Method for detecting organic impurities in levosimendan

Abstract

The invention relates to a method for detecting organic impurities in levosimendan, which comprises the following steps: detecting a solution to be detected of a levosimendan raw material medicine or preparation by adopting a high performance liquid chromatography, and eluting by taking pentafluorophenyl silica gel as a chromatographic column of a filler, 5-30mmol / L monopotassium phosphate solution as a mobile phase A and methanol as a mobile phase B. By utilizing the method, a plurality of groups of impurities which have similar chemical structures, molecular weights and polarities and are difficult to separate are well separated; the separation degree between the organic impurities and the separation degree between the organic impurities and the levosimendan peak reach 1.5 or above, and the recovery rate meets the requirement of 80.0%-120.0%; the sensitivity is high, the quantitation limit of 13 organic impurities is within 200ppm, and better quality control on the levosimendan raw material medicine is realized.

Description

Technical Field

[0001] The present invention belongs to the field of analytical detection methods for impurities in bulk drugs, and particularly relates to a method for detecting organic impurities in levosimendan. Background Art

[0002] Levosimendan is a calcium sensitizer and KATP channel opener that acts by altering calcium-binding information transmission. Levosimendan binds directly to troponin, stabilizing the conformation of the myocardial fibrin necessary for calcium-induced myocardial contraction, thereby increasing myocardial contractility without significant changes in heart rate or myocardial oxygen consumption. At the same time, this product has a strong vasodilatory effect, dilating blood vessels by activating ATP-sensitive potassium channels. This product mainly dilates peripheral veins, reducing the cardiac preload, which is beneficial for the treatment of heart failure. When this product is used at high doses, it has a certain phosphodiesterase inhibitory effect, which can increase the intracellular cAMP concentration in myocardial cells and exert an additional positive inotropic effect.

[0003] In the mainstream synthesis process of levosimendan, 6-(4-aminophenyl)-4,5-dihydro-5-methyl-3(2H)-pyridazinone and malononitrile are used as starting materials for the reaction to synthesize levosimendan. It is easy to introduce impurities with genotoxic warning structures. Genotoxic impurities have carcinogenic, mutagenic, and teratogenic properties. According to ICH guidelines, genotoxic impurities in bulk drugs should be strictly controlled. Thirteen organic impurities of levosimendan are listed in Table 0.

[0004] Table 0 List of Levosimendan Impurities

[0005]

[0006]

[0007]

[0008] Among the organic impurities of levosimendan, impurities D, I, III, and III-IB have genotoxic impurity warning structures. Impurity D is a by-product generated during the synthesis of crude levosimendan and contains an azo warning structure; impurity I is a by-product generated during the synthesis of levosimendan intermediates and contains a halogenated benzene warning structure; impurities III and III-IB have aniline warning structures. According to the limit calculation based on the TTC value of 1.5 μg / day, the maximum daily dose of levosimendan is 17.9 mg, and the impurity limit = TTC / maximum daily dose = 1.5 (μg / day) / 17.9 mg × 1000 = 83.80 ppm. The research limit is "should not exceed 83 ppm".

[0009] In the detection of levosimendan related substances, the chemical structures, molecular weights and polarities of impurity III and impurity III-IA are close, making them difficult to separate; the chemical structures, molecular weights and polarities of levosimendan and impurity K are close, making them difficult to separate; the chemical structures, molecular weights and polarities of impurity F and impurity III-IA are close, making them difficult to separate.

[0010] In the prior art, most of the analysis and detection of levosimendan related substances use a reversed-phase C18 column. Chinese Patent Application CN118191195A discloses a method for detecting levosimendan related substances by high performance liquid chromatography. It uses a reversed-phase C18 column, selects acetonitrile as the organic phase for the mobile phase, and uses a weakly acidic buffer salt with a pH of 4.8 - 5.2 to adjust the retention behavior, and detects impurity C, impurity D, impurity E, impurity III-ID, and impurity III in Table 0. This detection method has limited separation performance for impurities with similar polarities in levosimendan. Summary of the Invention

[0011] The purpose of the present invention is to provide a method for detecting organic impurities in levosimendan, which has good accuracy, high recovery rate, and the recovery rates of each impurity are between 98% and 103%. It has good sensitivity, and the quantitative limits of 13 organic impurities are within 200 ppm. The peak emergence between levosimendan and each impurity to be detected does not interfere with each other, and the resolution is high, with the resolution of each impurity being above 1.6. Under forced degradation experiments, the material balance under each condition is between 98.0% and 102.0%, and the specificity is good, realizing better quality control of levosimendan raw materials.

