Method for improving quality control of dopamine hydrochloride injection

Abstract

The invention relates to a method for improving quality control of a dopamine hydrochloride injection, which comprises the following steps: injecting a test solution of the dopamine hydrochloride injection into a high performance liquid chromatograph to separate and detect related substances in the dopamine hydrochloride injection, gradient elution is adopted, and the elution time is controlled within 35-45 minutes, and the mobile phase B accounts for 80-100% of the total volume of the mobile phase. The method disclosed by the invention can be used for simultaneously detecting seven impurities in the dopamine hydrochloride injection, especially impurity F and [4-(2-aminoethyl) phenyl] phenyl ketone, and has good sensitivity and separation degree.

Description

Technical Field

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

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

[0003] Dopamine hydrochloride is a precursor of norepinephrine biosynthesis and one of the central neurotransmitters. It has the function of stimulating α-receptors, β-receptors and dopamine receptors. It is clinically used for various types of shock, especially for patients with shock accompanied by weakened cardiac contractility and renal insufficiency.

[0004] Injection is a common dosage form of dopamine hydrochloride. The detection of related substances in injection is one of the key items of drug quality standards. In this regard, in the "Registration Standards for Imported Drugs" (Standard No. JX20150157) on dopamine hydrochloride, it is clearly stipulated in the content of related substance inspection that "if there are impurity peaks in the chromatogram of the test solution, the single impurity shall not be larger than the main peak area of ​​the control solution (0.10%), and the sum of the areas of the impurity peaks shall not be larger than twice the main peak area 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 individual impurities are an important aspect of controlling drug quality.

[0005] 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 solution, resulting in reduced drug efficacy; the presence of impurities may affect the content determination of the main drug, resulting in the actual drug concentration not being consistent with the labeled concentration, and failing to achieve the expected therapeutic effect; therefore, being able to detect impurities well and thus control the amount of impurities is crucial for drug quality and safety.

[0006] At present, the "Registration Standards for Imported Drugs" (standard number JX20150157) discloses the use of high performance liquid chromatography to detect relevant substances, and only records two specific impurities; however, the impurities of dopamine hydrochloride injection include not only 5-(2-aminoethyl)-2-methoxyphenol (impurity A) and 4-(2-aminoethyl)-2-methoxyphenol (impurity B), but also some other impurities, such as 2-(3,4-dimethoxyphenyl)ethane-1-amine (impurity C), 5,6-dihydroxyindole, etc.

[0007] In order to improve the quality control level of dopamine hydrochloride injection, we need to develop a method that can detect more impurities at the same time to further improve the safety of clinical use of dopamine hydrochloride injection. Summary of the invention

[0008] The purpose of the present invention is to provide a high performance liquid chromatography method, which has good sensitivity and separation, 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, and has good detection performance of the main component dopamine hydrochloride.

[0009] The object of the present invention is achieved by the following method: A method for improving the quality control of dopamine hydrochloride injection, comprising: The test solution of dopamine hydrochloride injection was injected into a high performance liquid chromatograph to separate and detect the related substances in dopamine hydrochloride injection, wherein: Mobile phase A is a mixed solution composed of sodium octane sulfonate-citric acid buffer solution, methanol, and acetonitrile in a volume ratio of (80-95):(3-10):(4-9); mobile phase B is a mixed solution composed of sodium octane sulfonate-citric acid buffer solution, methanol, and acetonitrile in a volume ratio of (65-75):(5-15):(15-25); The total flow rate of the mobile phase is 0.8-1.2 ml / min, and gradient elution is adopted, wherein the elution time is controlled within the range of 35-45 minutes, and the volume percentage of mobile phase B in the total volume of the mobile phase is 80-100%.

[0010] 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 the "Imported Drug Registration Standard" (standard number JX20150157), the method of the present invention further sets the gradient elution conditions of 25 to 60 minutes, especially the volume percentages of mobile phases A and B at 35 to 45 minutes are within a specific numerical range, especially the volume percentage of mobile phase B is more than 80%, thereby achieving the purpose of simultaneously separating and detecting multiple related substances of dopamine hydrochloride, the related substances including 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 accurately.

