A method for detecting the enantiomers of metaraminol bitartrate in an injection

Abstract

The application belongs to the technical field of medical detection, and particularly relates to a method for detecting metaraminol enantiomers in injection. The method comprises detecting metaraminol enantiomers in injection by high performance liquid chromatography, wherein the detection conditions of the high performance liquid chromatography comprise: a chromatographic column adopts a packing material with a polysaccharide derivative bonded on the surface of silica gel as a stationary phase; and a mixed solution of n-hexane, anhydrous ethanol and ethylenediamine is used as a mobile phase. The method has good separation degree and high separation efficiency. Through complete methodology verification, the method can effectively, accurately and stably detect the content of metaraminol enantiomers, and is beneficial to improving the safety of clinical medication.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical testing technology, specifically relating to a method for detecting the enantiomers of meta-hydroxylamine bitartrate in injection solutions. Background Technology

[0002] Metaraminol bitartrate is an α-adrenergic receptor agonist used in the early treatment of shock and to prevent acute hypotension during spinal anesthesia. Its structure contains two chiral carbon atoms, resulting in four configurations: (1R, 2S), (1S, 2R), (1R, 2R), and (1S, 2S). The (1R, 2S) configuration is the pharmaceutically effective configuration, while the (1S, 2R) configuration is its enantiomer.

[0003] Because optical isomers exhibit different physiological activities, these differences reflect the stringent requirements on the stereostructure for drug binding to receptors. To ensure drug safety, controlling the enantiomer content in drugs with multiple chiral centers is crucial. Therefore, for metaraminol bitartrate injection, the detection of enantiomers of related substances (1S, 2R) configurations is particularly critical in drug quality control.

[0004] Chinese patent CN106596794A discloses a high-performance liquid chromatography (HPLC) method for separating and determining m-hydroxylamine bitartrate and its isomers, used for detecting m-hydroxylamine bitartrate and its isomers in m-hydroxylamine bitartrate raw materials. While this method can achieve the separation and determination of m-hydroxylamine bitartrate and its isomers, it lacks methodological validation. The specificity, precision, accuracy, and robustness of this method are unclear, potentially leading to systematic biases, poor data reproducibility, and an inability to reliably reflect the true quality of the sample.

[0005] Furthermore, directly applying the separation and detection methods for isomers in active pharmaceutical ingredients to the detection of injectable products may reduce the separation effect of meta-tartrate and isomers due to the potential influence of the injectable matrix (such as sodium chloride, sodium metabisulfite, and other excipients) on the separation system.

[0006] Therefore, there is a need to develop a new method for detecting the enantiomers or meta-hydroxylamine isomers of meta-hydroxylamine bitartrate injection in order to overcome the shortcomings of existing detection methods. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is how to efficiently and accurately detect the content of the enantiomer of metaraminol bitartrate in metaraminol bitartrate injection.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, this application provides a method for detecting the enantiomers of m-hydroxylamine bitartrate in an injection solution, comprising using high-performance liquid chromatography (HPLC) to detect the enantiomers of m-hydroxylamine bitartrate in the injection solution, wherein the detection conditions of the HPLC include: The chromatographic column uses a stationary phase consisting of silica gel with polysaccharide derivatives bonded to its surface. A mixed solution of n-hexane, anhydrous ethanol, and ethylenediamine was used as the mobile phase.

[0009] In some embodiments of this application, the polysaccharide derivative is amylose-tris(3,5-dimethylphenylcarbamate).

[0010] In some embodiments of this application, the volume ratio of n-hexane, anhydrous ethanol and ethylenediamine in the mobile phase is (650-750):(250-350):(2-3).

[0011] In some embodiments of this application, isocratic elution is performed using the mobile phase during high-performance liquid chromatography detection.

[0012] In some embodiments of this application, the detection wavelength is 270-280 nm.

[0013] In some embodiments of this application, the method includes the following steps: Prepare a system suitability solution, wherein the system suitability solution includes m-hydroxylamine and impurity D; Prepare the test solution; High-performance liquid chromatography (HPLC) was performed on both the system suitability solution and the test solution.

