Unknown impurity in low concentration epinephrine prefilled injection and its separation, identification and detection method

Abstract

This application provides a method for separating, identifying, and detecting unknown impurities in a low-concentration pre-filled epinephrine injection, belonging to the field of pharmaceutical synthesis technology. The method involves adding epinephrine raw material and an antioxidant to the pre-filled epinephrine injection, and conducting a high-temperature reaction in a closed environment to accelerate the formation of unknown impurities, resulting in a reaction solution enriched with the unknown impurities. The resulting reaction solution is then separated and purified by preparative high-performance liquid chromatography (HPLC), and the eluent is collected to obtain a solution containing the unknown impurities. Mass spectrometry and nuclear magnetic resonance (NMR) measurements are performed using LC-MS and 1H NMR spectroscopy to obtain the results. 1 HNMR spectroscopy confirmed the structure of the unknown impurity. HPLC was used to determine the content of this impurity in the product, ensuring the safety of this low-concentration pre-filled epinephrine injection formulation.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical synthesis technology, specifically to an unknown impurity in a low-concentration pre-filled epinephrine injection solution and its separation, identification, and detection methods. Background Technology

[0002] Low-concentration pre-filled epinephrine injection is indicated primarily for severe respiratory distress caused by bronchospasm. It can rapidly relieve anaphylactic shock caused by drugs, prolong the duration of action of infiltration anesthesia, and is a primary resuscitation drug for cardiopulmonary resuscitation in cases of cardiac arrest from various causes. Its specification is 10ml:1mg (0.1mg / ml), a concentration suitable for emergency resuscitation in both children and adults. The structural formula of epinephrine is as follows: .

[0003] The main active ingredient in low-concentration pre-filled epinephrine injection is epinephrine, and the impurities mainly come from process impurities and degradation impurities of epinephrine.

[0004] The low-concentration pre-filled epinephrine injection solution of this invention has a concentration of 0.1 mg / ml. According to the British Pharmacopoeia, the United States Pharmacopoeia, and the Chinese Pharmacopoeia, possible impurities in epinephrine include epinephrine red, norepinephrine, epinephrine, R-benzylepinephrine, benzylepinephrine, epinephrine sulfite, D-epinephrine, 1-(4-hydroxyphenyl)-2-(methylamino)ethanol, 4-[2-(methylamino)ethyl]phenyl-1,2-diol, 4-(1-methoxy-2-(methylamino)ethyl)-1,2-phenylenediol, and epinephrine sulfonate. Following the analytical methods of the pharmacopoeia, these known impurities can be accurately analyzed and evaluated using HPLC with reference standards, ensuring the quality of the product.

[0005] During long-term stability studies and high-temperature and light-exposed tests, a new unknown impurity was discovered in low-concentration pre-filled epinephrine injection. This impurity slowly increased with time and temperature, indicating a degradation impurity in this concentration of injection. Separating and identifying this unknown impurity will improve the impurity profile of the low-concentration pre-filled epinephrine injection. Furthermore, an HPLC method was developed for its detection and analysis to ensure the quality and safety of this formulation. Summary of the Invention

[0006] In view of the technical problems existing in the background art, this application provides a method for separating and identifying unknown impurities in a low-concentration pre-filled epinephrine injection. The method involves decomposing and enriching the low-concentration pre-filled epinephrine injection, separating and purifying the decomposed solution using liquid chromatography, collecting the eluent to obtain a preparative solution containing the unknown impurities, concentrating it, and then performing structural identification.

[0007] This application provides a method for identifying an unknown impurity in a low-concentration pre-filled epinephrine injection solution, the structural formula of which is: .