[0011]

[0012] The method of the present invention has good sensitivity and separation, especially for impurity F, the linear range is 0.08-3.5 μg / ml, the quantitative limit is 0.0875 μg / ml, the detection limit is 0.0438 μg / ml, it has a lower detection concentration and a lower quantitative determination concentration, and has good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the HPLC chromatogram of the dopamine hydrochloride system suitability solution measured according to the chromatographic conditions of the embodiment.

[0014] Figure 2 It is a HPLC chromatogram of the test solution measured according to the chromatographic conditions of the embodiment.

[0015] Figure 3 It is an HPLC chromatogram of a test solution to which seven impurity reference substances were added, measured according to the chromatographic conditions of the embodiment.

[0016] Figure 4 It is a standard curve diagram of impurity F measured by external standard method according to the chromatographic conditions of the embodiment.

[0017] Figure 5 It is the HPLC chromatogram of the test solution with the impurity F reference substance added, measured according to the chromatographic conditions of Comparative Example 2.

[0018] Figure 6 It is the HPLC chromatogram of the test solution with the impurity F reference substance added, measured according to the chromatographic conditions of Comparative Example 3. DETAILED DESCRIPTION

[0019] In the present invention, "test sample" refers to dopamine hydrochloride injection containing the related substances to be determined.

[0020] In the present invention, "related substances of dopamine hydrochloride injection" include impurity A, impurity B, impurity C, impurity E, impurity F, impurity H and 5,6-dihydroxyindole impurity, and the structural formulas of these impurities are as follows: Impurity A, Impurity B, Impurity C, Impurity E, Impurity F, Impurity H, 5,6-Dihydroxyindole.

[0021] In the present invention, all numerical values ​​used to express amount, percentage or ratio should be understood to be modified by the term "about" in all cases. In this regard, the term "about" used herein can include the range of ±5%, ±4%, ±3%, ±2%, ±1% or ±0.5% of the numerical value.

[0022] In the present invention, in order to more clearly show the impurity peaks, the HPLC chromatogram shown in the attached figure is an enlarged view of the image marked in blue in the upper right corner of the figure.

[0023] The invention provides a method for improving the quality control of dopamine hydrochloride injection, comprising: injecting a test solution of the dopamine hydrochloride injection into a high performance liquid chromatograph to separate and detect related substances in the dopamine hydrochloride injection, wherein mobile phases A and B are mixed solutions composed of a sodium octane sulfonate-citric acid buffer solution, methanol and acetonitrile in different volume proportions; the total flow rate of the mobile phase is 0.8-1.2 ml / min, gradient elution is adopted, wherein within the elution time range of 35 to 45 minutes, the volume percentage of the mobile phase B in the total volume of the mobile phase is 80-100%.

[0024] Unexpectedly, according to experimental data (as shown in the comparative example below), impurity F could not be detected when the volume percentage of mobile phase B was below 70% at 35 to 45 minutes.

[0025] According to the provisions on the maximum content of a single impurity recorded in the "Registration Standards for Imported Drugs" (Standard No. JX20150157), "If there is an impurity peak in the chromatogram of the test solution, the single impurity shall not be greater than the main peak area of ​​the reference solution (0.10%)", the proposed content limit of a single impurity is 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, using JEOL JNM-ECZ400S / L1 instrument, internal standard is 1,3,5-trimethoxybenzene, solvent is deuterated dimethyl sulfoxide DMSO-d6, sample peak used for quantification: chemical shift δ is 3.014-3.124, 4H; internal standard peak used for quantification: chemical shift δ is 3.710, 9H).

[0026] Preferably, in the method of the present invention, a gradient elution method is used, and elution is performed according to the gradient elution program shown in Table 2 below:

[0027] The total volume percentage of mobile phase A and B is 100%.

[0028] In the present invention, the numerical range of the volume percentage of the mobile phase refers to that the volume percentage of the mobile phase can be selected as the value of any end point of the range, or any value within the range; for example, in the above Table 2, when the elution time is 35 minutes, the volume ratio range of mobile phase A is 0~20%, which means that at 35 minutes, the volume percentage of mobile phase A is a specific value, which can be an endpoint value of 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.