[0014] In some embodiments of this application, the content of impurity D in the system suitability solution is 1-3 μg / mL, and the content of intermediate hydroxylamine in the system suitability solution is 0.1-1.0 mg.

[0015] In some embodiments of this application, the system suitability solution is prepared by the following steps: 1) Preparation of reference stock solution: Take an appropriate amount of the bitartrate of impurity D, dissolve it in ethanol and dilute it with ethanol to prepare a solution containing 5-20 μg of impurity D per 1 ml; 2) Take appropriate amounts of the reference stock solution and metaraminol bitartrate injection, dissolve and dilute with ethanol to prepare a mixed solution containing 0.1-1.0 mg metaraminol and 1-3 μg impurity D per 1 ml.

[0016] In some embodiments of this application, the flow rate of the mobile phase is 0.5-2 ml / min; and / or the column temperature is 25-40°C.

[0017] Secondly, this application provides the application of the method described in the first aspect above in the field of quality control of m-hydroxylamine bitartrate and its preparations. Beneficial effects

[0018] The method described in this application exhibits excellent separation and high separation efficiency. Through comprehensive methodological validation, it is demonstrated more scientifically and objectively that the detection method described in this application can effectively, accurately, and stably detect the content of enantiomers, which is beneficial to improving the safety of clinical drug use. Attached Figure Description

[0019] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0020] Figure 1 This is a high-performance liquid chromatogram of the system adaptability experiment in Example 1. Detailed Implementation

[0021] The present invention will now be described in detail with reference to embodiments. The principles and features of the present invention are described below with reference to embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other. The embodiments given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0022] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0023] In this application, v / v% refers to volume percentage.

[0024] Because the meta-hydroxylamine in metaraminol bitartrate contains two chiral carbon atoms, it exists in four configurations: (1R, 2S), (1S, 2R), (1R, 2R), and (1S, 2S). (1R, 2S) is the effective configuration, while (1S, 2R) is its enantiomer. Although various methods are used to control enantiomers during the preparation of metaraminol bitartrate, the final metaraminol bitartrate raw material still contains a certain amount of enantiomers, and correspondingly, the injection solution also contains a certain amount of enantiomers. Therefore, in order to control the quality of metaraminol bitartrate injection and improve medication safety, it is particularly important to efficiently and accurately detect the (1S, 2R) enantiomer configuration of metaraminol bitartrate in the injection solution.

[0025] In this application, m-hydroxylamine refers to m-hydroxylamine with the configuration (1R, 2S), which is the effective configuration. Impurity D refers to the enantiomer of m-hydroxylamine with the configuration (1S, 2R).

[0026] In one specific embodiment of this application, a method for detecting the enantiomers of m-hydroxylamine bitartrate in an injection solution is provided. The method includes high-performance liquid chromatography (HPLC) for detecting the enantiomers of m-hydroxylamine bitartrate in the injection solution. The detection conditions for HPLC include: The chromatographic column uses a stationary phase consisting of silica gel with polysaccharide derivatives bonded to its surface. A mixed solution of n-hexane, anhydrous ethanol, and ethylenediamine was used as the mobile phase.

[0027] In some embodiments of this application, the chromatographic column is a bonded chiral chromatographic column with linear starch-tris(3,5-dimethylphenylcarbamate) covalently bonded to the surface of silica gel as the packing material.

[0028] In some embodiments of this application, the volume ratio of n-hexane, anhydrous ethanol and ethylenediamine in the mobile phase is (650-750):(250-350):(2-3).

[0029] In some embodiments of this application, the detection wavelength is 270-280nm, which provides better detection sensitivity.

[0030] In some embodiments of this application, the metaraminol bitartrate injection includes metaraminol bitartrate and excipients, wherein the excipients include sodium chloride.

[0031] In some embodiments of this application, the excipients also include sodium metabisulfite.

[0032] In some embodiments of this application, the sodium chloride content in the metaraminol bitartrate injection is 5-10 mg / mL, and preferably, the sodium metabisulfite content is 1-5 mg / mL.

