Method for improving quality control of dopamine hydrochloride injection

Abstract

The present invention relates to a method for improving the quality control of dopamine hydrochloride injection, which comprises: injecting a test solution of dopamine hydrochloride injection into a high performance liquid chromatograph to separate and detect related substances in dopamine hydrochloride injection, wherein gradient elution is adopted, and within the elution time range of 35 to 45 minutes, the volume percentage of mobile phase B in the total volume of the mobile phase is 80-100%. The method of the present invention can simultaneously detect 7 impurities in dopamine hydrochloride injection, especially impurity F, [4-(2-aminoethyl)phenyl]phenylmethanone, with good sensitivity and resolution.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and relates to a method for improving the quality control of dopamine hydrochloride injection, including separating and detecting the related substances of dopamine hydrochloride injection. Background Art

[0002] Dopamine hydrochloride, with the chemical name 4-(2-aminoethyl)-1,2-benzenediol hydrochloride, has the structural formula

[0003] 。

[0004] Dopamine hydrochloride is a precursor for the biosynthesis of norepinephrine and is one of the central neurotransmitters. It has the effects of exciting α-receptors, β-receptors, and dopamine receptors, and is clinically used for various types of shock, especially for those with weakened cardiac contractility and renal insufficiency during shock.

[0005] Injection is a common dosage form of dopamine hydrochloride. The detection of related substances in the injection is one of the key items in the drug quality standard. In this regard, in the "Imported Drug Registration Standard" (standard number JX20150157) regarding dopamine hydrochloride, for the content of related substance inspection, it is clearly stipulated that "if there are impurity peaks in the chromatogram of the test solution, the area of a single impurity shall not be greater than the area of the main peak of the control solution (0.10%), and the sum of the areas of each impurity peak shall not be greater than 2 times the area of the main peak of the control solution (0.2%)"; it can be seen that the detection of as many impurities as possible and the separation and detection of a single impurity are important aspects of controlling drug quality.

[0006] In addition, impurities have many adverse effects on the safety and effectiveness of drugs. For example, impurities may cause adverse reactions such as allergic reactions and toxic reactions; impurities may interact with the main drug in the injection, resulting in a decrease in drug efficacy; the presence of impurities may affect the determination of the content of the main drug, leading to a discrepancy between the actual drug concentration and the labeled concentration, and thus unable to achieve the expected therapeutic effect; therefore, being able to detect impurities well and thereby control the amount of impurities is crucial for drug quality and safety.

[0007] Currently, the "Imported Drug Registration Standard" (standard number JX20150157) discloses the use of high-performance liquid chromatography for the detection of related substances, and only records 2 specific impurities; however, in addition to 5-(2-aminoethyl)-2-methoxyphenol (impurity A) and 4-(2-aminoethyl)-2-methoxyphenol (impurity B), the impurities in dopamine hydrochloride injection also include some other impurities, such as 2-(3,4-dimethoxyphenyl)ethan-1-amine (impurity C), 5,6-dihydroxyindole, etc.

[0008] In order to improve the quality control level of dopamine hydrochloride injection, we need to develop a method that can detect more impurities simultaneously to further enhance the safety of clinical use of dopamine hydrochloride injection. Summary of the Invention

[0009] The object of the present invention is to provide a high performance liquid chromatography method with good sensitivity and resolution, which can simultaneously detect related substances in dopamine hydrochloride injection, including impurity A, impurity B, impurity C, impurity E, impurity F, impurity H and 5,6-dihydroxyindole impurity, especially impurity F, while the detection performance of the main component dopamine hydrochloride is good.

[0010] The object of the present invention is achieved by the following method: A method for improving the quality control of dopamine hydrochloride injection, which includes:

[0011] Inject the test solution of dopamine hydrochloride injection into a high performance liquid chromatograph to separate and detect the related substances in dopamine hydrochloride injection, wherein,

[0012] Mobile phase A is a mixed solution composed of a buffer solution of sodium octanesulfonate - citric acid, methanol, and acetonitrile in a volume ratio of (80 - 95):(3 - 10):(4 - 9); Mobile phase B is a mixed solution composed of a buffer solution of sodium octanesulfonate - citric acid, methanol, and acetonitrile in a volume ratio of (65 - 75):(5 - 15):(15 - 25);

[0013] The total flow rate of the mobile phase is 0.8 - 1.2 ml / min, and gradient elution is adopted, wherein within the elution time range of 35 to 45 minutes, the volume percentage of mobile phase B in the total volume of the mobile phase is 80 to 100%.

[0014] Unexpectedly, compared with the prior art, for example, compared with the gradient elution program (as shown in Table 1 below) adopted in the method of "Imported Drug Registration Standard" (Standard No. JX20150157), the method of the present invention achieves the purpose of simultaneously separating and detecting multiple related substances of dopamine hydrochloride by further setting the gradient elution conditions of 25 to 60 minutes, especially when the volume percentages of mobile phase A and B at 35 to 45 minutes are within a specific numerical range, especially when the volume percentage of mobile phase B is more than 80%. The related substances include impurity A, impurity B, impurity C, impurity E, impurity F, impurity H and 5,6-dihydroxyindole, especially impurity F; On the contrary, the prior art cannot detect impurity F, so the prior art cannot control the quality of the drug more precisely.

