GC-MS (Gas Chromatography-Mass Spectrometer) method for determining contents of camphor, isoborneol and L-borneol in concentrated solution for edaravone dextroborneol injection

Abstract

The invention discloses a GC-MS (Gas Chromatography-Mass Spectrometer) method for determining the contents of camphor, isoborneol and L-borneol in a concentrated solution for edaravone dextroborneol injection, and belongs to the technical field of pharmaceutical analysis. The method comprises the following steps: preparing a test solution, an impurity stock solution, a reference solution and a system applicability solution by adopting a gas chromatography-mass spectrometry technology; a capillary column using beta cyclodextrin as a stationary liquid is used as a chromatographic column; respectively and precisely measuring the reference solution and the test solution for direct sample injection, recording a chromatogram, and calculating the contents of camphor, isoborneol and L-borneol by peak areas according to an external standard method; the impurities in the concentrated solution for injection of edaravone dextroborneol comprise camphor, isoborneol and L-borneol. The method is high in specificity, sensitivity, accuracy, stability and durability, the quality of the concentrated solution for edaravone dextroborneol injection can be effectively controlled, and medication safety is guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to the determination of camphor, isoborneol, and levoborneol content in concentrated edaravone dexborneol injection solution by GC-MS. Background Technology

[0002] Edaravone dexborneol concentrated solution for injection is a key drug in the clinical treatment of acute ischemic stroke. Its main component, edaravone, can scavenge free radicals, while dexborneol can improve cerebral microcirculation; the two work synergistically to exert their therapeutic effect. However, during the production, storage, and transportation of the drug, impurities such as camphor, isoborneol, and levoborneol may be generated due to the introduction of raw materials and degradation reactions. Among these, camphor has potential neurotoxicity, while isoborneol and levoborneol affect the stability and efficacy of the drug. Therefore, it is crucial to establish an accurate, reliable, and sensitive detection method to strictly control the levels of these three impurities.

[0003] Currently, the main methods for detecting volatile impurities in drugs include gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). GC has advantages such as high separation efficiency and fast analysis speed, but it struggles to effectively separate structurally similar isoborneol and levoborneol (which are isomers), and peak overlap can lead to inaccurate quantification. GC-MS, on the other hand, combines the high separation capability of GC with the high sensitivity and selectivity of mass spectrometry. It can accurately capture characteristic ions of impurities through selected ion monitoring (SIM) mode, effectively eliminating matrix interference. Chinese invention patent CN112697939A discloses a method for determining multiple components in a chest-opening aerosol based on GC-MS / MS. While it demonstrates advantages in simultaneous quantification of multiple components, it faces key technical bottlenecks in practical applications, limiting its completeness and versatility. First, there are significant shortcomings in chromatographic separation. The chromatograms of isoborneol and α-humulene are completely co-eluted, and the isoborneol isomers fail to achieve baseline separation. Although co-elution interference can be resolved with the high selectivity of GC-MS / MS, for ordinary GC-MS, this co-elution problem leads to mixed mass spectra, making qualitative and quantitative analysis extremely difficult. Second, existing methods are mainly developed for aerosol dosage forms. For complex matrix-based concentrated solutions for injection, matrix effects may interfere with the chromatographic behavior and mass spectrometric response of the target compound, posing a significant challenge to the specificity of the method.

[0004] While some methods for detecting drug impurities have been reported in the existing technology, there are few specific methods for detecting camphor, isoborneol, and levoborneol in edaravone dexborneol concentrated injection solution. Furthermore, some methods suffer from poor specificity, low sensitivity, and poor accuracy, making it difficult to meet the stringent requirements of drug quality control. Therefore, developing a detection method with strong specificity, high sensitivity, good accuracy, excellent stability, and robustness is of great significance for ensuring the quality and safety of edaravone dexborneol concentrated injection solution. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a GC-MS method for determining the content of camphor, isoborneol, and levoborneol in concentrated edaravone dexborneol injection solution. This method can rapidly, effectively, accurately, and reliably separate and detect camphor, isoborneol, and levoborneol in edaravone dexborneol injection solution, which is beneficial to improving the product quality of edaravone dexborneol injection solution and enhancing patient medication safety.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] The contents of camphor, isoborneol, and levonorgestrel in concentrated edaravone dexborneol injection solution were determined by GC-MS using gas chromatography-mass spectrometry, including the following steps:

[0008] 1) Prepare the test solution, impurity stock solution, reference solution and system suitability solution;

[0009] 2) Set the chromatographic conditions: use a capillary column with β-cyclodextrin as the stationary phase; use a mass spectrometer detector; use an electron impact ion source with an ion source temperature of 260℃ and a quadrupole temperature of 180℃; use a solvent delay of 10 minutes; use selected ion monitoring with a quantitative ion of 95 and an electron energy of 70 eV.

