CN113933414A - Method for detecting organic impurities in irbesartan amlodipine besylate composite preparation - Google Patents

Method for detecting organic impurities in irbesartan amlodipine besylate composite preparation Download PDF

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CN113933414A
CN113933414A CN202111144844.XA CN202111144844A CN113933414A CN 113933414 A CN113933414 A CN 113933414A CN 202111144844 A CN202111144844 A CN 202111144844A CN 113933414 A CN113933414 A CN 113933414A
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amlodipine
impurity
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irbesartan
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张议
李霞
王玲兰
朱婧
张子轩
章海云
谭莎
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Qianjin Pharmaceutical Co ltd
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Abstract

The invention belongs to the technical field of biochemical detection, and particularly relates to a method for detecting organic impurities in an irbesartan amlodipine besylate composite preparation. The method can simultaneously determine at least 9 impurities in the irbesartan amlodipine besylate composite preparation under the condition of a chromatogram, the separation degree of each chromatogram peak and a main peak or other impurity peaks is good, the quantitative limit also meets the analysis requirement, the sensitivity is high, the recovery rate, the precision and the stability are high, the specificity is strong, the reproducibility is good, the accuracy is high, the method is used for monitoring the impurities in the production process of the irbesartan amlodipine besylate composite preparation, and the quality of the composite preparation is favorably controlled and improved; meanwhile, the method is simple to operate, short in detection time and very suitable for quality monitoring of the medicines in large-scale industrial production.

Description

Method for detecting organic impurities in irbesartan amlodipine besylate composite preparation
Technical Field
The invention belongs to the technical field of biochemical detection. More particularly, relates to a method for detecting organic impurities in an irbesartan amlodipine besylate composite preparation.
Background
The irbesartan is an Angiotensin II (Ang II) receptor inhibitor, inhibits the conversion of AngI into Ang II, can specifically antagonize Angiotensin converting enzyme 1 receptor (AT1), has 8500 times of antagonism on AT1 compared with AT2, inhibits vasoconstriction and release of aldosterone by selectively blocking the combination of Ang II and AT1 receptor, and produces the effect of lowering blood pressure. Amlodipine besylate is a dihydropyridine calcium antagonist and can inhibit calcium ions from entering vascular smooth muscle and cardiac muscle through a membrane, so that the smooth muscle of coronary artery and peripheral blood vessels is relaxed. Irbesartan and amlodipine besylate are often used in combination or prepared into a composite preparation clinically to treat diseases such as hypertension and the like (high aromatic, gaofang, the curative effect of irbesartan and amlodipine in combination on treating senile hypertension with type 2 diabetes [ J ] practical medicines and clinics, 2012,15(10): 628-629.).
In the quality monitoring of actual production, certain organic impurities exist in the production process of the irbesartan amlodipine besylate composite preparation, the purity and the drug effect of a final product can be influenced, and certain potential safety hazards exist. At present, for quality monitoring of irbesartan amlodipine besylate composite preparations, impurities from irbesartan sources or amlodipine besylate sources can only be detected in a targeted manner by adopting different methods, more time and energy are needed, and quality monitoring of the irbesartan amlodipine besylate composite preparations is greatly limited. Therefore, there is an urgent need to provide a method that can simultaneously detect various organic impurities in the irbesartan amlodipine besylate composite preparation.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art that a method for simultaneously detecting various organic impurities in an irbesartan amlodipine besylate composite preparation is lacked, and provides a method for simultaneously detecting various organic impurities in the irbesartan amlodipine besylate composite preparation, which has the advantages of good separation degree, high accuracy and simple operation.
The invention aims to provide a method for detecting organic impurities in an irbesartan amlodipine besylate composite preparation.