[0015]

[0016] The method of the present invention has good sensitivity and resolution. Especially for impurity F, its linear range is 0.08 - 3.5 μg / ml, the limit of quantitation is 0.0875 μg / ml, and the limit of detection is 0.0438 μg / ml. It has a low detection concentration and a low concentration for quantitative determination, and the accuracy is good. Description of the Drawings

[0017] Figure 1 It is the HPLC chromatogram of the dopamine hydrochloride system suitability solution determined according to the chromatographic conditions of the example.

[0018] Figure 2 It is the HPLC chromatogram of the test solution determined according to the chromatographic conditions of the example.

[0019] Figure 3 It is the HPLC chromatogram of the test solution added with 7 kinds of impurity reference substances determined according to the chromatographic conditions of the example.

[0020] Figure 4 It is the standard curve graph of impurity F determined by the external standard method according to the chromatographic conditions of the example.

[0021] Figure 5 It is the HPLC chromatogram of the test solution added with the impurity F reference substance determined according to the chromatographic conditions of Comparative Example 2.

[0022] Figure 6 It is the HPLC chromatogram of the test solution added with the impurity F reference substance determined according to the chromatographic conditions of Comparative Example 3. Detailed Description of the Invention

[0023] In the present invention, the "test sample" refers to the dopamine hydrochloride injection for which the related substances are to be determined.

[0024] In the present invention, the "related substances of dopamine hydrochloride injection" include impurity A, impurity B, impurity C, impurity E, impurity F, impurity H, and 5,6 - dihydroxyindole impurity. The structural formulas of these impurities are as follows:

[0025] Impurity A, Impurity B,

[0026] Impurity C, Impurity E,

[0027] Impurity F, Impurity H,

[0028] 5,6 - Dihydroxyindole.

[0029] In the present invention, all numerical values representing amounts, percentages or ratios should be understood to be modified by the term "about" in all cases. In this regard, the term "about" used herein may include a range of ±5%, ±4%, ±3%, ±2%, ±1% or ±0.5% of the stated numerical value.

[0030] In the present invention, in order to more clearly show the impurity peaks, the HPLC chromatogram shown in the attached drawing is an enlarged view of the image marked in blue in the upper right figure.

[0031] The present invention provides a method for improving the quality control of dopamine hydrochloride injection, which includes: injecting the test solution of dopamine hydrochloride injection into a high performance liquid chromatograph to separate and detect the related substances in dopamine hydrochloride injection, wherein mobile phases A and B are mixed solutions composed of buffer solutions of sodium octanesulfonate - citric acid, methanol, and acetonitrile in different volume ratios; the total flow rate of the mobile phase is 0.8 - 1.2 ml / min, and gradient elution is adopted, wherein within the elution time range of 35 to 45 minutes, the volume percentage of mobile phase B in the total volume of the mobile phase is 80 - 100%.

[0032] Unexpectedly, according to the experimental data (shown in the comparative examples below), when the volume percentage of mobile phase B is below 70% at 35 to 45 minutes, impurity F cannot be detected.

[0033] According to the regulation on the maximum content of individual impurities recorded in the "Import Drug Registration Standard" (standard number JX20150157) "If there are impurity peaks in the chromatogram of the test solution, the content of individual impurities shall not be greater than the peak area of the control solution (0.10%)", the content limit of individual impurities is set at 0.1%. In the present invention, the nuclear magnetic quantitative purity of the impurity F reference substance is 85.1% (determined by high - resolution quantitative NMR measurement method, using JEOL JNM - ECZ400S / L1 instrument, with 1,3,5 - trimethoxybenzene as the internal standard, deuterated dimethyl sulfoxide DMSO - d6 as the solvent, sample peaks for quantitative analysis: chemical shift δ is 3.014 - 3.124, 4H; internal standard peaks for quantitative analysis: chemical shift δ is 3.710, 9H).

[0034] Preferably, in the method of the present invention, gradient elution is adopted, and elution is carried out according to the gradient elution program shown in Table 2 below.

[0035]

[0036] The sum of the volume percentages of mobile phases A and B is 100%.

[0037] In the present invention, regarding the numerical range of the volume percentage of the mobile phase, it means that the volume percentage of the mobile phase can be selected as the value at either endpoint of this range, or any value within this range; for example, in Table 2 above, when the elution time is 35 minutes, the volume ratio range of mobile phase A is 0 to 20%, which means that at 35 minutes, the volume percentage of mobile phase A is a specific value, which can be the endpoint value 0% or 20%, or any value within the range of 0% to 20%, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, etc.