[0010] 3) Accurately measure the reference solution and the test solution separately and inject them directly. Record the chromatograms and calculate the contents of camphor, isoborneol and levonorgestrel by peak area using the external standard method.

[0011] The impurities in the concentrated edaravone dexborneol injection solution include camphor, isoborneol, and levoborneol.

[0012] Further, in step 1), the preparation process of the test solution is as follows: accurately measure 5 ml of the concentrated edaravone dexborneol injection solution test sample, place it in a 10 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, shake well, and use it as the test solution.

[0013] Further, in step 1), the preparation process of the impurity stock solution (1) is as follows: take about 12.5 mg of levorotatory borneol reference standard, accurately weigh it, put it in a 25 ml volumetric flask, add 50% ethanol to dissolve and dilute to the mark, and shake well; accurately measure 5 ml, put it in a 50 ml volumetric flask, dilute to the mark with 50% ethanol, shake well, and prepare a solution containing about 50 μg of levorotatory borneol per ml, as the impurity stock solution (1); the preparation process of the impurity stock solution (2) is as follows: take about 10 mg of camphor reference standard and about 15 mg of isoborneol reference standard, accurately weigh them, put them in the same 100 ml volumetric flask, add 50% ethanol to dissolve and dilute to the mark, and shake well; accurately measure 5 ml, put it in a 50 ml volumetric flask, dilute to the mark with 50% ethanol, and shake well to prepare a mixed solution containing about 10 μg of camphor and 15 μg of isoborneol per ml, as the impurity stock solution (2).

[0014] Further, in step 1), the preparation process of the reference solution is as follows: accurately measure 1 ml each of impurity stock solution (1) and impurity stock solution (2), place them in the same 20 ml volumetric flask, dilute to the mark with 50% ethanol, shake well, and prepare a mixed solution containing approximately 0.5 μg camphor, 0.75 μg isoborneol and 2.5 μg levoborneol per 1 ml, which is used as the reference solution.

[0015] Further, in step 1), the preparation process of the system suitability solution is as follows: accurately measure 5 ml of the concentrated edaravone dexborneol injection sample, place it in a 10 ml volumetric flask, add 0.5 ml of impurity stock solution (1), dilute to the mark with anhydrous ethanol, shake well, and use it as the system suitability solution.

[0016] Furthermore, in step 2), the initial temperature is 45~70℃.

[0017] Furthermore, in step 2), the injection port temperature is 295~305℃.

[0018] Furthermore, in step 2), the carrier gas is high-purity helium, and the carrier gas flow rate is 1.0~2.2 ml / min.

[0019] Further, in step 2), the split ratio is 5:1 and the injection volume is 1 μl.

[0020] Further, in step 3), the limit judgment is as follows: if the camphor content in the test sample is ≤0.2%, isoborneol ≤0.3%, and L-borneol ≤1.0%, then the sample is judged to meet the quality requirements.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] (1) The method of the present invention, under the GC-MS detection method, camphor, isoborneol and levoborneol in edaravone dexborneol injection can be well separated, the excipient peak and solvent peak do not interfere with the detection of main components and impurities, and the peak purity of the destroyed samples under each condition is greater than 990 after peak purity detection, and the impurities generated under each condition do not interfere with the detection of impurities and main components, and the specificity of related substances detection is good.

[0023] (2) Compared with the prior art, the present invention has no effect on the separation and examination results of camphor, isoborneol and levoborneol in edaravone dexborneol injection when the chromatographic conditions and parameters change slightly, and has good durability.

[0024] (3) The present invention uses high performance liquid chromatography to quickly, effectively, accurately and reliably separate and detect camphor, isoborneol and levoborneol in edaravone dexborneol injection, which is beneficial to improve the product quality of edaravone dexborneol injection and improve the safety of patients taking the medication. Attached Figure Description

[0025] Figure 1 This is a solution spectrum illustrating the system suitability of Example 2 of this application;

[0026] Figure 2 This is the chromatogram of the reference solution from Example 2 of this application;

[0027] Figure 3 This is the camphor chromatogram of Example 2 of this application;

[0028] Figure 4 This is the chromatogram of isoborneol from Example 2 of this application;

[0029] Figure 5 The chromatogram of levorotatory borneol in Example 2 of this application is shown below.

