A method for separating and detecting enantiomers in a valsartan-amlodipine pharmaceutical composition

Abstract

The invention discloses a method for separating and detecting enantiomers in a valsartan amlodipine pharmaceutical composition, and belongs to the field of pharmaceutical analysis. The method uses a reversed-phase chromatographic column with C18 as a filler, uses acetonitrile-water containing triethanolamine as a mobile phase, and utilizes isocratic elution to carry out HPLC separation and determination of the valsartan enantiomers in the valsartan amlodipine pharmaceutical composition. The reversed-phase chromatography system used in the present invention has the advantages of stable chromatographic medium performance, wide pH tolerance range, strong separating power, easy operation, etc., and the inventive method has good specificity, good accuracy, low requirements for instrumentation, good sensitivity, good injection precision and repeatability, good actual detection effect, and can better control the product quality of the valsartan amlodipine pharmaceutical composition.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and particularly relates to a method for separating and detecting enantiomers in a valsartan and amlodipine pharmaceutical composition. Background Art

[0002] Valsartan and amlodipine tablets are a compound preparation of valsartan and amlodipine besylate, suitable for the treatment of essential hypertension, and are primarily used for hypertensive patients who have not responded well to treatment with amlodipine or valsartan alone. Valsartan is N-(1-pentanoyl)-N-[4-[2-(1H-tetrazol-5-yl)phenyl]benzyl]-L-valine. Valsartan contains one chiral center and exists as a pair of enantiomers. Since the drug activity of the R-enantiomer is much lower than that of the S-enantiomer, the limit of the R-enantiomer (valsartan enantiomer) needs to be controlled.

[0003]

[0004] In the existing technology, the 2020 edition of the "Chinese Pharmacopoeia" Part IV uses an α-acid glycoprotein column (AGP) as a chromatographic column and pH 7.0 phosphate buffer-isopropanol (98:2) as a mobile phase to separate and detect enantiomers in valsartan raw materials at a wavelength of 227nm; this method has problems such as strict use conditions of the AGP column and a short service life.

[0005] Chinese patent publication CN114689731A discloses a method for detecting valsartan enantiomers. The detection conditions are: an OD chromatographic column, n-hexane as mobile phase A, ethanol containing 0.1% trifluoroacetic acid as mobile phase B, a flow rate of 0.5-1.0 mL / min, a column temperature of 20-40°C, and a detection wavelength of 210-270 nm. This method is a normal-phase liquid chromatography method. OD chromatographic columns are expensive, have low impurity tolerance, and a short service life. Furthermore, gradient elution is used, making the method relatively complex. Furthermore, HPLC methods based on chiral chromatographic columns have poor reproducibility, making inter-laboratory method transfer difficult.

[0006] Chinese patent publication CN115248262A discloses a method for separating and detecting valsartan and its enantiomers. This method utilizes a CHIRALPAK IA chromatographic column with a normal phase mixed solvent of n-hexane and isopropanol as the mobile phase. Separation and detection are performed at a flow rate of 0.8 to 1.2 mL / min, a column temperature of 33 to 37°C, and a detection wavelength of 227 nm. The CHIRALPAK IA column utilizes starch-3,5-dimethylphenylcarbamate bonded to the silica gel surface as a filler. This expensive method is also applicable only to the detection of individual enantiomers in valsartan.

[0007] In order to overcome the above-mentioned shortcomings of the existing technology, it is necessary to develop an HPLC method based on a C18 reversed-phase chromatography column that is easy to implement, has strong anti-interference ability, is simple in method, highly reproducible, and easy to transfer methodology between laboratories for the separation and detection of enantiomers in valsartan and amlodipine pharmaceutical compositions. Summary of the Invention

[0008] In order to solve the above technical problems, the present invention provides a method for separating and detecting enantiomers in a valsartan amlodipine pharmaceutical composition based on a C18 reverse phase chromatographic column. The method has high separation degree, is simple and easy to operate, and can better control the product quality of the valsartan amlodipine pharmaceutical composition.

[0009] The specific technical solutions adopted are as follows:

[0010] The invention discloses a method for separating and detecting enantiomers in a valsartan-amlodipine pharmaceutical composition. The method uses a reverse phase chromatographic column filled with C18 and acetonitrile-water containing triethanolamine as a mobile phase to perform HPLC separation and determination of the valsartan enantiomers in the valsartan-amlodipine pharmaceutical composition by isocratic elution.