[0029] Preferably, the gradient elution program is shown in Table 3 below:

[0030] More preferably, the gradient elution program is as shown in Table 4 below:

[0031] The method of the present invention is beneficial to further improve the separation degree of related substances of dopamine hydrochloride injection, especially 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 by adopting the above-mentioned specific gradient elution program, especially the elution conditions at 30 to 46 minutes, especially the volume percentage of mobile phase A and B at 35 to 45 minutes, especially the volume percentage of mobile phase B is 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 100%.

[0032] In an optional embodiment, the gradient elution procedure of Table 4 is followed, except that at 35 minutes, it is 10% A, 90% B; at 45 minutes, it is 10% A, 90% B; in another optional embodiment, the gradient elution procedure of Table 4 is followed, except that at 35 minutes, it is 5% A, 95% B; at 45 minutes, it is 5% A, 95% B.

[0033] Preferably, mobile phase A is a mixture of sodium octane sulfonate-citric acid buffer solution, methanol, and acetonitrile in a volume ratio of (80-95):(3-10):(4-9), preferably 88:5:7; mobile phase B is a mixture of sodium octane sulfonate-citric acid buffer solution, methanol, and acetonitrile in a volume ratio of (65-75):(5-15):(15-25), preferably 70:10:20.

[0034] 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.

[0035] Preferably, the mass ratio of sodium octane sulfonate in mobile phase B to mobile phase A is less than or equal to 4:3 and greater than 1:1, preferably 1.3 to 1.1, more preferably 1.3 to 1.2, and most preferably 1.25.

[0036] In a specific embodiment, in the buffer solution of sodium octane sulfonate and citric acid contained in mobile phases A and B, based on the total volume of the buffer solution, the mass concentration of sodium octane sulfonate in mobile phase B is greater than the mass concentration in mobile phase A, and the ratio is less than or equal to 4:3 and greater than 1:1, preferably 1.3-1.1, more preferably 1.3-1.2, and most preferably 1.25.

[0037] Preferably, in the sodium octane sulfonate-citric acid buffer solution of mobile phase A, the mass concentration of sodium octane sulfonate is 1.0-1.5 g / L, preferably 1.2-1.25 g / L; in the sodium octane sulfonate-citric acid buffer solution of mobile phase B, the mass concentration of sodium octane sulfonate 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 separation of the method of the present invention.

[0038] Preferably, in the sodium octane sulfonate-citric acid buffer solution 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 the mass concentrations may be the same or different, preferably the same; the volume in the mass concentration is based on the total volume of the buffer solution.

[0039] Preferably, in the sodium octane sulfonate-citric acid buffer solution of mobile phase A, the mass concentration ratio of citric acid to sodium octane sulfonate is 9:1 to 11.5:1, preferably 9.5:1 to 11:1; in the sodium octane sulfonate and citric acid buffer solution of mobile phase B, the mass concentration ratio of citric acid to sodium octane sulfonate is 7.5:1 to 8.5:1, preferably 8:1 to 8.4:1.

[0040] In the present invention, the sodium octane sulfonate-citric acid buffer solution is an aqueous solution composed of citric acid, sodium octane sulfonate, 1 mol / L sodium hydroxide and 0.1 mol / L hydrochloric acid.

[0041] In a specific embodiment of the present invention, in the sodium octane sulfonate-citrate buffer solution of mobile phase A and B, the volume usage of 1 mol / L sodium hydroxide is 110~130ml, preferably 118~122ml, relative to 1L of buffer solution, and the volume usage can be the same or different; relative to 1L of buffer solution, the volume usage of 0.1 mol / L hydrochloric acid is 390~410ml, preferably 388~402ml, and the volume usage can be the same or different.

[0042] In another specific embodiment of the present invention, the preparation method of the sodium octane sulfonate-citric acid buffer solution is: take 12.6 g of citric acid and 1.2-1.56 g of sodium octane sulfonate, add 120 ml of 1 mol / L sodium hydroxide solution to dissolve them, add water to 600 ml, and then add 400 ml of 0.1 mol / L hydrochloric acid solution; preferably, the sodium octane sulfonate in mobile phase A is 1.23 g, and the sodium octane sulfonate in mobile phase B is 1.54 g.