[0033] In some embodiments of this application, the method includes the following steps: Prepare a system suitability solution, wherein the system suitability solution includes m-hydroxylamine and impurity D; Prepare the test solution; High-performance liquid chromatography (HPLC) was used to analyze the system suitability solution and the test solution.

[0034] In some embodiments, the content of impurity D in the system suitability solution is 1-3 μg / mL, and the content of intermediate hydroxylamine in the system suitability solution is 0.1-1.0 mg.

[0035] In some embodiments of this application, the test sample is a solution containing metaraminol bitartrate injection, wherein the solvent of the test sample solution includes ethanol; preferably, the concentration of metaraminol in the test sample solution is 0.1-1.5 mg / ml.

[0036] In some embodiments of this application, the method further includes the steps of preparing a system suitability solution and performing high-performance liquid chromatography analysis on the system suitability solution, wherein the system suitability solution is prepared by the following steps: 1) Preparation of reference stock solution: Take an appropriate amount of the bitartrate of impurity D, add ethanol to dissolve it, and dilute with ethanol to prepare a solution containing 5-20 μg of impurity D per 1 ml; 2) Take appropriate amounts of the reference stock solution and metaraminol bitartrate injection, dissolve and dilute with ethanol to prepare a mixed solution containing 0.1-1.0 mg metaraminol and 1-3 μg impurity D per 1 ml.

[0037] This application also provides the application of the above-mentioned detection method in the field of quality control of m-hydroxylamine bitartrate and its preparations.

[0038] The detection method for related substances in the meta-hydroxylamine bitartrate injection of the present invention will be specifically described below through specific embodiments. In the embodiments, all original reagent materials are commercially available, and experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0039] Table 1. Reagents used in the examples and comparative examples Ditartrate of impurity D 98.76 SINCO PHARMACHEM m-hydroxylamine bitartrate 99.8 USP metaraminol bitartrate injection 1 mL / vial, each vial contains 10 mg of metaraminol. Hainan Aike Pharmaceutical Co., Ltd. Table 2. Instruments used in the examples High Performance Liquid Chromatography Vanquish Thermo Fisher Example 1

[0040] Determination method for enantiomers of meta-tartrate The content of m-hydroxylamine bitartrate enantiomers in m-hydroxylamine bitartrate injection was determined by high performance liquid chromatography.

[0041] 1. Chromatographic conditions: High Performance Liquid Chromatography (HPLC): Chromatographic column: The stationary phase was a silica gel packing material with linear starch-tris(3,5-dimethylphenylcarbamate) bonded to its surface (CHIRALPAK AD-H, 4.6 mm × 250 mm, particle size 5 μm). Mobile phase: n-hexane: anhydrous ethanol: ethylenediamine = 700:300:2.8 (volume ratio), isocratic elution was used.

[0042] The flow rate was 1.0 ml / min, the column temperature was 30 ℃, the detection wavelength was 274 nm, the detector was a UV-Vis detector, and the injection volume was 20 μl.

[0043] 2. Solution preparation 1) Blank solution: Use anhydrous ethanol as the blank solution.

[0044] 2) Preparation of test solution Mix 5 vials of metaraminol bitartrate injection, accurately measure 1 mL (approximately equivalent to 10 mg of metaraminol), add it to a 20 mL volumetric flask, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant to prepare a solution containing approximately 0.5 mg of metaraminol per 1 mL.

[0045] 3) Preparation of reference standard stock solution Take an appropriate amount of the reference standard of impurity D (metahydroxylamine bitartrate enantiomer), dissolve it in anhydrous ethanol, and dilute it with anhydrous ethanol to a solution containing 10 μg of impurity D per mL (conversion factor between impurity D and impurity D bitartrate: 0.5270).

[0046] 4) System suitability solution: Accurately measure 1 ml of metaraminol bitartrate injection (approximately equivalent to 10 mg of metaraminol), place it in a 20 ml volumetric flask, add 2.0 ml of reference stock solution, dilute to the mark with ethanol, shake well, centrifuge and take the supernatant to prepare a mixed solution containing approximately 0.5 mg of metaraminol and 1 μg of impurity D (enantiomer) per 1 ml.