[0011] Compared with α-acid glycoprotein columns or OD chromatography columns used in the prior art, C18 chromatography columns have a long service life, good versatility, and a wide pH tolerance range. The present invention utilizes a common and easily available C18 chromatography column to achieve separation and determination of valsartan enantiomers, and has good separation and detection effects. In addition, the present invention uses acetonitrile-water containing triethanolamine as a mobile phase, and triethanolamine can combine with valsartan enantiomers through non-covalent bonds such as hydrogen bonds to form reversible diastereomeric complexes, thereby achieving separation of valsartan and valsartan enantiomers.

[0012] Preferably, the C18-filled reverse phase chromatography column is Welch Xtimate C18, 150×4.6 mm, 3 μm. This type of column has better performance and can tolerate a wider pH range of 1.0 to 12.5.

[0013] Preferably, the volume ratio of acetonitrile to water in the mobile phase is 45-50:50-55.

[0014] More preferably, the volume of triethanolamine in the mobile phase accounts for 0.2-0.4% of the total volume of acetonitrile and water. When the triethanolamine content in the mobile phase is within the above range, the detection effect is good.

[0015] The chromatographic conditions of the HPLC include:

[0016] Mobile phase flow rate 0.8-1.0 mL / min;

[0017] Column temperature 28-30°C;

[0018] The detector used was an ultraviolet detector with a detection wavelength of 230 nm.

[0019] Further preferably, the HPLC chromatographic conditions include: mobile phase flow rate of 0.8 mL / min; chromatographic column temperature of 30°C.

[0020] Take an appropriate amount of the valsartan and amlodipine pharmaceutical composition, grind it into powder, dissolve it in the mobile phase, and filter it to prepare a test solution with a valsartan concentration of 0.4-0.6 mg / mL, and then perform HPLC separation and determination.

[0021] Preferably, the injection volume of the test solution is 5 to 10 μL.

[0022] More preferably, the injection volume of the test solution is 10 μL.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The method of the present invention uses a common and easily available C18 chromatographic column to achieve the separation and detection of enantiomers in the valsartan amlodipine pharmaceutical composition, and the separation and detection effect is good. The reversed-phase chromatography system used has the advantages of stable chromatographic medium performance, wide pH tolerance range, strong separation ability, and easy operation, which can more conveniently achieve quality control of the valsartan amlodipine pharmaceutical composition.

[0025] (2) The present invention uses acetonitrile-water containing triethanolamine as the mobile phase, wherein triethanolamine can form a reversible diastereomeric complex with the valsartan enantiomers through non-covalent bonds such as hydrogen bonds, so that valsartan and the valsartan enantiomers are separated due to different retention times and distributions on the stationary phase. The detection method of the present invention has a quantitative limit of 0.1467 μg / mL and a detection limit of 0.0734 μg / mL. It has good specificity and accuracy, low requirements on instruments and equipment, good sensitivity, good injection precision and repeatability, and good actual detection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the chromatogram of the system suitability solution in Example 1.

[0027] Figure 2 The chromatogram of the test solution in Example 1.

[0028] Figure 3 This is the chromatogram of the system suitability solution in Comparative Example 1.

[0029] Figure 4 This is the chromatogram of the system suitability solution in Comparative Example 2. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the following examples and accompanying drawings. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0031] In the following examples, the high performance liquid chromatograph used was Agilent 1260, and the materials and reagents used, unless otherwise specified, were all commercially available.

[0032] 1. Test sample

[0033]

[0034] 2. Reference substances

[0035] name batch number source Valsartan enantiomer reference 100836-201802 China Inspection and Quarantine Agency Valsartan reference substance 100651-202006 China Inspection and Quarantine Agency Amlodipine besylate reference substance 100374-201605 China Inspection and Quarantine Agency

[0036] 3. Standard regulations

[0037] (1) System applicability

[0038] In the system suitability solution chromatogram, the separation degree between the valsartan peak and the valsartan enantiomer peak should be no less than 2.0.