[0038] Preferably, the gradient elution program is as shown in Table 3 below:

[0039]

[0040] More preferably, the gradient elution program is as shown in Table 4 below:

[0041]

[0042] By adopting the above specific gradient elution program in the method of the present invention, especially the elution conditions at 30 to 46 minutes, especially the volume percentages of mobile phases A and B at 35 to 45 minutes, especially when the volume percentage of mobile phase B is more than 80%, preferably more than 90%, more preferably more than 95%, and most preferably 100%, it is beneficial to further improve the resolution of related substances in dopamine hydrochloride injection, especially to improve the detection effect of impurity F, including optimizing the separation effect, improving the accuracy, and reducing the quantitative limit and detection limit of impurity F.

[0043] In an alternative embodiment, it is carried out according to the gradient elution program in Table 4 above, with the difference that at 35 minutes, it is 10% A and 90% B; at 45 minutes, it is 10% A and 90% B; in another alternative embodiment, it is carried out according to the gradient elution program in Table 4 above, with the difference that at 35 minutes, it is 5% A and 95% B; at 45 minutes, it is 5% A and 95% B.

[0044] Preferably, mobile phase A is a mixed solution composed of a buffer solution of sodium octanesulfonate - citric acid, methanol, and acetonitrile in a volume ratio of (80 - 95) : (3 - 10) : (4 - 9), preferably 88 : 5 : 7; mobile phase B is a mixed solution composed of a sodium octanesulfonate - citric acid buffer solution, methanol, and acetonitrile in a volume ratio of (65 - 75) : (5 - 15) : (15 - 25), preferably 70 : 10 : 20.

[0045] In particular, the related substances of dopamine hydrochloride injection that can be detected by the method of the present invention include impurity F, [4-(2-aminoethyl)phenyl]phenylmethanone.

[0046] Preferably, the mass ratio of sodium octanesulfonate in mobile phase B to mobile phase A is less than or equal to 4:3 to greater than 1:1, preferably 1.3 - 1.1, more preferably 1.3 - 1.2, and most preferably 1.25.

[0047] In a specific embodiment, in the buffer solution of sodium octanesulfonate and citric acid contained in mobile phases A and B, based on the total volume of the buffer solution, the mass concentration of sodium octanesulfonate in mobile phase B is greater than that in mobile phase A, and the ratio is less than or equal to 4:3 to greater than 1:1, preferably 1.3 - 1.1, more preferably 1.3 - 1.2, and most preferably 1.25.

[0048] Preferably, in the sodium octanesulfonate-citric acid buffer solution of mobile phase A, the mass concentration of sodium octanesulfonate is 1.0 - 1.5 g / L, preferably 1.2 - 1.25 g / L; in the sodium octanesulfonate-citric acid buffer solution of mobile phase B, the mass concentration of sodium octanesulfonate is 1.2 - 2.0 g / L, preferably 1.52 - 1.56 g / L, based on the total volume of the buffer solution; thereby further improving the sensitivity and resolution of the method of the present invention.

[0049] Preferably, in the sodium octanesulfonate-citric acid buffer solutions of mobile phases A and B, the mass concentration of citric acid is 12 - 13 g / L, preferably 12.5 - 12.7 g / L, and this mass concentration can be the same or different, preferably the mass concentrations are the same; the volume in the mass concentration is based on the total volume of the buffer solution.

[0050] Preferably, in the sodium octanesulfonate-citric acid buffer solution of mobile phase A, the mass concentration ratio of citric acid to sodium octanesulfonate is 9:1 to 11.5:1, preferably 9.5:1 to 11:1; in the sodium octanesulfonate and citric acid buffer solution of mobile phase B, the mass concentration ratio of citric acid to sodium octanesulfonate is 7.5:1 to 8.5:1, preferably 8:1 to 8.4:1.

[0051] In the present invention, the buffer solution of sodium octanesulfonate-citric acid is an aqueous solution composed of citric acid, sodium octanesulfonate, 1 mol / L sodium hydroxide, and 0.1 mol / L hydrochloric acid.

[0052] In a specific embodiment of the present invention, in the sodium octanesulfonate-citric acid buffer solution of mobile phases A and B, with respect to 1 L of the buffer solution, the volume of 1 mol / L sodium hydroxide used is 110 - 130 ml, preferably 118 - 122 ml, and these volume usages can be the same or different; with respect to 1 L of the buffer solution, the volume of 0.1 mol / L hydrochloric acid used is 390 - 410 ml, preferably 388 - 402 ml, and these volume usages can be the same or different.

[0053] In another specific embodiment of the present invention, the preparation method of the sodium octanesulfonate-citric acid buffer solution is as follows: Take 12.6 g of citric acid and 1.2 - 1.56 g of sodium octanesulfonate, add 120 ml of 1 mol / L sodium hydroxide solution to dissolve it, add water to 600 ml, and then add 400 ml of 0.1 mol / L hydrochloric acid solution; preferably, the amount of sodium octanesulfonate in mobile phase A is 1.23 g, and the amount of sodium octanesulfonate in mobile phase B is 1.54 g.

[0054] In the method of the present invention, the total flow rate of the mobile phase is 0.8 - 1.2 ml / min, preferably 0.9 - 1.1 ml / min, and more preferably 1 ml / min.

[0055] In the method of the present invention, an ultraviolet detector is used, and the detection wavelength is 279 - 281 nm, preferably 280 nm.