[0030] Figure 6 This is the chromatogram of the blank solution from Example 2 of this application;

[0031] Figure 7 This is the blank excipient chromatogram of Example 2 of this application;

[0032] Figure 8 This is the photodestruction chromatogram of Example 2 of this application;

[0033] Figure 9 This is the high-temperature degradation chromatogram of Example 2 of this application;

[0034] Figure 10 This is the acid-destruction chromatogram of Example 2 of this application;

[0035] Figure 11 This is the chromatogram of alkali destruction in Example 2 of this application;

[0036] Figure 12 This is the chromatogram of oxidation damage in Example 2 of this application;

[0037] Figure 13 This is the chromatogram of the 0h impurity reference solution from Example 2 of this application;

[0038] Figure 14 The spectrum of the impurity reference solution in Example 2 of this application is shown at 41.5h.

[0039] Figure 15 This is the chromatogram of the 0h test solution from Example 2 of this application;

[0040] Figure 16 The spectrum of the test solution after 41.5 hours in Example 2 of this application;

[0041] Figure 17 This is the camphor standard curve diagram for this application;

[0042] Figure 18 This is the standard curve diagram of isorhamnet in this application;

[0043] Figure 19 This is the standard curve diagram of levorotatory borneol in this application;

[0044] Figure 20 This is a chromatogram of the label recovery rate of Example 2 of this application;

[0045] Figure 21 This is the original condition chromatogram of the durability of Example 2 of this application;

[0046] Figure 22 This is the chromatogram of the robustness conditions (injection temperature 295°C) for Example 2 of this application;

[0047] Figure 23 This is the chromatogram of the robustness conditions (injection temperature 305°C) for Example 2 of this application;

[0048] Figure 24 The chromatogram is for the robustness conditions (flow rate 0.8 ml / min) of Example 2 of this application;

[0049] Figure 25 The chromatogram is for the robustness conditions (flow rate 1.2 ml / min) of Example 2 of this application;

[0050] Figure 26 This is a chromatogram of the durability conditions (starting temperature 65°C) for Example 2 of this application;

[0051] Figure 27 This is a chromatogram of the durability conditions (starting temperature 75°C) for Example 2 of this application. Detailed Implementation

[0052] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0053] Example 1

[0054] The determination of camphor, isoborneol, and levonorgestrel content in concentrated edaravone dexborneol injection solution by GC-MS includes the following steps:

[0055] 1) Instruments and samples were selected as follows: The GC-MS instrument was an Agilent 7890B-5977B; the edaravone batch numbers for the edaravone dexborneol injection concentrated solution were 2404001, 2411001, and 2411002, sourced from Jiangsu Zhengda Fenghai Pharmaceutical Co., Ltd.; the dexborneol batch numbers were 5200-1-240501, 5200-1-240502, and 5200-1-240503, sourced from Hubei Tianshu Pharmaceutical Co., Ltd. Information on camphor, isoborneol, and levoborneol is shown in Table 1.

[0056]

[0057] 2) Preparation of test solution and reference solution:

[0058] The preparation process of the test solution is as follows: accurately measure 5 ml of concentrated edaravone and dexborneol injection solution (specification: 5 ml contains 10 mg of edaravone and 2.5 mg of dexborneol), place it in a 10 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, shake well, and prepare a solution containing approximately 1 mg of edaravone and 0.25 mg of dexborneol per ml.

[0059] The preparation process of the impurity stock solution is as follows:

[0060] ① Impurity stock solution (1): Weigh approximately 25 mg of levorotatory borneol reference standard accurately, place it in a 500 ml volumetric flask, dissolve and dilute to the mark with 50% ethanol, shake well, and prepare a solution containing 50 μg per ml.

[0061] ② Impurity stock solution (2): Weigh about 5 mg of camphor reference standard and about 7.5 mg of isoborneol reference standard accurately, place them in a 500 ml volumetric flask, add 50% ethanol to dissolve and dilute to the mark, shake well, and prepare a mixed solution containing 10 μg of camphor and 15 μg of isoborneol per 1 ml.

[0062] The preparation process of the reference solution is as follows: accurately measure 1 ml each of the impurity stock solution (1) and (2), place them in the same 20 ml volumetric flask, dilute to the mark with 50% ethanol, shake well, and prepare a solution containing approximately 0.5 ug of camphor, 0.75 ug of isoborneol and 2.5 ug of levoborneol per 1 ml.