[0039] (2) Calculation formula for the enantiomer content in the test solution:

[0040]

[0041] Where: W 对 The weight of the reference substance (mg)

[0042] W 供 The amount of the test sample (mg)

[0043] A 对 Peak area of ​​reference solution

[0044] A 供 Peak area of ​​valsartan enantiomers in the test solution

[0045] D 供 Dilution factor of the test solution

[0046] D 对 Dilution multiple of the reference substance

[0047] C 对％ Content of reference substance

[0048] Example 1

[0049] (1) Solution preparation

[0050] Diluent: acetonitrile-water (45:55), add 4 mL of triethanolamine to 1 L of acetonitrile-water;

[0051] Test solution: Take the valsartan and amlodipine pharmaceutical composition, grind it into powder, accurately weigh 111.83 mg of the fine powder, place it in a 100 mL volumetric flask, add an appropriate amount of diluent, sonicate for 15 minutes to dissolve the main component, let it cool to room temperature, then add diluent to the scale, shake well and filter to prepare the test solution. The valsartan concentration in the test solution is about 0.52 mg / mL;

[0052] Reference substance stock solution: Take 10.12 mg of valsartan enantiomer reference substance, place it in a 50 mL volumetric flask, add an appropriate amount of diluent, sonicate to dissolve, let cool to room temperature, then add diluent to the scale, and shake well to prepare the reference substance stock solution;

[0053] Reference solution: Accurately measure 0.5 mL of reference stock solution, place it in a 20 mL volumetric flask, add diluent to the mark, and shake well to prepare the reference solution;

[0054] System suitability solution: Weigh 10.41 mg each of valsartan reference substance and amlodipine besylate reference substance into a 20 mL volumetric flask, add appropriate amount of diluent to dissolve, add 0.5 mL of reference substance stock solution, add diluent to the scale, and shake well to make the system suitability solution.

[0055] (2) Chromatographic conditions

[0056] Column: Welch Xtimate C18, 150 × 4.6 mm, 3 μm;

[0057] Mobile phase: acetonitrile-water (45:55), add 4 mL of triethanolamine to 1 L of acetonitrile-water;

[0058] Column temperature: 30°C;

[0059] Flow rate: 0.8 mL / min;

[0060] The detection was carried out using an ultraviolet detector at a wavelength of 230 nm;

[0061] (3) Sample testing

[0062] Accurately measure 10 μL of the system suitability solution and inject it into the high performance liquid chromatograph. Perform HPLC detection according to the above chromatographic conditions and record the chromatogram ( Figure 1 );

[0063] Accurately measure 10 μL of the test solution and the reference solution and inject them into the high performance liquid chromatograph. Perform HPLC detection according to the above chromatographic conditions, and then calculate the content of valsartan enantiomers in the test sample.

[0064] (4) Experimental results (4.1) System applicability

[0065]

[0066] Combining the system suitability solution chromatogram and the results in the above table, it can be seen that the separation degree between the valsartan peak and the valsartan enantiomer peak is greater than 2.0, indicating that the method of the present invention can be used for the separation and detection of enantiomers in the valsartan and amlodipine pharmaceutical compositions.

[0067] (4.2) Chromatograms of test solution and reference solution

[0068]

[0069] The chromatogram of the test solution is as follows Figure 2 As shown, valsartan and valsartan enantiomers eluted in sequence with good separation. The enantiomer content in the test solution was calculated to be 0.15%, indicating that the enantiomer content in the test solution met the company's internal control standards.

[0070] Example 2

[0071] (1) Solution preparation

[0072] Diluent: acetonitrile-water (50:50), add 2 mL of triethanolamine to 1 L of acetonitrile-water;

[0073] Test solution: Take the valsartan and amlodipine pharmaceutical composition, grind it into powder, accurately weigh 111.69 mg of the fine powder, place it in a 100 mL volumetric flask, add an appropriate amount of diluent, sonicate for 15 minutes to dissolve the main component, let it cool to room temperature, then add diluent to the scale, shake well and filter to prepare the test solution. The valsartan concentration in the test solution is about 0.49 mg / mL;

[0074] The same reference substance stock solution and reference substance solution as in Example 1 were used.