[0056] In the method of the present invention, the chromatographic column is a chromatographic column filled with octadecylsilyl silica gel; in an embodiment, the chromatographic column is a chromatographic column filled with octadecylsilyl silica gel, such as the Waters Nova-Pak C18 chromatographic column.

[0057] In the method of the present invention, the injection volume is 5 - 15 μl, the column temperature is 25 - 35 °C, preferably 30 °C.

[0058] In a specific embodiment of the present invention, the chromatographic column is Waters Nova-Pak C18, the column temperature is 30 °C, the total flow rate is 1.0 ml / min, and the injection volume is 10 μl.

[0059] In the method of the present invention, the preparation of all the solutions used is carried out using mobile phase A in the high performance liquid chromatography detection method of the present invention as the diluent.

[0060] Preferably, the test solution of the dopamine hydrochloride injection injected is a solution of dopamine hydrochloride with a concentration of 1.5 - 2.5 mg / ml diluted with mobile phase A.

[0061] The preparation method of the test solution of dopamine hydrochloride injection (referred to as the test solution for short) is as follows: Pipette a certain amount of dopamine hydrochloride injection and quantitatively dilute it with mobile phase A to obtain a test solution containing 1.5 - 2.5 mg of dopamine hydrochloride per 1 ml.

[0062] In a specific embodiment of the present invention, the dopamine hydrochloride injection is formulated into a test solution with a concentration of 1.5 - 2.5 mg / ml, preferably 1.8 - 2.2 mg / ml, and more preferably 2 mg / ml.

[0063] In the method of the present invention, an auto - control solution of the test solution is also used, and the concentration of dopamine hydrochloride in it is 0.1% of the concentration of the test solution.

[0064] In the method of the present invention, a system suitability solution containing a dopamine hydrochloride reference substance, an impurity A reference substance, and an impurity B reference substance is also used. Specifically, in the said system suitability solution, the mass concentration of dopamine hydrochloride is 1.5 - 2.5 mg / ml, the mass concentration of impurity A is 16 - 28 μg / ml, and the mass concentration of impurity B is 16 - 28 μg / ml. Preferably, the mass ratio of dopamine hydrochloride, impurity A, and impurity B is (90 - 70):1:1. Using the above - mentioned system suitability solution and observing the separation effect between the dopamine hydrochloride peak and the impurity B peak, as well as the retention times of the dopamine hydrochloride peak, impurity B, and impurity A, the chromatographic system can be tested and adjusted, and the comprehensive characteristics in aspects such as electrical signals, analysis operations, and samples can be comprehensively detected.

[0065] In the method of the present invention, a sensitivity solution is also used to detect the sensitivity of the method of the present invention. In the present invention, the mass concentration of dopamine hydrochloride in the said sensitivity solution is 0.005% of the mass concentration of dopamine hydrochloride in the test solution. In a specific embodiment, the mass concentration of dopamine hydrochloride in the said sensitivity solution is 0.1 μg / ml.

[0066] In the method of the present invention, a test solution added with 7 impurity reference substances is also used to detect the separation effect of the method of the present invention on dopamine hydrochloride and related substances. In the present invention, in the test solution added with 7 impurity reference substances, the mass concentration of dopamine hydrochloride is 1.5 - 2.5 mg / ml, and the concentrations of impurity A, impurity B, impurity C, impurity E, impurity F, impurity H, and 5,6 - dihydroxyindole are 1.4 - 2.8 μg / mL respectively.

[0067] In the present invention, a single-standard solution of dopamine hydrochloride related substances is also used to determine the peak positions of each impurity, i.e., the retention times. The single-standard solution is prepared by quantitatively diluting a certain amount of impurity A, impurity B, impurity C, impurity E, impurity F, impurity H, and 5,6-dihydroxyindole stock solution with a diluent respectively. Preferably, in the single-standard solution of dopamine hydrochloride related substances, the mass concentrations of these impurities are 1.4 - 2.8 μg / mL, preferably 1.8 - 2.2 μg / mL, especially 2 μg / mL.

[0068] The method of the present invention is applied to the quality control of dopamine hydrochloride injection to conduct quality control during the preparation and storage processes and improve the safety of the drug. Examples

[0069] The method of the present invention will be described and explained in more detail below with specific examples. Those skilled in the art will understand that the purpose of providing these examples is only for illustrative purposes and does not constitute any limitation to the scope of the present invention.

[0070] In the following examples, various processes and methods not described in detail are conventional methods well known in the art, and reagents without indicating the source and specifications are of commercially available analytical pure or chromatographic pure grade.

[0071] I. General description:

[0072] 1. Solvents, reference substances, and test samples

[0073] 1.1 Solvents

[0074] Methanol: Chromatographic pure, Tianjin Concord Technology Co., Ltd.

[0075] Acetonitrile: Chromatographic pure, Tianjin Concord Technology Co., Ltd.

[0076] 1.2 Reference substances

[0077] Impurity A: 5-(2-Aminoethyl)-2-methoxyphenol, HPLC purity 98.8%, Beijing Kangpai Medical Technology Co., Ltd.

