CZ12233U1 - Extremely pure reference standard for testing purity and stability of amlodipine maleate - Google Patents

Description

High purity reference standard for testing the purity and stability of amlodipine maleate

Technical field

The present invention addresses the use of two new chemical compounds as reference standards for determining the purity or stability of substances and pharmaceutical compositions containing amlodipine maleate.

BACKGROUND OF THE INVENTION

Pharmaceutical products are controlled by state authorities in most countries. For example, U.S. Pat. The Food & Drug Administration (FDA) usually requires applicants to demonstrate the safety and efficacy of the pharmaceutical during the assessment / approval process, and the follow-up of the drug continues after approval. Similar requirements exist in many European countries and worldwide. In order to comply with the safety requirements, inspection authorities usually require a production specification that sets the maximum quantity of each identified impurity as well as the maximum quantity for all unidentified impurities. Once approved, each manufactured batch or kit of pharmaceutical product is tested to confirm that the specification is met. In addition, stability testing of the pharmaceutical product is performed to demonstrate that the composition does not change significantly over time, i.e., over the stated shelf life. It is important that the pharmaceutical product does not deviate from its approved specification prior to administration to the patient in a manner that could cause a reduction in safety or efficacy. In practice, the retention of samples from each commercial batch released to the public has proved to be effective so that stability and any defect can be detected and corrected.

Thus, the purity of the pharmaceuticals is tested both during manufacture and subsequently during its useful life. The product is usually tested by comparing analytical results with relevant reference standards results. In the case of impurity detection, this normally means determining the pharmaceutical product and comparing the result with the result obtained for the highly pure form of the impurity detected in the same assay. Sources of potential impurities in the pharmaceutically active agent or composition include:

- residual quantities of synthetic precursors

- by-product of the preparation and processing of the active substance

residual solvents

- degradation products occurring during storage, including products resulting from interactions with excipients in pharmaceutical compositions

- isomers of the active substance

- trace contaminants from, for example, instruments or the environment.

Without identifying a potential impurity and without a synthetic route leading to the preparation of its reference standard, it is difficult or impossible to successfully identify or otherwise monitor its content in the pharmaceutical product.

Amlodipine besylate is a very successful drug marketed by Pfizer under the trade name NORVASC for the treatment of high blood pressure and angina. Amlodipine itself was first patented in EP 089 167 and the corresponding US patent 4,572,909. These patents cite 2 - [- (2-aminoethoxy) methyl] -4- (2-chlorophenyl) -3-ethoxycarbonyl- 5-methoxycarbonyl-6-methyl-1,4-dihydropyridine. This compound, currently commonly known as amlodipine, has the following formula:

- 1 CZ 12233 Ul

Examples 8, 11, 12 and 22 in EP 089 167 illustrate the preparation of amlodipine in the form of the maleate salt. Although a wide variety of suitable mixed acid salts exist, the maleate salt is mentioned as the most suitable mixed acid salt. The following patent EP 244 944 and the corresponding patent US 4,879,303 were directed to the preparation of the besylate (or benzenesulfonate) salt of amlodipine. It is reported that the besylate salt has advantages over other known salts, for example, an easy method of preparation. Amlodipine besylate quality standards are described in Pharmeuropa 10 (2), 187 (1998).

A review of available parts of NORVASC (amlodipine besylate) New Drug Application (NDA) filed in U.S. Pat. The Food & Drug Administration by Pfizer reveals that during development there was a shift from the original amlodipine maleate to amlodipine besylate. (See “Review of NDA Original” for

NDA # 19-787 of October 10, 1990, accessible by the FDA under the Freedom of Information Act).

This diversion was probably due to problems with tablet preparation and stability. However, specific examples of stability or tablet problems are not publicly available among the information provided by the FDA.

Amlodipine maleate is still a useful salt of amlodipine. It would therefore be desirable to identify and solve the stability problems of this chemical compound and to develop a method for controlling its production.

The essence of the technical solution

The present invention is concerned with the discovery of two new impurities associated with amlodipine maleate substances and pharmaceutical compositions containing the compound. One aspect of the present invention relates to a process which comprises determining a high purity amlodipinaspartate or amlodipine maleate amide under a set of conditions to obtain an analytical result for a reference standard under said set of conditions. Another aspect of the present invention relates to a process for testing the purity and stability of an amlodipine maleate sample or pharmaceutical dosage form comprising amlodipine maleate, comprising analyzing a sample of amlodipine maleate substance or amlodipine maleate composition for the presence of amlodipine maleate or amlodipine maleate. For example, a method of controlling the purity or stability of an amlodipine maleate substance or amlodipine maleate composition comprising analyzing an amlodipine maleate substance or amlodipine maleate composition providing an analytical result and comparing the analytical result with a corresponding analytical result for a reference standard of amlodipine maleate or amlodipine maleate indicating the purity or stability of the substance amlodipine maleate sufficient. Another aspect of the present invention relates to a process for the production of pharmaceutically acceptable doses of amlodipine maleate in solid form. The process comprises mixing amlodipine maleate with at least one pharmaceutically acceptable substance to form a mixture, either a) filling the mixture into capsules, or b) compressing the mixture into tablets to prepare pharmaceutical dosage forms of solid amlodipine maleate, taking a sample from said pharmaceuticals. doses of amlodipine maleate in solid form, analyzing the sample to obtain an analytical result of the sample, comparing the analytical result of the sample with the corresponding analytical result for the reference standard of amlodipine or amlodipine maleate determining the relative amount of amlodipine or amlodipine maleate relative to that amlodipine maleate;