[0012] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0013] A method for detecting organic impurities in levosimendan, comprising the following steps:

[0014] Perform high performance liquid chromatography detection on the test solution of levosimendan raw materials or preparations. Use a chromatographic column filled with pentafluorophenyl silica gel, and elute with a 5 - 30 mmol / L potassium dihydrogen phosphate solution with a pH of 2.5 - 4.0 as mobile phase A and methanol as mobile phase B; detection wavelength: 260 - 300 nm; flow rate: adjust the flow rate until the column pressure is 2000 psi - 3000 psi; chromatographic column temperature: 25°C - 35°C.

[0015] Preferably, the organic impurities include: impurity III, impurity III-IA, and impurity III-IB, and the structural formulas are as follows:

[0016]

[0017] Furthermore, the organic impurities include: impurity III, impurity III-IA, impurity III-IB, impurity K, and impurity F, and the structural formulas are as follows:

[0018]

[0019] Furthermore, the organic impurities further include: impurity B, impurity C, impurity D, impurity E, impurity III-ID, impurity H, and the structural formulas are as follows:

[0020]

[0021]

[0022] Still further, the organic impurities further include impurity I and impurity L, and the structural formulas are as follows:

[0023]

[0024] Furthermore, the chromatographic column is selected from the Thermo Fisher Hypersil Gold PFP series or the Welch Ultimate PFP series.

[0025] Also, the particle size of the chromatographic column packing is ≤5 μm, the length is ≥25 cm, and the inner diameter is ≤4.6 mm.

[0026] Furthermore, in the mobile phase A, the pH is adjusted to 2.5 - 4.0 with phosphoric acid.

[0027] Preferably, the elution process is as follows: isocratic elution is performed with 80% - 90% by volume of mobile phase A and 10 - 20% by volume of mobile phase B. After the target peak is eluted, elution is performed with 30 - 50% by volume of mobile phase A and 50 - 70% by volume of mobile phase B for 15 - 25 minutes. After no more chromatographic peaks are eluted, the initial mobile phase ratio is restored for equilibration, and the equilibration time is not less than 10 minutes.

[0028] Also, the elution process is as follows: isocratic elution is performed with 85% - 90% by volume of mobile phase A and 10 - 15% by volume of mobile phase B. After the target peak is eluted, elution is performed with 35% - 40% by volume of mobile phase A and 60% - 65% by volume of mobile phase B for 15 - 25 minutes. After no more chromatographic peaks are eluted, the initial mobile phase ratio is restored for equilibration, and the equilibration time is not less than 15 minutes.

[0029] Also, the levosimendan preparation is levosimendan injection or levosimendan for injection, the concentration of levosimendan in the test solution is 0.5 - 1.5 mg / ml, and the injection volume is 5 - 20 μl.

[0030] The inventor unexpectedly found that in high performance liquid chromatography (HPLC) detection, a chromatographic column filled with pentafluorophenyl silica gel (PFP) can improve the separation effect of impurity III, impurity III-IA, impurity K, impurity F and levosimendan in a reverse liquid chromatography system, making the peak shape sharper and more symmetrical with high sensitivity. This may be because the benzene ring structure in this type of chromatographic column packing can undergo π-π stacking interaction with the aromatic ring in the analyte, which is particularly suitable for separating these polycyclic aromatic hydrocarbons, substituted benzenes or drug molecules. In addition, the three-dimensional structure of the pentafluorophenyl group may enhance the ability to distinguish stereoisomers or structural analogs (such as chiral molecules, isomers) among these levosimendan related substances, and cooperate with the specific mobile phase of the present invention, thus obtaining a good separation effect.

[0031] The present invention selects a mobile phase combination of 5-30 mmol / L potassium dihydrogen phosphate solution and methanol, and develops an elution gradient for the retention ability of each impurity, so that all impurities can be eluted and peak out. If the content of potassium dihydrogen phosphate in the mobile phase is too high, the baseline noise will be too large. If the content of potassium dihydrogen phosphate in the mobile phase is too low, it will affect the resolution between levosimendan and impurity I.