[0047] System suitability requirements: In the system suitability solution chromatogram, the elution order is impurity D (enantiomer) and m-hydroxylamine, and the resolution between the two peaks should be greater than 2.0.

[0048] 5) Detection of meta-hydroxylamine bitartrate and its enantiomers Take 20 μl each of the test solution and the system suitability solution, inject them into the high-performance liquid chromatograph, and record the chromatogram for 28 minutes. The chromatographic analysis results are shown in Table 3.

[0049] Table 3 Chromatogram Retention Times Impurity D 5.846 - metaraminol 17.894 18.49 Peaks smaller than the main peak area in the chromatogram of the test solution are negligible. The limit for impurity D is ≤0.2%. Impurity D was not detected in the test solution.

[0050] It should be noted that since the bitartrate salt of impurity D exists in solution as impurity D, the content of the m-hydroxylamine bitartrate enantiomer can be accurately reflected by measuring the content of impurity D.

[0051] The method for detecting the enantiomers of m-hydroxylamine bitartrate in the above-mentioned m-hydroxylamine bitartrate injection was validated.

[0052] 1. Method specificity (1) Solution preparation Blank solution: Use anhydrous ethanol as a blank solution.

[0053] Blank excipient solution: Weigh 850.5 mg of sodium chloride and 200.5 mg of sodium metabisulfite into a 100 mL volumetric flask, dissolve and dilute with water to the mark, and shake well to obtain the blank excipient stock solution. Accurately measure 1 mL of the blank excipient stock solution into a 20 mL volumetric flask, dilute to the mark with anhydrous ethanol, shake well, centrifuge, and collect the supernatant.

[0054] Test solution: Mix 5 vials of metaraminol bitartrate injection, accurately measure 1 ml (approximately equivalent to 10 mg of metaraminol), place it in a 20 ml volumetric flask, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant to prepare a solution containing approximately 0.5 mg of metaraminol per 1 mL.

[0055] Preparation of reference standard stock solution Take 1.012 mg of the reference standard of impurity D (enantiomer of m-hydroxylamine bitartrate), place it in a 50 mL volumetric flask, add anhydrous ethanol to dissolve it, and dilute to the mark with anhydrous ethanol. Shake well.

[0056] Preparation of impurity D localization solution Accurately measure 2 ml of the reference standard stock solution and place it in a 20 ml volumetric flask. Dilute to the mark with anhydrous ethanol and shake well.

[0057] System suitability solution: Accurately measure 1 mL of metaraminol bitartrate injection (approximately equivalent to containing 10 mg of metaraminol), place it in a 20 mL volumetric flask, add 2 mL of 2 mL of reference stock solution, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant to prepare a mixed solution containing approximately 0.5 mg of metaraminol and 1 µg of impurity D per mL.

[0058] Accurately measure 20 µl of each of the above solutions and inject them into the liquid chromatograph for analysis.

[0059] (2) Acceptable standards The blank solution and blank excipient solution should not interfere with the elution of m-hydroxylamine and impurity D (enantiomer); In the system suitability solution chromatogram, the elution order is impurity D (enantiomer) and m-hydroxylamine, and the resolution between the two peaks should be greater than 2.0.

[0060] (3) Experimental results System adaptability test results are as follows Figure 1 As shown. By Figure 1 It can be seen that the blank solution and blank excipient solution do not interfere with the elution of m-hydroxylamine and impurity D. The elution order is impurity D and m-hydroxylamine, and the resolution between the two peaks is 18.49 > 2.0. In summary, this method has good specificity.

[0061] 2. Limit of Detection and Limit of Quantification (1) Solution preparation Blank solution: Use anhydrous ethanol as a blank solution.

[0062] System suitability solution: Accurately measure 1 mL of metaraminol bitartrate injection (approximately equivalent to containing 10 mg of metaraminol), place it in a 20 mL volumetric flask, add 2 mL of 2 mL of reference stock solution, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant to prepare a mixed solution containing approximately 0.5 mg of metaraminol and 1 µg of impurity D per mL.

