CN107764910B - Method for measuring content of active ingredients of Sacubitril valsartan trisodium semipentahydrate capsule - Google Patents

Abstract

The invention discloses a method for determining the content of the active ingredients of sacubitril-valsartan trisodium hemipentahydrate capsules. Sacubitril-valsartan trisodium hemi-pentahydrate reference substance, take sacubitril-valsartan trisodium hemi-pentahydrate capsule samples to prepare sacubitril-valsartan with a concentration of 50μg/mL Trisodium hemipentahydrate test sample; then formulate high performance liquid chromatography conditions as follows: C ₁₈ chromatographic column (4.6mm × 250mm, 5 μm), mobile phase is 0.2% aqueous diisopropylamine solution—N-methylpyrrolidone, The detection wavelength was 250 nm, the flow rate was 0.8 mL/min, the column temperature was 30 °C, and the injection volume was 10 μL; finally, the reference substance and the test solution were precisely drawn and injected into the liquid chromatograph for measurement. The invention provides a content determination method for the active ingredients of sacubitril valsartan trisodium hemipentahydrate capsules, which is convenient, accurate and reliable, and can truly reflect the quality of the product.

Description

Content determination of active ingredients in a sacubitril-valsartan trisodium hemipentahydrate capsule method

technical field

The invention relates to the field of medicine, in particular to a method for determining the content of active ingredients of sacubitril-valsartan trisodium hemipentahydrate capsules.

Background technique

Sacubitril and Valsartan TrisodiumHemipentahydrate is trisodium (4-{[(1S,3R)-1-([1,1'-biphenyl]-4-methylene yl)-4-ethoxy-3-methyl-4-oxobutyl]amino}-4-oxobutyric acid)(N-pentanoyl-N-{[2'-(1H-tetrazolium) -5-yl)[1,1'-biphenyl]-4-yl]methyl}-L-valine) hemipentahydrate, the molecular weight is 1916.018g/mol, and the structural formula is as follows:

Sacubitril valsartan trisodium hemipentahydrate is composed of the neprilysin inhibitor Sacubitril (NEP inhibitor)-Sacubitril and the angiotensin Ⅱ receptor blocker (Angiotensin Ⅱ receptor blocker)-Valsartan valsartan. 1:1 equimolar ratio, is an orally bioavailable, dual-acting angiotensin receptor-neprilysin inhibitor for the treatment of hypertension and heart failure to reduce risk Cardiovascular death and hospitalization for chronic heart failure (NYHA II-IV) and reduced ejection fraction in heart failure patients. The drug was successfully developed by Novartis Pharmaceuticals Co., Ltd. It is the first cardiovascular drug in the history of EU drug regulation to obtain the qualification for accelerated evaluation. Decay treatment.

Currently, sacubitril-valsartan trisodium hemipentahydrate is a known cardiovascular drug. However, in the prior art, there are few reports on the content determination method of sacubitril-valsartan trisodium hemipentahydrate capsules. As a pharmaceutical preparation, it is very necessary to strictly control its content when it leaves the factory. The present invention provides a convenient, accurate, efficient and inexpensive detection method in order to solve the problem of detecting the content of active components of sacubitril valsartan trisodium hemipentahydrate before delivery or use.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for determining the content of the active ingredients of sacubitril-valsartan trisodium hemipentahydrate capsules, which is convenient, accurate and reliable, and can truly reflect the quality of the product.

In order to achieve the above-mentioned purpose, the solution of the present invention is:

A method for determining the content of active ingredients of sacubitril-valsartan trisodium hemipentahydrate capsules, comprising the following steps:

(1) Preparation of reference substance: take an appropriate amount of sacubitril valsartan trisodium hemipentahydrate reference substance;

(2) Preparation of test product: take 10-15 mg of sacubitril-valsartan trisodium hemipentahydrate capsule sample content, extract with methanol to obtain filtrate, and take the filtrate and place it in a volumetric flask for later use;

(3) HPLC conditions: C ₁₈ 4.6mm×250mm, 5μm chromatographic column, mobile phase is 0.2% diisopropylamine aqueous solution-N-methylpyrrolidone, detection wavelength is 250nm; flow rate is 0.8mL/min, The column temperature was 30 °C, and the injection volume was 10 μL;

(4) Determination method: Precisely draw the reference substance and the test solution and inject it into the liquid chromatograph for determination.