The above purpose of the invention is realized by the following technical scheme:
a method for detecting organic impurities in an irbesartan amlodipine besylate composite preparation adopts a high performance liquid chromatography to detect, wherein the high performance liquid chromatography uses 0.01-0.02 mol/L phosphate buffer solution with the pH value of 2.5-3.4 as a mobile phase A and 80-100% methanol as a mobile phase B, and the detection is carried out under the following gradient elution conditions:
0 → (5 to 7) min, mobile phase a: 100 → 100%; (5-7) → (20-22) min, mobile phase A: 100 → 60%; (20-22) → (30-32) min, mobile phase A: 60 → 60%; (30-32) → (30.1-32.1) min, and a mobile phase A: 60 → 100%; (30.1-32.1) → (35-37) min, and a mobile phase A: 100 → 100%.
Further, the phosphate is dipotassium hydrogen phosphate and/or potassium dihydrogen phosphate.
Preferably, the mobile phase B is 100% methanol.
Furthermore, in the high performance liquid chromatography, a chromatographic column is filled with octadecylsilane chemically bonded silica, the detection wavelength is 230-240 nm, the column temperature is 30-40 ℃, the flow rate is 0.9-1.1 ml/min, and the sample injection amount is 10-20 mu L. Preferably, in the high performance liquid chromatography, the detection wavelength is 237nm, the column temperature is 40 ℃, the flow rate is 1.0ml/min, and the sample injection amount is 10 muL.
More preferably, the column is Wondasil C18, 150mm by 4.6mm, 3.5 μm.
Further, organic impurities in the irbesartan amlodipine besylate composite preparation comprise:
irbesartan impurity i: 1-pentanoylaminocyclopentanecarboxylic acid [2' - (1H-tetrazol-5-yl) -biphenyl-4-ylmethyl ] -amide, CAS No.: 748812-53-5, the structural formula is:
Figure BDA0003285045970000021
amlodipine EP impurity a: 3-ethyl 5-methyl (4RS) -4- (2-chlorophenyl) -2- [ [2- (1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) ethoxy ] methyl ] 6-methyl-1, 4-dihydropyridine-3, 5-dicarboxylate, CAS number: 88150-62-3, the structural formula is:
Figure BDA0003285045970000022
amlodipine EP impurity B: 3-ethyl 5-methyl (4RS) -4- (2-chlorophenyl) -6-methyl-2- [ [2- [2- (methylcarbamoyl) benzamido ] ethoxy ] methyl ] -1, 4-dihydropyridine-3, 5-dicarboxylate with CAS number: 721958-72-1, the structural formula is:
Figure BDA0003285045970000031
amlodipine EP impurity D: 3-ethyl 5-methyl 2- [ (2-aminoethoxy) methyl ] -4- (2-chlorophenyl) -6-methylpyridine-3, 5-dicarboxylate with CAS number: 113994-41-5, the structural formula is:
Figure BDA0003285045970000032
amlodipine EP impurity E: (4RS) -2- [ (2-aminoethoxy) methyl ] -4- (2-chlorophenyl) -6-methyl-1, 4-dihydropyridine-3, 5-dicarboxylic acid ethyl ester, CAS number: 140171-65-9, the structural formula is:
Figure BDA0003285045970000033
amlodipine EP impurity F: (4RS) -2- [ (2-aminoethoxy) methyl ] -4- (2-chlorophenyl) -6-methyl-1, 4-dihydropyridine-3, 5-dicarboxylic acid methyl ester, CAS number: 140171-66-0, the structural formula is:
Figure BDA0003285045970000034
amlodipine EP impurity G: 4- (2-chlorophenyl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarboxylic acid dimethyl ester, CAS No.: 43067-01-2, structural formula is:
Figure BDA0003285045970000041
amlodipine EP impurity H: 2- [ [2- [ [ (4RS) -4- (2-chlorophenyl) -3- (ethoxycarbonyl) -5- (methoxycarbonyl) -6-methyl-1, 4-dihydropyridin-2-yl ] methoxy ] ethyl ] carbamoyl ] benzoic acid, CAS number: 318465-73-5, the structural formula is:
Figure BDA0003285045970000042
amlodipine USP impurity 1: 5-Ethyl-7-methyl-6- (2-chlorophenyl) -8-methyl-3, 4,6, 7-tetrahydro-2H-benzo [ b ] [1,4] oxazine-5, 7-dicarboxylate, CAS number: N/A, the structural formula is as follows:
Figure BDA0003285045970000043
furthermore, in the high performance liquid chromatography, methanol is used as a solvent for preparing a reference substance.