[0075] (2) Chromatographic conditions

[0076] Column: Welch Xtimate C18, 150 × 4.6 mm, 3 μm;

[0077] Mobile phase: acetonitrile-water (50:50), add 2 mL of triethanolamine to 1 L of acetonitrile-water;

[0078] Column temperature: 28°C;

[0079] Flow rate: 1.0 mL / min;

[0080] The detection was carried out using an ultraviolet detector at a wavelength of 230 nm;

[0081] (3) Sample testing

[0082] Accurately measure 5 μL of the test solution and the reference solution and inject them into the high performance liquid chromatograph. Perform HPLC detection according to the above chromatographic conditions, and then calculate the content of valsartan enantiomers in the test sample.

[0083] (4) Experimental results (4.1) Chromatograms of test solution and reference solution

[0084]

[0085]

[0086] The enantiomer content in the test solution was calculated from the above data to be 0.16%, indicating that the enantiomer content in the test solution met the company's internal control standards.

[0087] Example 3

[0088] (1) Solution preparation

[0089] Diluent: acetonitrile-water (50:50), add 3 mL of triethanolamine to 1 L of acetonitrile-water;

[0090] Test solution: Take the valsartan and amlodipine pharmaceutical composition, grind it into powder, accurately weigh 113.75 mg, place it in a 100 mL volumetric flask, add an appropriate amount of diluent, sonicate for 15 minutes to dissolve the main component, let it cool to room temperature, then add diluent to the scale, shake well and filter to prepare the test solution. The valsartan concentration in the test solution is about 0.56 mg / mL;

[0091] The same reference substance stock solution and reference substance solution as in Example 1 were used.

[0092] (2) Chromatographic conditions

[0093] Column: Welch Xtimate C18, 150 × 4.6 mm, 3 μm;

[0094] Mobile phase: acetonitrile-water (50:50), add 3 mL of triethanolamine to 1 L of acetonitrile-water;

[0095] Column temperature: 30°C;

[0096] Flow rate: 1.0 mL / min;

[0097] The detection was carried out using an ultraviolet detector at a wavelength of 230 nm;

[0098] (3) Sample testing

[0099] Accurately measure 10 μL of the test solution and the reference solution and inject them into the high performance liquid chromatograph. Perform HPLC detection according to the above chromatographic conditions, and then calculate the content of valsartan enantiomers in the test sample.

[0100] (4) Experimental results (4.1) Chromatograms of test solution and reference solution

[0101]

[0102] The enantiomer content in the test solution was calculated from the above data to be 0.12%, indicating that the enantiomer content in the test solution met the company's internal control standards.

[0103] Comparative Example 1

[0104] The detection method of Comparative Example 1 is the same as that of Example 1, except that:

[0105] (1) Triethanolamine was not added to acetonitrile-water in the diluent and mobile phase;

[0106] (2) No test solution and reference solution were prepared, but amlodipine besylate localizing solution and valsartan localizing solution were added: approximately 10 mg of amlodipine besylate reference solution and valsartan reference solution were weighed respectively, and amlodipine besylate localizing solution and valsartan localizing solution were prepared according to the method for preparing reference solution;

[0107] (3) The test sample solutions are different. In this comparative example, the amlodipine besylate positioning solution, valsartan positioning solution, reference substance stock solution and system suitability solution are mainly tested;

[0108] The other steps were the same as those in the test conditions and comparative example 1.

[0109] The experimental results of this comparative example are as follows:

[0110]

[0111] From the results in the above table and Figure 3 It can be seen that using this method, the peaks of each substance elute earlier, and the valsartan enantiomer peak and the valsartan peak in the system suitability solution cannot be effectively separated, which proves the necessity of adding triethanolamine to the mobile phase and that this chromatographic condition is not applicable.

[0112] Comparative Example 2

[0113] The detection method of Comparative Example 2 is the same as that of Comparative Example 1, except that the diluent and mobile phase are methanol-water (42:58), and 4 mL of triethanolamine is added to 1 L of methanol-water; the other steps and detection conditions are the same as those of Comparative Example 1.

[0114] The experimental results of this comparative example are as follows:

[0115]

[0116] From the results in the above table and Figure 4 It can be seen that using this method, the peaks of each substance are eluted earlier, and the valsartan enantiomer peak and the valsartan peak in the system suitability solution cannot be effectively separated. This chromatographic condition is not applicable.