The relative amount of amlodipinaspartate or amlodipine maleate amide does not exceed a predetermined / approved limit, for example 1.0 wt. %, preferably 0.1 wt. % based on the amount of amlodipine maleate.

Amlodipine maleate has the following chemical formula:

J300H (

ΌΟΟΗ

It has been found that at least two previously unidentified impurities should be taken into account in amlodipine maleate quality testing. Both impurities result from side reactions during the preparation of amlodipine maleate and / or are formed during storage. These impurities are specific to amlodipine maleate because they are formed in the presence of maleic acid and are therefore not detected or monitored in conjunction with amlodipine besylate. These chemical compounds are referred to herein as amlodipinepartate and amlodipine maleate amide. The formula of both compounds is shown below. "Amlodipinaspartát"

"Amlodipinmaleinamid"

Both amlodipinaspartate and amlodipine maleainamide are calcium channel blockers and can thus be used as pharmaceutically active substances. These novel chemical compounds and their uses in medicaments are described in more detail in the US patent entitled "Chemical compound amlodipine malinamide, pharmaceutical composition, process for their preparation and use in the treatment of angina and high blood pressure", the entire contents of which are incorporated herein by reference.

These compounds are formed by the reaction between amlodipine and maleic acid. Amlodipinaspartate is formed by the Michael-type addition reaction between the amlodipine amino group and the maleic acid double bond. Amlodipine maleainamide is formed by the amidation reaction of the amino group of amlodipine with the carboxylic acid group of maleic acid.

Chemical reactions between amlodipine and maleic acid leading to the formation of impurities of amlodipine aspartate or amlodipine maleate amide may occur as a by-product during the preparation of amlodipine maleate salt. Amlodipine maleate may further undergo subsequent conversion / decomposition by the aforementioned Michael-type addition or amidation reaction during processing into the resulting pharmaceutical form or during subsequent storage, resulting in the formation of impurities of amlodipine aspartate and amlodipine maleate, respectively. Of these two impurities are

UL1 forms more easily amlodipinaspartate, especially during storage, and is therefore a more important impurity requiring monitoring. Although these compounds may not be a nuisance in the pharmaceutical composition, they themselves act as calcium channel blockers, current regulatory practices usually require consistent control of such "impurities". Now that these impurities have been identified and prepared in a sufficiently pure state, amlodipine aspartate and amlodipine maleate can be used as reference indicators for routine analysis of amlodipine maleate or pharmaceutical doses containing this compound, both during development work to solve the aforementioned stability problems or during industrial production of amlodipine maleate and pharmaceutical forms based thereon.

The preparation of amlodipinaspartate for use as a reference standard or indicator can be achieved by bringing the amlodipine free base or its salt and maleic acid into intimate contact with each other under conditions suitable for the Michael-type addition. This reaction is illustrated below.

Conditions of higher pH, higher temperature and longer reaction times or contact times usually lead to the addition reaction. A pH of greater than 7, preferably greater than 7.5, is preferred. At lower pHs, there is a tendency to inhibit the addition and hinder the reaction. Further, it is preferred to carry out the reaction in the melt phase or in solution. "Melt phase" refers to a state in which amlodipine is melted for at least the time that maleic acid is present. When the reaction is carried out in solution, a temperature of at least 60 ° C, more often at least 80 ° C, and most preferably in the range of 85 ° C to 110 ° C is preferred. Solvents suitable for the addition reaction include polar aprotic solvents such as Ν, Ν-dimethylformamide, alcohols such as ethanol and isopropanol, esters such as ethyl acetate, and hydrocarbons such as toluene.

Amlodipine and maleic acid are usually mixed in approximately stoichiometric ratios, namely 0.9: 1 to 1: 0.9. A common method of carrying out the reaction is to melt amlodipine maleate. Although maleic acid in this case is previously combined with amlodipine, this is considered to be included in the field of the invention. Alternatively, the reaction may be carried out by contacting the free base of amlodipine and maleic acid in a solvent or melt after prior dry blending.