[0043] 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, more preferably 1 ml / min.

[0044] In the method of the present invention, an ultraviolet detector is used, and the detection wavelength is 279-281 nm, preferably 280 nm.

[0045] In the method of the present invention, the chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as a filler; in an embodiment, the chromatographic column is a chromatographic column with octadecylsilane-based silica gel as a filler, such as a Waters Nova-Pak C18 chromatographic column.

[0046] In the method of the present invention, the injection volume is 5-15 μl, and the column temperature is 25-35°C, preferably 30°C.

[0047] 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.

[0048] In the method of the present invention, the solutions used are prepared by using the mobile phase A in the high performance liquid chromatography detection method of the present invention as the diluent.

[0049] Preferably, the test solution of the injected dopamine hydrochloride injection is a dopamine hydrochloride solution diluted with mobile phase A with a concentration of 1.5-2.5 mg / ml.

[0050] The preparation method of the test solution of dopamine hydrochloride injection (referred to as the test solution) is as follows: a certain amount of dopamine hydrochloride injection is taken, and mobile phase A is added for quantitative dilution to obtain 1.5-2.5 mg of dopamine hydrochloride per 1 ml of the test solution.

[0051] In a specific embodiment of the present invention, dopamine hydrochloride injection is prepared into a test solution with a concentration of 1.5-2.5 mg / ml, preferably 1.8-2.2 mg / ml, more preferably 2 mg / ml.

[0052] In the method of the present invention, a self-control solution of the test solution is also used, and its dopamine hydrochloride concentration is 0.1% of the concentration of the test solution.

[0053] In the method of the present invention, a system suitability solution comprising a dopamine hydrochloride reference substance, an impurity A reference substance and an impurity B reference substance is also used. Specifically, in the 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 time of the dopamine hydrochloride peak, impurity B and impurity A, the chromatographic system can be tested and adjusted, and the comprehensive characteristics of electrical signals, analytical operations and samples can be fully detected.

[0054] 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 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 sensitivity solution is 0.1 μg / ml.

[0055] In the method of the present invention, a test solution to which 7 impurity reference substances are added 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 to which 7 impurity reference substances are added, 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.

[0056] In the present invention, a single-label solution of a dopamine hydrochloride-related substance is also used to determine the peak position of each impurity, i.e., the retention time. The single-label 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. Preferably, in the single-label solution of a dopamine hydrochloride-related substance, the mass concentrations of these impurities are 1.4-2.8 μg / mL, preferably 1.8-2.2 μg / mL, and particularly 2 μg / mL.

[0057] The method of the present invention is applied to the quality control of dopamine hydrochloride injection, so as to perform quality control during the preparation and storage process and improve the safety of the medicine. Example

[0058] The method of the present invention will be described and illustrated in more detail below in conjunction with specific embodiments. It will be understood by those skilled in the art that the purpose of providing these embodiments is only for illustrative purposes and does not constitute any limitation to the scope of the present invention.

[0059] In the following examples, various processes and methods not described in detail are conventional methods known in the art, and the reagents used without indicating the source and specifications are all commercially available analytical grade or chromatographic grade.

[0060] I. General Description: 1. Solvents, reference substances and test substances 1.1 Solvent Methanol: chromatographic grade, Tianjin Concord Technology Co., Ltd.

[0061] Acetonitrile: chromatographic grade, Tianjin Kangkede Technology Co., Ltd.

[0062] 1.2 Reference substances Impurity A: 5-(2-aminoethyl)-2-methoxyphenol, HPLC purity 98.8%, Beijing Kangpai Pharmaceutical Technology Co., Ltd.

[0063] Impurity B: 4-(2-aminoethyl)-2-methoxyphenol, HPLC purity 98.8%, batch number DBAR220827B1, Guangzhou Aici Pharmaceutical Technology Co., Ltd.

[0064] Impurity C: 2-(3,4-dimethoxyphenyl)ethane-1-amine, HPLC purity 99.2%, batch number DBAR220828C1, Guangzhou Aici Pharmaceutical Technology Co., Ltd.

[0065] 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.