[0063] The preparation process of the system suitability solution is as follows: accurately measure 5 ml of the test solution (specification: 5 ml contains 10 mg of edaravone and 2.5 mg of dextromethorphan), place it in a 10 ml volumetric flask, add 0.5 ml of impurity stock solution (1), dilute to the mark with anhydrous ethanol, shake well, and prepare a mixed solution containing approximately 1 mg of edaravone, 0.25 mg of dextromethorphan and 2.5 μg of levoborneol per 1 ml.

[0064] 3) Set the conditions for gas chromatography-mass spectrometry:

[0065] ① Chromatographic column: A β-cyclodextrin stationary phase capillary column (Agilent Cyclosil-β) with dimensions of 30m × 0.25mm × 0.25μm was used.

[0066] ② Column temperature program: Start at 70℃ and maintain for 10 minutes, then increase the temperature to 200℃ at a rate of 7℃ / min, and run for 7 minutes after reaching 250℃.

[0067] ③ Sample injection system: Inlet temperature 300℃, split ratio 5:1, injection volume 1μl.

[0068] ④ Carrier gas system: The carrier gas is high-purity helium, with a flow rate of 1.0 ml / min.

[0069] ⑤ Mass spectrometry system: The ion source is an EI source (electron energy 70eV), the ion source temperature is 260℃, the quadrupole temperature is 180℃; the solvent delay is 10 minutes; the SIM mode is used to monitor and quantify ions with m / z=95.

[0070] 4) Detection and Calculation

[0071] (1) System suitability verification: Inject the system suitability solution and the reference solution to confirm that the separation degree between the L-borneol peak and the dextroborneol peak is ≥1.5, the elution order of camphor, isoborneol and L-borneol is correct and the separation degree is ≥1.5, and the signal-to-noise ratio of each component peak is ≥30. Only after meeting the system suitability requirements can the sample be tested.

[0072] (2) Sample detection: Accurately measure 1 μl of the reference solution and the test solution respectively and inject them into the standard curve. Record the chromatogram. Establish an external standard curve based on the peak area and concentration of each impurity in the reference solution; then calculate the content based on the peak area of ​​each impurity in the test solution and substitute it into the standard curve.

[0073] (3) Limit judgment: If the camphor content in the test sample is ≤0.2% (calculated as dexborneol), isoborneol ≤0.3%, and levoborneol ≤1.0%, the sample is judged to meet the quality requirements.

[0074] Example 2

[0075] The detection method of Example 1 was validated by verifying system suitability, destructive testing, limit of quantitation, limit of detection, linearity, precision, accuracy, and solution stability.

[0076] 1. System Applicability

[0077] Blank solvent: 50% ethanol

[0078] Blank excipient solution: Accurately measure 5 ml of blank excipient solution (batch number: 20241203, source: Jiangsu Lianhuan Pharmaceutical Co., Ltd.), place it in a 10 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, shake well, and the solution is ready.

[0079] Test solution: Accurately measure 5 ml of concentrated edaravone and dexborneol injection solution (specification: 5 ml contains 10 mg edaravone and 2.5 mg dexborneol), place it in a 10 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, shake well, and prepare a solution containing approximately 1 mg edaravone and 0.25 mg dexborneol per ml.

[0080] The preparation process of the impurity stock solution is as follows:

[0081] ① Impurity stock solution (1): Weigh approximately 25 mg of levorotatory borneol reference standard accurately, place it in a 500 ml volumetric flask, dissolve and dilute to the mark with 50% ethanol, shake well, and prepare a solution containing 50 μg per ml.

[0082] ② Impurity stock solution (2): Weigh about 5 mg of camphor reference standard and about 7.5 mg of isoborneol reference standard accurately, place them in a 500 ml volumetric flask, add 50% ethanol to dissolve and dilute to the mark, shake well, and prepare a mixed solution containing 10 μg of camphor and 15 μg of isoborneol per 1 ml.

[0083] The preparation process of the reference solution is as follows: accurately measure 1 ml each of impurity stock solution (1) and (2), place them in the same 20 ml volumetric flask, dilute to the mark with diluent 2, shake well, and prepare a solution containing approximately 0.5 ug of camphor, 0.75 ug of isoborneol and 2.5 ug of levoborneol per 1 ml.

[0084] The preparation process of the system suitability solution is as follows: accurately measure 5 ml of the test solution (specification: 5 ml contains 10 mg of edaravone and 2.5 mg of dextromethorphan), place it in a 10 ml volumetric flask, add 0.5 ml of impurity stock solution (1), dilute to the mark with anhydrous ethanol, shake well, and prepare a mixed solution containing approximately 1 mg of edaravone, 0.25 mg of dextromethorphan and 2.5 μg of levoborneol per 1 ml.