Amlodipine maleainamide can be prepared by reacting amlodipine or a salt thereof with a carbonic acid-activated maleic acid. This reaction is essentially an amidation reaction and is therefore supported by the presence of an acid catalyst, an elevated temperature, etc. Other amidation conditions are well known to those skilled in the art. The term "activated carbonyl group maleic acid" refers to maleic acid or a derivative thereof having a suitably activated carbonyl group to facilitate amidation reaction with amlodipine. In some methods, carbonyl activation is achieved by the presence of an acid catalyst, usually a Lewis acid such as aluminum chloride or phosphoric acid. Although preferred is a process wherein maleic anhydride is used which provides an activated carbonyl group without the need for a catalyst. Maleic acid in itself and in the absence of a catalyst or activator is not a carbonyl-activated maleic acid and will not readily form amlodipine maleainamide even in the presence of amlodipine. The reaction with maleic anhydride is shown below.

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The amlodipine maleate amide formation reaction can generally be carried out by bringing the amlodipine free base or salt thereof into direct contact with maleic acid. It is preferred to carry out the reaction in a suitable solvent at an elevated temperature in the range of 25 to 100 ° C, more often in the range of 35 to 50 ° C. Solvents suitable for this addition reaction include polar aprotic solvents such as Ν, Ν-dimethylformamide, alcohols such as ethanol or isopropanol, esters such as ethyl acetate, and hydrocarbons such as toluene. Amlodipine and carbonyl-activated maleic acid are usually mixed in approximately stoichiometric ratios, namely 0.9: 1 to 1: 0.9.

Amlodipine maleate and amlodipine maleate can both be used as reference standards (or benchmarks) in determining the presence and / or content of impurities in a sample of amlodipine maleate substance or a pharmaceutical composition containing amlodipine maleate. As a reference standard, amlodipinaspartate or amlodipine maleate should be in sufficiently pure form, usually more than 80%, more often more than 90%. High levels of purity are achievable but not necessary. The amlodipinaspartate and amlodipine maleainamide prepared as described above can, if necessary, be further purified to achieve the desired degree of purity. Purification can be accomplished by standard methods, for example, recrystallization from a suitable solvent, or stirring, optionally while heating, a suspension of the compound in a suitable liquid followed by removal of the liquid phase.

The assay utilized in the present invention is not particularly limited and includes any methods that can detect or otherwise detect the presence of a target compound. In general, assays can be divided into methods based on physical separation of the target compound (s) from the sample and non-separation or observation-based methods such as IR and NMR, although other methods are possible. Separation-based assays are preferred.

Examples of this type of assay include thin layer chromatography (TLC) and high performance liquid chromatography (HPLC).

Amlodipinepartate or amlodipine maleate amide are determined by the given conditions, providing an analytical result of the reference standard. The “analytical result of a reference standard” may be a quantitative or a qualitative result and may be in the form of numerical, graphical, pictorial, etc.

In some cases, the result may be stored electronically for later comparison.

Amlodipine maleate material to be analyzed for the presence of amlodipine malate and amlodipine maleate includes amlodipine maleate substances and amlodipine maleate compositions. The term "amlodipine maleate" is used herein to refer to a material that contains almost only amlodipine maleate. Examples of such substances include crude amlodipine maleate obtained during synthesis as well as purified amlodipine maleate. "Amlodipine maleate formulations" include mixtures, solutions, suspensions, etc. that contain amlodipine maleate. Examples of formulations include a mixed powder formulation used in tableting techniques to prepare tablets as well as precursors and final dosage forms. Prior to performing the assay, the composition may be further processed, e.g., by crushing tablets resulting in powder formation, such modifications are also included within the composition. The amlodipine maleate itself may be in any suitable form, including a crystalline state or an amorphous state.

A preferred amlodipine maleate formulation to be tested is a pharmaceutical dosage in solid form. The pharmaceutical dosage in solid form includes tablets, capsules, sachets, etc.,

Which contain amlodipine maleate in a pharmaceutically effective amount and at least one pharmaceutically acceptable excipient such as a binder, filler, solvent, lubricant, disintegrant, etc. Such compositions are prepared according to well known methods, including wet granulation, dry granulation and direct granulation. compression into tablets and mixing and filling for capsule preparation,

In both cases, the composition is prepared by admixing amlodipine maleate with at least one pharmaceutically acceptable excipient. The mixture is then further processed, which involves filling the mixture into capsules or compressing the mixture into tablets. It should be noted that mixing or granulating methods include both wet and dry processing methods. Pharmaceutical compositions containing amlodipine maleate having a pH in the range of 5.5 to 7, as measured in 20% by weight, are preferred. aqueous suspension, and thus exhibit improved stability, as described in more detail in the US patent entitled "Pharmaceutical compositions containing amlodipine maleate, a process for their preparation and use in the treatment of angina and high blood pressure", the entire contents of which are incorporated herein by reference.