[0066] Impurity F: [4-(2-aminoethyl)phenyl]phenyl ketone, HPLC purity 99.2%, batch number DBAR230528F1, Guangzhou Aici Pharmaceutical Technology Co., Ltd.

[0067] 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.

[0068] 5,6-Dihydroxyindole: purity 99.5%, HPLC purity 99.5%, batch number DBAR230510D1, Guangzhou Aici Pharmaceutical Technology Co., Ltd.

[0069] 1.3 Test products Dopamine hydrochloride injection: Henan Runhong Pharmaceutical Co., Ltd., specification 2.5 ml: 50 mg.

[0070] 2. Instruments Ultrapure water machine: WP-UP-YJ-10, Sichuan Water Treatment Equipment Co., Ltd.

[0071] High performance liquid chromatography-ultraviolet detector (purchased from Thermo Fisher Scientific Inc., model: Ultimate 3000).

[0072] Electronic balance: AP135W (one hundred thousandth balance), Shimadzu.

[0073] Electronic balance: MSA6-6S-OCE-DM (one millionth balance), Sartorius.

[0074] II. Example: Separation and Reliability Test 1. Chromatographic conditions Detection wavelength: 280 nm; Column: Waters Nova-Pak C18, 3.9 × 150 mm, 4 μm; Column temperature: 30°C; Total flow rate: 1.0 mL / min; Injection volume: 10 μL; Mobile phase A: Sodium octane sulfonate-citric acid buffer solution (12.6 g citric acid and 1.23 g sodium octane sulfonate were dissolved in 1 mol / L, 120 ml sodium hydroxide solution, water was added to 600 ml, and then 0.1 mol / L, 400 ml hydrochloric acid solution was added) with methanol and acetonitrile in a volume ratio of 88:5:7; Mobile phase B: Sodium octane sulfonate-citric acid buffer solution (take 12.6 g citric acid and 1.54 g sodium octane sulfonate, add 1 mol / L, 120 ml sodium hydroxide solution to dissolve, add water to 600 ml, and then add 0.1 mol / L, 400 ml hydrochloric acid solution) and methanol and acetonitrile in a volume ratio of 70:10:20; The elution was performed according to the gradient elution program in Table 4 above.

[0075] 2. Solution Preparation (1) Dilution Prepare Mobile Phase A as described in Section II, Item 1 above.

[0076] (2) Blank solution Prepare Mobile Phase A as described in Section II, Item 1 above.

[0077] (3) Test solution Take 1.0 ml of dopamine hydrochloride injection, place it in a 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and the test solution of dopamine hydrochloride injection is obtained (the concentration of dopamine hydrochloride is about 2 mg / ml).

[0078] (4) Self-control solution (0.1% self-control) Accurately measure 1.0 ml of the test solution, place it in a 100 ml volumetric flask, add diluent to dilute 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 dilute to the scale, shake well, and you will get the control solution of dopamine hydrochloride injection (dopamine hydrochloride concentration is about 2 μg / ml).

[0079] (5) System suitability solution Take appropriate amounts of dopamine hydrochloride reference substance, impurity A reference substance and impurity B reference substance, add diluent to dissolve and dilute to make a mixed solution containing 2 mg of dopamine hydrochloride, 24 μg of impurity A and impurity B per 1 ml, shake well, and use as the system suitability solution.

[0080] (6) Sensitivity solution Accurately measure an appropriate amount of the self-control solution prepared above, add diluent to dilute, shake well, and prepare a solution containing 0.1 μg of dopamine hydrochloride per 1 ml.

[0081] (7) Stock solution of dopamine hydrochloride-related substances Weigh 10 mg of impurity A, impurity B, impurity C, impurity E, impurity F and impurity H reference substances respectively, place them in 10 ml volumetric flasks respectively, add diluent to dissolve and make up to the scale, shake well to obtain the stock solutions of impurity A, impurity B, impurity C, impurity E, impurity F and impurity H respectively.

[0082] Weigh 10 mg of 5,6-dihydroxyindole, place it in a 10 ml volumetric flask, add 2 ml of methanol to dissolve it, and then dilute to the mark with diluent. Shake well to obtain the stock solution of 5,6-dihydroxyindole.