[0078] Impurity B: 4-(2-Aminoethyl)-2-methoxyphenol, HPLC purity 98.8%, batch number DBAR220827B1, Guangzhou Aiqixi Medical Technology Co., Ltd.

[0079] Impurity C: 2-(3,4-Dimethoxyphenyl)ethan-1-amine, HPLC purity 99.2%, batch number DBAR220828C1, Guangzhou Aiqixi Medical Technology Co., Ltd.

[0080] Impurity E: {1-[2-(3,4-dihydroxyphenyl)ethyl]-3-hydroxy-2,5-dioxotetrahydro-1H-pyrrol-3-yl}acetic acid, HPLC purity 99.6%, batch number DBAR230516E1, Guangzhou Aici Pharmaceutical Technology Co., Ltd.

[0081] Impurity F: [4-(2-aminoethyl)phenyl]phenyl ketone, HPLC purity 99.2%, batch number DBAR230528F1, Guangzhou Aici Pharmaceutical Technology Co., Ltd.

[0082] Impurity H: 3-({[2-(3,4-dihydroxyphenyl)ethyl]amino}carbonyl)-3-hydroxyglutaric acid, HPLC purity 93.8%, batch number DBAR230607H1, Guangzhou Aici Pharmaceutical Technology Co., Ltd.

[0083] 5,6-Dihydroxyindole: purity 99.5%, HPLC purity 99.5%, batch number DBAR230510D1, Guangzhou Aici Pharmaceutical Technology Co., Ltd.

[0084] 1.3 Test products

[0085] Dopamine hydrochloride injection: Henan Runhong Pharmaceutical Co., Ltd., specification 2.5 ml: 50 mg.

[0086] 2. Instruments

[0087] Ultrapure water machine: WP-UP-YJ-10, Sichuan Water Treatment Equipment Co., Ltd.

[0088] High performance liquid chromatography-ultraviolet detector (purchased from Thermo Fisher Scientific Inc., model: Ultimate 3000).

[0089] Electronic balance: AP135W (one hundred thousandth balance), Shimadzu.

[0090] Electronic balance: MSA6-6S-OCE-DM (one millionth balance), Sartorius.

[0091] II. Example: Separation and Reliability Test

[0092] 1. Chromatographic conditions

[0093] Detection wavelength: 280 nm;

[0094] Column: Waters Nova-Pak C18, 3.9 × 150 mm, 4 μm;

[0095] Column temperature: 30°C;

[0096] Total flow rate: 1.0 mL / min;

[0097] Injection volume: 10 μL;

[0098] Mobile phase A: Prepared by mixing sodium octanesulfonate-citric acid buffer solution (take 12.6 g of citric acid and 1.23 g of sodium octanesulfonate, add 120 ml of 1 mol / L sodium hydroxide solution to dissolve, add water to 600 ml, and then add 400 ml of 0.1 mol / L hydrochloric acid solution), methanol and acetonitrile in a volume ratio of 88:5:7;

[0099] Mobile phase B: Prepared by mixing sodium octanesulfonate-citric acid buffer solution (take 12.6 g of citric acid and 1.54 g of sodium octanesulfonate, add 120 ml of 1 mol / L sodium hydroxide solution to dissolve, add water to 600 ml, and then add 400 ml of 0.1 mol / L hydrochloric acid solution), methanol and acetonitrile in a volume ratio of 70:10:20;

[0100] Perform elution according to the gradient elution program in Table 4 above.

[0101] 2. Solution preparation

[0102] (1) Diluent

[0103] Prepared according to mobile phase A described in item 1 of Part II above.

[0104] (2) Blank solution

[0105] Prepared according to mobile phase A described in item 1 of Part II above.

[0106] (3) Test solution

[0107] Take 1.0 ml of dopamine hydrochloride injection, place it in a 10 ml volumetric flask, add diluent to the scale, shake well, and obtain the test solution of dopamine hydrochloride injection (the concentration of dopamine hydrochloride is about 2 mg / ml).

[0108] (4) Self-control solution (0.1% self-control)

[0109] Accurately measure 1.0 ml of the test solution, place it in a 100 ml volumetric flask, add diluent to the scale, shake well, and then accurately measure 1.0 ml of the above solution, place it in a 10 ml volumetric flask, add diluent to the scale, shake well, and obtain the control solution of dopamine hydrochloride injection (the concentration of dopamine hydrochloride is about 2 μg / ml).

[0110] (5) System suitability solution

[0111] Weigh appropriate amounts of dopamine hydrochloride reference substance, impurity A reference substance and impurity B reference substance, dissolve and dilute them with the diluent to prepare a mixed solution containing 2 mg of dopamine hydrochloride, 24 μg of impurity A and 24 μg of impurity B per 1 ml respectively. Shake well to obtain the system suitability solution.

[0112] (6)Sensitivity solution

[0113] Precisely measure an appropriate amount of the self - reference solution prepared above, add the diluent and dilute, shake well to prepare a solution containing 0.1 μg of dopamine hydrochloride per 1 ml.