[0008] This application provides a method for separating unknown impurities in a low-concentration pre-filled epinephrine injection solution, comprising the following steps: S1. Add additional adrenaline raw material and antioxidant to a low-concentration pre-filled adrenaline injection solution, and carry out a high-temperature reaction in a closed environment to accelerate the generation of unknown impurities, thereby obtaining a reaction solution enriched with unknown impurities. S2. The reaction solution enriched with unknown impurities is separated and purified by preparative high performance liquid chromatography, and the eluent is collected to obtain a solution containing unknown impurities; The conditions for the preparative high-performance liquid chromatography include: Chromatographic column: C18 silica gel column; Mobile phase A: selected from water, 0.1%-1% aqueous solution of heptafluorobutyric acid or aqueous solution of trifluoroacetic acid; Mobile phase B: Acetonitrile; The volume ratio of mobile phase A to mobile phase B during injection is (80:20) to (20:80); The elution flow rate was 20 mL / min to 30 mL / min.

[0009] The antioxidant is added in the following manner: every 8 hours, the antioxidant is added; the amount of antioxidant added is 0.5g per 100ml of reaction solution enriched with unknown impurities.

[0010] When the compound elutes, the retention time of the peak in the solution containing unknown impurities is 5-7 minutes.

[0011] In some embodiments, in step S1, the amount of adrenaline raw material added is 2g per 100ml of reaction solution enriched with unknown impurities.

[0012] In some embodiments, the method further includes a step of concentrating the collected eluent containing unknown impurities; the concentration is a vacuum concentration, the concentration temperature is 40°C to 50°C, the vacuum degree is ≤-0.095 MPa, and the concentration is reduced to 1 / 2 to 2 / 3 of the original volume.

[0013] Furthermore, the concentration of unknown impurities in the concentrate obtained by concentrating the solution containing unknown impurities (area normalized) is 8-10%.

[0014] In some embodiments, the temperature of the high-temperature reaction is 40°C to 80°C; and the duration of the high-temperature reaction is 48 hours to 72 hours.

[0015] In some embodiments, the antioxidant is sodium metabisulfite or sodium bisulfite.

[0016] This application provides a method for identifying unknown impurities in a low-concentration pre-filled epinephrine injection solution. The method involves identifying the unknown impurities obtained by the aforementioned separation method by first performing LC-MS analysis on the unknown impurities to obtain their molecular weight and mass spectrometry fragment information; then, using nuclear magnetic resonance spectroscopy to analyze the unknown impurities... 1 1H NMR analysis was performed to obtain its proton NMR data; by combining molecular weight and mass spectrometry fragment information with the proton NMR data, the chemical structure of the unknown impurity was determined.

[0017] Furthermore, the LC-MS analysis conditions are as follows: Mobile phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile; Elution method: Isogradient elution; Column: Unisil PFP 4.6 250mm, 5μm; column temperature 25℃, absorption wavelength 210.

[0018] Washing procedure: Mass spectrometry parameters: Ion source ESI, scan type Full Ms / ddMS², auxiliary gas heating temperature 300-400℃, capillary temperature 300-400℃, sheath gas flow rate 50-80 ml / min, auxiliary gas flow rate 15-25 ml / min, resolution 35000-70000, spray voltage 3-4 kV, NCE 20V, 30V, 40V.

[0019] Furthermore, 1 The HNMR analysis conditions were as follows: Instrument: Superconducting pulsed Fourier transform nuclear magnetic resonance spectrometer; Test method: Solvent: D2O, Reference: D2O ( 1 H spectrum).

[0020] This application provides a method for detecting unknown impurities in a low-concentration pre-filled epinephrine injection solution, employing high-performance liquid chromatography (HPLC). The chromatographic conditions for the HPLC method include: Chromatographic column: A chromatographic column packed with octadecylsilane-bonded silica gel; Buffer solution: an aqueous solution of sodium octane sulfonate and potassium dihydrogen phosphate, or an aqueous solution of sodium heptane sulfonate and potassium dihydrogen phosphate, adjusted to pH 3.2-3.6 with phosphoric acid; Mobile phase A: the buffer solution; Mobile phase B: composed of the buffer solution and acetonitrile at a volume ratio of 65-75:25-35; Washing procedure: Detection wavelength: 200 nm to 220 nm.

[0021] Furthermore, the relative retention time of the unknown impurity under the chromatographic conditions is 0.72, with a deviation of ±0.2.