For quality control reasons, it is preferred that the pharmaceutical composition be prepared from amlo15 dipin maleate, which is characterized by a low content of amlodipinaspartate and amlodipine maleate amide. A suitable process for the preparation of amlodipine and its conversion to a maleate salt characterized by minimal impurity formation and good yields is described in more detail in the US patent "Amlodipine maleate chemical compound, pharmaceutical composition, process for their preparation and use for the treatment of angina and high blood pressure". here attached by reference.

The pharmaceutical compositions are conveniently manufactured in batches or manufacturing kits. Production kits typically comprise from 100,000 to 1,000,000 or more tablets or capsules. The production kit should be checked to confirm that the content of amlodipine aspartate or amlodipine maleate is within specification, the so-called quality control test. A sample is taken from the manufacturing kit (eg 10 to 100 capsules or tablets) and the presence of amlodipine aspartate and amlodipine 25 maleinamide is analyzed and the content of these compounds is also determined. Pharmaceutical compositions containing amlodipine maleate should contain less than 1%, preferably less than 0.5%, preferably less than 0.2% amlodipine maleate or amlodipine maleate, based on the amount of amlodipine maleate. Generally, except for all samples taken, the entire production kit will be sold or otherwise released by the manufacturer unless an unacceptable content of amlodipinepartate or amlodipine maleate 30 amide is found. In this case, the kit will not be sold or released, that is, neither commercially used nor used in clinical trials.

The same strategy can be applied to production sets of substances containing amlodipine maleate. A sample (e.g. 0.5 to 2 g of material) is taken from the production kit and the presence of amlodipinaspartate and amlodipine maleate amide is analyzed and the content of these compounds is determined. Amlodipine maleate-containing substances should contain less than 1%, preferably less than 0.5%, preferably less than 0.2%, of amlodipine maleate or amlodipine maleate, based on the amount of amlodipine maleate. In general, the entire production kit, except for all samples taken, will be sold or otherwise released by the manufacturer unless an unacceptable content of amlodipinepartate or amlodipine maleate amide is found. In this case, the manufacturing kit will not be sold or released, that is, neither commercially used nor used in the manufacture of pharmaceutical products.

Analysis of amlodipine maleate-containing materials generally provides an analytical result of the sample. The “analytical result of a sample” is usually somehow compared to the analytical result for an amlodipinepartate or amlodipine maleate reference standard. The comparison can be made manually, for example by visual observation and / or by an automated procedure. The analytical result for the reference standard may be obtained simultaneously with the analytical result of the sample, ie immediately before, during, or immediately after the analysis of the amlodipine maleate sample, or may be obtained earlier, even several months to years earlier. In some procedures, the analytical result for a reference standard is stored electronically and used with a computer program to determine the presence of amlodipinepartate or amlodipine maleate amide and to determine the content of these compounds. This more recent procedure involves calibrating the equipment according to the analytical results of the reference standards or the results derived therefrom and / or by so-called internal standardization. All such comparisons,

-633 12233 Ul direct, indirect, manual or automated are included in the uniform designation “comparison”.

The analytical procedure used to determine the analytical results of the reference standards is usually the same analytical procedure under the same conditions that was used to test the amlodipine maleate-containing material, although this is not necessarily required.

The present solution will be described below with reference to two preferred analytical techniques, namely TLC and HPLC. In TLC, samples of test material containing amlodipine maleate and reference standard (s) containing a known amount of amlodipine maleate and / or amlodipine maleate amide are developed on a suitable chromatographic plate using a suitable developing liquid (mobile phase) under given conditions. These conditions include the solvent, the concentration of the amlodipine derivative in the solvent, and the amount of solution applied to the plate. The choice of suitable solvents and concentrations is well documented in the description of techniques. The analytical results obtained under such conditions may include an Rf value, i.e. the ratio between the distance traveled by the material concerned and the distance traveled by the solvent and / or the size of the spot formed in the chromatogram.

It is preferred that a reference standard be applied to the same chromatographic plate to which the test sample is applied at the same time, allowing simultaneous comparison. In other procedures, the reference standard is already defined and is simply compared with the developed chromatogram of the sample.

Thus, one of the procedures for testing the purity and / or degradation stability of a sample containing amlodipine maleate comprises the steps of:

(a) dissolving the sample containing amlodipine maleate in a solvent leading to the formation of a sample solution

(b) dissolving a sample of amlodipine aspartate and / or amlodipine maleate amide in a solvent giving rise to a reference solution

c) analyzing the sample solution and the reference solution by thin layer chromatography to obtain a TLC chromatogram for each solution

(d) calculation of the intensity of all secondary spots obtained from the sample solution whose Rf value corresponds to the Rf value of the reference indicator using the intensity of the spots of amlodipinepartate or amlodipine maleate amide in the chromatogram of the reference solution. It should be noted that the reference solution may be a "mixed" reference solution which may contain, in addition to amlodipinaspartate, other reference material of known purity, ie containing a known amount of amlodipine and / or amlodipine maleate of known purity, etc.