[0083] (8) Single-label solution of dopamine hydrochloride-related substances Accurately 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 the above step (7), place them in 10 ml volumetric flasks, dilute to the mark with diluent, and shake well to obtain.

[0084] (9) Add the test solution containing 7 impurity reference substances Accurately measure 1.0 ml of the test sample dopamine hydrochloride injection and place it in a 10 ml volumetric flask. Accurately 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 the above (7) and place them in the above volumetric flasks. Add diluent to dilute to the scale and shake well to obtain the solution.

[0085] (10) Add impurity F reference substance to the test solution Accurately measure 1.0 ml of the test sample dopamine hydrochloride injection and place it in a 10 ml volumetric flask. Accurately measure 0.02 ml of the stock solution of impurity F prepared in (7) above and place it in the above volumetric flask. Add diluent to dilute to the scale and shake well to obtain.

[0086] 3. Testing The determination was carried out according to the high performance liquid chromatography method in accordance with the general rules 0512 of the fourth volume of the 2020 edition of the Chinese Pharmacopoeia. The method of the present invention was verified according to the general rules 9101 of the fourth volume of the 2020 edition of the Chinese Pharmacopoeia.

[0087] 3.1 System suitability experiment The blank solution, system suitability solution, and sensitivity solution prepared in item 2 of the above-mentioned Example II were respectively injected into the liquid chromatograph, and the chromatogram was recorded according to the chromatographic conditions described in item 1 of the above-mentioned Example II. The chromatogram of the system suitability solution can be found in Figure 1 , calculate the separation degree of dopamine hydrochloride and related substances in the chromatogram. The results are shown in Table 5 below.

[0088]

[0089] Note: / means there is no next peak and no test data.

[0090] The above results show that the separation between dopamine hydrochloride and impurity B and the separation between impurity B and impurity A in the system applicability solution are greater than 5.0, and there are no interference peaks in the blank solution. The signal-to-noise ratio of the sensitivity solution is greater than 10, which meets the requirements; indicating that the system applicability and sensitivity of the method provided by the present invention are good.

[0091] 3.2 Determination of separation The test solution prepared in item 2 of the above-mentioned Example II and the self-control solution, blank solution, single-label solution of dopamine hydrochloride related substances, and the test solution with seven impurity reference substances added were respectively injected into a liquid chromatograph, and the chromatograms were recorded according to the chromatographic conditions of item 1 of the above-mentioned Example II. The chromatograms of the test solution and the test solution with seven impurity reference substances are shown in FIG. Figure 2 and Figure 3 , calculate the separation degree of dopamine hydrochloride and related substances in the chromatogram. The results are shown in Table 6 below.

[0092] Table 6 Separation results of dopamine hydrochloride and its related substances

[0093] Note: na means not determined.

[0094] *Indicates the separation degree of impurity F from the previous peak.

[0095] It was determined that the blank solution had no interfering peaks, and in the test solution to which the seven impurity reference substances were 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 of which were above 980; this indicates that the purity of the main component peak is good and does not contain peaks of other impurities; that is, the method of the present invention has no effect on the detection performance of the main component, and the separation effect of the main component is good.

[0096] As can be seen from the above table, in the test solution to which the seven impurity reference substances are added, the separation between the chromatographic peak of each impurity and its adjacent chromatographic peak is above 1.5, which indicates that the method for determining the related substances of dopamine hydrochloride injection provided by the present invention has good specificity and can simultaneously detect and separate the 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 ).

[0097] 3.3 Linearity experiment of impurity F A series of solutions with different concentrations of impurity F: Accurately weigh the impurity F reference substance, add an appropriate amount of diluent, and prepare solutions containing impurity F of different concentrations as shown in Table 7 below; then inject these solutions into a liquid chromatograph, and record the chromatogram according to the chromatographic conditions described in Item 1 of the above II Example; 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 follows: Figure 4 As shown, the linear equation is: y=0.4492x-0.0052, R 2 =0.9997.

[0098] Table 7 Impurity F linearity results

[0099] The results show that the concentration of impurity F determined by the high performance liquid chromatography provided by the present invention is in the range of 0.08 to 3.5 μg / ml, and the concentration of impurity F has a good linear relationship with the chromatographic peak area.