[0085] Camphor positioning solution: Weigh approximately 10 mg of camphor accurately, place it in a 100 ml volumetric flask, dilute to the mark with 50% ethanol, shake well, accurately measure 5 ml, place it in a 50 ml volumetric flask, shake well, and the solution is ready.

[0086] Isorhones localization solution: Weigh approximately 15 mg of isorhones accurately, place it in a 100 ml volumetric flask, dilute to the mark with 50% ethanol, shake well, accurately measure 5 ml, place it in a 50 ml volumetric flask, shake well, and the solution is ready.

[0087] L-borneol localization solution: Weigh approximately 50 mg of L-borneol accurately, place it in a 100 ml volumetric flask, dilute to the mark with 50% ethanol, shake well, accurately measure 5 ml, place it in a 50 ml volumetric flask, shake well, and the solution is ready.

[0088] Undamaged solution: Accurately transfer 5 ml of this product into a 10 ml volumetric flask, dilute to the mark with diluent 1, and shake well to obtain the solution.

[0089] Acid-base blank excipient solution: Accurately transfer 5 ml of blank excipient solution into a 10 ml volumetric flask, add 1 ml of 1 mol / L hydrochloric acid, shake well and let stand for 1 h, then add 1 ml of 1 mol / L sodium hydroxide to neutralize, dilute to the mark with diluent 1, and shake well to obtain the solution.

[0090] Acid-degraded solution: Accurately transfer 5 ml of this product into a 10 ml volumetric flask, add 1 ml of 1 mol / L hydrochloric acid, shake well and let stand for 1 h, then add 1 ml of 1 mol / L sodium hydroxide to neutralize, dilute to the mark with diluent 1, shake well, and the solution is ready.

[0091] Alkali-degraded solution: Accurately transfer 5 ml of this product into a 10 ml volumetric flask, add 1 ml of 1 mol / L sodium hydroxide, shake well and let stand for 1 h, then add 1 ml of 1 mol / L hydrochloric acid to neutralize, dilute to the mark with diluent 1, shake well, and the solution is ready.

[0092] Oxidized blank excipient solution: Accurately transfer 5 ml of blank excipient solution into a 10 ml volumetric flask, add 1 ml of 6% hydrogen peroxide, shake well and let stand for 1 hour, dilute with diluent 1 to the mark, shake well, and the solution is ready.

[0093] Oxidative Degradation Solution: Accurately transfer 5 ml of this product into a 10 ml volumetric flask, add 1 ml of 6% hydrogen peroxide, shake well, let stand for 1 hour, dilute with diluent 1 to the mark, shake well, and the solution is ready.

[0094] High-temperature destructive solution: Accurately transfer 5 ml of the sample heated in an 80°C water bath for 8 hours into a 10 ml volumetric flask, add diluent 1 to dilute to the mark, and shake well to obtain the solution.

[0095] Photodegradation solution: Accurately transfer 5 ml of the sample exposed to light for 10 days into a 10 ml volumetric flask, add diluent 1 to dilute to the mark, and shake well to obtain the solution.

[0096] 1 μL each of the blank solvent, excipient blank solution, test sample solution, destruction solution of each test sample, and localization solution of each impurity was injected into the gas chromatography-mass spectrometry (GC-MS) instrument. Chromatograms were recorded to investigate the interference of each solution on impurity detection. The results are shown in Table 2 below. Figure 1-12 As shown.

[0097] Table 2 System Suitability Test Results

[0098]

[0099] From Table 2 and Figure 1-7 It was found that the blank solvent and excipient blank solution showed no chromatographic peaks at the retention times of the three impurities; the retention times of each impurity in the reference solution were consistent with those in the positioning solution (camphor 19.43 min, isoborneol 21.14 min, levoborneol 21.46 min); the resolution between the levoborneol peak and the isoborneol peak was 3.8, and the resolution between the levoborneol peak and the dextroborneol peak was 3.5, both ≥1.5. This method exhibits good specificity and is free from interference. Figure 8-12 It can be seen that the peaks of degradation products and impurities in the test sample destruction solution do not overlap, and impurities do not interfere with the detection of impurities under various destruction conditions of excipient blank and test sample solution.

[0100] 2. Solution stability

[0101] Reference solution: Take the solution from the system suitability section.