Further, in the high performance liquid chromatography, the preparation method of the sample to be detected comprises the following steps: dispersing a sample to be detected by using a phosphate buffer solution, adding methanol to a constant volume, and taking a subsequent filtrate for determination.
Furthermore, the quantitative limit of the amlodipine EP impurity A, the amlodipine EP impurity B, the amlodipine EP impurity E, the amlodipine EP impurity F, the amlodipine EP impurity G and the amlodipine EP impurity H is (1.3-2.7) ng.
Furthermore, the content of the impurity D containing amlodipine EP in the irbesartan amlodipine besylate composite preparation is not more than 0.5% (correction factor 2), the content of other single impurities is not more than 0.2%, and the content of total impurities is not more than 1.5%.
The invention has the following beneficial effects:
the detection method can simultaneously detect at least 9 impurities in the irbesartan amlodipine besylate composite preparation under the same chromatographic condition, has good separation degree of each chromatographic peak and a main peak or other impurity peaks, can completely separate impurities from different sources, has high sensitivity, higher recovery rate, precision and stability, strong specificity, good reproducibility and high accuracy, can be used for monitoring the impurities in the production process of the irbesartan amlodipine besylate composite preparation, and is favorable for controlling and improving the quality of the irbesartan amlodipine besylate composite preparation; meanwhile, the method is simple to operate, short in detection time and very suitable for quality monitoring of the medicines in large-scale industrial production.
Drawings
FIG. 1 is a chromatogram of a solution of a standard test sample in the specificity test in example 1 of the present invention.
FIG. 2 is a PDA scanning spectrum of the maximum absorption wavelength of each component contained in the impurity mixed localization solution in the specificity determination of example 1 of the present invention.
FIG. 3 is a chromatogram for detecting a labeled test solution in example 2 of the present invention.
FIG. 4 is a chromatogram for detection of a solution of the labeled test sample in comparative example 1.
FIG. 5 is a chromatogram for detection of a solution to be tested in comparative example 2.
FIG. 6 is a chromatogram for detection of a solution of the labeled test sample in comparative example 3 according to the present invention.
FIG. 7 is a chromatogram for detection of a solution of a labeled test sample in comparative example 4.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Wherein, the potassium dihydrogen phosphate is analytically pure (national drug group chemical reagent limited), the methanol is chromatographically pure (SPECTRUM), irbesartan (China food and drug testing institute), amlodipine besylate (China food and drug testing institute), and irbesartan amlodipine besylate tablets (big Japan Sumitomo pharmaceutical Co., Ltd., specification irbesartan 100 mg/amlodipine 10 mg).
