Method for determining related impurities in quinidine sulfate by ultra-high performance liquid chromatography

Abstract

The invention relates to a method for determining related impurities in quinidine sulfate by ultra-high performance liquid chromatography. The method comprises the following steps: taking quinidine sulfate, quinine sulfate, dihydroquinidine and cinchonine reference substances, precisely weighing, placing in the same measuring flask, adding a mobile phase, dissolving and diluting to prepare a solution as a system applicable solution; taking 10mg of quinidine sulfate, precisely weighing the quinidine sulfate, placing the quinidine sulfate in a 10ml measuring flask, adding the mobile phase to dissolve and dilute the quinidine sulfate to a scale, and uniformly shaking the quinidine sulfate to obtain a test solution; precisely measuring 20L of the test solution and 20L of the reference solution, respectively injecting into a liquid chromatograph, and recording chromatograms; the method has the advantages that the related impurities in the quinidine sulfate are detected by adopting the ultra-high performance liquid chromatography, the separation effect is good, the detection time can be effectively shortened, the reagent does not need to be separated through ions, the method is simple, and misjudgment is avoided, so that the related impurities of a preparation can be effectively controlled, and the detection efficiency is improved. And the detection cost of research and development production links is reduced.

Description

Technical Field

[0001] This invention relates to the field of quality control methods for quinidine sulfate, and specifically to a method for determining impurities in quinidine sulfate using ultra-high performance liquid chromatography. Background Technology

[0002] Quinidine sulfate is a class Ia antiarrhythmic drug that acts directly on cell membranes, primarily inhibiting the transmembrane movement of sodium ions, affecting phase 0 of the action potential, suppressing myocardial automaticity, especially that of ectopic excitation points, reducing conduction velocity, prolonging the effective refractory period, and decreasing excitability. Its prolongation of the atrial refractory period is more pronounced than that of the ventricles, shortening the refractory period of the atrioventricular junction, and increasing the fibrillation threshold of the atrial and ventricular myocardium. Secondly, it inhibits calcium ion influx, reducing myocardial contractility. It also indirectly affects the heart through its anticholinergic effects.

[0003] Quinidine sulfate is rarely used in single-agent formulations, but in combination formulations, it increases dextromethorphan plasma levels by competitively inhibiting cytochrome P450 2D6. Cytochrome P450 2D6 catalyzes the main biotransformation pathway of dextromethorphan, and can be combined with drugs such as dextromethorphan to treat mental illnesses.

[0004] The original testing standard for quinidine sulfate used high performance liquid chromatography (HPLC) to detect its content. However, this method is time-consuming and the impurities are optical isomers of natural products, which are difficult to separate. Separation with ion-pairing reagents is necessary, which is a complex method and may lead to misjudgments. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for determining impurities in quinidine sulfate by ultra-high performance liquid chromatography (UHPLC). This method can solve the problems of long detection time, difficulty in separating natural optical isomers of impurities, the need for separation by ion-pairing reagents, complexity of the method, and the possibility of misjudgment in the existing technology.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention includes the following steps: S1. Weigh quinidine sulfate, quinine sulfate containing dihydroquinine, dihydroquinidine, and cinchonine reference standards accurately, place them in the same volumetric flask, add mobile phase to dissolve and dilute to prepare a solution with the concentration of each substance in the solution being 0.2 mg / ml, which is used as the system suitability solution. S2. Weigh 10 mg of quinidine sulfate accurately, place it in a 10 ml volumetric flask, add the mobile phase to dissolve and dilute to the mark, shake well, and use it as the test solution. S3. Accurately measure 20µL each of the test solution and the system suitability solution, inject them into the liquid chromatograph, and record the chromatograms.

[0007] Furthermore, the mobile phase is an acetonitrile-buffered saline solution.

[0008] Furthermore, the buffer salt solution is one or more of sodium formate, sodium acetate, ammonium formate, and ammonium acetate, and the buffer concentration is 0.1%-0.7%.

[0009] Further, in step S3, the chromatographic conditions of the liquid chromatograph are as follows: octadecylsilane-bonded silica gel (BEH-C18, 50mm × 2.1mm, 1.7μm particle size) is used as the packing material, the column temperature is 45℃, reversed-phase ultra-high performance liquid chromatography isocratic elution is used, and the flow rate is 0.5ml / min; the detector used is an ultraviolet detector with a detection wavelength of 316nm.

[0010] Furthermore, the gradient elution uses acetonitrile and buffer salt solution as the mobile phase, and the mobile phase gradient process is as follows: Time / min 0.5% Sodium Acetate Buffer Acetonitrile% 0.01 93 7 4.00 93 7 10.00 15 85 12.00 93 7 15.00 93 7

[0011] Furthermore, in step S3, if the chromatogram of the test solution contains a chromatographic peak with the same retention time as the chromatogram of the system suitability solution, the content of each impurity is calculated by peak area using the external standard method, and the calculation formula is as follows: .