[0063] Impurity D stock solution: Accurately weigh 2.042 mg of impurity D bitartrate reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with ethanol, and shake well.

[0064] m-hydroxylamine bitartrate stock solution: Accurately weigh 2.031 mg of m-hydroxylamine bitartrate reference standard, place it in a 10 ml volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.

[0065] Mixed reference standard stock solution: Accurately measure 2 ml each of impurity D stock solution and m-hydroxylamine bitartrate stock solution, place them in the same 20 ml volumetric flask, dilute to the mark with anhydrous ethanol, and shake well.

[0066] Limit of Quantitation Solution: Accurately measure 1 ml of the mixed reference standard stock solution, place it in a 50 ml volumetric flask, dilute to the mark with anhydrous ethanol, and shake well.

[0067] Detection limit solution: Accurately measure 5 ml of the quantitation limit solution and place it in a 10 ml volumetric flask. Dilute to the mark with anhydrous ethanol and shake well.

[0068] (2) Acceptable standards: In the limit of quantitation (LOQ) solution, the signal-to-noise ratio (SNR) of the peak height for each component should be ≥10; in the limit of detection (LOD) solution, the SNR of the peak height for each component should be ≥3. The LOQ should be determined repeatedly for 6 times, and the RSD of the peak area for each component should not exceed 20%, and the RSD of the retention time should not exceed 2%.

[0069] (3) Experimental conclusions The results for the limit of detection solution and the limit of quantitation solution are summarized in Table 4: Table 4 Results for Limit of Detection (LOD) and Limit of Quantification (LOQ) Solutions

[0070] As shown in Table 4, the S / N values ​​of intermediate hydroxylamine and impurity D in the detection limit solution are all greater than 3; the S / N values ​​of intermediate hydroxylamine and impurity D in the quantitation limit solution are all greater than 10; and in 6 consecutive determinations of the quantitation limit solution, the RSD of the retention time of intermediate hydroxylamine and impurity D is less than 2%, and the RSD of the peak area is less than 20%, which meets the requirements.

[0071] 3. Linearity and Range Linearity of test sample concentration: Five reference solutions with different concentrations were prepared within a certain concentration range (20-120% of the test sample concentration), and the peak area was measured for each solution. Linear regression analysis was performed with concentration as the abscissa (X) and peak area as the ordinate (Y).

[0072] Solution preparation Blank solution: Use anhydrous ethanol as a blank solution.

[0073] System suitability solution: Accurately weigh 1.012 mg of impurity D bitartrate reference standard and place it in a 50 ml volumetric flask. Dissolve and dilute to the mark with anhydrous ethanol, and shake well to obtain the reference standard stock solution. Accurately measure 1 ml of meta-hydroxylamine bitartrate injection and place it in a 20 ml volumetric flask. Add 2.0 ml of the reference standard stock solution, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant.

[0074] Linear reference stock solution: Accurately weigh 25.42 mg of m-hydroxylamine bitartrate reference standard, place it in a 5 ml volumetric flask, add 0.5 ml of water to dissolve it, dilute to the mark with anhydrous ethanol, and shake well.

[0075] (2) Acceptable standards Within the above concentration range, the correlation coefficient r is greater than or equal to 0.990, and the response value of |Y-axis intercept| / 100% is less than or equal to 25%.

[0076] (3) Experimental conclusions Within the concentration range of 0.1070 mg / ml to 0.6417 mg / ml, the linear equation for metaraminol was y = 14384327.7955x - 240.4818, with a linear correlation coefficient of 0.9999 > 0.990 and a response value of |Y-intercept| / 100% of 0.0033% < 25%, indicating a good linear relationship.

[0077] 4. Impurity Limit Linearity (1) Solution preparation Blank solution: Use anhydrous ethanol as a blank solution.

[0078] System suitability solutions: See “System suitability solutions” under the repeatability section below.

[0079] Impurity D stock solution: Accurately weigh 2.042 mg of impurity D bitartrate reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with anhydrous ethanol, and shake well.