Irbesartan impurity I is 1-pentanoyl amino cyclopentane carboxylic acid [2' - (1H-tetrazol-5-yl) -biphenyl-4-ylmethyl ] -amide, and the source is Chinese food and drug testing institute;
amlodipine EP impurity A is 3-ethyl 5-methyl (4RS) -4- (2-chlorophenyl) -2- [ [2- (1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) ethoxy ] methyl ] 6-methyl-1, 4-dihydropyridine-3, 5-dicarboxylate from PCL;
amlodipine EP impurity B is 3-ethyl 5-methyl (4RS) -4- (2-chlorophenyl) -6-methyl-2- [ [2- [2- (methylcarbamoyl) benzoylamino ] ethoxy ] methyl ] -1, 4-dihydropyridine-3, 5-dicarboxylate with source of PCL;
amlodipine EP impurity D is 3-ethyl 5-methyl 2- [ (2-aminoethoxy) methyl ] -4- (2-chlorophenyl) -6-methylpyridine-3, 5-dicarboxylic acid ester, and the source is PCL;
amlodipine EP impurity E is (4RS) -2- [ (2-aminoethoxy) methyl ] -4- (2-chlorphenyl) -6-methyl-1, 4-dihydropyridine-3, 5-dicarboxylic acid ethyl ester, and the source is PCL;
amlodipine EP impurity F is (4RS) -2- [ (2-aminoethoxy) methyl ] -4- (2-chlorphenyl) -6-methyl-1, 4-dihydropyridine-3, 5-dicarboxylic acid methyl ester, and the source is PCL;
amlodipine EP impurity G is 4- (2-chlorphenyl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dimethyl dicarboxylate, and the source is PCL;
amlodipine EP impurity H is 2- [ [2- [ [ (4RS) -4- (2-chlorophenyl) -3- (ethoxycarbonyl) -5- (methoxycarbonyl) -6-methyl-1, 4-dihydropyridin-2-yl ] methoxy ] ethyl ] carbamoyl ] benzoic acid, derived from PCL;
amlodipine USP impurity 1 is 5-ethyl 7-methyl 6- (2-chlorophenyl) -8-methyl-3, 4,6, 7-tetrahydro-2H-benzo [ b ] [1,4] oxazine-5, 7-dicarboxylate with PCL as source.
The remaining reagents and materials used in the following examples are all commercially available unless otherwise specified.
Example 1 a method for detecting organic impurities in irbesartan amlodipine besylate composite preparation
The method for detecting organic impurities in the irbesartan amlodipine besylate composite preparation comprises the following steps:
chromatographic conditions are as follows: column Wondasil C18(150 mm. times.4.6 mm, 3.5 μm) packed with octadecylsilane bonded silica gel; linear gradient elution was performed according to Table 1 using 0.02mol/L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid) -methanol (50:50) as mobile phase A and methanol as mobile phase B; the flow rate is 1.0 mL/min; the column temperature was 40 ℃; the detection wavelength is 237 nm; the amount of sample was 10. mu.L.
Table 1 example 1 linear gradient elution conditions
Figure BDA0003285045970000061
Figure BDA0003285045970000071
The chromatographic conditions were verified for specificity, solution stability, and quantitation limits as follows:
1. specificity
Solution preparation:
amlodipine EP impurity A, B, E, F, G, H and USP impurity 1 localization solution (about 2.5 μ g/ml): taking 2.5mg of each impurity reference substance, respectively placing into a 20ml measuring flask, adding methanol to dissolve and dilute to scale, and shaking up to obtain impurity reference substance stock solution; precisely measuring 0.5ml of stock solution, placing in a 25ml measuring flask, adding solvent to dissolve and dilute to scale, and shaking.
② irbesartan impurity I positioning solution (about 15 mug/ml): weighing about 4mg of irbesartan impurity I reference substance, placing the reference substance into a 20ml measuring flask, adding methanol to dissolve and dilute the reference substance to scale, and shaking the reference substance uniformly to serve as irbesartan impurity I reference substance stock solution; and (3) taking 100 mu l of irbesartan impurity I reference substance stock solution, putting the irbesartan impurity I reference substance stock solution into a 1.5ml sample injection vial, adding 1ml of solvent, and shaking up to obtain the irbesartan sodium liquid.
③ amlodipine EP impurity D localization solution (about 15 μ g/ml): weighing about 1.5mg of an amlodipine EP impurity D reference substance, placing the reference substance into a 20ml measuring flask, adding methanol to dissolve and dilute the reference substance to a scale, and shaking the reference substance uniformly to serve as irbesartan impurity I reference substance stock solution; taking 75 mu l of amlodipine EP impurity D reference substance stock solution, putting the amlodipine EP impurity D reference substance stock solution into a 1.5ml sample injection vial, adding 1ml of solvent, and shaking up to obtain the amlodipine EP impurity D reference substance.