[0032] In the present invention, by adding an organic solvent methanol to the potassium dihydrogen phosphate solution and adjusting the elution ratio, on the basis of separating impurity III, impurity III-IA, impurity F, impurity K and levosimendan well, the successful elution and separation of impurity B, impurity C, impurity D, impurity E, impurity III-ID, impurity H, impurity I, impurity L, impurity III-IB are realized.

[0033] In the elution process of the present invention, isocratic elution is carried out with 80%-90% mobile phase A and 10-20% mobile phase B by volume percentage, and the resolution is better and the peak shape is better. During isocratic elution, if the proportion of the organic solvent is too large, the retention ability of each component may become poor, affecting the resolution; if the proportion of the organic phase is too small, chromatographic peak tailing or fronting may occur, having a negative impact on the peak shape.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention uses a chromatographic column filled with pentafluorophenyl silica gel and a specific mobile phase system to separate and detect impurities III and impurity III-IA, impurity K, impurity F and levosimendan, etc., whose chemical structures, molecular weights and polarities are close and difficult to separate from each other among the levosimendan related impurities, enhancing the separation effect between these impurities, and the resolution of each impurity is above 1.6, better controlling the quality of the product.

[0036] In the present invention, by adding organic solvent methanol into potassium dihydrogen phosphate solution and controlling the proportion of mobile phase, on the basis of separating impurity III, impurity III-IA, impurity F, impurity K and levosimendan well, the successful elution and separation of impurity B, impurity C, impurity D, impurity E, impurity F, impurity III-ID, impurity H, impurity I, impurity L and impurity III-IB are further realized. The recovery rate is high, and the recovery rates of 13 organic impurities of levosimendan all meet the requirement of 80.0% - 120.0%.

[0037] The detection method of the present invention has good specificity, with sharp and symmetrical peak shapes, high sensitivity of the substances to be detected. The proportion of organic impurities equivalent to the quantitation limit of the test substance is all within 200 ppm. Among them, the proportion of impurity D, impurity I, impurity III and impurity III-IB equivalent to the quantitation limit of the test substance is within 50 ppm, providing a reliable test basis for the quality detection of levosimendan raw material drug and its preparations. It can better control each organic impurity introduced and generated in the synthesis process of levosimendan. Description of the Drawings

[0038] Figure 1 It is the chromatogram of the diluent of the test sample (1) in Example 1 of the present invention.

[0039] Figure 2 It is the chromatogram of the system suitability solution of the test sample (2) in Example 1 of the present invention.

[0040] Figure 3 It is the chromatogram of the reference solution of the test sample (3) in Example 1 of the present invention.

[0041] Figure 4 It is the chromatogram of the system suitability solution of the test sample (4) in Example 1 of the present invention.

[0042] Figure 5 It is the chromatogram of the reference solution of the test sample (5) in Example 1 of the present invention.

[0043] Figure 6 It is the chromatogram of the reference solution of impurity III, impurity III-IA, III-IB, impurity F, impurity K and levosimendan of the test sample (6) in Example 1 of the present invention.

[0044] Figure 7 It is the chromatogram of the system suitability solution in Example 2 of the present invention.

[0045] Figure 8 It is the chromatogram of the reference solution of impurity III, impurity III-IA, III-IB, impurity I, impurity L and levosimendan in Example 2 of the present invention.

[0046] Figure 9It is the chromatogram of the system suitability solution in Comparative Example 1.

[0047] Figure 10 It is the chromatogram of the system suitability solution in Comparative Example 2. Detailed implementation manners

[0048] The present invention will be further described below in conjunction with specific embodiments.

[0049] For the experimental methods without specific conditions indicated in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product instruction manual; in the chromatograms of the examples and comparative examples, the unit of the peak elution time is minutes (min).

[0050] The reference substances used in the embodiments of the present invention are shown in Table 1.