[0102] Test solution: Take the sample from the system suitability section.

[0103] The reference solution and the test solution were placed at room temperature, and injections were performed at 0 h, 8 h, 17.5 h, 25.5 h, 33.5 h, and 41.5 h, respectively. The RSD of the peak area of ​​each impurity was examined. The results are shown in Table 3 below. Figure 13-16 As shown.

[0104] Table 3 Results of solution stability test

[0105]

[0106] From Table 3 and Figure 13-16 The peak area RSDs of camphor, isoborneol, and levorotatory borneol in the reference solution were 6.7%, 8.4%, and 8.5%, respectively; the peak area RSDs of the three impurities in the test solution were 6.8%, 6.5%, and 7.8%, respectively, all ≤9.2%. The solution showed good stability within 41.5 hours at room temperature, meeting the requirements of actual detection operations.

[0107] 3. Limit of Detection and Limit of Quantification

[0108] The signal-to-noise ratio (S / N) method was used. The impurity reference solution under the system suitability section was gradually diluted, and the S / N ratio at different concentrations was determined to establish the limit of detection (S / N≥3) and the limit of quantitation (S / N≥10). Simultaneously, six limit of quantitation solutions were prepared, and the RSD of peak area and retention time was examined. The results are shown in Tables 4-5 below.

[0109] Table 4 Detection results of the limit of quantitation

[0110]

[0111] Table 5 Detection Limit Results

[0112]

[0113] As shown in Tables 4-5, the limits of quantitation (LOQ) for camphor, isoborneol 1, isoborneol 2, and levorotatory borneol are 0.1108 μg / ml, 0.1545 μg / ml, 0.1545 μg / ml, and 0.4811 μg / ml, respectively, while the limits of detection (LODs) are 0.0554 μg / ml, 0.0772 μg / ml, 0.0772 μg / ml, and 0.2405 μg / ml, respectively. The method exhibits high sensitivity, capable of detecting impurities as low as 0.0554 μg / ml, and demonstrates good quantitative accuracy.

[0114] 4. Linearity and Range

[0115] A series of linear solutions with impurity quantitation limit concentrations of 50%, 80%, 100%, 120%, 150%, and 300% were prepared. After injection, linear regression was performed with concentration (x) on the x-axis and peak area (y) on the y-axis. The correlation coefficient (r), intercept / 100% response, and response factor RSD were calculated. The results are shown in Tables 6-8 and 6-8. Figure 17-19 As shown.

[0116] Table 6 Results of the camphor linear relationship test

[0117]

[0118] Table 7 Results of linear relationship test of isoborneol

[0119]

[0120] Table 8 Results of the linear relationship test of borneol

[0121]

[0122] From Table 6-8 and Figure 17-19It can be seen that, when plotting the peak area A as the ordinate and the concentration C as the abscissa, the linear regression equation for camphor is A=8035.6919C-140.5214, with a linear range of 0.1108~1.6613μg / ml; the linear equation for isoborneol is A=11867.3214C-410.5373, with a linear range of 0.1545~2.3170μg / ml; and the linear equation for levorotatory borneol is A=16200.2339C-1905.2786, with a linear range of 0.4811~7.2162μg / ml. Within the concentration range of the limit of quantitation to 300%, the linear correlation coefficient r of the three impurities was at least 0.9997 and greater than 0.990, the intercept / 100% response was at most 4.94% and less than 25%, and the RSD of the response factor was at most 5.2% and less than 10%, which met the validation requirements and showed a good linear relationship.

[0123] 5. Accuracy

[0124] Recovery solutions at three concentration levels (50%, 100%, and 150%) were prepared, with three replicates for each level. The impurity content in each recovery solution was determined, and the recovery rate was calculated as (measured amount - original amount) / added amount × 100%. The average recovery rate and RSD for each concentration level were statistically analyzed. The results are shown in Tables 9-11 and 9-150. Figure 20 .

[0125] Table 9. Camphor Accuracy Test Results

[0126]

[0127] Table 10 Results of the accuracy test of isoborneol

[0128]

[0129] Table 11 Results of the accuracy test of borneol

[0130]

[0131] From Table 9-11 and Figure 20 It can be seen that the average recovery rates of the three impurities at concentration levels of 50%, 100%, and 150% are all within the range of 80% to 120%. The maximum RSD of the recovery rate data is 5.6% (L-borneol), which is less than the acceptable standard of 10%, indicating that the method has good accuracy and can accurately determine the true content of impurities in the test sample.