[0080] m-hydroxylamine bitartrate stock solution: Accurately weigh 2.031 mg of m-hydroxylamine bitartrate reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with anhydrous ethanol, and shake well.

[0081] Mixed reference standard stock solution: Accurately measure 2 ml each of impurity D stock solution and m-hydroxylamine bitartrate stock solution, place them in the same 20 ml volumetric flask, dilute to the mark with anhydrous ethanol, and shake well.

[0082] Experimental Methods: Six reference solutions of different concentrations were prepared within a concentration range from the limit of quantitation to a certain concentration range (200% of the limit concentration). The peak areas of each solution were measured. Linear regression analysis was performed with concentration as the abscissa (X) and peak area as the ordinate (Y). The results are shown in Table 5. Table 5

[0083] The results show that, within the corresponding concentration ranges, the peak area of ​​m-hydroxylamine and impurity D exhibits a good linear relationship with their concentrations.

[0084] 5. Precision 5.1 Repeatability (1) Experimental method: Prepare spiked test solution according to the test method of meta-hydroxylamine bitartrate enantiomer, prepare 6 parallel samples, calculate the content of impurity D, and examine the RSD of the 6 determination results.

[0085] (2) Solution preparation Blank solution: i.e., anhydrous ethanol.

[0086] Reference stock solution: Accurately weigh 1.048 mg of the tartrate reference standard of impurity D, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with anhydrous ethanol, and shake well.

[0087] System suitability solution: Accurately measure 1 ml of metaraminol bitartrate injection (approximately equivalent to containing 10 mg of metaraminol), place it in a 20 ml volumetric flask, add 2.0 ml of reference stock solution, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant.

[0088] 100% spiked test solution: Mix 20 vials of metaraminol tartrate injection, accurately measure 1 ml (approximately equivalent to containing 10 mg metaraminol), place in a 20 ml volumetric flask, add 2.0 ml of reference stock solution, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant, and prepare 6 parallel aliquots.

[0089] (3) Acceptable standards The RSD of impurity D in 6 spiked test solutions should not exceed 3%.

[0090] (4) Experimental conclusions The RSD of impurity D content in the 6 spiked test solutions was 1.2% < 3%, which meets the requirements, indicating that the method has good repeatability.

[0091] 5.2 Intermediate Precision (1) Experimental method: Another analyst prepared new solutions at different times and with different instruments for testing. The content of impurity D was calculated according to the experiment under the repeatability item. The calculation results were compared with the repeatability test results, and the RSD of 12 results was calculated.

[0092] (2) Solution preparation Blank solution: i.e., anhydrous ethanol.

[0093] Reference stock solution: Accurately weigh 1.027 mg of the tartrate reference standard of impurity D, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with anhydrous ethanol, and shake well.

[0094] System suitability solution: Accurately measure 1 ml of metaraminol tartrate injection (approximately equivalent to containing 10 mg of metaraminol), place it in a 20 ml volumetric flask, add 2.0 ml of reference stock solution, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant.

[0095] 100% spiked test solution: Mix 20 vials of metaraminol tartrate injection, accurately measure 1 ml (approximately equivalent to 10 mg of metaraminol), place in a 20 ml volumetric flask, add 2.0 ml of reference stock solution, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant, and prepare 6 parallel aliquots.

[0096] (3) Acceptable standards The RSD of impurity D in 6 spiked test solutions should not exceed 3%; The RSD of impurity D content in 12 spiked test solutions should not exceed 6%.

[0097] (4) Experimental conclusions In the intermediate precision test, the RSD of impurity D in 6 spiked test solutions was 0.7% < 3%, which meets the requirements. In the repeatability and intermediate precision tests, the RSD of impurity D in 12 spiked test solutions was 2.3% < 6%, which meets the requirements; the intermediate precision of this method is good.

[0098] 6. Accuracy (1) Experimental method: Take a sample with known impurity content and add test solution containing 50%, 100% and 150% of the impurity limit, respectively. Prepare 3 copies of each concentration, for a total of 9 copies. Take the above 9 test solutions for determination and calculate the amount of impurity D and the recovery rate in the test sample.