In step (1), the concentration of the sacubitril-valsartan trisodium hemipentahydrate reference substance is 50ug/mL.

In step (2), first place the sacubitril valsartan trisodium hemipentahydrate capsule sample content in a 20 mL volumetric flask, add methanol to dissolve, ultrasonicate for 5 min, filter, let the filtrate cool to room temperature, and then add The mobile phase was diluted to the mark, the mixed solution was shaken well, 1 mL of the mixed solution was accurately measured and placed in a 10 mL volumetric flask, and finally diluted to the mark with methanol, and the mixture was shaken to obtain the test product.

In step (2), first place the sacubitril-valsartan trisodium hemipentahydrate capsule sample content in a 20mL volumetric flask, add methanol to shake to dissolve, filter, let the filtrate cool to room temperature, and then add the Dilute the mobile phase to the mark, shake the mixed solution, accurately measure 1 mL of the mixed solution and place it in a 10 mL volumetric flask, and finally dilute to the mark with methanol and shake well to obtain the test product.

In step (3), the pH value of the mobile phase is 2.0 to 4.0, the pH value of the mobile phase is adjusted with acid, preferably the pH value is 3.0, and the mobile phase 0.2% diisopropylamine aqueous solution—N-methyl pyrrolidone=60:40, the acid includes but is not limited to phosphoric acid, acetic acid, crotonic acid, formic acid, citric acid, tartaric acid, sodium hydrogen sulfate, potassium hydrogen sulfate, hydrochloric acid, sulfuric acid, carbonic acid, perchloric acid, malic acid , citric acid, salicylic acid or caffeic acid, preferably phosphoric acid, acetic acid, crotonic acid, formic acid, citric acid, tartaric acid, sodium bisulfate or potassium bisulfate, and most preferably crotonic acid. The concentration of the crotonic acid is 5-20%, preferably the concentration is 10%. The preferred 10% crotonic acid in the present invention can make the chromatographic peaks in the sacubitril-valsartan trisodium hemipentahydrate capsules have good separation and improve the peak tailing phenomenon.

In step (4), the determined content of sacubitril valsartan trisodium hemipentahydrate is not less than 73.5 mg/granule.

The best method for determining the content of the active ingredients of sacubitril-valsartan trisodium hemipentahydrate capsules of the present invention comprises the following steps:

(1) Preparation of reference substance: Precisely weigh an appropriate amount of Sacubitril-Valsartan Trisodium Hemipentahydrate reference substance, add methanol to prepare a sacubitril-valsartan trisodium hemipentahydrate containing 50 μg per 1ml solution;

(2) Preparation of test product: take 20 sacubitril-valsartan trisodium hemipentahydrate capsules, put them in a mortar and grind them through a No. 4 sieve, take sacubitril-valsartan trisodium hemipentahydrate An appropriate amount of the sample content (equivalent to 10 mg of sacubitril-valsartan trisodium hemipentahydrate), accurately weighed, placed in a 20 mL volumetric flask, shaken with methanol to dissolve, filtered, and the filtrate was allowed to cool to room temperature , add the mobile phase 0.2% diisopropylamine aqueous solution-N-methylpyrrolidone to dilute to the mark, shake well, accurately measure 1mL and place it in a 10mL volumetric flask, dilute to the mark with methanol, shake well, and use it as the test solution;

(3) HPLC conditions:

OJ-RH C₁₈色谱柱(4.6mm×250mm,5μm)；流动相：0.2％二异丙基胺水溶液—N-甲基吡咯烷酮＝60:40，用10％丁烯酸调节pH值至3.0；检测波长：250nm；流速:0.8mL/min；柱温：30℃；进样量：10μL；理论板数按沙库比曲缬沙坦三钠半五水合物峰计算应不小于6000；进样方式：自动进样；Column:

OJ-RH C ₁₈ chromatographic column (4.6mm×250mm, 5μm); mobile phase: 0.2% diisopropylamine aqueous solution-N-methylpyrrolidone=60:40, adjust pH to 3.0 with 10% crotonic acid; Detection wavelength: 250 nm; flow rate: 0.8 mL/min; column temperature: 30 °C; injection volume: 10 μL; Method: automatic injection;

(4) Determination method: Precisely draw 10 μL each of the reference solution and the test solution, inject them into a liquid chromatograph, and measure.