Similarly, HPLC analysis may be described. The analytical result for the reference standard may include a resolution factor, a response factor, a retention time and / or peak area corresponding to amlodipine aspartate or amlodiopine maleate amide. The procedure for testing the purity and / or degradation stability of a sample containing amlodipine maleate includes, for example, the steps of:

(a) dissolving the sample containing amlodipine maleate in a solvent resulting in the formation of one or more sample solutions

(b) dissolving a sample of amlodipine aspartate and / or amlodipine maleate amide in a solvent giving rise to a reference solution

(c) injection of sample solution and reference solutions on the HPLC column; and

(d) determining the peak area of each solution and calculating the amlodipinepartate and / or amlodipine maleate amide content using these values.

In this procedure, it may be necessary or desirable to apply a solution determining the suitability of the system to the HPLC column to determine the resolution factor between amlodipine maleate and

12233 U1 of any other compound present in the sample. In such a case, the method comprises the additional step b ') of dissolving the amlodipine maleate and a suitable external standard (s) to form a system suitability solution and injecting a system suitability solution onto the HPLC column to determine the resolution factor (s). This method is useful to verify that the column is still operating to its specifications. The solution determining the suitability of the system may be a solution of amlodipine aspartate or amlodipine maleate amide, but is not limited to these compounds, and may contain any substance that determines whether the column is still working as designed.

Giant. 1 demonstrates that HPLC can be used to determine amlodipinaspartate and amlodipine maleate 10 amide in addition to amlodipine. The artificial mixture of amlodipine, amlodipine aspartate and amlodipine maleainamide was analyzed by HPLC. The results are shown as the HPLC chromatogram in Fig. 1. Note that the peaks corresponding to amlodipinaspartate and amlodipine maleate amide are visible.

As an alternative to determining a reference indicator sample in each analysis, a parameter known as the response factor (R) may be used. The response factor is a predetermined ratio of the numerical result (eg peak area in HPLC) obtained by analyzing a sample of amlodipine aspartate or amlodipine maleate using a given analytical method and the corresponding numerical result obtained by analyzing the same amount of pure amlodipine maleate of equal concentration. Known resolution factors for amlodipine aspartate or amlodipine maleate can be used to calculate the quantity of the relevant indicator in the sample under investigation. In this way, as is well known, the relative amount of impurity relative to the amount of amlodipine maleate can be determined in the sample.

In the above procedures, the solvent required to dissolve the amlodipine maleate sample is to be understood as the solvent required only to dissolve the amlodipine maleate substance and impurities of interest. Other ingredients, such as inert fillers in pharmaceutical compositions, may not be soluble in the solvent system and may not be "dissolved" in the sense of the preceding "dissolution" step, as is customary for methods of analyzing pharmaceutical compositions.

The present solution also allows the use of a compound selected from amlodipine maleate of formula 1 or amlodipine maleate amide of formula 2 as a reference indicator in the analysis of purity or decomposition stability of a bulk sample of amlodipine maleate or a bulk sample of a pharmaceutical dose containing amlodipine maleate. Such analytical testing of a drug substance or form containing amlodipine maleate serves primarily to confirm that compounds 1 or 2 are not present (i.e., are present in an amount below the assay limit) or are present in amounts below the maximum permitted limit characterizing the pharmaceutical the quality of products containing amlodipine maleate, ie the quality of products marketed or sold as medicines.

BRIEF DESCRIPTION OF THE DRAWINGS

Giant. 1 shows an HPLC chromatogram of an artificial mixture of amlodipine maleate, amlodipinepartate and amlodipine maleate amide.

Giant. 2 shows the 1 H-NMR spectrum of the amlodipinaspartate compound prepared in Example 1.

Giant. 3 shows the 13C-NMR spectrum of the amlodipinaspartate compound prepared in Example 1.

Giant. 4 shows the 1 H-NMR spectrum of the amlodipine maleinamide compound prepared in Example

2.

Giant. 5 shows an HPLC chromatogram of amlodipine maleate tablets containing less than 0.2 wt. amlodipine maleate or amlodipinaspartate.

Giant. 6 shows an HPLC chromatogram of amlodipine maleate tablets containing more than 0.2 wt. amlodipine maleate or amlodipinaspartate.

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Giant. 7 shows a scanned TLC chromatogram of amlodipine maleate contaminated with amlodipinepartate and amlodipine maleate amide.