[0051] Table 1

[0052]

[0053] System suitability experiment for the detection method of organic impurities in levosimendan raw material drug in Example 1. The high performance liquid chromatography detection conditions are as follows:

[0054] High performance liquid chromatograph:

[0055] Detector: UV detector;

[0056] Chromatographic column: Welch Ultimate PFP series pentafluorophenyl silica gel chromatographic column;

[0057] Mobile phase A: 5 - 30 mmol / L potassium dihydrogen phosphate, adjusted to pH 3.5 with phosphoric acid;

[0058] Mobile phase B: methanol;

[0059] Column temperature: room temperature;

[0060] Flow rate: Adjust the flow rate so that the column pressure is between 2000 psi and 3000 psi;

[0061] Detection wavelength: 260 - 300 nm;

[0062] Injection volume: 5 - 20 μl.

[0063] Elution program: Isocratic elution is carried out with 80% - 90% (by volume) of mobile phase A and 10% - 20% (by volume) of mobile phase B. After the target peak is eluted, elution is carried out with 40% (by volume) of mobile phase A and 60% (by volume) of mobile phase B for 15 - 25 minutes. After no more chromatographic peaks are eluted, the initial mobile phase ratio is restored for equilibration, and the equilibration time is not less than 10 minutes.

[0064] 1. Solution preparation

[0065] Diluent: Mobile phase A: Mobile phase B = 40:60, by volume.

[0066] Test solution: Weigh an appropriate amount of levosimendan raw material accurately, dissolve and dilute it with the diluent to prepare a solution containing about 1.0 mg per 1 ml, shake well, and it is ready.

[0067] Reference solution: Weigh appropriate amounts of reference substances of impurity B, impurity C, impurity D, impurity E, impurity F, impurity III-ID, impurity H, impurity I, impurity K, impurity L, impurity III, impurity III-IA, and impurity III-IB accurately, place them in the same volumetric flask, dissolve and dilute with the diluent to prepare a mixed solution containing about 1 μg of impurity B, impurity C, impurity E, impurity F, impurity H, impurity K, impurity L, and impurity III-IA per 1 ml, and containing about 0.05 μg of impurity D, impurity I, impurity III, and impurity III-IB per 1 ml, shake well, and it is ready.

[0068] Impurity reference solution: Weigh appropriate amounts of reference substances of impurity B, impurity C, impurity D, impurity E, impurity F, impurity III-ID, impurity H, impurity I, impurity K, impurity L, impurity III, impurity III-IA, and impurity III-IB accurately, place them in different volumetric flasks, dissolve and dilute with the diluent respectively to prepare impurity reference solutions containing about 5 μg of a single impurity per 1 ml, shake well, and they are ready.

[0069] System suitability solution: Weigh appropriate amounts of levosimendan raw material, reference substances of impurity B, impurity C, impurity D, impurity E, impurity F, impurity III-ID, impurity H, impurity I, impurity K, impurity L, impurity III, impurity III-IA, and impurity III-IB, dissolve them with the diluent to prepare a solution with a levosimendan concentration of 1.0 mg / ml and impurity concentrations of 1 μg / ml.

[0070] 2. Sample detection

[0071] Take 6 groups of samples to be tested, which are (1) 20 μl of diluent, (2) 10 μl of system suitability solution, (3) 20 μl of reference solution, (4) 20 μl of system suitability solution, (5) 20 μl of reference solution, (6) 20 μl of impurity III reference solution, impurity III-IA reference solution, impurity III-IB reference solution, impurity F reference solution, impurity K reference solution, and levosimendan test solution respectively. Inject the samples to be tested into the high-performance liquid chromatograph for injection detection and elution. The elution process: Isocratic elution is carried out with 80% - 90% of mobile phase A. After the target peak is eluted, elute with 60% of mobile phase B for 15 - 25 minutes. After observing that no more chromatographic peaks are eluted, restore to the initial ratio and equilibrate for no less than 10 minutes.

[0072] Among them, when detecting sample (3), the concentration of potassium dihydrogen phosphate in mobile phase A is 5 mmol / L; when detecting sample (5), the concentration of potassium dihydrogen phosphate in mobile phase A is 15 mmol / L; when detecting sample (6), the chromatographic conditions for each impurity control solution are the same, and the detection results are processed by Empower software as an overlay map; except for detecting sample (3), when detecting the remaining samples, the concentration of potassium dihydrogen phosphate in mobile phase A is 30 mmol / L; record the chromatograms of each sample to be detected.