[0022] The beneficial effects of this application are as follows: This application discloses a method for separating, identifying, and detecting unknown impurities in a low-concentration pre-filled epinephrine injection solution. Using epinephrine injection solution as raw material, this application accelerates the generation of unknown impurities through high temperature and the addition of antioxidants to achieve enrichment. The concentrate is then obtained through preparative liquid chromatography-phase separation and purification, and subsequently subjected to LC-MS and... 1 HNMR determination confirmed the molecular weight and structure of the impurity, improving the impurity profile of the pre-filled adrenaline injection. At the same time, an HPLC detection method for the impurity was developed and validated to ensure drug safety.

[0023] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of this application, the accompanying drawings used in this application will be briefly described below. Obviously, the drawings described below are merely some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort.

[0025] Figure 1 This is a mass spectrum of an unknown impurity in the low-concentration pre-filled epinephrine injection solution of this application.

[0026] Figure 2 For the unknown impurities in the low-concentration pre-filled epinephrine injection solution of this application 1 1H NMR spectrum.

[0027] Figure 3 This is an HPLC chromatogram of unknown impurities in the low-concentration pre-filled epinephrine injection solution of this application. Detailed Implementation

[0028] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0030] This application provides an embodiment of an unknown impurity in a low-concentration pre-filled epinephrine injection solution, the structural formula of which is: .

[0031] This application provides a method for separating unknown impurities from a low-concentration pre-filled epinephrine injection solution, comprising the following steps: S1. Add additional adrenaline raw material and antioxidant to a low-concentration pre-filled adrenaline injection solution, and carry out a high-temperature reaction in a closed environment to accelerate the generation of unknown impurities, thereby obtaining a reaction solution enriched with unknown impurities; wherein, the amount of adrenaline raw material added is 2g per 100ml of reaction solution enriched with unknown impurities.

[0032] The antioxidant is added as follows: add antioxidant every 8 hours, and add 0.5g of antioxidant per 100ml of reaction solution enriched with unknown impurities.

[0033] The high-temperature reaction temperature is 40°C to 80°C; the high-temperature reaction time is 48 hours to 72 hours.

[0034] The antioxidant is sodium metabisulfite or sodium bisulfite. Sodium metabisulfite is preferred.

[0035] S2. The reaction solution enriched with unknown impurities is separated and purified by preparative high performance liquid chromatography, and the eluent is collected to obtain a solution containing unknown impurities; The conditions for the preparative high-performance liquid chromatography include: Chromatographic column: C18 silica gel column; Mobile phase A: selected from water, 0.1%-1% aqueous solution of heptafluorobutyric acid or aqueous solution of trifluoroacetic acid; Mobile phase B: Acetonitrile; The volume ratio of mobile phase A to mobile phase B during injection is (80:20) to (20:80); The elution flow rate was 20 mL / min to 30 mL / min.

[0036] When the compound elutes, the retention time of the peak in the solution containing unknown impurities is 5-7 minutes.

[0037] The method for separating unknown impurities from low-concentration pre-filled epinephrine injection solution further includes a step of concentrating the collected eluent containing the unknown impurities; the concentration is vacuum concentration, the concentration temperature is 40°C to 50°C, and the vacuum degree is ≤-0.095 MPa. Preferably, the concentration is carried out to 1 / 2 to 2 / 3 of the volume of the solution containing the unknown impurities.

[0038] The concentration of unknown impurities in the concentrate obtained by concentrating the solution containing unknown impurities is 8-10%.

[0039] This method involves the destruction and enrichment of low-concentration pre-filled epinephrine injection solutions, followed by LC-Mass synthesis to collect unknown impurity products, which are then analyzed by mass spectrometry and proton NMR. 1 The structure of the unknown impurity was determined by HNMR, and the content of the unknown impurity in the pre-filled epinephrine injection was detected by HPLC.

[0040] This application provides a method for identifying unknown impurities in a low-concentration pre-filled epinephrine injection solution. First, LC-MS analysis is performed on the unknown impurities to obtain their molecular weight and mass spectrometry fragment information; then, nuclear magnetic resonance spectroscopy is used to analyze the unknown impurities. 1 1H NMR analysis was performed to obtain its proton NMR data; by combining molecular weight and mass spectrometry fragment information with the proton NMR data, the chemical structure of the unknown impurity was determined.