[0100] 3.4 Detection accuracy of impurity F Preparation of accuracy solution 1: Accurately pipette 1 ml of dopamine hydrochloride injection and place it in a 10 ml volumetric flask. Add an appropriate amount of impurity F reference substance and dilute to volume to prepare an accuracy solution equivalent to 50% of the impurity F limit content. Prepare three replicates in parallel. The impurity F concentration of the actual prepared solution is the "added impurity concentration" described in the following tables for accuracy 1-1, 1-2, and 1-3.

[0101] Preparation of accuracy solution 2: Accurately pipette 1 ml of dopamine hydrochloride injection and place it in a 10 ml volumetric flask. Add an appropriate amount of impurity F reference substance and dilute to volume to prepare an accuracy solution equivalent to 100% of the impurity F limit content. Prepare three replicates in parallel. The impurity F concentration of the actual prepared solution is the "added impurity concentration" as described in the following tables for accuracy 2-1, 2-2, and 2-3.

[0102] Preparation of accuracy solution 3: Accurately pipette 1 ml of dopamine hydrochloride injection and place it in a 10 ml volumetric flask. Add an appropriate amount of impurity F reference substance and dilute to volume to prepare an accuracy solution equivalent to 150% of the impurity F limit content. Prepare three copies in parallel. The impurity F concentration of the actual prepared solution is the "added impurity concentration" described in the following tables for accuracy 3-1, 3-2, and 3-3.

[0103] Then, the accuracy solution and two parallel test solutions (prepared as in item 2 of the above-mentioned Example II) were respectively introduced into a liquid chromatograph, and the chromatogram was recorded 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.

[0104]

[0105] The results in the above table show that the average recovery rate of impurity F at each level of concentration is 97.5% to 100.3%, and the RSD (%) of the recovery rate is 2.48%, which is in compliance with the regulations. This shows that the accuracy of the high performance liquid chromatography method provided by the present invention for determining impurity F is good.

[0106] 3.5 Determination of the limit of quantification of impurity F According to the linear experiment in Item 3.3 of the above-mentioned Example II, the signal-to-noise ratio (S / N) of the solution of impurity F at the lowest concentration (0.0875 μg / ml) is about 10:1; the solution was injected 6 times in parallel, and the chromatogram was recorded according to the chromatographic conditions described in Item 1 of the above-mentioned Example II. The quantitative limit results of impurity F are shown in Table 9 below.

[0107]

[0108] The results in the table above show that the peak area RSD is within 10%, and the S / N is ≥10, which is in compliance with the regulations; the quantitative limit of impurity F is thus determined to be 0.0875 μg / ml, which is equivalent to 0.0044% of the concentration of the test sample dopamine hydrochloride. This shows that the method of the present invention can be used to perform quantitative determination at a lower concentration of impurity F, and has good sensitivity for the detection of impurity F.

[0109] 3.6 Determination of detection limit of impurity F According to the signal-to-noise ratio of the impurity F solution at the quantitative limit, the impurity F solution at the quantitative limit concentration was taken, diluted with diluent, and the chromatogram was recorded according to the chromatographic conditions described in item 1 of the above-mentioned II Example section, and the signal-to-noise ratio (S / N) was about 3:1, and the detection limit solution of impurity F was obtained. The sample was injected 6 times in parallel, and the detection limit results of impurity F are shown in Table 10 below.

[0110]

[0111] The results in the table above show that the peak area RSD is within 10%, and the S / N is ≥3, which is in compliance with the regulations; the quantitative limit of impurity F is thus determined to be 0.0438 μg / ml, which is equivalent to 0.0022% of the concentration of the test sample dopamine hydrochloride. This shows that the method of the present invention can detect a low lower limit of the content of impurity F, and has good sensitivity for the detection of impurity F.

[0112] III. Comparative Examples Comparative Example 1 According to the detection method for related substances of dopamine hydrochloride recorded in the "Imported Drug Registration Standard" (Standard No.: JX20150157), compared with the chromatographic conditions of the above embodiment, the only difference is that the elution time is 25 minutes; the specific gradient elution program is shown in Table 11 Comparative Example 1. As a result, impurity F cannot be detected. On the contrary, the embodiment of the present invention can detect impurity F (retention time is about 41.7 minutes, see Figure 3 ).