[0132] 6. Sample injection precision

[0133] Prepare the reference solution according to the prescribed method, accurately measure 1 μl and inject it into the gas chromatograph-mass spectrometer. Inject six times consecutively, record the retention time and peak area of ​​each impurity, and calculate the RSD value. The results are shown in Table 12.

[0134] Table 12 Results of Sample Injection Precision Test

[0135]

[0136] As shown in Table 12, the RSD of the retention time of each impurity peak is 0.1%, which is less than 1%; the maximum RSD of the peak area is 3.9% (isoborneol 1 and isoborneol 2), which is less than 10%, meeting the acceptable standard. This indicates that the instrument has good sample injection precision and the measurement results are reproducible and stable.

[0137] 7. Repeatability

[0138] Six labeled test solutions (containing known impurities) and two reference solutions were prepared in parallel. The solutions were injected and analyzed under the set chromatographic conditions. The content of each impurity in the six test solutions was calculated using the external standard method, and the RSD values ​​were statistically analyzed. The results are shown in Table 13.

[0139] Table 13 Repeatability Test Results

[0140]

[0141] As shown in Table 13, the maximum RSD of each impurity content in the six labeled test solutions was 0.9% (camphor, isoborneol), which is much less than 10%, indicating that the method has excellent repeatability and the results of multiple measurements under the same experimental conditions are consistent.

[0142] 8. Intermediate precision

[0143] Another analyst independently established a chromatographic system on different dates, prepared six parallel labeled test solutions and two reference solutions, and determined them using the same method. Combining the results of the six determinations under the repeatability section, the RSD values ​​of each impurity in the 12 samples were calculated to investigate the influence of random variations such as personnel and time on the determination results. The results are shown in Table 14.

[0144] Table 14 Results of intermediate precision test

[0145]

[0146] As shown in Table 14, the maximum RSD of each impurity content in the 12 labeled test solutions was 6.9% (camphor), which is less than the acceptable standard of 10%. This indicates that the intermediate precision of this method is good, and it is not greatly affected by external factors such as personnel and time. The test results are stable and reliable.

[0147] 9. Durability

[0148] Based on the established chromatographic conditions, key parameters were fine-tuned to examine the stability of the method after the changes. Adjusted parameters included: injection port temperature (300±5℃), initial temperature (50±5℃), and carrier gas flow rate (2.0±0.2 ml / min), with a total of six variations. The content of various impurities in the test solution was determined, and system suitability indicators (resolution, signal-to-noise ratio) were also examined. The results are shown in Tables 15-16 and 16. Figure 21-27 .

[0149] Table 15 Durability Test Content

[0150]

[0151] Table 16 Durability Test Results

[0152]

[0153] From Table 15-16 and Figure 21-27 It can be seen that, regarding system suitability: under various changing conditions, the minimum resolution between the L-borneol and D-borneol peaks in the system suitability solution is 2.6, and the minimum resolution between the L-borneol and isoborneol peaks in the reference solution is 2.9, both greater than 1.5; the minimum signal-to-noise ratio of each component peak is 33.88, greater than 30, meeting the requirements; regarding content differences: the maximum differences in the content of each impurity in the test sample compared to the original conditions are: camphor 0.010%, isoborneol 0.003%, and L-borneol 0.041%, all meeting the acceptable standard of "difference ≤ 0.02% when content < 0.1%, and difference ≤ 0.2% when content ~ 1.0%". After fine-tuning the chromatographic parameters, the system suitability indicators and impurity content determination results all meet the requirements, indicating that this method has good robustness, is suitable for routine detection in different laboratories and with different instruments, and has strong anti-interference ability.

[0154] 10. Testing Implementation Cases

[0155] Multiple batches of this product (20250401, 20250402, 20250403) were tested, and the test results are shown in Table 17 below.

[0156] Table 17 Results of Multiple Batch Tests

[0157]

[0158] As shown in Table 17, multiple batches of samples tested demonstrated that the blank solution and blank excipient solution did not interfere with the detection of camphor, isoborneol, and L-borneol, and all three substances met the requirements. Therefore, this method can accurately determine the amounts of camphor, isoborneol, and L-borneol in concentrated edaravone dexborneol injection solution, and is also generally applicable to the detection of camphor, isoborneol, and L-borneol in other products.