[0012] The advantages of this invention are as follows: it uses ultra-high performance liquid chromatography (UHPLC) to detect relevant impurities in quinidine sulfate, achieving good separation results, effectively shortening detection time, and improving detection efficiency. It eliminates the need for separation using ion-pairing reagents, making the method simple and avoiding misjudgments. Therefore, this invention can effectively control relevant impurities in the formulation, reduce detection costs in the R&D and production stages, and rigorously validate the method to ensure its scientific rigor, meeting the needs of R&D and production. It is of indispensable significance for the quality control of quinidine sulfate. Attached Figure Description

[0013] Figure 1 The sample chromatogram of method 1 in the embodiments of the present invention; Figure 2 The sample chromatogram of method 2 in the embodiments of the present invention; Figure 3 This is a system applicability map of method 2 in the embodiments of the present invention. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments will enable those skilled in the art to more fully understand this invention, but do not limit the invention to the scope of the described embodiments.

[0015] Quinidine sulfate is a natural alkaloid extracted from cinchona bark, and its structure is as follows: ; This product is listed in the pharmacopoeias of many countries, including the European Pharmacopoeia, the United States Pharmacopoeia, and the Chinese Pharmacopoeia. The detection methods in these pharmacopoeias all employ high-performance liquid chromatography (HPLC), which is time-consuming and the impurities are optical isomers of natural products, making separation difficult. In some cases, separation using ion-pairing reagents is necessary, which is complex and may lead to misjudgments. To reduce detection costs in the research and development and production process, this invention improves the method for determining impurities in quinidine sulfate using ultra-high-performance liquid chromatography (UHPLC), which can effectively shorten the detection time and improve detection efficiency.

[0016] This specific implementation method adopts the following technical solution, including the following steps: S1. Weigh accurately the reference standards of quinidine sulfate, quinine sulfate (including dihydroquinine), dihydroquinidine, and cinchonine, place them in the same volumetric flask, add the mobile phase to dissolve and dilute to prepare a solution with the concentration of each substance in the solution being 0.2 mg / ml, which is used as the system suitability solution. The mobile phase is an acetonitrile-buffered salt solution, and the buffered salt solution is one or more of sodium formate, sodium acetate, ammonium formate, and ammonium acetate. The buffer concentration is 0.1%-0.7%, preferably 0.5%.

[0017] S2. Weigh 10 mg of quinidine sulfate accurately, place it in a 10 ml volumetric flask, add the mobile phase to dissolve and dilute to the mark, shake well, and use it as the test solution. S3. Accurately measure 20µL each of the test solution and the system suitability solution, inject them into the liquid chromatograph, and record the chromatograms.

[0018] The chromatographic conditions of the liquid chromatograph were as follows: octadecylsilane-bonded silica gel (BEH-C18), 50 mm × 2.1 mm, with a particle size of 1.7 μm, a column temperature of 45 ℃, and isocratic elution using reversed-phase ultra-high performance liquid chromatography at a flow rate of 0.5 ml / min. The detector used was an ultraviolet detector with a detection wavelength of 316 nm.

[0019] The gradient elution uses acetonitrile and buffer salt solution as the mobile phase, eluted according to the following table: Time / min 0.5% Sodium Acetate Buffer Acetonitrile% 0.01 93 7 4.00 93 7 10.00 15 85 12.00 93 7 15.00 93 7

[0020] If the chromatogram of the test solution contains a peak with the same retention time as the chromatogram of the system suitability solution, the content of each impurity shall be calculated by peak area using the external standard method, as follows: .

[0021] This invention explored the mobile phase system by using acetonitrile and buffer salt solution for gradient elution, adjusting the gradient, and finally establishing the high-performance liquid chromatography system of this invention, and conducted methodological validation.

[0022] (1) Exploration of the mobile phase Based on the conversion calculation method provided by the column supplier, the following mobile phase gradient ratio was explored: Method 1: Time / min 0.5% Sodium Acetate Buffer Acetonitrile% 0.01 90 10 3.00 90 10 7.00 20 80 10.00 90 10 15.00 90 10 Method 2: Time / min 0.5% sodium acetate buffer Acetonitrile% 0.01 93 7 4.00 93 7 10.00 15 85 12.00 93 7 15.00 93 7 The chromatograms of the test samples for Method 1 and Method 2 are attached. Figure 1 and attached Figure 2 The results show that Method 2 has a longer retention time for the principal components and a better separation effect.