System applicability solution: weighing about 3mg of irbesartan impurity I reference substance, placing the reference substance into a 20ml measuring flask, adding methanol to dissolve and dilute the reference substance to scale, and shaking the reference substance uniformly to serve as irbesartan impurity I reference substance stock solution; taking about 5mg of amlodipine EP impurity D, precisely weighing, placing in a 100ml measuring flask, adding methanol for dissolving, diluting to scale, shaking uniformly, and taking as a reference stock solution of amlodipine EP impurity D; respectively taking about 50mg of irbesartan and about 17.5mg of amlodipine besylate, precisely weighing, placing into the same 50ml measuring flask, precisely adding 0.5ml of irbesartan impurity I reference substance stock solution and 0.375ml of amlodipine EP impurity D reference substance stock solution respectively, adding methanol to dissolve and dilute to scale, and shaking uniformly to obtain the irbesartan amlodipine besylate.
Impurity mixing and positioning solution: and (4) taking 1ml of the system applicability solution, putting the system applicability solution into a 1.5ml sample injection small bottle, adding 20 mul of each of the obtained amlodipine EP impurity A, B, E, F, G, H and the USP impurity 1 reference substance stock solution, and shaking up to obtain the amlodipine.
Sixthly, the stock solution of the test sample: taking 5 irbesartan amlodipine besylate tablets, precisely weighing, placing into a 100ml measuring flask, adding a proper amount of 0.02mol/L phosphate buffer solution (pH3.0), and performing ultrasonic dispersion; adding appropriate amount of methanol, shaking vigorously to dissolve, diluting with methanol and fixing volume, filtering, and collecting subsequent filtrate.
Seventh, solvent: methanol-0.02 mol/L phosphate buffer (pH3.0) (60: 40).
The test article solution: taking 5.0ml of the test sample stock solution, placing the test sample stock solution in a 25ml measuring flask, adding the solvent to dilute the test sample to scale, and shaking the test sample evenly.
Ninthly, adding a standard sample solution: taking 5 irbesartan amlodipine besylate tablets, precisely weighing, placing into a 100ml measuring flask, adding a proper amount of 0.02mol/L phosphate buffer solution (pH3.0), and performing ultrasonic dispersion; adding a proper amount of methanol, violently shaking to dissolve, precisely adding 300 mu l of the stock solution of the amlodipine EP impurity D reference obtained by the third step, 5mL of the stock solution of the irbesartan impurity I reference obtained by the third step and 200 mu l of each of the stock solutions of the amlodipine impurities (amlodipine EP impurity A, B, E, F, G, H and USP impurity 1) reference obtained by the third step, diluting with methanol, fixing the volume, filtering, and taking subsequent filtrate.
Control solution for r: taking about 10.5mg of amlodipine besylate reference substance, precisely weighing, placing in a 25ml measuring flask, dissolving and diluting to scale with methanol, and shaking uniformly to serve as amlodipine besylate reference substance stock solution; taking an irbesartan reference substance of about 20mg, precisely weighing, placing in a 20ml measuring flask, dissolving and diluting to scale with methanol, and shaking uniformly to serve as irbesartan reference substance stock solution; precisely measuring 5ml of irbesartan reference substance stock solution and 1ml of amlodipine besylate reference substance stock solution, putting the irbesartan reference substance stock solution and the amlodipine besylate reference substance stock solution into the same 50ml measuring flask, diluting the irbesartan reference substance stock solution to a scale by using a solvent, and shaking the mixture uniformly; precisely measuring 1ml, placing in a 20ml measuring flask, diluting with solvent to scale, and shaking.
Measurement and results:
taking blank solvent (namely, solvent), impurity mixed positioning solution, irbesartan impurity I positioning solution, amlodipine EP impurity D positioning solution, solutions for reference products (R) and (r) to-be-tested products and solutions for plus-standard to-be-tested products, respectively 10 mu L, injecting into a liquid chromatograph, and recording chromatogram, wherein the result is shown in figure 1.
Among them, system applicability requires: in a system applicability solution chromatogram, the separation degree among an amlodipine EP impurity D, amlodipine, irbesartan and an irbesartan impurity I is required, and the number of theoretical plates is not less than 3000 according to the irbesartan and the amlodipine.
