation

Accurately weighing 20mg of each of the impurity I and the impurity II, placing the impurity I and the impurity II in the same 100ml measuring flask, adding a solvent to dissolve and dilute the impurity I and the impurity II to a scale, and shaking up; precisely measuring 1ml, placing in a 10ml measuring flask, adding a solvent to dilute to a scale, and shaking up to obtain a linear stock solution.

Precisely measuring linear stock solutions of 1ml, 2ml, 3ml, 5ml, 7.5ml, 10ml and 15ml, respectively placing the stock solutions into different 100ml measuring bottles, adding a solvent to dilute the stock solutions to a scale, and shaking the stock solutions uniformly to be respectively used as linear solutions with limits of 20%, 40%, 60%, 100%, 150%, 200% and 300%. According to the chromatographic condition detection, a linear equation and a correlation coefficient are calculated.

(2) Chromatographic conditions

The chromatographic conditions were the same as in example 1

(3) Test results

Test results show that the correlation coefficients of the linear equations are both greater than 0.999 and the linear relationship meets the requirements when the impurity I and the impurity II are in the concentration range of 300% limited by quantitative limit. The test results are shown in tables 3 and 4, the linear solution typical graph is shown in FIG. 7, and the working curve graph is shown in FIGS. 8 and 9.

TABLE 3 Linear test results for impurity I

TABLE 4 Linear test results for impurity II

Example 4 accuracy

(1) Sample solution preparation

Control solution: taking a proper amount of impurity I and impurity II, accurately weighing, placing in a same measuring flask, adding a solvent to dissolve and dilute to prepare a solution with the concentration of each of impurity I and impurity II being 0.1 mu g/ml.

Accuracy solution: taking 200mg of a sample, 3 parts in total, precisely weighing, respectively placing in 10ml measuring flasks, adding a solvent for dissolving, diluting to a scale, and shaking up to obtain a blank solution; and taking 200mg of the test sample, 9 parts in total, taking 3 parts in each group, 3 groups in total, respectively placing the test sample in 10ml measuring bottles, respectively adding 1ml, 2.5ml and 10ml of the reference substance solution into each group, then adding the solvent for dissolving and diluting to a scale, shaking up, and respectively using the test sample as the accuracy test solutions of 20%, 100% and 200% of the limit. The above solutions were tested according to the chromatographic conditions described in the present invention.

(2) Chromatographic conditions

The chromatographic conditions were the same as in example 1

(3) Test results

And calculating the detected amount of the impurities I and II in each group of samples according to the following formula, deducting the data of the contents of the impurities I and II in the blank samples to obtain the actual detected amount, calculating the ratio of the actual detected amount to the theoretical added amount, taking the result as the recovery rate data, and counting the range, the average value and the Relative Standard Deviation (RSD) of the recovery rate for evaluating the accuracy of the method. Test results show that the recovery rate of the solution with different concentrations of impurity I and impurity II is 95.0-110.0%, the RSD is less than 3.0%, and the accuracy of the method is good. The test results are shown in Table 5.

In the formula: a. the_{Sample (A)}Is the peak area of the impurity I or the impurity II in the test solution;

C_{sample (A)}The concentration (mg/ml) of valsartan in the test solution;

A_{to pair}The peak area of the impurity I or the impurity II in the impurity reference substance solution is shown;

C_{to pair}The concentration (mg/ml) of impurity I or impurity II in the impurity control solution is shown.

TABLE 5 accuracy results

EXAMPLE 5 durability

(1) Sample preparation

Test solution: same as example 1

(2) Chromatographic conditions

Keeping other parameters consistent with the chromatographic conditions in example 1, setting the flow rate to be 0.9ml/min and 1.1ml/min, the column temperature to be 35 ℃, the column temperature to be 45 ℃, and the concentration of trifluoroacetic acid in the mobile phases A and B to be 0.05 percent and 0.15 percent respectively, detecting the sample solution, and recording the separation degree of impurities I and II and adjacent impurity peaks in the sample solution spectrum under each condition.

(3) Test results

The test result shows that the flow speed, the column temperature and the concentration of the mobile phase trifluoroacetic acid are changed in a certain range, the degrees of separation of the impurities I and II from adjacent impurity peaks are larger than 1.5, and the durability of the method meets the requirement.

Table 4 durability results

Chromatographic peak name	Unknown impurity 1	Impurity I	Unknown impurity 2	Impurity II	Unknown impurity 3
						Normal condition	－	3.21	2.39	1.92	3.28
The content of mobile phase trifluoroacetic acid is 0.05 percent	－	3.26	2.43	1.97	3.03
						The content of mobile phase trifluoroacetic acid is 0.15 percent	－	3.22	2.28	1.86	3.17
Column temperature 35 deg.C	－	2.97	3.48	2.39	3.22
						Column temperature 45 deg.C	－	2.79	4.50	1.53	2.73
Flow rate 0.9ml/min	－	1.85	3.70	2.14	3.20
						Flow rate 1.1ml/min	－	2.44	4.27	1.79	2.86

In conclusion, the method provided by the invention is simple to operate, high in sensitivity and good in specificity, can accurately determine the potential genotoxic impurities in the valsartan, and is a prospective method for further improving and innovating the process improvement and quality standard of the valsartan raw material medicament and preparation.

Claims (1)

1. A method for analyzing and determining genotoxic impurities in valsartan by using an HPLC method is characterized in that,

(1) chromatographic conditions are as follows: taking phenyl silane bonded silica gel as a stationary phase, trifluoroacetic acid aqueous solution as a mobile phase A, and trifluoroacetic acid acetonitrile solution as a mobile phase B, and performing gradient elution;

(2) preparation of sample solution: preparing a sample to be detected into a sample solution by using an acetonitrile aqueous solution as a solvent;

(3) separation and analysis: injecting the sample solution into a high performance liquid chromatograph to complete the determination of the valsartan genotoxic impurities,

wherein the genotoxic impurities are 4 ' -bromomethyl-2-cyanobiphenyl and 4 ', 4 ' -dibromomethyl-2-cyanobiphenyl,

wherein, the chromatographic column using phenyl silane bonded silica gel as a stationary phase has the particle size of 5 μm, the length of 250mm and the inner diameter of 4.6 mm; the temperature of the chromatographic column is 40 ℃, the detection wavelength is 254nm, and the sample injection volume is 20 mu l;

in the aqueous trifluoroacetic acid solution as mobile phase a, the concentration of trifluoroacetic acid was 0.1%; the concentration of trifluoroacetic acid in the trifluoroacetic acid acetonitrile solution as the mobile phase B is 0.1%;

the flow rate of the mobile phase was 1.0ml/min,

the concentration of acetonitrile in the acetonitrile water solution is 90 percent,

the elution gradient was:

time (min) Mobile phase A (%) Mobile phase B (%) 0 60 40 3 60 40 12 20 80 22 20 80 23 60 40 30 60 40

。

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