[0041] The conditions for LC-MS analysis are as follows: Mobile phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile; Elution method: Isogradient elution; Mass spectrometry parameters: Ion source ESI, scan type Full Ms / ddMS², auxiliary gas heating temperature 300-400℃, capillary temperature 300-400℃, sheath gas flow rate 50-80 ml / min, auxiliary gas flow rate 15-25 ml / min, resolution 35000-70000, spray voltage 3-4 kV, NCE 20V, 30V, 40V.

[0042] This application provides a method for detecting unknown impurities in a low-concentration pre-filled epinephrine injection solution, wherein the chromatographic conditions of the high-performance liquid chromatography (HPLC) method include: Chromatographic column: A chromatographic column packed with octadecylsilane-bonded silica gel; the column dimensions are 4.6 mm × 150 mm × 3.0 μm.

[0043] Buffer solution: an aqueous solution of sodium octane sulfonate and potassium dihydrogen phosphate, or an aqueous solution of sodium heptane sulfonate and potassium dihydrogen phosphate, adjusted to pH 3.2-3.6 with phosphoric acid; Mobile phase A: the buffer solution; Mobile phase B: composed of the buffer solution and acetonitrile at a volume ratio of 65-75:25-35; Washing procedure: Detection wavelength: 200 nm to 220 nm. Preferably, the detection wavelength for high performance liquid chromatography is 210 nm.

[0044] Relative retention time and deviation of unknown impurities: relative retention time is 0.72; deviation is ±0.2.

[0045] The detection method of this application uses adrenaline as a starting material to destroy and enrich the unknown impurity, and then purifies it by preparative liquid chromatography for LC-MS and 1H NMR spectroscopy to obtain mass spectrometry and... 1 HNMR spectroscopy confirmed the structure of the unknown impurity. HPLC was used to determine the content of this impurity in the product, ensuring the safety of this low-concentration pre-filled epinephrine injection formulation.

[0046] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0047] Impurity enrichment: Example 1 100 ml of a low-concentration adrenaline solution (0.1 mg / ml) was added to a 150 ml single-necked flask, followed by 2 g of adrenaline raw material. The mixture was stirred and dissolved at 60 °C, and the reaction was maintained at this temperature. 0.5 g of sodium metabisulfite was added every 8 hours. The reaction time was 48 hours. After the reaction was complete, the mixture was cooled to room temperature. Analysis showed that the normalized area content of this impurity after degradation was 0.8%.

[0048] Example 2 Compared with Example 1, the difference is that the reaction time is changed from 48h to 72h. Other experimental parameters and conditions are basically the same as those in Example 1, and will not be repeated here.

[0049] Sampling analysis shows that the normalized content of this impurity after destruction is 1%.

[0050] Example 3 Compared with Example 1, the difference is that the reaction temperature is changed from 60°C to 50°C. Other experimental parameters and conditions are basically the same as those in Example 1, and will not be repeated here.

[0051] Sampling analysis showed that the normalized content of this impurity after destruction was 0.6%.

[0052] Example 4 Compared with Example 1, the difference is that the reaction temperature was changed from 60℃ to 50℃ and the reaction time was changed from 48h to 72h. Other experimental parameters and conditions are basically the same as those in Example 1, and will not be repeated here.

[0053] Sampling analysis showed that the normalized content of this impurity after destruction was 0.8%.

[0054] Impurity separation, purification, and concentration: Example 5 Take 100 ml of the reaction solution enriched with impurities obtained in Example 1 and purify it using preparative liquid chromatography (Agilent 1260 Infinity II chromatograph). The chromatographic column is a C18 silica column, and the sample loading method is liquid chromatography. The mobile phase A is 0.2% trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile. The peak time of the impurities is monitored online using dual-band ultraviolet wavelengths λ=205nm and 210nm. During the injection process, the ratio of mobile phase A to B is 50:50 (v:v), and the elution rate of the mobile phase is 25 ml / min.