[0159] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining the content of camphor, isoborneol, and levonorhalil in concentrated edaravone dexborneol injection solution by GC-MS, characterized in that... The gas chromatography-mass spectrometry (GC-MS) method includes the following steps: 1) Prepare the test solution, impurity stock solution, reference solution and system suitability solution; 2) Set the chromatographic conditions: use a capillary column with β-cyclodextrin as the stationary phase; use a mass spectrometer detector; use an electron impact ion source with an ion source temperature of 260℃ and a quadrupole temperature of 180℃; use a solvent delay of 10 minutes; use selected ion monitoring with a quantitative ion of 95 and an electron energy of 70 eV. 3) Accurately measure the reference solution and the test solution separately and inject them directly. Record the chromatograms and calculate the contents of camphor, isoborneol and levonorgestrel by peak area using the external standard method. The impurities in the concentrated edaravone dexborneol injection solution include camphor, isoborneol, and levoborneol.

2. The method for determining the content of camphor, isoborneol, and levonorhalil in edaravone dexborneol concentrated solution for injection using GC-MS according to claim 1, characterized in that: In step 1), the preparation process of the test solution is as follows: accurately measure 5 ml of the concentrated edaravone dexborneol injection solution test sample, place it in a 10 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, shake well, and use it as the test solution.

3. The method for determining the content of camphor, isoborneol, and levonorhalil in edaravone dexborneol concentrated solution for injection using GC-MS according to claim 1, characterized in that: In step 1), the preparation process of impurity stock solution (1) is as follows: take about 12.5 mg of levorotatory borneol reference standard, accurately weigh it, put it in a 25 ml volumetric flask, add 50% ethanol to dissolve and dilute to the mark, and shake well; accurately measure 5 ml, put it in a 50 ml volumetric flask, dilute to the mark with 50% ethanol, shake well, and prepare a solution containing about 50 μg of levorotatory borneol per ml, as impurity stock solution (1); the preparation process of impurity stock solution (2) is as follows: take about 10 mg of camphor reference standard and about 15 mg of isoborneol reference standard, accurately weigh them, put them in the same 100 ml volumetric flask, add 50% ethanol to dissolve and dilute to the mark, and shake well; accurately measure 5 ml, put it in a 50 ml volumetric flask, dilute to the mark with 50% ethanol, and shake well to prepare a mixed solution containing about 10 μg of camphor and 15 μg of isoborneol per ml, as impurity stock solution (2).

4. The method for determining the content of camphor, isoborneol, and levonorhalil in edaravone dexborneol concentrated solution for injection using GC-MS according to claim 1, characterized in that: In step 1), the preparation process of the reference solution is as follows: accurately measure 1 ml each of impurity stock solution (1) and impurity stock solution (2), place them in the same 20 ml volumetric flask, dilute to the mark with 50% ethanol, shake well, and prepare a mixed solution containing approximately 0.5 μg camphor, 0.75 μg isoborneol and 2.5 μg levoborneol per 1 ml, which is used as the reference solution.

5. The method for determining the content of camphor, isoborneol, and levonorhalil in edaravone dexborneol concentrated solution for injection using GC-MS according to claim 1, characterized in that: In step 1), the preparation process of the system suitability solution is as follows: accurately measure 5 ml of the concentrated edaravone dexborneol injection sample, place it in a 10 ml volumetric flask, add 0.5 ml of impurity stock solution (1), dilute to the mark with anhydrous ethanol, shake well, and use it as the system suitability solution.

6. The method for determining the content of camphor, isoborneol, and levonorhalil in edaravone dexborneol concentrated solution for injection using GC-MS according to claim 1, characterized in that: In step 2), the initial temperature is 45~70℃.

7. The method for determining the content of camphor, isoborneol, and levonorhalil in edaravone dexborneol concentrated solution for injection using GC-MS according to claim 1, characterized in that: In step 2), the injection port temperature is 295~305℃.

8. The method for determining the content of camphor, isoborneol, and levonorhalil in edaravone dexborneol concentrated solution for injection using GC-MS according to claim 1, characterized in that: In step 2), the carrier gas is high-purity helium, and the carrier gas flow rate is 1.0~2.2 ml / min.

9. The method for determining the content of camphor, isoborneol, and levonorhalil in edaravone dexborneol concentrated solution for injection using GC-MS according to claim 1, characterized in that: In step 2), the split ratio is 5:1 and the injection volume is 1 μl.

10. The method for determining the content of camphor, isoborneol, and levonorhalil in edaravone dexborneol concentrated solution for injection using GC-MS according to claim 1, characterized in that: In step 3), the limit judgment is as follows: if the camphor content in the test sample is ≤0.2%, isoborneol ≤0.3%, and L-borneol ≤1.0%, then the sample is judged to meet the quality requirements.

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