From fig. 1, it can be seen that the blank solvent does not interfere with the detection, and irbesartan, amlodipine besylate and impurities are well separated and do not interfere with the detection, and the specificity meets the requirements; the system applicability also meets the requirements.
Taking 10 mu L of impurity mixed positioning solution, injecting into a liquid chromatograph, scanning at 200-400 nm by using a PDA detector, and recording a chromatogram, wherein the result is shown in figure 2 and table 2.
TABLE 2 impurity mix positioning solution positioning results
Figure BDA0003285045970000081
Figure BDA0003285045970000091
As can be seen from the above table, irbesartan, amlodipine and various impurities have large absorption at a wavelength of 237nm, so 237nm is selected as a detection wavelength.
2. Stability of solution
Placing the special sample solution with the added mark at room temperature for 0, 8, 12, 24, 36 and 48 hours, precisely measuring 10 mu L of the special sample solution, respectively injecting the special sample solution into a liquid chromatograph, recording a chromatogram, and calculating and analyzing, wherein the result is shown in tables 3-4.
TABLE 3 stability results of spiked test article solutions Irbesartan
Figure BDA0003285045970000092
Table 4 stability results of spiked test article solutions-amlodipine
Figure BDA0003285045970000101
Note: the absolute change amount is an absolute change amount compared with 0 h.
As can be seen from the table, compared with 0h, the absolute change rates of the impurity contents of irbesartan and amlodipine besylate in the standard sample solution are respectively 0.01 percent and 0.05 percent at most, which are not more than 0.05 percent, the absolute change rates of the total impurity contents are respectively 0.01 percent and 0.02 percent at most, which are not more than 0.1 percent at most, no impurity which is more than 0.05 percent is newly added, and the solution stability meets the requirement.
3. Limit of quantification
Taking a proper amount of the ammoniacal amlodipine EP A, B, E, F, G, H stock solution with specificity, diluting the stock solution with methanol until the S/N is about 10, and determining the stock solution as a sample to be tested for the impurity quantitative limit, wherein the specific result is shown in Table 5.
TABLE 5 limit of impurity quantitation
Figure BDA0003285045970000102
Figure BDA0003285045970000111
Example 2 method for detecting organic impurities in irbesartan amlodipine besylate composite preparation
The difference from example 1 is that the linear gradient elution conditions of example 2 are shown in Table 6, and the rest of the parameters and operations are shown in example 1, and ninthly plus standard test solution in specificity is shown in FIG. 3.
As can be seen from the figure, the separation degrees of irbesartan, amlodipine, irbesartan impurity I, amlodipine EP impurity A, amlodipine EP impurity B, amlodipine EP impurity D, amlodipine EP impurity E, amlodipine EP impurity F, amlodipine EP impurity G, amlodipine EP impurity H and amlodipine USP impurity 1 are all more than 1.5, and the separation effect is good.
Comparative example 1
Comparative example 1 differs from example 1 in that the mobile phase of comparative example 1 was changed to 0.02mol/L potassium dihydrogen phosphate solution (adjusted to pH3.0 with phosphoric acid) as mobile phase a, methanol as mobile phase B, and in that the ratio of mobile phase a: mobile phase B was eluted isocratically at 40:60, with the remaining parameters being referenced to the ninthly spiked test sample solution of example 1 specificity. (the detection method of comparative example 1 refers to the method for measuring the content of amlodipine besylate contained in the Japanese pharmacopoeia, 17 th edition)
As shown in fig. 4, it can be seen that amlodipine EP impurity H and amlodipine EP impurity B cannot be separated, and amlodipine impurity a does not peak, and the specificity is difficult to meet the requirement.
The results of comparative example 1 were compared with those of example 1 and are shown in tables 7 to 8.
TABLE 7 comparison of System suitability of comparative example 1 versus example 1
Figure BDA0003285045970000112
As can be seen from the table, the applicability of the system of example 1 and the system of comparative example 1 are both in accordance with the regulations (the separation degree of each peak is more than 1.5, and the theoretical plate numbers of the peaks of irbesartan and amlodipine are both more than 3000), but the theoretical plate number of example 1 is higher, the tailing factor is smaller, and the peak pattern is better.