Examples

Example 1 - Procedure for the preparation of amlodipinaspartate

16 g of Amlodipine and 12 g of Amlodipine maleate were melted in a 300 ml flask. The melt was cooled to room temperature and dissolved in 300 mL of dichloromethane. The mixture was extracted with 300 mL of 1M NaOH solution. The organic layer was removed and the aqueous layer was acidified with 55 mL of 6M HCl solution. The mixture was extracted with 300 mL of dichloromethane. The layers were separated and the organic layer was dried over Na ₂ SO ₄ . The mixture was evaporated to dryness and the solid residue 10 was recrystallized from ethanol. The solid obtained was dried in a vacuum oven at 40 ° C to give 4.7 g of an off-white product.

Yield: 4.7 g (39%)

Melting point: 178 ° C to 183 ° C (decomposition)

Purity: more than 90%

1H NMR Spectrum:

The 1 H-NMR spectrum was measured at 30.05 ° C on a Bruker Advance-400 instrument in deuterated dimethylsulfoxide at 400 MHz. The spectrum is shown in Figure 2.

δ assignment 20 May 1.12 (t, 3H, J 1, 12 = 7.0Hz, 3xH-12); 2.36 (s, 3H, 3xH-15); 2.87 (m, -2H, 2xH-3 '); 3.24 (m, -2H, 2xH-9); 3.52 (s, 33H, 3xH-14); 25 3.76 (bs, 2H, 2xH-8); 4.00 (m, 3H, 2xH-11 + H-2); 4.65 (m, 2H, 2 * H-7), 5.33 (s, 1H, H-4); 7.13 (dt, 1H, J 3 ', 4' = J 4 ', 5' = 7.6Hz, J 4 ', 6' = 1.8Hz, H-4 '); 30 7.26 (m, 2H, H-3 ' + H-5 '); 7.37 (d, 1H, J 5 ', 6' = 7.8Hz, H-6 '); 8.61 (s, ΙΗ, ΝΗ).

13C - NMR Spectrum:

The 13 C-NMR spectrum was measured at 30.05 ° C on a Bruker Advance-400 35 instrument in deuterated dimethylsulfoxide at 100.6 MHz. The spectrum is shown in Figure 3.

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δ assignment δ assignment 14.18 (C-12); 66.64, 66.69 (C-7, C-8); 18.33 (C-15); 101.92, 102.40 (C-3, C-5); 35.77 (C-3); 127.54 (C-5 '); 36.85 (C-4); 127.88 (C-4 '); 45,63,45,74 (C-9); 129.07 (C-2 '); 50.58 (C-14); 131.11 (C-6); 56.42 (C-2); 166.43 (C-10); 59.50 (C-11); 167.28 170.55, 171.49 (C-13); (C-1, C-4).

Example 2 - Preparation of amlodipine maleamide g Amlodipine was dissolved in 50 ml of ethyl acetate and heated to 60 ° C. To this mixture was added 1.15 g of maleic anhydride and the mixture was shaken until a clear solution was obtained. The mixture was then cooled to room temperature and allowed to stand overnight. The mixture was then evaporated to dryness and then dried in a vacuum oven at 25 ° C for 3 hours to give a yellow solid.

Yield: 6.1 g (99%)

Melting point: 83 ° C to 86 ° C Purity:> 95% (HPLC)

1H NMR Spectrum:

The 1 H-NMR spectrum was measured at 29.85 ° C on a Bruker Advance-400 instrument in deuterated dimethylsulfoxide at 400 MHz. The spectrum is shown in Fig. 4.

δ assignment 1.12 (t, 3H, J 1.12 = 7.0Hz, 12-H3) 2.31 (s, 3H, 15H3) 3.44 (bq, ~ 2H, 9-H2) 3.52 (s, ~ 3H, 14H3) 3.58 (bt, ~ 2 H, 8-H2) 3.99 (m, ~ 2H, 11-H 2) 4.62 (AB q, 2H, 7-H 2) 5.32 (s, 1 H, 4-H) 6.27 (d, ~ 1H, J4,5 = 12.4Hz, 4-H) 6.43 (d, ~ 1H, J 4.5 = 12.4Hz, 5-H) 7.13 (dt, 1H, J 3 ', 4' = J 4 ', 5' = 7.7Hz, J 4 ', 6' = 1.6Hz, 4'-H) 7.23 (dt, 1H, J4 ', 5' = J5'6 '= 7.7Hz, J3', 5 '= 1.0Hz, 5'-H) 7.28 (dd, 1H, J 3 '4' = 7.7Hz, J 3 ', 5' = 1.0Hz, 3'-H) 7.34 (dd, 1H, J4 ', 6' = 1.6Hz, J5'6 '= 7.7Hz, 6'-H) 8.49 (s, ~ 1H, 1H) 9.24 (bt, - 1H, 9'-NH).