[0114] (7)Stock solution of related substances of dopamine hydrochloride

[0115] Weigh 10 mg of reference substances of impurity A, impurity B, impurity C, impurity E, impurity F and impurity H respectively, place them in 10 - ml volumetric flasks, dissolve with the diluent and make up to the mark, shake well to obtain the stock solutions of impurity A, impurity B, impurity C, impurity E, impurity F and impurity H respectively.

[0116] Weigh 10 mg of 5,6 - dihydroxyindole, place it in a 10 - ml volumetric flask, dissolve it with 2 ml of methanol and then make up to the mark with the diluent, shake well to obtain the stock solution of 5,6 - dihydroxyindole.

[0117] (8)Single - standard solutions of related substances of dopamine hydrochloride

[0118] Precisely measure 0.02 ml of the stock solutions of impurity A, impurity B, impurity C, impurity E, impurity F, impurity H and 5,6 - dihydroxyindole prepared in (7) above respectively, place them in 10 - ml volumetric flasks, add the diluent and dilute to the mark, shake well to obtain the solutions.

[0119] (9)Test solution added with 7 impurity reference substances

[0120] Precisely measure 1.0 ml of the test sample dopamine hydrochloride injection, place it in a 10 - ml volumetric flask, precisely measure 0.02 ml of the stock solutions of impurity A, impurity B, impurity C, impurity E, impurity F, impurity H and 5,6 - dihydroxyindole prepared in (7) above respectively and place them in the above volumetric flask, add the diluent and dilute to the mark, shake well to obtain the solution.

[0121] (10)Test solution added with impurity F reference substance

[0122] Precisely measure 1.0 ml of the test sample dopamine hydrochloride injection, place it in a 10 - ml volumetric flask, precisely measure 0.02 ml of the stock solution of impurity F prepared in (7) above and place it in the above volumetric flask, add the diluent and dilute to the mark, shake well to obtain the solution.

[0123] 3. Testing

[0124] Determined by the high performance liquid chromatography method in General Rules 0512 of Part IV of the Chinese Pharmacopoeia (2020 Edition). The method of the present invention was verified according to the General Guidelines for Validation of Analytical Methods in General Rules 9101 of Part IV of the Chinese Pharmacopoeia (2020 Edition).

[0125] 3.1 System suitability test

[0126] Inject the blank solution, system suitability solution, and sensitivity solution prepared in Item 2 of the above-mentioned Example II into the liquid chromatograph respectively, and record the chromatogram according to the chromatographic conditions described in Item 1 of the above-mentioned Example II. See the chromatogram of the system suitability solution in Figure 1 , and calculate the resolution of dopamine hydrochloride and each related substance in the chromatogram. The results are shown in Table 5 below.

[0127]

[0128] Note: / indicates that there is no next peak and no test data.

[0129] The above results show that the resolution between dopamine hydrochloride and impurity B and the resolution between impurity B and impurity A in the system suitability solution are > 5.0, there are no interfering peaks in the blank solution, and the signal-to-noise ratio of the sensitivity solution is greater than 10, meeting the requirements; indicating that the system suitability and sensitivity of the method provided by the present invention are good.

[0130] 3.2 Resolution determination

[0131] Inject the test solution, self-control solution, blank solution, single standard solution of dopamine hydrochloride related substances, and test solution added with 7 impurity reference substances prepared in Item 2 of the above-mentioned Example II into the liquid chromatograph respectively, and record the chromatogram according to the chromatographic conditions described in Item 1 of the above-mentioned Example II. See the chromatograms of the test solution and the test solution added with 7 impurity reference substances in Figure 2 and Figure 3 , and calculate the resolution of dopamine hydrochloride and each related substance in the chromatogram. The results are shown in Table 6 below.

[0132] Table 6 Resolution results of dopamine hydrochloride and its related substances

[0133]

[0134] Note: n.a. indicates not determined.

[0135] * indicates the resolution between impurity F and the previous peak.

[0136] It was determined that there were no interfering peaks in the blank solution. Moreover, in the test solution with 7 impurity reference substances added, the test solution, and the self-control solution, the peak matching degrees of the main component (i.e., dopamine hydrochloride) were 1000, 1000, and 982 respectively, all above 980. This indicates that the purity of the main component peak is good and does not contain the peaks of other impurities. That is to say, the method of the present invention has no impact on the detection performance of the main component, and the separation effect of the main component is good.

[0137] As can be seen from the above table, in the test solution with 7 impurity reference substances added, the resolution between the chromatographic peaks of each impurity and its adjacent chromatographic peaks is above 1.5. This indicates that the method for determining related substances of dopamine hydrochloride injection provided by the present invention has good specificity and can simultaneously detect and separate related substances of dopamine hydrochloride, including impurity A, impurity B, impurity C, impurity E, impurity F, impurity H, and 5,6-dihydroxyindole, especially impurity F (retention time is about 41.7 minutes, see Figure 3 ).