In order to prove the accuracy and reliability of the content determination method of the active ingredient of the sacubitril-valsartan trisodium hemipentahydrate capsule of the present invention, the following test method is used to verify.

1. Instruments and reagents

The reagents used in the present invention can be purchased from the market or can be prepared by the method described in the present invention.

Reagents: N-methylpyrrolidone and water are of chromatographic grade, and crotonic acid and diisopropylamine solutions are of analytical grade.

Sample: provided by Beijing Rundekang Pharmaceutical Technology Co., Ltd.

Reference substance: Sacubitril valsartan trisodium hemipentahydrate reference substance (batch number: J08373P1) was provided by Novartis Pharmaceuticals Co., Ltd. for content determination.

Instruments: Agilent 1100 high performance liquid chromatograph (Agilent company), DV215CD electronic balance (OHausdiscovery company), UV-1750 UV-Vis spectrophotometer (Japan Shimadzu company), 410HT-ultrasonic cleaning instrument (Guangzhou Xingshuo Instrument Co., Ltd. ).

2. Chromatographic conditions

OJ-RH C₁₈色谱柱(4.6mm×250mm,5μm)；流动相：0.2％二异丙基胺水溶液—N-甲基吡咯烷酮＝60:40，用10％丁烯酸调节pH值至3.0；检测波长：250nm；流速:0.8mL/min；柱温：30℃；进样量：10μL；理论板数按沙库比曲缬沙坦三钠半五水合物峰计算应不小于6000；进样方式：自动进样。Column:

OJ-RH C ₁₈ chromatographic column (4.6mm×250mm, 5μm); mobile phase: 0.2% diisopropylamine aqueous solution-N-methylpyrrolidone=60:40, adjust pH to 3.0 with 10% crotonic acid; Detection wavelength: 250 nm; flow rate: 0.8 mL/min; column temperature: 30 °C; injection volume: 10 μL; Method: automatic injection.

3. Preparation of reference solution: Precisely weigh an appropriate amount of Sacubitril-Valsartan trisodium hemipentahydrate reference substance, add methanol to prepare a solution containing 50 μg of Sacubitril-Valsartan trisodium hemipentahydrate per 1 ml. solution.

4. Preparation of the test solution: take 20 capsules of sacubitril-valsartan trisodium hemipentahydrate, put them in a mortar and grind them through a No. 4 sieve, take sacubitril-valsartan trisodium hemipentahydrate An appropriate amount of the sample content (equivalent to 10 mg of sacubitril-valsartan trisodium hemipentahydrate), accurately weighed, placed in a 20 mL volumetric flask, shaken with methanol to dissolve, filtered, and the filtrate was allowed to cool to room temperature , add the mobile phase to dilute to the mark, shake well, accurately measure 1mL and place it in a 10mL volumetric flask, dilute it with methanol to the mark, and shake well to obtain sacubitril valsartan trisodium half with a concentration of 50μg/mL. Pentahydrate test solution.

5. Determination method: Precisely draw 10 μL of the reference solution and the test solution, respectively, inject them into a liquid chromatograph, and measure.

6. Negative sample interference determination:

Negative samples lacking sacubitril-valsartan trisodium hemipentahydrate were prepared according to the prescription of sacubitril-valsartan trisodium hemipentahydrate capsules. Prepare the negative control solution according to the preparation method of the test solution under the content determination item of the capsules, and measure it according to the determination method under the content determination item of sacubitril valsartan trisodium hemipentahydrate capsules, and record the chromatogram (such as figure 1). The results showed that in sacubitril valsartan trisodium hemipentahydrate (lactose, microcrystalline cellulose, sodium carboxymethyl cellulose, sodium lauryl sulfate, magnesium stearate and povidone, etc.) At the peak, other excipients in the prescription did not appear peaks, so the excipients did not interfere with the determination of sacubitril valsartan trisodium hemipentahydrate.