Example 3 - Determination of amlodipine maleate, amlodipinepartate and amlodipine maleate amide in amlodipine maleate by HPLC

1. Reference Solution 1 was prepared by transferring approximately 32 mg of amlodipine maleate reference standard (of known purity) to a 50 ml volumetric flask and dissolving in a mixture of 0.02 M acetate buffer pH 5.0 and acetonitrile (1: 1 v / v). ).

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2. Reference solution 2 was prepared by transferring approximately 32 mg of amlodipine maleate mixed standard (a mixture of 4 parts amlodipine maleate, 1 part amlodipine maleate and 1 part amlodipine maleate of high purity) to a 50 ml volumetric flask and dissolving in a mixture of 0.02M acetate buffer pH 5.0 and acetonitrile (1: 1 v / v).

3. A sample solution was prepared by transferring approximately 32 mg of the analyzed sample of amlodipine maleate to a 50 mL volumetric flask and dissolving in a mixture of 0.01 M acetate buffer pH 5.0 and acetonitrile (1: 1 v / v).

4. Chromatography procedure

(a) conditions of chromatography:

Analytical column: Symmetry Shield C8 (Waters), 100 x 4.6 mm, dp = 3.5 mm

Protective column: Sentry Guard column, Symmetry C8 (Waters), 20 x 3.9 mm, dp = 5 mm

Mobile phase: A) 0.05M perchlorate buffer pH 2.75 and acetonitrile 65:35 (v / v)

B) 0.05 M perchlorate buffer pH 2.75 and acetonitrile 35:65 (v / v)

Perchlorate buffer pH 2.75: Dissolve 7 g of NaClO ₄ · H ₂ 0 (0.05 M) and 1.74 g of K ₂ HPO ₄ (0.01 M) in 1,000 ml of water. Adjust the pH to 2.75 by adding phosphoric acid.

Flow rate: 1.0 mL / min, gradient

Gradient Program: Time (min) %AND % B 0.0 100 ALIGN! 0 4.0 100 ALIGN! 0 14.0 35 65 21.0 0 100 ALIGN! 28.0 0 100 ALIGN! 29.0 100 ALIGN! 0 32.0 100 ALIGN! 0 Detection: UV, 237 nm Column temperature: 45 ° C

Automatic dispenser: 4 ° C, protected from light

Injection volume: 10 μΐ

(b) injection procedure:

After a permanent baseline was reached, reference solution 2 was applied and the resolution factor between amlodipine, amlodipinepartate and amlodipine maleainamide was calculated. In addition, the symmetry factor and number of theoretical plates were calculated using conventional European Pharmacopoeia methods to calculate system suitability parameters.

Values obtained:

Resolution: amlo-aspartate / amlodipine = 15.6, amlodipine / amlo-maleinamide = 20.1.

Symmetry factor: amlodipinaspartate 1.2, amlodipine 1.3, amlodipine maleainamide 1.2.

Number of theoretical plates: amlodipinepartate 6387, amlodipine 22852, amlodipine maleinamide 56676.

Reference solution 1 and sample solution were then loaded onto the column.

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5. Calculations

Amlodipine maleate content and the respective impurities in the analyzed sample were calculated by comparing the peak areas of compounds from the sample solution chromatogram with the corresponding peak areas obtained from the chromatograms of reference solutions 1 and 2, considering the mass and purity of the reference samples and predetermined relative response factors (RRF). The RFF values and assay limits for each impurity, expressed as a percentage of amlodipine, are shown in the following table:

Compound RRF Limit determination (%) Aspartate 0.78 0.01 Amid 1.01 0.02

Example 4 - Determination of amlodipine maleate and amlodipinaspartate in tablets containing amlodipine maleate in an amount corresponding to 2.5 mg amlodipine free base by HPLC The assay is performed as described in Example 3 with the following changes:

The sample solution was prepared by transferring 5 tablets of amlodipine to a 25 mL volumetric flask. 20 ml of a mixture of 0.02 M acetate buffer pH 5.0 and acetonitrile (1: 1 v / v) was added and the mixture was stirred until all tablets were completely disintegrated. The mixture was then placed in an ultrasonic water bath for 5 minutes. The flask was made up to volume with 0.02 M acetate buffer pH 5.0 and acetonitrile (1: 1 v / v) and mixed. Approximately 10 ml of the obtained solution was centrifuged at 4000 rpm for 10 minutes. The supernatant was used for analysis.

Almost the same procedure can be used to analyze tablets containing 5 or 10 mg amlodipine maleate, as opposed to 2.5 mg as above, except that a 50 ml or 100 ml volumetric flask is used.