TABLE 8 comparison of the results of the measurement of the solutions of the test sample added with the standard of comparative example 1 and example 1
Name (R) Example 1 degree of separation Comparative example 1 degree of separation
Amlodipine EP impurity D / /
Amlodipine EP impurity F 5.78 1.96
Amlodipine (I) salt 6.00 3.76
Irbesartan impurity I 6.52 4.13
Amlodipine EP impurity G 1.80 7.92
Irbesartan 8.17 2.49
Amlodipine EP impurity H 5.07 /
Amlodipine EP impurity B 6.87 /
Amlodipine USP impurity 1 2.82 /
Amlodipine EP impurity A 6.27 Does not peak
As can be seen from the table, in example 1, more impurities can be detected simultaneously under the same condition, the specificity is stronger, and the separation degree is better.
To further increase the degree of separation and improve the peak profile, 0.02mol/L potassium dihydrogen phosphate solution (adjusted to pH3.0 with phosphoric acid) was used as mobile phase A, methanol was used as mobile phase B, and in mobile phase A: and (4) isocratic elution is carried out under the condition that the mobile phase B is 60:40, and the rest parameters are unchanged, so that ninc labeled test sample solution in specificity is detected. As a result, irbesartan is precipitated under the mobile phase and cannot be detected.
Comparative example 2
Comparative example 2 is different from example 1 in that the linear gradient elution conditions of comparative example 2 are changed as shown in table 9, and ninthly plus standard test sample solution among specificity is examined with reference to example 1 for the remaining parameters, and the results are shown in fig. 5.
TABLE 9 Linear gradient elution conditions for comparative example 2
Figure BDA0003285045970000121
Figure BDA0003285045970000131
As can be seen from the figure, the peak separation of the irbesartan impurity I and the amlodipine EP impurity E is not good, and the impurities cannot be accurately detected.
Comparative example 3
Comparative example 3 differs from example 1 in that mobile phase a of comparative example 3 was changed to 0.02mol/L potassium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) -methanol (50:50), the remaining parameters were measured with reference to example 1, and the ninc plus standard test sample solution of example 1 was tested with the results shown in fig. 6 and table 10.
TABLE 10 statistics of test results for comparative example 3
Retention time/min RRT (calculated as amlodipine) Attribution Degree of separation
3.257 0.48 Amlodipine EP impurity D /
4.759 0.70 Amlodipine EP impurity F 5.75
6.751 1.00 Amlodipine (I) salt 5.87
10.272 1.52 Irbesartan impurity I 7.29
12.283 1.82 Irbesartan 4.04
As can be seen from the figure and the table, the amlodipine impurity E, the amlodipine EP impurity G, the amlodipine EP impurity B, the amlodipine EP impurity H, the amlodipine USP impurity 1 and the amlodipine EP impurity a all do not show peaks, and cannot meet the detection requirement.
Comparative example 4
Comparative example 4 differs from example 1 in that mobile phase a of comparative example 3 was changed to 0.02mol/L potassium dihydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid) -methanol (50:50), the remaining parameters were measured with reference to example 1, and the ninx plus standard test sample solution of example 1 was specified, and the results are shown in fig. 7 and table 11.
TABLE 11 statistics of test results for comparative example 4
Figure BDA0003285045970000132
Figure BDA0003285045970000141
As can be seen from the figures and tables, the separation degree of amlodipine EP impurity E from the adjacent peak is 1.35, less than 1.5, and the separation degree is not good.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. The method for detecting organic impurities in the irbesartan amlodipine besylate composite preparation is characterized in that high performance liquid chromatography is adopted for detection, wherein the high performance liquid chromatography takes 0.01-0.02 mol/L phosphate buffer solution with the pH value of 2.5-3.4 as a mobile phase A and takes 50-100% methanol as a mobile phase B, and the detection is carried out under the following gradient elution conditions:
0 → (5 to 7) min, mobile phase a: 100 → 100%; (5-7) → (20-22) min, mobile phase A: 100 → 60%; (20-22) → (30-32) min, mobile phase A: 60 → 60%; (30-32) → (30.1-32.1) min, and a mobile phase A: 60 → 100%; (30.1-32.1) → (35-37) min, and a mobile phase A: 100 → 100%.