7. Investigation of linear relationship

Accurately weigh about 20 mg of sacubitril-valsartan trisodium hemipentahydrate, accurately weigh it, place it in a 20 mL volumetric flask, add methanol and shake to dissolve and dilute to the mark, shake well, and accurately weigh 10 mL and place it in a 20 mL volumetric flask. In a 100mL volumetric flask, dilute to the mark with methanol, shake well, and use it as a stock solution.

Precisely measure 1mL, 2mL, 5mL, 10mL, 12mL, and 15mL of the stock solution and place them in a 20mL volumetric flask, dilute to the mark with 50% acetonitrile aqueous solution, shake well, and use it as a linear test solution. Taking the peak area of sacubitril-valsartan trisodium hemipentahydrate (A) as the ordinate and the sacubitril-valsartan trisodium hemipentahydrate solution concentration (C) as the abscissa, the sacubitril was obtained. Regression equation of valsartan trisodium hemipentahydrate-valsartan A=15128C+3.4183 (R ² =0.9999, Figure 2-A), sacubitril valsartan trisodium hemipentahydrate-sacubit Qu's regression equation A=25460C+6.8811 (R ² =0.9999, Figure 2-B), the results show that valsartan has good linearity in the range of 0.0025-0.0374mg/mL, and sacubitril is in the range of 0.0023-0.0349mg/mL The linearity is good in the range, and the linearity of sacubitril-valsartan trisodium hemipentahydrate is good in the range of 0.0023-0.0349 mg/mL.

8. Strong degradation test

Take an appropriate amount of sacubitril-valsartan trisodium hemipentahydrate reference substance, and test under the following conditions respectively: (1) alkali destruction: add an appropriate amount of 0.1mol/L sodium hydroxide solution to dissolve, and place at room temperature for 0.5 hours, Add acid to neutralize, add mobile phase to the specified volume, as alkali degradation product solution; (2) Acid destruction: add 0.1mol/L hydrochloric acid solution to dissolve, leave at room temperature for 2 hours, add alkali to neutralize, add mobile phase to specified volume volume, as acid degradation product solution; (3) high temperature destruction: placed in 80 ℃ water bath for 2 hours, allowed to cool to room temperature, diluted to the mark with mobile phase, as high temperature degradation product solution; (4) photodestruction: placed under illuminator Place for 12 hours, dilute to the mark with mobile phase, and use as photodegradation product solution; (5) Oxidative destruction: add 1 mL of 30% hydrogen peroxide, place for about 2 hours, dilute to mark with mobile phase, and use as oxidative destruction product solution. Measurements were carried out under the above-mentioned chromatographic conditions, respectively. As shown in Figure 3, sacubitril valsartan trisodium hemipentahydrate is destroyed by acid, alkali, oxidation, high temperature and light, the peak purities of valsartan and sacubitril meet the requirements, and the peaks of degradation products do not interfere Determination of principal components.

9. Stability test

About 10 mg of sacubitril-valsartan trisodium hemipentahydrate capsules were taken and processed according to the content determination method to prepare the test solution. 7, 8, 9, 10h for sampling and determination, and record the chromatogram. The results are shown in Table 1. The test solution was placed at room temperature for about 10 hours, and the change rate of the peak area of valsartan and Sacubitus peaks was less than 2%, indicating that the product was stable at room temperature for 10 hours.

Table 1 Sacubitril Valsartan Trisodium Hemipentahydrate Capsule Solution Stability Test Results

10. Repeated experiments

Take the same batch of sacubitril valsartan trisodium hemipentahydrate capsules, prepare 6 parts of the test solution according to the preparation method of the test solution, carry out content determination, record the chromatogram, and calculate the sacubit ratio according to the external standard method Trivalsartan trisodium hemipentahydrate content. The results are shown in Table 2. Under the chromatographic conditions, the average content of 6 samples of sacubitril and valsartan was 98.6%, and the RSD (n=6) was 0.49%; the results showed that the method had good repeatability.

Table 2 Repeatability test results of sacubitril valsartan trisodium hemipentahydrate capsules

11. Recovery rate experiment

Accurately weigh about 8 mg, 10 mg, and 12 mg of sacubitril-valsartan trisodium hemipentahydrate and place them in a 20-mL volumetric flask, add methanol and shake to dissolve, and dilute to the mark, shake well, and accurately measure 1 mL. Place in a 10mL volumetric flask, dilute to the mark with methanol, shake well, and use as 80%, 100%, and 120% concentration of the test solution. 3 copies of each concentration were prepared in parallel, and the chromatogram was recorded according to the content determination method.