[0023] Method 2 was used to examine the system's suitability and the separation degree of various impurities in the solution. The results are shown in the appendix. Figure 3 .

[0024] (2) Methodological validation ①Exclusivity Acceptable standard: The blank solvent should not cause interference.

[0025] Verification results: The solvent does not interfere with the determination of impurities related to quinidine sulfate.

[0026] ② Sample injection precision Acceptable criteria: The RSD% of peak area and retention time after 6 repeated injections is not greater than 2.

[0027] Verification results: Experimental results show that when the test sample and system suitability solution are injected continuously for 6 injections, the RSD of retention time and peak area are both less than 2.0%, indicating that the injection precision of this method is good.

[0028] ③ Detection limit Acceptable criteria: The limit of detection is the concentration with a signal-to-noise ratio (S / N) of ≈3, and the limit of quantitation is the concentration with a signal-to-noise ratio (S / N) of ≈10.

[0029] Validation results: The detection limits for each impurity are approximately 0.5–1.2 μg / ml (0.025%–0.06%). This method has good sensitivity and meets the detection requirements.

[0030] ④ Linearity and Range Acceptable standard: The correlation coefficient r of linear regression is not less than 0.995.

[0031] Verification results: Within the concentration range of 0.5–10 μg / ml, the concentration of each impurity showed a good linear relationship with the peak area.

[0032] ⑤ Solution stability Acceptable standard: The RSD% of the main peak area before and after sample placement is not greater than 2.

[0033] Verification results: Experimental results show that the test solution, control solution, and system suitability solution are stable after being placed at room temperature for 30 hours.

[0034] ⑥ Repeatability Acceptable standard: The RSD% of the content and recovery results of six determinations of impurities near the limit concentration is not greater than 2%.

[0035] Validation results: Experimental results show that when the same batch of samples is measured in parallel for 6 times, the RSD% of the impurity detection results is no greater than 2%. This method has good repeatability.

[0036] ⑦ Durability Acceptable standard: After changing the testing conditions, the separation degree between the main peak and the isomer under each condition meets the requirements, and the detection content of the isomer does not change significantly.

[0037] Validation results: When the relative flow rate changed by ±0.02 ml / min, the column temperature changed by ±5℃, and the proportion of organic phase in the mobile phase changed by ±1%, the system applicability and detection results did not change significantly. This method has good robustness.

[0038] The validation results show that all indicators of this method meet the requirements of the 2020 edition of the Chinese Pharmacopoeia and are suitable for detection.

[0039] The foregoing has shown and described the basic principles and main features of the present invention, as well as its advantages. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for determining impurities in quinidine sulfate using ultra-high performance liquid chromatography, characterized in that: Includes the following steps: S1. Weigh quinidine sulfate, quinine sulfate containing dihydroquinine, dihydroquinidine, and cinchonine reference standards accurately, place them in the same volumetric flask, add mobile phase to dissolve and dilute to prepare a solution with the concentration of each substance in the solution being 0.2 mg / ml, which is used as the system suitability solution. S2. Weigh 10 mg of quinidine sulfate accurately, place it in a 10 ml volumetric flask, add the mobile phase to dissolve and dilute to the mark, shake well, and use it as the test solution. S3. Accurately measure 20µL each of the test solution and the system suitability solution, inject them into the liquid chromatograph, and record the chromatograms. In step S3, the chromatographic conditions of the liquid chromatograph are as follows: octadecylsilane-bonded silica gel (BEH-C18, 50mm × 2.1mm, 1.7μm particle size) is used as the packing material, the column temperature is 45℃, reversed-phase ultra-high performance liquid chromatography isocratic elution is used, and the flow rate is 0.5ml / min; the detector used is an ultraviolet detector with a detection wavelength of 316nm.

2. The method for determining impurities in quinidine sulfate by ultra-high performance liquid chromatography according to claim 1, characterized in that: The mobile phase is an acetonitrile-buffered salt solution.

3. The method for determining impurities in quinidine sulfate by ultra-high performance liquid chromatography according to claim 2, characterized in that: The buffer salt solution is one or more of sodium formate, sodium acetate, ammonium formate, and ammonium acetate, and the buffer concentration is 0.1%-0.7%.

4. The method for determining impurities in quinidine sulfate by ultra-high performance liquid chromatography according to claim 1, characterized in that: The gradient elution uses acetonitrile and buffer salt solution as the mobile phase, and the gradient process of the mobile phase is as follows: 。 5. The method for determining impurities in quinidine sulfate by ultra-high performance liquid chromatography according to claim 1, characterized in that: In step S3, if the chromatogram of the test solution contains a chromatographic peak with the same retention time as the chromatogram of the system suitability solution, the content of each impurity is calculated by peak area using the external standard method, and the calculation formula is as follows: 。

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