2. The method of claim 1, wherein the phosphate is dipotassium hydrogen phosphate and/or potassium dihydrogen phosphate.
3. The method of claim 2, wherein the mobile phase B is 100% methanol.
4. The method according to claim 1, wherein the HPLC method comprises packing a column with octadecylsilane chemically bonded silica, and detecting the wavelength at 230-240 nm, the column temperature at 30-40 ℃, and the flow rate at 0.9-1.1 ml/min.
5. The method of claim 4, wherein the chromatographic column is Wondasil C18, 150mm x 4.6mm, 3.5 μm.
6. The method of any one of claims 1 to 5, wherein the organic impurities comprise:
irbesartan impurity i: 1-pentanoylaminocyclopentanecarboxylic acid [2' - (1H-tetrazol-5-yl) -biphenyl-4-ylmethyl ] -amide;
amlodipine EP impurity a: 3-ethyl 5-methyl (4RS) -4- (2-chlorophenyl) -2- [ [2- (1, 3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) ethoxy ] methyl ] 6-methyl-1, 4-dihydropyridine-3, 5-dicarboxylate;
amlodipine EP impurity B: 3-ethyl 5-methyl (4RS) -4- (2-chlorophenyl) -6-methyl-2- [ [2- [2- (methylcarbamoyl) benzoylamino ] ethoxy ] methyl ] -1, 4-dihydropyridine-3, 5-dicarboxylate;
amlodipine EP impurity D: 3-ethyl 5-methyl 2- [ (2-aminoethoxy) methyl ] -4- (2-chlorophenyl) -6-methylpyridine-3, 5-dicarboxylate;
amlodipine EP impurity E: (4RS) -2- [ (2-aminoethoxy) methyl ] -4- (2-chlorophenyl) -6-methyl-1, 4-dihydropyridine-3, 5-dicarboxylic acid ethyl ester;
amlodipine EP impurity F: (4RS) -2- [ (2-aminoethoxy) methyl ] -4- (2-chlorophenyl) -6-methyl-1, 4-dihydropyridine-3, 5-dicarboxylic acid methyl ester;
amlodipine EP impurity G: 4- (2-chlorophenyl) -2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarboxylic acid dimethyl ester;
amlodipine EP impurity H: 2- [ [2- [ [ (4RS) -4- (2-chlorophenyl) -3- (ethoxycarbonyl) -5- (methoxycarbonyl) -6-methyl-1, 4-dihydropyridin-2-yl ] methoxy ] ethyl ] carbamoyl ] benzoic acid;
amlodipine USP impurity 1: 5-Ethyl-7-methyl-6- (2-chlorophenyl) -8-methyl-3, 4,6, 7-tetrahydro-2H-benzo [ b ] [1,4] oxazine-5, 7-dicarboxylate.
7. The method of claim 6, wherein the reference is prepared by high performance liquid chromatography using methanol as a solvent.
8. The method of claim 6, wherein the sample to be tested is prepared by the following steps: dispersing a sample to be detected by using a phosphate buffer solution, adding methanol to a constant volume, and taking a subsequent filtrate for determination.
9. The method according to claim 6, wherein the limit of quantification of the amlodipine EP impurity A, the amlodipine EP impurity B, the amlodipine EP impurity E, the amlodipine EP impurity F, the amlodipine EP impurity G and the amlodipine EP impurity H is (1.3-2.7) ng.
10. The method as claimed in claim 6, wherein the content of amlodipine besylate EP impurity D in the irbesartan amlodipine besylate composite preparation is not more than 0.5%, the content of other single impurities is not more than 0.2%, and the content of total impurities is not more than 1.5%.
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