[0113] Comparative Example 2 Comparative Example 2 was carried out according to the method of the embodiment (see 3.2 Determination of separation degree), except that the test solution with impurity F reference substance added as described in item 2 of the above II embodiment section was used as the loading solution and injected into the liquid chromatograph for gradient elution, wherein the volume ratio of mobile phase A and B at 35 to 60 minutes was changed to 40% A and 60% B. The elution conditions and results are shown in Tables 11 and 11 below. Figure 5 shown.

[0114] Comparative Example 3 Comparative Example 3 was carried out according to the method of the embodiment (see 3.2 Determination of Separation), except that the test solution with the reference substance of impurity F added as described in item 2 of the above-mentioned Example II was used as the loading solution and injected into the liquid chromatograph for gradient elution, wherein 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 shown in Tables 11 and 11 below. Figure 6 shown.

[0115]

[0116] Note: * indicates that the elution time of comparative example 1 (i.e. "Registration Standard for Imported Drugs" (Standard No.: JX20150157)) is 25 minutes.

[0117] Depend on Figure 5-6 It can be seen that no other peaks appeared after the main peak (main component dopamine hydrochloride, retention time of about 5.1 min), which indicates that impurity F cannot be detected using the chromatographic conditions of Comparative Examples 2-3; on the contrary, impurity F can be detected by the specific gradient elution conditions of the method of the present invention, especially the elution conditions at 30 to 46 minutes, especially when the volume ratio of mobile phase B is 100% at 35 to 45 minutes.

[0118] In summary, the advantages of the method of the present invention are that it has good sensitivity and separation, and 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 impurities, especially impurity F, and the detection performance of the main component dopamine hydrochloride is good. In addition, using the method of the present invention, the linear range of the concentration of impurity F is 0.08~3.5 μg / ml, the limit of quantification 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 limit of quantification and detection limit. Therefore, the separation and reliability of the method of the present invention are excellent.

Claims

1. A method for improving the quality control of dopamine hydrochloride injection, comprising: The test solution of dopamine hydrochloride injection was injected into a high performance liquid chromatograph to separate and detect the related substances in dopamine hydrochloride injection, wherein: Mobile phase A is a mixed solution composed of sodium octane sulfonate-citric acid buffer solution, methanol, and acetonitrile in a volume ratio of (80-95):(3-10):(4-9); mobile phase B is a mixed solution composed of sodium octane sulfonate-citric acid buffer solution, methanol, and acetonitrile in a volume ratio of (65-75):(5-15):(15-25); The total flow rate of the mobile phase is 0.8 to 1.2 ml / min, and gradient elution is adopted, wherein the elution time is controlled within the range of 35 to 45 minutes, and the volume percentage of mobile phase B in the total volume of the mobile phase is 80% to 100%.

2. The method according to claim 1, wherein the gradient elution is performed according to the following gradient elution program: The total volume percentage of mobile phase A and B is 100%.

3. The method according to claim 1, wherein the gradient elution is performed according to the following gradient elution program: 。 4. The method according to claim 1, wherein the related substance comprises impurity F, [4-(2-aminoethyl)phenyl]phenyl ketone, having the following structural formula: Impurity F.

5. The method according to claim 1, wherein the test solution of dopamine hydrochloride injection injected is a solution of dopamine hydrochloride diluted with mobile phase A with a concentration of 1.5 to 2.5 mg / ml.

6. The method according to claim 1, wherein: In the sodium octane sulfonate-citric acid buffer solution of mobile phase A, the mass concentration of sodium octane sulfonate is 1.0 to 1.5 g / L, and in the sodium octane sulfonate-citric acid buffer solution of mobile phase B, the mass concentration of sodium octane sulfonate is 1.2 to 2.0 g / L.

7. The method according to claim 1, comprising using an ultraviolet detector with a detection wavelength of 279 to 281 nm.

8. The method according to claim 1, wherein the chromatographic column is a chromatographic column using octadecylsilane bonded silica gel as a filler.

9. The method according to claim 8, wherein the chromatographic column is a Waters Nova-Pak C18 chromatographic column.

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

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