[0099] Recovery rate (%) = (Measured amount - Original amount of each impurity in the sample) / Added amount × 100% (2) Solution preparation Blank solution: i.e., anhydrous ethanol.

[0100] Reference stock solution: Accurately weigh 1.048 mg of the bitartrate (enantiomer) reference standard of impurity D, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with anhydrous ethanol, and shake well.

[0101] Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with anhydrous ethanol, and mix well.

[0102] Mixed test sample: Take 20 vials of m-hydroxylamine bitartrate injection, mix well, System suitability solution: See “System suitability solution” under the repeatability test section above.

[0103] Test solution: Accurately measure 1 ml of the mixed test solution (approximately equivalent to 10 mg of m-hydroxylamine), place it in a 20 ml volumetric flask, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant.

[0104] 50% spiked test solution: Accurately measure 1 ml of the mixed test solution (approximately equivalent to 10 mg of m-hydroxylamine), place it in a 20 ml volumetric flask, add 1.0 ml of the reference stock solution, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant. Prepare 3 parallel aliquots.

[0105] 100% spiked test solution: Accurately measure 1 ml of the mixed test solution (approximately equivalent to 10 mg of m-hydroxylamine), place it in a 20 ml volumetric flask, add 2.0 ml of the reference stock solution, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant. Prepare 3 parallel aliquots.

[0106] 150% spiked test solution: Accurately measure 1 ml of the mixed test solution (approximately equivalent to 10 mg of m-hydroxylamine), place it in a 20 ml volumetric flask, add 3.0 ml of the reference stock solution, dilute to the mark with anhydrous ethanol, shake well, centrifuge and collect the supernatant. Prepare 3 parallel aliquots.

[0107] (3) Acceptable standards At all concentration levels, the recovery rate of impurity D should be in the range of 90% to 108%, with an RSD of no more than 3%.

[0108] (4) Experimental conclusions At various concentration levels, the recovery rate of impurity D ranged from 100.00% to 105.15%. Within the range of 90% to 108%, the RSD of the recovery rate was 1.4% (<3%), indicating that the method has good accuracy.

[0109] 7. Solution stability (1) Experimental method: Take the spiked test solution, place it in a 15℃ injection tray, and take samples at different times to test the stability of the solution.

[0110] (2) Solution preparation Blank solution: Use anhydrous ethanol as a blank solution.

[0111] System suitability solution: See “System suitability solution” under the repeatability test section above.

[0112] 100% spiked test solution: See the first "100% spiked test solution" under the repeatability test section above.

[0113] (3) Acceptable standards Stability of 100% spiked test solution: The absolute value of the change in impurity content is within 0.05% compared with 0h.

[0114] (4) Experimental conclusions When the 100% spiked test solution was placed at 15℃ for 38 hours, the maximum absolute change in impurity D content compared to 0 hours was 0.005% < 0.05%, indicating that the 100% spiked test solution had good stability within 38 hours at 15℃.

[0115] 8. Durability (1) Experimental method: The solution was prepared according to the test method for the enantiomers of meta-hydroxylamine bitartrate. The chromatographic conditions were slightly varied, and the determination was performed once under each condition. The experimental conditions are shown in Table 6.

[0116] Table 6 Durability Test Conditions Standard conditions 30 1.0 274 70 Column temperature -2 28 1.0 274 70 Column temperature +2 32 1.0 274 70 Flow rate +10% 32 1.1 274 70 Flow rate -10% 32 0.9 274 70 hexane ratio -2% 32 1.0 274 68 hexane ratio +2% 32 1.0 274 72 (2) Solution preparation Blank solution: Use anhydrous ethanol as a blank solution.

[0117] System suitability solution: See “System suitability solution” under the repeatability test section above.

[0118] 100% spiked test solution: See the first “100% spiked test solution” under the accuracy test section above.