[0055] Through tracking and confirmation of the unknown impurity, it was found that the peak elution time of this unknown impurity in the preparative liquid phase was approximately 6 minutes.

[0056] The eluent containing this impurity was collected online, approximately 180 ml. The eluent was concentrated under vacuum at 40℃~50℃, with a vacuum degree ≤ -0.095 MPa; it was concentrated to 1 / 10 of its original volume, i.e., 18 ml, and then cooled to room temperature to obtain a concentrated solution containing this unknown degradation impurity.

[0057] Example 6 Take 100 ml of the reaction solution enriched with impurities obtained in Example 1 and purify it using preparative liquid chromatography (Agilent 1260 Infinity II chromatograph). The chromatographic column is a C18 silica column, and the sample loading method is liquid chromatography. The mobile phase A is 0.3% trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile. The peak time of the impurities is monitored online using dual-band ultraviolet wavelengths λ=205nm and 210nm. During the injection process, the ratio of mobile phase A to B is 70:30 (v:v), and the elution rate of the mobile phase is 25 ml / min.

[0058] Through tracking and confirmation of the unknown impurity, it was found that the peak elution time of this unknown impurity in the preparative liquid phase was approximately 5.8 min.

[0059] The eluent containing this impurity was collected online, approximately 190 ml. The eluent was concentrated under vacuum at 40℃~50℃, with a vacuum degree ≤ -0.095 MPa; it was concentrated to 1 / 10 of its original volume, i.e., 19 ml, and then cooled to room temperature to obtain a concentrated solution containing this unknown degradation impurity.

[0060] Example 7 Take 150 ml of the reaction solution enriched with impurities obtained in Example 1, and purify it using preparative liquid chromatography (Agilent 1260 Infinity II chromatograph). The chromatographic column is a C18 silica column, and the sample loading method is liquid chromatography. The mobile phase A is 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile. The peak time of the impurities is monitored online using dual-band ultraviolet wavelengths λ=205 nm and 210 nm. During the injection process, the ratio of mobile phase A to B is 65:35 (v:v), and the elution rate of the mobile phase is 25 ml / min.

[0061] Through tracking and confirmation of the unknown impurity, it was found that the peak elution time of this unknown impurity in the preparative liquid phase was approximately 6.3 min.

[0062] The eluent containing this impurity was collected online, approximately 160 ml. The eluent was concentrated under vacuum at 40℃~50℃, with a vacuum degree ≤ -0.095 MPa; it was concentrated to 1 / 10 of its original volume, i.e., 16 ml, and then cooled to room temperature to obtain a concentrated solution containing this unknown degradation impurity.

[0063] Example 8 Take 150 ml of the reaction solution enriched with impurities obtained in Example 1, and purify it using preparative liquid chromatography (Agilent 1260 Infinity II chromatograph). The chromatographic column is a C18 silica column, and the sample loading method is liquid chromatography. The mobile phase A is 0.15% trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile. The peak time of the impurities is monitored online using dual-band ultraviolet wavelengths λ=205nm and 210nm. During the injection process, the ratio of mobile phase A to B is 35:65 (v:v), and the elution rate of the mobile phase is 25 ml / min.

[0064] Through tracking and confirmation of the unknown impurity, it was found that the peak elution time of this unknown impurity in the preparative liquid phase was approximately 6.5 min.

[0065] Collect approximately 150 ml of the eluent containing this impurity online. Concentrate the eluent under vacuum at 40℃~50℃, with a vacuum degree ≤ -0.095MPa; concentrate to 1 / 10 of the original volume, i.e., 15 ml, and cool to room temperature to obtain a concentrated solution containing this unknown degradation impurity.

[0066] Structural confirmation The structure of the unknown impurity in the concentrate obtained in the above-mentioned process was identified. Qualitative analysis of the unknown impurity was performed using LC-MS (Liquid-Mass Spectrometry). Further analysis was conducted using high-resolution mass spectrometry and two-dimensional mass spectrometry fragmentation. 1 The molecular structure of the unknown impurity was confirmed by 1H NMR spectroscopy.