Precisely weigh about 10 mg of Sacubitril and Valsartan reference substance respectively and put them in a 20 mL volumetric flask, add mobile phase and shake to dissolve, and dilute to the mark, shake well, and then accurately measure 1 mL and place it in a 10 mL volumetric flask. , diluted to the mark with mobile phase, shake well, as the reference solution. 3 copies of each concentration were prepared in parallel, and the chromatogram was recorded according to the content determination method.

The results are shown in Table 3 and Table 4. Under the proposed chromatographic conditions, the average recoveries of the two component peaks of sacubitril-valsartan trisodium hemipentahydrate are between 98.0% and 101.0%, and the RSD value is less than 1.0 %, the recovery rate meets the regulations.

Table 3 Sacubitril Valsartan Trisodium Hemipentahydrate-Valsartan Accuracy Results

Table 4 Sacubitril Valsartan Trisodium Hemipentahydrate-Sacubitril Accuracy Results

12. Durability test

Determination under the conditions of detection wavelength 250nm±2nm, flow rate 1.0mL/min±0.1mL/min, column temperature 30℃±5℃, mobile phase pH 3.0±0.1, water phase ratio 50%±5% and different chromatographic columns The content of sacubitril-valsartan trisodium hemipentahydrate, the results show that the RSD of the data repeated three times under each change condition is less than 2%, and the durability of the chromatographic conditions is good.

13. Intermediate precision test

Different test personnel prepare the test sample and reference substance solution on different test instruments, precisely measure 10 μL of the test sample and reference substance solution and inject it into the liquid chromatograph, inject 6 times continuously, and record the chromatogram. Compared with the content data of 6 test solutions in 10. Repeated experiments, the RSD values of valsartan, sacubitril, sacubitril and valsartan trisodium hemipentahydrate were 0.66%, 0.64%, 0.59%. In the same laboratory, samples were tested at different times, by different analysts, and by different equipment, and the sample test results were not much different.

In conclusion, the present invention provides a method for determining the content of the active ingredients of sacubitril-valsartan trisodium hemipentahydrate capsules, which is convenient, accurate and reliable, and can truly reflect the quality of the product.

Description of drawings

Figure 1 is the experimental spectrum of the interference determination of negative samples, the Y axis is the peak intensity, the X axis is the peak time, wherein Figure 1 (A) corresponds to the negative sample, Figure 1 (B) corresponds to the control substance;

Fig. 2 is the linear spectrum fitted by the linear regression of the test example, wherein Fig. 2(A) corresponds to the linear spectrum of sacubitril valsartan trisodium hemipentahydrate-valsartan, and Fig. 2(B) corresponds to the sacubitril valsartan trisodium hemipentahydrate-valsartan Linear spectrum of kubitril-valsartan trisodium hemipentahydrate-sacubitril;

Figure 3 is the chromatogram of the strong degradation test of the test example, the Y axis is the peak intensity, the X axis is the peak time, wherein Figure 3 (A) corresponds to alkali damage, Figure 3 (B) corresponds to acid damage, Figure 3 (C) Corresponding to high temperature damage, Fig. 3(D) corresponds to light damage, and Fig. 3(E) corresponds to oxidative damage.

Detailed ways

In order to further explain the technical solutions of the present invention, the present invention will be described in detail below through specific embodiments.

Example 1

A method for determining the content of active ingredients of sacubitril-valsartan trisodium hemipentahydrate capsules, comprising the following steps:

(1) Preparation of reference substance: Precisely weigh an appropriate amount of Sacubitril-Valsartan Trisodium Hemipentahydrate reference substance, add methanol to prepare a sacubitril-valsartan trisodium hemipentahydrate containing 50 μg per 1ml solution;