Example 5

A sample was taken from a set of 10 mg amlodipine maleate tablets and the presence of amlodipinepartate and amlodipine maleate amide was determined by HPLC. In order for these tablets to be considered "acceptable", it is required that the amount of amlodipine aspartate or amlodipine maleate present does not exceed 0.2% by weight. The HPLC chromatogram is shown in Fig. 5. The assay showed that the tablets contained 0.17 wt. amlodipine aspartate and 0.01% amlodipine maleate amide relative to the peak area corresponding to amlodipine, and the set of tablets is therefore acceptable and approved.

Comparison

A sample was taken from a different set of 10 mg amlodipine maleate tablets than used above and the presence of amlodipinepartate and amlodipine maleate amide was determined by HPLC. For these tablets to be considered "acceptable", the amount of amlodipine aspartate or amlodipine maleate amide required is not to exceed 0.2% by weight. The HPLC chromatogram is shown in Figure 6. The assay showed that the tablets contained 0.42% by weight. % of amlodipinaspartate and 0.02 wt. amlodipine maleaamide, relative to the peak area corresponding to amlodipine, and the tablet set is therefore unacceptable and has not been approved for dispensing.

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Example 6 - Determination of amlodipine maleate and amlodipine maleate amide in amlodipine maleate drug substance by TLC

1. Description of the method

Solvent: Water and methanol 50:50 by volume.

Sample solution A: Transfer approximately 51 mg of crude amlodipine maleate into a brown 10 ml vial. Dissolve in 4 ml solvent and mix well (equivalent to 10 mg amlodipine per ml).

Amlo-aspartate, 0.3% solution B: Transfer approximately 6 mg of amlodipinaspartate to a 25 ml graduated flask. Add 20 ml of solvent and dissolve the solid phase using an ultrasonic water bath.

Make up to the mark and mix well (stock solution B). Pipette 0.5 ml of stock solution B into a 10 ml vial. Add 3.5 ml of solvent and mix well (equivalent to 0.03 mg amlodipine per ml).

Amlo-maleinamide, 0.3% solution C: Transfer approximately 6 mg of amlodipine maleinamide to a 25 ml graduated flask. Add 20 ml of solvent and dissolve using an ultrasonic water bath.

Make up to the mark with the solvent and mix well (stock solution C). Pipette 0.5 ml of stock solution C into the vial. Add 3.5 ml of solvent and mix well (equivalent to 0.03 mg amlodipine per ml).

Mixed Standard D: Transfer approximately 51 mg of crude amlodipine maleate into a brown vial. Dissolve in 3 ml solvent. Add 0.5 ml of stock solution B and 0.5 ml of stock solution C and mix well.

Plates: HPTLC plates coated with silica gel

F254 20 x 10 cm (Merck)

Sample volume: 1.0 μΐ

Band: 10 mm

Application rate: 4 sec / μΐ

Distance from bottom edge of HPTLC plate: 15 mm Distance between samples: 10 mm

Mobile phase:

Chamber saturation:

Developing:

Air drying: Detection:

Methyl isobutyl ketone, methanol and acetic acid in a 70: 20: 10 by volume ratio

Developing system filled with 20 ml of mobile phase and saturated for 5 minutes

Separation path 9 cm, horizontal arrangement min, at room temperature

UV 366 nm

2. Evaluation of chromatograms

a) After all four samples were applied to the plate, the TLC plate was air dried and then examined under UV light at 366 nm. The experiment was not valid until the chromatogram obtained from Mixed Standard D showed three clearly separated spots.

(b) The intensity of any secondary spot in the chromatograms of solution A was calculated to have a Rf value of 40 corresponding to that obtained for the principal spot in the chromatograms of solution B (corresponding to 0.3% amylpinaspartate).

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(c) The intensity of any secondary spot in the chromatogram obtained with solution A was calculated to have an Rf value equal to that obtained for the principal spot in the chromatogram obtained with solution C (corresponding to 0.3% amlodipine maleate amide).

3. Results

An example chromatogram of a mixture of amlodipine maleate, amlodipine aspartate and amlodipine maleate amide obtained by scanning on a Camag TLC scanner is shown in Figure 7.

Compound Rf

Amlodipinaspartate 0,06

Amlodipine maleate 0.19 Amlodipine maleate 0.76.

It will be apparent to those skilled in the art that the particular concepts and procedures described herein can be readily inferred and practiced to be practiced and practiced without departing from the original meaning and scope of the invention as defined herein. following claims.

Claims (1)

15 PROTECTION REQUIREMENTS A high purity reference standard for testing the purity or stability of amlodipine maleate pharmaceutical compositions, i.e. an impurity content of amlodipine maleate or amlodipine maleate of less than 0.2% by weight, characterized in that it contains more than 80%, preferably more than 90% by weight. amlodipinepartate or amlodipine maleate amide.