[0073] The detection spectra of each sample are shown respectively in Figures 1 - 6 , where ZXMA is the levosimendan peak; the recovery rates of each impurity are shown in Table 2, and the retention times and resolutions of each impurity in the system suitability solution are shown in Table 3.

[0074] Table 2 Recovery Rates of Each Impurity

[0075]

[0076]

[0077]

[0078] As can be seen from Table 2, the recovery rates of each impurity are between 98% and 103%.

[0079] From Figure 1 , it can be known that the blank solvent has no interference on the substances to be detected.

[0080] From Figure 2 , it can be known that the separation of the chromatographic peaks of 14 components to be detected from the adjacent impurity peaks is good, and the retention times and resolutions of each impurity in the system suitability solution are shown in Table 3.

[0081] Table 3

[0082] Name RT (min) Resolution Tailing factor Theoretical plate number Impurity III 9.630 / 0.96 11727 Impurity III - IB 13.258 10.06 0.87 25872 Impurity F 14.599 4.24 0.92 41272 Impurity III - IA 15.101 1.74 0.92 46556 Impurity B 17.860 10.24 0.97 70633 Impurity C 19.419 6.35 0.95 96557 Impurity III - ID 24.642 21.23 0.94 155922 Impurity I 26.179 6.6 0.95 246192 Impurity K 27.181 5.27 1.00 438663 Impurity L 27.903 4.41 1.01 463114 Impurity D 28.724 4.72 1.03 361709 Levosimendan 29.517 3.76 0.97 256712 Impurity E 30.745 4.89 0.95 231563 Impurity H 31.785 3.72 1.05 139362

[0083] As can be seen from Table 3, the resolution of each impurity is above 1.7, the tailing factor is less than 1.2, the peak shape symmetry is good, the theoretical plate number is not less than 5000, and the performance of the chromatographic column and the system suitability are good.

[0084] From Figures 2 - 3 and Figure 5 , it can be known that in the range of 5 - 30 mmol / L potassium dihydrogen phosphate concentration, the retention times of each component to be detected do not change significantly. When the salt concentration in the mobile phase increases within this range, noise appears in the baseline, but it does not interfere with the detection of target impurities.

[0085] From Figure 4 , it can be known that in the range of 5 - 20 μl injection volume, when the injection volume is adjusted, each component to be detected maintains an extremely baseline separation effect.

[0086] It can be seen from Figure 6 that impurities III, III-IA, III-IB, F, and K can be well separated from levosimendan, with sharp peak shapes and no tailing.

[0087] 3. Forced degradation experiment

[0088] Perform a forced degradation experiment on the levosimendan bulk drug. According to the ICH guidelines, degrade the levosimendan bulk drug. Use the levosimendan bulk drug test solution at a concentration of 1.0 mg / ml as the undegraded control. The degradation directions include acid, base, oxidation, high temperature, and light conditions. After degradation, detect the test samples according to the detection conditions of this example. The specific degradation experiments are as follows:

[0089] Acid degradation (liquid): Weigh 25.31 mg of levosimendan bulk drug, place it in a 25-ml volumetric flask, add 2.0 ml of 1 mol / l hydrochloric acid aqueous solution, let it stand at room temperature for 6 hours, then add 2.0 ml of 1 mol / l sodium hydroxide aqueous solution to neutralize, and then add an appropriate amount of acetonitrile, ultrasonically dissolve and dilute to the mark, and shake well to obtain the solution.

[0090] Base degradation (liquid): Weigh 24.95 mg of levosimendan bulk drug, place it in a 25-ml volumetric flask, add 1.0 ml of 1 mol / l sodium hydroxide aqueous solution, let it stand at room temperature for 1.5 hours, then add 1.0 ml of 1 mol / L hydrochloric acid aqueous solution to neutralize, and then add an appropriate amount of acetonitrile, ultrasonically dissolve and dilute to the mark, and shake well to obtain the solution.

[0091] High temperature degradation (liquid): Weigh 25.18 mg of levosimendan bulk drug, place it in a headspace vial, add 20 ml of acetonitrile, ultrasonically dissolve, then place it in a water bath at 80 °C and heat for 4 hours, take it out, cool to room temperature, transfer it to a 25-ml volumetric flask, add acetonitrile to dilute to the mark, and shake well to obtain the solution.