[0067] The analysis conditions for LC-MS are as follows: Liquid chromatography parameters: Instrument: Liquid chromatography-mass spectrometry (LC-MS) Mobile phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile; Elution method: Isogradient elution; Column: Unisil PFP 4.6 250mm, 5μm; column temperature 25℃, absorption wavelength 210.

[0068] Washing procedure: Mass spectrometry parameters: Ion source ESI, scan type Full Ms / ddMS², auxiliary gas heating temperature 300-400℃, capillary temperature 300-400℃, sheath gas flow rate 50-80 ml / min, auxiliary gas flow rate 15-25 ml / min, resolution 35000-70000, spray voltage 3-4 kV, NCE 20V, 30V, 40V.

[0069] Hydrogen spectrum 1 The HNMR analysis conditions were as follows: Instrument: Superconducting pulsed Fourier transform nuclear magnetic resonance spectrometer; Test method: Solvent: D₂O, Reference: D₂O ( 1 H spectrum).

[0070] After testing, the measured molecular weight of the impurity was 200.09110, and the inferred high-resolution elemental composition is C9H. 14 O4N. The specific mass spectrum is shown below. Figure 1 As shown.

[0071] Table 1 shows the secondary mass spectrometry fragments and their possible attributions: Table 1 It can be seen that the molecular ion peak of the impurity compound is m / z 200 [M+H]. + Its precise mass is 200.09110, and its elemental composition is C9H. 14 O4N has one more oxygen atom than adrenaline.

[0072] use 1 The structures of the impurity compounds were identified by HNMR. 1 The HNMR analysis conditions were as follows: Instrument: Superconducting pulsed Fourier transform nuclear magnetic resonance spectrometer; Test method: Solvent: D2O, Reference: D2O 1 H spectrum), internal standard: δ TMS 0.

[0073] 1 HNMR spectrum as follows Figure 2 As shown, the impurity compounds in D2O 1 H-NMR data are shown in Table 2: .

[0074] Table 2 Impurity Compounds 1 H-NMR data Analysis of the possible attributions of fragments from secondary mass spectrometry and proton spectrometry. 1 HNMR analysis revealed that the main difference between this impurity compound and adrenaline was that the impurity element had an additional oxygen atom, which, combined with... 1 The complete overlap of the C1-H and C5-H chemical shifts in the ¹H NMR spectrum confirms the presence of symmetrical structural units in this impurity. Therefore, the unknown impurity is confirmed to be a novel compound not previously reported in the literature, and its molecular structure is as follows: .

[0075] HPLC detection method for unknown impurities: Example 9: Detection of unknown impurities in low-concentration adrenaline injection: 1. Preparation of HPLC mobile phase Buffer solution: Take 5.0g potassium dihydrogen phosphate and 4.0g sodium octane sulfonate, add 1000ml of water, and adjust the pH to 3.4 with phosphoric acid.

[0076] Mobile phase A: buffer solution; The volume ratio of mobile phase B to mobile phase A to acetonitrile is 70:30.

[0077] 2. Preparation of the detection solution Test solution: Take the low-concentration epinephrine injection of this product (concentration 0.1 mg / mL).

[0078] Reference solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with mobile phase A to prepare a solution containing 1 μg of adrenaline per 1 ml.

[0079] System suitability solutions: Dilute the epinephrine stock solution with mobile phase A to prepare a solution containing 100 μg of epinephrine per 1 ml.

[0080] 3. Chromatographic conditions The chromatographic column is made of octadecylsilane-bonded silica gel, with dimensions of 4.6 mm × 150 mm × 3.0 μm.

[0081] Washing procedure: The detection wavelength was 210 nm, the column temperature was 40 °C, the injection plate temperature was 8 °C, and the injection volume was 20 μL.

[0082] 4. Testing The system suitability solution, reference solution, and test solution were injected sequentially, and detected under the set HPLC conditions. The results are as follows: Figure 3 As shown.

[0083] The analytical results for the unknown impurities are as follows: The results above show that the retention times of adrenaline and unknown impurities differed significantly, indicating that they could be effectively separated.