[0117] Method Analysis

[0118] In order to verify the separation and detection method of the enantiomers in the valsartan and amlodipine pharmaceutical composition of the present invention, a systematic method validation was performed, including precision, accuracy, repeatability and limit of quantification, and the validation results are as follows:

[0119] (1) Intermediate precision: The method of quantitatively adding impurities to the test solution was adopted. The RSD of the enantiomer content in 6 test solutions was 1.7%, which was less than 10%, indicating that the intermediate precision of this method was good.

[0120] name Enantiomers (%) Test solution 1 1.2189 Test solution 2 1.1795 Test solution 3 1.2203 Test solution 4 1.2001 Test solution 5 1.2374 Test solution 6 1.2251 average value 1.2136 RSD (%) 1.7

[0121] (2) Accuracy: The recoveries and average values ​​of the enantiomers in 9 test solutions at 3 concentrations were all between 90% and 108%, with RSDs less than 5.0%, indicating that the method has good accuracy and is suitable for the detection of enantiomers in valsartan and amlodipine pharmaceutical compositions.

[0122]

[0123] (3) Repeatability: The method of quantitatively adding impurities to the test solution was adopted. The RSD of the enantiomer content in 6 test solutions was 0.8%, which was less than 10%, indicating that the method had good repeatability.

[0124] name Enantiomers (%) Test solution 1 1.2018 Test solution 2 1.2050 Test solution 3 1.2216 Test solution 4 1.2186 Test solution 5 1.2128 Test solution 6 1.2269 average value 1.2145 RSD (%) 0.8

[0125] (4) Limit of quantification: The valsartan enantiomer limit of quantification solution was injected six times continuously, and the RSD of the retention time was less than 1.0%, and the RSD of the peak area was less than 5.0%, indicating good sensitivity and the ability to ensure accurate determination of the enantiomers.

[0126] Number of injections Retention time (min) Peak area (A) 1 7.634 5864 2 7.671 6014 3 7.664 5667 4 7.641 5915 5 7.656 5580 6 7.675 5438 RSD (%) 0.2 3.8

[0127] In summary, the results of systematic method validation, including system suitability, precision, accuracy, repeatability, and limit of quantitation, indicate that the method for separation and detection of enantiomers in the valsartan and amlodipine pharmaceutical composition of the present invention meets the requirements for enantiomer detection of this product and can effectively evaluate the quality of this product.

[0128] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for separating and detecting enantiomers in a valsartan and amlodipine pharmaceutical composition, characterized in that: The valsartan enantiomers in the valsartan and amlodipine pharmaceutical composition were separated and determined by HPLC using a reverse phase chromatographic column filled with C18 and acetonitrile-water containing triethanolamine as the mobile phase using isocratic elution; Take an appropriate amount of valsartan and amlodipine pharmaceutical composition, grind it into powder, dissolve it in mobile phase, and filter it to prepare a test solution with a valsartan concentration of 0.4-0.6 mg / mL, and then perform HPLC separation and determination; The volume ratio of acetonitrile to water in the mobile phase is 45-50:50-55; the volume of triethanolamine in the mobile phase accounts for 0.2-0.4% of the total volume of acetonitrile and water; The chromatographic conditions of the HPLC include: Mobile phase flow rate 0.8-1.0 mL / min; Column temperature 28-30°C; The detector used was an ultraviolet detector with a detection wavelength of 230 nm.

2. The method for separating and detecting enantiomers in the valsartan and amlodipine pharmaceutical composition according to claim 1, wherein The reverse phase chromatography column with C18 as filler is Welch Xtimate C18, 150×4.6 mm, 3 μm.

3. The method for separating and detecting enantiomers in the valsartan and amlodipine pharmaceutical composition according to claim 1, wherein The HPLC chromatographic conditions include: mobile phase flow rate of 0.8 mL / min; chromatographic column temperature of 30°C.

4. The method for separating and detecting enantiomers in the valsartan and amlodipine pharmaceutical composition according to claim 1, wherein: The injection volume of the test solution is 5-10 μL.

5. The method for separating and detecting enantiomers in the valsartan and amlodipine pharmaceutical composition according to claim 1, wherein: The injection volume of the test solution was 10 μL.

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