[0119] (3) Acceptable standards Minor changes in chromatographic conditions (column temperature ±2℃, flow rate ±10%, n-hexane ratio change ±2%, etc.): ① In the system suitability solution chromatogram, the elution order is impurity D (enantiomer) and m-hydroxylamine, and the resolution between the two peaks should be greater than 2.0; ② Compared with standard conditions, the absolute change in the content of impurities in the 100% spiked test solution should be within 0.05%.

[0120] (4) Experimental conclusions Minor changes in chromatographic conditions (column temperature ±2℃, flow rate ±10%, n-hexane ratio change ±2%): In the system suitability solution chromatogram, the elution order is impurity D and m-hydroxylamine, with the minimum resolution between the two peaks being 18.24 > 2.0; Compared with standard conditions, the absolute change in impurity content in the 100% spiked test solution was no more than 0.006%, which is within 0.05%, indicating that the method has good robustness.

[0121] Comparative Example 1 The difference compared to Example 1 is as follows: Mobile phase: n-hexane: anhydrous ethanol: isopropylamine = 80:20:0.2 (volume ratio), isocratic elution was used. The chromatographic analysis results are shown in Table 7.

[0122] Table 7 Chromatogram Retention Times Impurity D 12.831 - metaraminol 27.534 Meets pharmacopoeia requirements Therefore, the peak elution time of m-hydroxylamine in Comparative Example 1 is around 27 minutes, while the peak elution time of m-hydroxylamine in the detection method of this application is around 18 minutes, indicating higher separation efficiency. Furthermore, the separation degree between m-hydroxylamine and impurity D (enantiomer) in the method of this application reaches over 18%, demonstrating better separation performance. Method validation results show that the method of this application has good specificity, high accuracy, good stability, and good robustness, and can effectively, accurately, and stably detect enantiomer content, which is beneficial to improving the safety of clinical drug use.

[0123] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

[0124] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for detecting the enantiomer of m-hydroxylamine bitartrate in an injection solution, characterized in that, The method includes detecting the enantiomers of m-hydroxylamine bitartrate in the injection solution using high-performance liquid chromatography (HPLC), wherein the detection conditions for HPLC include: The chromatographic column uses a stationary phase consisting of silica gel with polysaccharide derivatives bonded to its surface. A mixed solution of n-hexane, anhydrous ethanol, and ethylenediamine was used as the mobile phase.

2. The method according to claim 1, characterized in that, The polysaccharide derivative is amylose-tris(3,5-dimethylphenylcarbamate).

3. The method according to claim 1 or 2, characterized in that, The volume ratio of n-hexane, anhydrous ethanol and ethylenediamine in the mobile phase is (650-750):(250-350):(2-3).

4. The method according to claim 3, characterized in that, When performing high performance liquid chromatography (HPLC) detection, the mobile phase isocratic elution is performed.

5. The method according to claim 1, characterized in that, The detection wavelength is 270-280nm.

6. The method according to claim 1, characterized in that, The method includes the following steps: Prepare a system suitability solution, wherein the system suitability solution includes m-hydroxylamine and impurity D; Prepare the test solution; High-performance liquid chromatography (HPLC) was performed on both the system suitability solution and the test solution.

7. The detection method according to claim 6, characterized in that, The content of impurity D in the system suitability solution is 1-3 μg / mL, and the content of intermediate hydroxylamine in the system suitability solution is 0.1-1.0 mg.

8. The detection method according to claim 7, characterized in that, The system suitability solution is prepared by the following steps: 1) Preparation of reference stock solution: Take an appropriate amount of the bitartrate of impurity D, dissolve it in ethanol and dilute it with ethanol to prepare a solution containing 5-20 μg of impurity D per 1 ml; 2) Take appropriate amounts of the reference stock solution and metaraminol bitartrate injection, dissolve and dilute with ethanol to prepare a mixed solution containing 0.1-1.0 mg metaraminol and 1-3 μg impurity D per 1 ml.

9. The detection method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.5-2 ml / min; and / or the column temperature is 25-40℃.

10. The application of the method according to any one of claims 1-9 in the field of quality control of meta-hydroxylamine bitartrate and its preparations.

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