[0084] 5. Methodological Examination Based on the results of the methodological study, the method was fully validated.

[0085] (1) System applicability and specificity The diluent, excipient solutions, and other impurities should not interfere with the elution of the unknown impurity peak; the ratio of the retention time of the unknown impurity in the test solution to the average retention time of the main peak in three consecutive injections of the unknown impurity reference solution should be between 0.95 and 1.05; the ratio of the retention time of the unknown impurity in the test solution to that of adrenaline is approximately 0.71. This method exhibits good system adaptability and specificity.

[0086] (2) Limit of detection and limit of quantitation In the limit of quantitation solution, the concentrations of the unknown impurity and adrenaline were both 0.05 μg / ml, equivalent to 0.05% of the main component in the test solution. After six consecutive injections, the signal-to-noise ratio of the unknown impurity and adrenaline peaks was the lowest at 13.1, and the peak area RSDs of the unknown impurity and adrenaline peaks were 1.5% and 3.2%, respectively.

[0087] In the detection limit solution, the concentrations of both the unknown impurity and adrenaline were 0.025 μg / ml, equivalent to 0.025% of the main component in the test sample solution. After three consecutive injections, the minimum signal-to-noise ratio for both the unknown impurity and adrenaline peaks was 11.6. Therefore, the detection limit and quantitation limit of this method meet the validation requirements.

[0088] (3) Linear The correlation coefficient between the unknown impurity and adrenaline was the smallest at 0.99993. The absolute values ​​of the Y-axis intercepts for both the unknown impurity and adrenaline were less than 10% of the Y value (100% concentration), indicating a good linear relationship.

[0089] (4) Accuracy The recovery rates of unknown impurities in the nine accuracy solutions were calculated using the external standard method, ranging from 101.8% to 103.3%, with an RSD of 0.6%. The recovery rates were calculated using the self-comparison method with correction factors, ranging from 108.1% to 119.0%, with an RSD of 4.6%.

[0090] The largest difference between the external standard method and the self-comparison method with correction factors was 15.7% in the recovery rate of unknown impurities in the nine accuracy solutions.

[0091] At the three concentration levels of 50%, 100%, and 150%, the recovery rate of unknown impurities should be between 80% and 120%, and the RSD should not exceed 5.0%. The difference between the recovery rate results of the external standard method and the self-control method with correction factor should not exceed 20%, and the accuracy meets the validation requirements.

[0092] (5) Precision (repeatability and intermediate precision) The highest content of unknown impurities in the 6 repeatability solutions was 0.13%, with an RSD of 4.9%, which is less than 10.0% and meets the requirements. The highest content of unknown impurities in the 6 intermediate precision solutions was 0.14%, with an RSD of 6.4%. The RSD of unknown impurities in the 12 solutions (including repeatability and intermediate precision solutions) was 6.8%, which is less than 15.0%, and the precision verification was successful.

[0093] (6) Solution stability Within 55 hours of standing at room temperature, the ratio of the peak area of ​​the unknown impurity in the test solution at each time point to that at 0 hours was between 1.03 and 1.04. The ratio of the peak area of ​​the unknown impurity in the test solution at each time point to that at 0 hours was between 0.9 and 1.1.

[0094] (7) Durability With minor variations in robustness conditions across all groups, neither mobile phase A nor the excipient solution interfered with the detection of unknown impurities. In the system suitability solution chromatograms, the lowest theoretical plate number for epinephrine was 29355, the lowest resolution between the norepinephrine and epinephrine peaks was 9.83, and the lowest resolution between adjacent impurity peaks was 1.94. Despite these minor variations in robustness conditions, the highest detected content of unknown impurities in the test solution was 0.14, with a maximum relative deviation of 7.1% from the original conditions, indicating good robustness of the method.