[0138] 3.3 Linear experiment of impurity F

[0139] Solution series of different concentrations of impurity F:

[0140] Accurately weigh the impurity F reference substance respectively, add an appropriate amount of diluent, and prepare solutions containing different concentrations of impurity F as shown in Table 7 below; then inject these solutions into the liquid chromatograph respectively, and record the chromatogram according to the chromatographic conditions described in item 1 of the above-mentioned II embodiment part; take the concentration of impurity F as the abscissa and the peak area of impurity F recorded in the chromatogram as the ordinate, and the linear relationship obtained is as Figure 4 shown, and the linear equation is: y = 0.4492x - 0.0052, R 2 = 0.9997.

[0141] Table 7 Linear results of impurity F

[0142]

[0143] This result indicates that by determination using the high performance liquid chromatography method provided by the present invention, within the range of 0.08 - 3.5 μg / ml of the impurity F concentration, the linear relationship between the impurity F concentration and the chromatographic peak area is good.

[0144] 3.4 Detection accuracy of impurity F

[0145] Preparation of accuracy solution 1: Accurately pipette 1 ml of dopamine hydrochloride injection into a 10-ml volumetric flask, add an appropriate amount of impurity F reference substance, and make up the volume with the diluent to prepare an accuracy solution equivalent to 50% of the limit content of impurity F. Prepare three portions in parallel. The concentration of impurity F in the actually prepared solution is the "added impurity concentration" as described in Accuracy 1-1, 1-2, and 1-3 in the following table.

[0146] Preparation of accuracy solution 2: Accurately pipette 1 ml of dopamine hydrochloride injection into a 10-ml volumetric flask, add an appropriate amount of impurity F reference substance, and make up the volume with the diluent to prepare an accuracy solution equivalent to 100% of the limit content of impurity F. Prepare three portions in parallel. The concentration of impurity F in the actually prepared solution is the "added impurity concentration" as described in Accuracy 2-1, 2-2, and 2-3 in the following table.

[0147] Preparation of accuracy solution 3: Accurately pipette 1 ml of dopamine hydrochloride injection into a 10-ml volumetric flask, add an appropriate amount of impurity F reference substance, and make up the volume with the diluent to prepare an accuracy solution equivalent to 150% of the limit content of impurity F. Prepare three portions in parallel. The concentration of impurity F in the actually prepared solution is the "added impurity concentration" as described in Accuracy 3-1, 3-2, and 3-3 in the following table.

[0148] Then, inject the above accuracy solutions and two parallel test sample solutions (prepared as described in item 2 of the above-mentioned Example II) into the liquid chromatograph respectively, and record the chromatograms according to the chromatographic conditions described in item 1 of the above-mentioned Example II. The accuracy results of impurity F are shown in Table 8 below.

[0149]

[0150] The results in the above table show that the average recovery rate of impurity F at each level concentration is 97.5% - 100.3%, and the RSD (%) of the recovery rate is 2.48%. The results meet the requirements. It shows that the accuracy of the high-performance liquid chromatography method for determining impurity F provided by the present invention is good.

[0151] 3.5 Determination of the quantitation limit of impurity F

[0152] According to the linear experiment in item 3.3 of the above-mentioned Example II, the signal-to-noise ratio (S / N) of the impurity F solution at the lowest concentration (0.0875 μg / ml) is about 10:1; inject this solution in parallel 6 times, and record the chromatograms according to the chromatographic conditions described in item 1 of the above-mentioned Example II. The quantitation limit results of impurity F are shown in Table 9 below.

[0153]

[0154] The results in the above table show that the RSD of peak areas are all within 10%, and the S / N are all ≥10. The results meet the requirements. Thus, the quantification limit of impurity F is determined to be 0.0875 μg / ml, which is equivalent to 0.0044% of the concentration of dopamine hydrochloride in the test sample. This indicates that the method of the present invention can perform quantitative determination at a relatively low concentration of impurity F and has good sensitivity for the detection of impurity F.

[0155] 3.6 Determination of the detection limit of impurity F

[0156] Based on the signal-to-noise ratio of the impurity F solution at the quantification limit, take the impurity F solution at the quantification limit concentration, dilute it with the diluent, and record the chromatogram according to the chromatographic conditions described in item 1 of the above-mentioned Example II. The signal-to-noise ratio (S / N) is about 3:1, and the detection limit solution of impurity F is obtained. Inject samples in parallel 6 times, and the detection limit results of impurity F are shown in Table 10 below.

[0157]

[0158] The results in the above table show that the RSD of peak areas is within 10%, and the S / N are all ≥3. The results meet the requirements. Thus, the quantification limit of impurity F is determined to be 0.0438 μg / ml, which is equivalent to 0.0022% of the concentration of dopamine hydrochloride in the test sample. This indicates that the method of the present invention can detect a relatively low lower limit of the content of impurity F and has good sensitivity for the detection of impurity F.