[0092] Light degradation (liquid): Weigh 25.23 mg of levosimendan bulk drug, place it in a 25-ml transparent volumetric flask, add acetonitrile, ultrasonically dissolve, dilute to the mark, shake well and seal, place it in a light stability test chamber (4500 lx ± 500 lx) for 24 hours, then take it out to obtain the solution.

[0093] Oxidation degradation (liquid): Weigh 25.10 mg of levosimendan bulk drug, place it in a 25-ml volumetric flask, add 1.0 ml of 6% hydrogen peroxide solution, let it stand at room temperature for 6 hours, then add acetonitrile, ultrasonically dissolve and dilute to the mark, and shake well to obtain the solution.

[0094] High-temperature degradation (solid): Weigh an appropriate amount of levosimendan raw material and spread it evenly in a weighing bottle. Place it in a constant-temperature forced-air drying oven at 105°C for 7 hours. Then, accurately weigh 25.24 mg of the levosimendan raw material and transfer it to a 25-ml volumetric flask. Add acetonitrile, sonicate to dissolve, and dilute to the mark. Shake well to obtain the solution.

[0095] Light degradation (solid): Weigh an appropriate amount of levosimendan raw material and spread it evenly in a weighing bottle. Place it in a light stability test chamber (4500 lx ± 500 lx) for 24 hours, then take it out. Accurately weigh 25.13 mg of the levosimendan raw material and transfer it to a 25-ml volumetric flask. Add acetonitrile, sonicate to dissolve, and dilute to the mark. Shake well to obtain the solution.

[0096] The results of the forced degradation experiment are shown in Table 4.

[0097] Table 4

[0098]

[0099]

[0100] As can be seen from Table 4, under the forced degradation experiment, the material balance of each condition is between 98.0% and 102.0%, indicating good method specificity.

[0101] Example 2

[0102] The high-performance liquid chromatography detection conditions are as follows:

[0103] High-performance liquid chromatograph:

[0104] Detector: UV detector;

[0105] Chromatographic column: Thermo Fisher Hypersil Gold PFP series, a chromatographic column filled with pentafluorophenyl silica gel;

[0106] Mobile phase A: 5 - 20 mmol / L potassium dihydrogen phosphate, adjusted to pH 2.5 with phosphoric acid;

[0107] Mobile phase B: Methanol;

[0108] Column temperature: Room temperature;

[0109] Flow rate: Adjust the flow rate so that the column pressure is between 2000 psi and 3000 psi;

[0110] Detection wavelength: 260 - 300 nm;

[0111] Injection volume: 20 μl.

[0112] Elution procedure: Isocratic elution is carried out with mobile phase A at 85% - 90% by volume and mobile phase B at 10 - 15%. After the target peak is eluted, elution is carried out with mobile phase A at 35% - 40% by volume and mobile phase B at 60% - 65% for 15 - 25 minutes. After no more chromatographic peaks are eluted, the initial mobile phase ratio is restored for equilibration, and the equilibration time is not less than 15 minutes.

[0113] The diluent, impurity reference solution, and system suitability solution are from Example 1.

[0114] System suitability solution: Appropriate amounts of levosimendan raw material, impurity B, impurity C, impurity D, impurity E, impurity F, impurity III-ID, impurity H, impurity I, impurity K, impurity L, impurity III, impurity III-IA, and impurity III-IB reference substances are taken and dissolved in the diluent to prepare a solution with a levosimendan concentration of 1.0 mg / ml and each impurity concentration of 1 μg / ml.

[0115] Inject the system suitability solution into the high-performance liquid chromatograph, perform sample injection and detection for elution. The separation of the 14 chromatographic peaks of the components to be detected from the adjacent impurity peaks is good. The resolution of each impurity is above 1.6, the tailing factor is less than 1.2, the peak shape symmetry is good, and the theoretical plate number is not less than 5000. The performance of the chromatographic column and the system suitability are good. For the detection results, see Figure 7 , and the retention time and resolution of each impurity in the system suitability solution are shown in Table 5.