[0095] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. An unknown impurity in a low-concentration pre-filled epinephrine injection solution, characterized in that, Its structural formula is: 。 2. A method for separating unknown impurities from a low-concentration pre-filled epinephrine injection solution, characterized in that, Includes the following steps: S1. Add additional adrenaline raw material and antioxidant to a low-concentration pre-filled adrenaline injection solution, and carry out a high-temperature reaction in a closed environment to accelerate the generation of unknown impurities, thereby obtaining a reaction solution enriched with unknown impurities. S2. The reaction solution enriched with unknown impurities is separated and purified by preparative high performance liquid chromatography, and the eluent is collected to obtain a solution containing unknown impurities; The conditions for the preparative high-performance liquid chromatography include: Chromatographic column: C18 silica gel column; Mobile phase A: selected from water, 0.1%-1% aqueous solution of heptafluorobutyric acid or aqueous solution of trifluoroacetic acid; Mobile phase B: Acetonitrile; The volume ratio of mobile phase A to mobile phase B during injection is (80:20) to (20:80); The elution flow rate was 20 mL / min to 30 mL / min.

3. The method for separating unknown impurities in a low-concentration pre-filled epinephrine injection solution according to claim 2, characterized in that, The antioxidant is added as follows: antioxidant is added once every 8 hours; the amount of antioxidant added is 0.5g per 100ml of reaction solution enriched with unknown impurities.

4. The method for separating unknown impurities in a low-concentration pre-filled epinephrine injection solution according to claim 2, characterized in that, In step S1, the amount of adrenaline raw material added is 2g per 100ml of reaction solution enriched with unknown impurities.

5. The method for separating unknown impurities in a low-concentration pre-filled epinephrine injection solution according to claim 2, characterized in that, It also includes a step of concentrating the collected eluent containing unknown impurities; the concentration is a vacuum concentration, the concentration temperature is 40°C to 50°C, and the vacuum degree is ≤-0.095 MPa.

6. The method for separating unknown impurities in a low-concentration pre-filled epinephrine injection solution according to claim 2, characterized in that, The high-temperature reaction is carried out at a temperature of 40°C to 80°C for 48 to 72 hours.

7. The method for separating unknown impurities in a low-concentration pre-filled epinephrine injection solution according to claim 2, characterized in that, The antioxidant is sodium metabisulfite or sodium bisulfite.

8. A method for identifying unknown impurities in a low-concentration pre-filled epinephrine injection solution, characterized in that, To identify unknown impurities in the low-concentration pre-filled epinephrine injection solution obtained by the separation method described in any one of claims 2-7, the unknown impurities are first analyzed by LC-MS using a liquid chromatography-mass spectrometry system to obtain their molecular weight and mass spectrometry fragment information. The unknown impurities were then analyzed using nuclear magnetic resonance spectroscopy. 1 1H NMR analysis was performed to obtain its proton NMR data; by combining molecular weight and mass spectrometry fragment information with the proton NMR data, the chemical structure of the unknown impurity was determined.

9. The method for identifying unknown impurities in a low-concentration pre-filled epinephrine injection solution according to claim 8, characterized in that, The conditions for the LC-MS analysis are as follows: Mobile phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile; Elution method: Isogradient elution; Mass spectrometry parameters: Ion source ESI, scan type Full Ms / ddMS², auxiliary gas heating temperature 300-400℃, capillary temperature 300-400℃, sheath gas flow rate 50-80 ml / min, auxiliary gas flow rate 15-25 ml / min, resolution 35000-70000, spray voltage 3-4 kV, NCE 20V, 30V, 40V.

10. A method for detecting unknown impurities in a low-concentration pre-filled epinephrine injection solution, characterized in that, High-performance liquid chromatography (HPLC) is used, and the chromatographic conditions of the HPLC include: Chromatographic column: A chromatographic column packed with octadecylsilane-bonded silica gel; Buffer solution: an aqueous solution of sodium octane sulfonate and potassium dihydrogen phosphate, or an aqueous solution of sodium heptane sulfonate and potassium dihydrogen phosphate, adjusted to pH 3.2-3.6 with phosphoric acid; Mobile phase A: the buffer solution; Mobile phase B: composed of the buffer solution and acetonitrile at a volume ratio of (65-75):(25-35); Washing procedure: Detection wavelength: 200 nm to 220 nm.

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