[0159] III. Comparative examples

[0160] Comparative example 1

[0161] According to the method for detecting related substances of dopamine hydrochloride recorded in the "Import Drug Registration Standard" (standard number: JX20150157), compared with the chromatographic conditions of the above-mentioned examples, the only difference is that the elution duration is 25 minutes. The specific gradient elution program is shown in Table 11 of Comparative example 1. As a result, impurity F cannot be detected. On the contrary, impurity F can be detected in the examples of the present invention (retention time is about 41.7 minutes, see Figure 3 )

[0162] Comparative example 2

[0163] Comparative example 2 was carried out according to the method of the example (see 3.2 Determination of resolution). The difference is that the test sample solution added with the impurity F reference substance described in item 2 of the above-mentioned Example II was used as the sample injection solution and injected into the liquid chromatograph for gradient elution, in which the volume ratio of mobile phase A and B from 35 to 60 minutes was changed to 40%A and 60%B. The elution conditions and results are shown in Table 11 and Figure 5 as shown

[0164] Comparative example 3

[0165] Comparative Example 3 was carried out according to the method of the Example (see 3.2 Determination of Resolution). The difference was that the test solution added with the reference substance of impurity F described in item 2 of the above-mentioned Example II was used as the sample solution, injected into the liquid chromatograph, and gradient elution was carried out, in which the volume ratio of mobile phase A and B at 35 to 45 minutes was changed to 30% A and 70% B. The elution conditions and results are as shown in Table 11 and Figure 6 shown below.

[0166]

[0167] Note: * indicates that the elution time of Comparative Example 1 (i.e., the "Import Drug Registration Standard" (standard number: JX20150157)) is 25 minutes.

[0168] It can be Figures 5 - 6 seen that no other peaks appeared after the main peak (the main component dopamine hydrochloride, retention time is about 5.1 min), which indicates that impurity F cannot be detected under the chromatographic conditions of Comparative Examples 2-3; on the contrary, through the specific gradient elution conditions of the method of the present invention, especially the elution conditions at 30 to 46 minutes, especially the volume ratio of mobile phase B is 100% at 35 to 45 minutes, impurity F can be detected.

[0169] In summary, the advantages of the method of the present invention are that it has good sensitivity and resolution, and can simultaneously detect the related substances in dopamine hydrochloride injection, including impurity A, impurity B, impurity C, impurity E, impurity F, impurity H and 5,6-dihydroxyindole impurity, especially impurity F, and at the same time, the detection performance of the main component dopamine hydrochloride is good. In addition, by using the method of the present invention, the linear range of the concentration of impurity F is 0.08~3.5 μg / ml, the quantitation limit is 0.0875 μg / ml, and the detection limit is 0.0438 μg / ml. The method of the present invention has good accuracy, as well as low quantitation limit and detection limit. Therefore, the resolution and reliability of the method of the present invention are excellent.

Claims

1. A method for improving the quality control of dopamine hydrochloride injection, which includes: Injecting the test solution of dopamine hydrochloride injection into a high performance liquid chromatograph to separate and detect the related substances in dopamine hydrochloride injection, wherein the related substances include: impurity F, [4-(2-aminoethyl)phenyl]phenylmethanone, impurity H, 3-({[2-(3,4-dihydroxyphenyl)ethyl]amino}carbonyl)-3-hydroxyglutaric acid; and impurity 5,6-dihydroxyindole; Wherein, The production unit of dopamine hydrochloride injection is Henan Runhong Pharmaceutical Co., Ltd.; Mobile phase A is a mixed solution composed of sodium octanesulfonate-citric acid buffer solution, methanol, and acetonitrile in a volume ratio of 88:5:7; mobile phase B is a mixed solution composed of sodium octanesulfonate-citric acid buffer solution, methanol, and acetonitrile in a volume ratio of 70:10:20; The preparation method of the sodium octanesulfonate-citric acid buffer solution of mobile phase A is: take 12.6 g of citric acid and 1.23 g of sodium octanesulfonate, add 1 mol / L, 120 ml of sodium hydroxide solution to dissolve it, add water to 600 ml, and then add 0.1 mol / L, 400 ml of hydrochloric acid solution; The preparation method of the sodium octanesulfonate-citric acid buffer solution of mobile phase B is: take 12.6 g of citric acid and 1.54 g of sodium octanesulfonate, add 1 mol / L, 120 ml of sodium hydroxide solution to dissolve it, add water to 600 ml, and then add 0.1 mol / L, 400 ml of hydrochloric acid solution; The total flow rate of the mobile phase is 1.0 ml / min, and gradient elution is adopted, wherein within the elution time range of 35 to 45 minutes, the volume percentage of mobile phase B in the total volume of the mobile phase is 80% to 100%; The said gradient elution is carried out according to the following gradient elution program, , The total volume percentage of mobile phase A and B is 100%; The detection wavelength is 280 nm; Wherein the chromatographic column used is Waters Nova-Pak C18, 4 μm.

2. The method according to claim 1, wherein the gradient elution is carried out according to the following gradient elution program, 。 3. The method according to claim 1, wherein the test solution of dopamine hydrochloride injection injected is a solution of dopamine hydrochloride with a concentration of 1.5 to 2.5 mg / ml diluted with mobile phase A.

4. The method according to claim 1, wherein the column temperature is 25 to 35 °C.

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