[0116] Table 5

[0117] Name RT (min) Resolution Impurity III 11.599 / Impurity F 15.574 15.70 Impurity III - IB 15.985 1.80 Impurity C 16.726 3.50 Impurity B 18.672 9.81 Impurity III - IA 20.418 9.74 Impurity III - ID 20.655 1.60 Impurity D 23.051 9.66 Impurity K 27.181 9.60 Impurity I 32.027 20.52 Impurity L 34.689 6.86 Levosimendan 36.211 3.73 Impurity E 37.464 3.33 Impurity H 39.280 4.69

[0118] According to 20 μl each of the impurity I reference solution, impurity L reference solution, impurity III-IA reference solution, impurity III-IB reference solution, impurity III reference solution, and levosimendan test solution prepared in Example 1, the samples to be detected are respectively injected into the high-performance liquid chromatograph for sample injection and detection, and elution is carried out. The detection conditions for different impurity reference solutions are the same. Record the detection results, and after processing by empower software, it is made into an overlay graph. For the detection results, see Figure 8 .

[0119] From Figure 8 it can be seen that there is good separation between impurity I, impurity L, impurity III-IA, impurity III-IB, impurity III and levosimendan, the peak shape is sharp, and there is no tailing.

[0120] Comparative Example 1

[0121] High-performance liquid chromatograph:

[0122] Detector: UV detector;

[0123] Chromatographic column: A chromatographic column packed with octadecylsilyl silica gel as the filler;

[0124] Mobile phase A: An aqueous solution of dipotassium hydrogen phosphate at 30 mmol / L, adjusted to pH 3.0 with phosphoric acid, used as mobile phase A.

[0125] Mobile phase B: Methanol;

[0126] Column temperature: Room temperature;

[0127] Flow rate: Adjust the flow rate so that the column pressure is between 2000 psi and 3000 psi;

[0128] Detection wavelength: 276 nm;

[0129] Sample injection volume: 20 μl.

[0130] 1. System suitability determination

[0131] Solution preparation conditions:

[0132] Diluent: Mobile phase A: Mobile phase B = 1:1, by volume ratio.

[0133] Test solution: Weigh an appropriate amount of levosimendan raw material accurately, dissolve and dilute it with the diluent to make a solution containing about 1 mg per 1 ml, shake well, and obtain.

[0134] Reference solution: Accurately measure appropriate amounts of impurities A - M respectively, dissolve and dilute them with the diluent to make a solution containing about 1 μg per 1 ml, shake well, and obtain.

[0135] System suitability solution: Weigh appropriate amounts of levosimendan raw material, impurity B, impurity C, impurity D, impurity E, impurity F, impurity III - ID, impurity H, impurity I, impurity K, impurity L, impurity III, impurity III - IA, and impurity III - IB reference substances respectively, dissolve and dilute them with the diluent to make a solution containing about 1 mg of levosimendan and 1 μg of each of the other impurities per 1 ml.

[0136] Take 4 samples to be tested, namely (1) diluent, (2) reference solution, (3) system suitability solution, and (4) test solution. Inject 20 μl of each of the 4 samples into the high - performance liquid chromatograph for injection detection and perform elution.

[0137] The test results are shown in Figure 8 , and it can be seen from Figure 8 that in the detection method of Comparative Example 1, only 11 chromatographic peaks were observed, and the 14 components to be detected could not be completely separated. Impurity III and III - IA could not be separated, and levosimendan, impurity I, and impurity L could not be separated, and the main peak had serious tailing.

[0138] Comparative Example 2

[0139] High performance liquid chromatograph:

[0140] Detector: UV detector;

[0141] Chromatographic column: A chromatographic column filled with pentafluorophenyl silica gel;

[0142] Mobile phase A: An aqueous solution of dipotassium hydrogen phosphate at 3 mmol / L, adjusted to pH 4.5 with phosphoric acid, serving as mobile phase A.

[0143] Mobile phase B: Acetonitrile

[0144] Column temperature: Room temperature;

[0145] Flow rate: 1.0 ml / min;

[0146] Detection wavelength: 276 nm;

[0147] Sample injection volume: 20 μl.

[0148] 1. System suitability determination

[0149] Solution preparation conditions:

[0150] Diluent: Mobile phase A: Mobile phase B = 1:1, by volume.

[0151] System suitability solution: Take appropriate amounts of levosimendan raw material, impurity B, impurity C, impurity D, impurity E, impurity F, impurity III-ID, impurity H, impurity I, impurity K, impurity L, impurity III, impurity III-IA, and impurity III-IB reference substances, dissolve and dilute with the diluent to prepare a solution containing approximately 1 mg of levosimendan and 1 μg of each of the remaining impurities per 1 ml.