(2) Preparation of test product: take 20 capsules of sacubitril-valsartan trisodium hemipentahydrate in different batches, grind them in a mortar and pass through a No. 4 sieve, and take sacubitril-valsartan trisodium The sample content of the hemipentahydrate capsule is appropriate (approximately equivalent to 10mg of sacubitril valsartan trisodium hemipentahydrate), accurately weighed, placed in a 20mL volumetric flask, shaken with methanol to dissolve, filtered, and the filtrate was placed in a 20mL volumetric flask. Cool to room temperature, add mobile phase to dilute to the mark, shake well, accurately measure 1mL and place it in a 10mL volumetric flask, dilute to the mark with methanol, shake well, and use it as the test solution;

(3) HPLC conditions:

Column: OJ-RH C ₁₈ chromatographic column (4.6mm×250mm, 5μm); mobile phase: 0.2% diisopropylamine aqueous solution-N-methylpyrrolidone=60:40, adjust pH to 3.0 with 10% crotonic acid; Detection wavelength: 250 nm; flow rate: 0.8 mL/min; column temperature: 30 °C; injection volume: 10 μL; Method: automatic injection;

(4) Determination method: Precisely draw 10 μL each of the reference solution and the test solution, inject 10 μL into the liquid chromatograph, measure, and calculate the valsartan, sacubitril, The content of sacubitril valsartan trisodium hemipentahydrate, the results are shown in Table 5.

Table 5 Sacubitril Valsartan Trisodium Hemipentahydrate Capsule Content Test Results

The above-mentioned embodiments and drawings do not limit the product form and style of the present invention, and any appropriate changes or modifications made by those of ordinary skill in the art should be regarded as not departing from the scope of the present invention.

Claims (4)

1. a content assay method for sacubitril valsartan trisodium hemipentahydrate capsules active ingredient, is characterized in that: comprise the steps: (1) Preparation of reference substance: take an appropriate amount of sacubitril valsartan trisodium hemipentahydrate reference substance; (2) Preparation of test product: take 10-15 mg of sacubitril-valsartan trisodium hemipentahydrate capsule sample content, extract with methanol to obtain filtrate, and take the filtrate and place it in a volumetric flask for later use; (3) HPLC conditions: C ₁₈ 4.6mm×250mm, 5μm column, mobile phase is 0.2% diisopropylamine aqueous solution-N-methylpyrrolidone=60:40 mobile phase, pH value of mobile phase is 2.0 to 4.0, the detection wavelength is 250 nm; the flow rate is 0.8 mL/min, the column temperature is 30 °C, and the injection volume is 10 μL; (4) Determination method: accurately draw the reference substance and the test solution and inject it into the liquid chromatograph for determination; Wherein, in step (2), the content of the sacubitril valsartan trisodium hemipentahydrate capsule sample was first placed in a 20 mL volumetric flask, dissolved in methanol, sonicated for 5 min, filtered, and the filtrate was allowed to cool to room temperature, Then add the mobile phase and dilute to the mark, shake the mixed solution, accurately measure 1 mL of the mixed solution and place it in a 10 mL measuring bottle, and finally dilute it to the mark with methanol, and shake well to obtain the test sample; Or in step (2), first place the sacubitril valsartan trisodium hemipentahydrate capsule sample content in a 20mL volumetric flask, add methanol to shake to dissolve, filter, let the filtrate cool to room temperature, and then add The mobile phase was diluted to the mark, the mixed solution was shaken well, 1 mL of the mixed solution was accurately measured and placed in a 10 mL volumetric flask, and finally diluted to the mark with methanol, and the mixture was shaken to obtain the test product. 2. the content assay method of a kind of sacubitril valsartan trisodium hemipentahydrate capsule active ingredient according to claim 1, is characterized in that: in step (1), described sacubitril valsaran The concentration of tantrisodium hemipentahydrate reference substance is 50ug/mL. 3. the content assay method of a kind of sacubitril valsartan trisodium hemipentahydrate capsule active ingredient according to claim 1, is characterized in that: in step (3), described mobile phase regulates pH with acid value, the acid is phosphoric acid, acetic acid, crotonic acid, formic acid, citric acid, tartaric acid, sodium hydrogen sulfate, potassium hydrogen sulfate, hydrochloric acid, sulfuric acid, carbonic acid, perchloric acid, malic acid, citric acid, salicylic acid or Caffeic acid. 4. the content determination method of a kind of sacubitril valsartan trisodium hemipentahydrate capsule according to claim 3, is characterized in that: described acid is 10% crotonic acid.

Images