[0152] Inject 20 μl of the system suitability solution into the high performance liquid chromatograph for sample injection and detection, and perform elution.

[0153] The detection results are shown in Figure 9 , and it can be seen from Figure 9 that in the detection method of Comparative Example 1, only 11 chromatographic peaks were observed, and the 14 components to be detected could not be completely separated. Impurity F and III-IA could not be separated, and levosimendan, impurity I, and impurity K could not be separated, and the main peak had serious tailing.

Claims

1. A method for detecting organic impurities in levosimendan, comprising the following steps: Detecting a test solution of levosimendan raw material drug or preparation by high performance liquid chromatography, using a chromatographic column filled with pentafluorophenyl silica gel, and eluting with a 5 - 30 mmol / L potassium dihydrogen phosphate solution with a pH of 2.5 - 4.0 as mobile phase A and methanol as mobile phase B; Detection wavelength: 260 - 300 nm; Flow rate: Adjust the flow rate so that the column pressure is 2000 psi - 3000 psi; Column temperature: 25°C - 35°C.

2. The detection method of organic impurities in levosimendan according to claim 1, wherein The organic impurities include: impurity III, impurity III - IA and impurity III - IB, and the structural formulas are as follows:

3. The detection method of organic impurities in levosimendan according to claim 1, wherein The organic impurities include: impurity III, impurity III - IA, impurity III - IB, impurity K and impurity F, and the structural formulas are as follows:

4. The detection method of organic impurities in levosimendan according to claim 2 or 3, characterized in that, The organic impurities also include: impurity B, impurity C, impurity D, impurity E, impurity III - ID and impurity H, and the structural formulas are as follows:

5. The detection method of organic impurities in levosimendan according to claim 4, characterized in that, The organic impurities also include impurity I and impurity L, and the structural formulas are as follows:

6. The detection method of organic impurities in levosimendan according to claim 1, wherein, The chromatographic column is selected from Thermo Fisher Hypersil Gold PFP series or Welch Ultimate PFP series.

7. The detection method of organic impurities in levosimendan according to claim 6, characterized in that, The particle size of the chromatographic column packing is ≤5 μm, the length is ≥25 cm, and the inner diameter is ≤4.6 mm.

8. The detection method of organic impurities in levosimendan according to claim 1, characterized in that, The elution process is: isocratic elution with 80% - 90% (by volume) of mobile phase A and 10% - 20% (by volume) of mobile phase B. After the target peak is eluted, elute with 30% - 50% (by volume) of mobile phase A and 50% - 70% (by volume) of mobile phase B for 15 - 25 minutes. After no more chromatographic peaks are eluted, restore the starting mobile phase ratio for equilibration, and the equilibration time is not less than 10 minutes.

9. The detection method of organic impurities in levosimendan according to claim 1, characterized in that, The detection conditions in the high performance liquid chromatography are as follows: Detector: UV detector; Chromatographic column: Welch Ultimate PFP series pentafluorophenyl silica gel chromatographic column; Mobile phase A: 5 - 30 mmol / L potassium dihydrogen phosphate, adjusted to pH 2.5 - 3.5 with phosphoric acid; Mobile phase B: methanol; Column temperature: room temperature; Flow rate: Adjust the flow rate so that the column pressure is between 2000 psi and 3000 psi; Detection wavelength: 260 - 300 nm; Injection volume: 5 - 20 μl; Elution program: isocratic elution with 80% - 90% (by volume) of mobile phase A and 10% - 20% (by volume) of mobile phase B. After the target peak is eluted, elute with 40% (by volume) of mobile phase A and 60% (by volume) of mobile phase B for 15 - 25 minutes. After no more chromatographic peaks are eluted, restore the starting mobile phase ratio for equilibration, and the equilibration time is not less than 10 minutes.

10. The detection method for organic impurities in levosimendan according to any one of claims 1-9, characterized in that, The levosimendan preparation is levosimendan injection or levosimendan for injection, and the concentration of levosimendan in the test solution is 0.5 - 1.5 mg / ml, and the injection volume is 5 - 20 μl.

Images