Method for detecting purity of omagazines tosylate for injection

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting the purity of omagazines tosylate for injection. The method mainly solves the technical problem that main components and key impurities such as epimers of the main components are difficult to effectively separate in existing chromatographic analysis. Optimized reversed-phase high performance liquid chromatography conditions are established, a phosphate buffer solution-acetonitrile gradient elution system with a specific pH value is adopted, precise weighing, system applicability verification and an automatic integration technology are combined, baseline separation of main components and impurities is achieved, and finally the content of the main components is accurately calculated through an area normalization method. The method has the advantages of good separation effect, strong specificity, high analysis efficiency and accurate and reliable result, and provides reliable technical guarantee for product quality control.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for detecting the purity of omalicycline tosylate for injection. Background Technology

[0002] In the field of pharmaceutical analysis, particularly in the purity determination of tetracycline antibiotics, high-performance liquid chromatography (HPLC) has become a routine analytical method. However, for the specific compound omalicycline tosylate, existing chromatographic analysis methods still have significant limitations.

[0003] Existing chromatographic conditions often fail to effectively separate the main component of omalicycline tosylate from its key impurities, such as epimers and dehydration impurities. Because these impurities are structurally highly similar to the main component, co-elution easily occurs under conventional chromatographic conditions, leading to insufficient resolution and failing to meet accurate quantification requirements.

[0004] Currently reported chromatographic methods suffer from instability. Even minor fluctuations in mobile phase pH or batch-to-batch variations in the column can cause retention time drift, thus affecting separation reproducibility. This lack of robustness poses a significant challenge to routine quality control.

[0005] Furthermore, existing analytical methods lack a systematic validation framework. Clear acceptance criteria have not been established for key indicators such as system applicability, detection sensitivity, and limit of quantitation, making it difficult to guarantee the accuracy and reliability of analytical results. This lack of a method validation framework poses certain risks during compliance reviews.

[0006] Therefore, we propose a method for detecting the purity of omalicycline tosylate for injection to address the problems mentioned above. Summary of the Invention

[0007] This invention relates to the field of pharmaceutical analytical chemistry, and specifically provides a systematic method for detecting the purity of omalicycline tosylate for injection. This method primarily solves the key technical problem that existing chromatographic analysis techniques cannot effectively separate the main component from its epimers and process impurities. This invention establishes an optimized reversed-phase high-performance liquid chromatography (RP-HPLC) system, employing a gradient elution system composed of a phosphate buffer solution at a specific pH and an organic phase. Combined with system suitability verification, precise sample preparation, and automated data processing, this invention achieves accurate separation and quantification of the main component and related impurities.

[0008] The core of this invention lies in providing a complete set of interconnected and synergistic technical solutions: flow based on precise control. The aqueous and organic phases required for chromatographic separation are prepared using a phase preparation process; efficient separation of each component is achieved through an optimized gradient elution program; system suitability verification is used to ensure the reliability of the analytical system; and finally, the area normalization method is used to accurately calculate the content of the principal component. This method has outstanding advantages such as good separation efficiency, strong specificity, and high reproducibility, providing comprehensive and reliable technical support for drug quality control and stability studies.

[0009] To achieve the above objective, a method for detecting the purity of omalicycline tosylate for injection is provided, the method comprising the following steps: S1: Prepare a phosphate buffer solution with a pH of 3.00±0.01, and establish a chromatographic analysis method for the omalicycline tosylate; S2: System suitability verification was performed on a solution containing a specific concentration of omalicycline tosylate as the main component, and the theoretical plate number of the chromatographic column and the resolution between the main peak and the key impurity peak were determined and judged. S3: Dissolve and dilute the raw material of omalicycline tosylate for injection to prepare the test solution of the test sample; S4: The sample solution is injected, separated, and signal acquired by an automated injection system under the set chromatographic conditions; S5: Use chromatographic data processing software to perform peak area integration on the acquired chromatographic signals to obtain peak area data for each component; S6: Apply the area normalization method to perform purity calculation on the peak area data to generate the purity analysis results of the omalicycline tosylate.

[0010] Preferably, the preparation of the phosphate buffer solution with a pH of 3.00 ± 0.01 in step S1 includes: The solution was prepared by precise weighing using superior grade potassium dihydrogen phosphate reagent. The pH value was precisely adjusted using a 0.1 mol / L dilute phosphoric acid solution under constant temperature conditions. The pH-adjusted solution is filtered through a microporous membrane to remove particulate matter; the filtered buffer solution is then subjected to ultrasonic degassing to obtain a phosphate buffer solution that meets the requirements for chromatographic analysis.

[0011] Preferably, the chromatographic analysis method established in S1 employs a gradient elution procedure, including: Configure a gradient scaling procedure that includes multiple time points; The flow parameters of the pump system are set according to the gradient program; The proportion of phosphate buffer solution is controlled by setting flow parameters; A chromatographic analysis method that optimizes the ratio variation conditions to obtain the best separation effect.

[0012] Preferably, the system suitability verification performed in step S2 includes: Prepare system-suitable solutions containing specific concentrations of the main component and related impurities; Chromatograms were obtained by performing chromatographic analysis on the system suitability solution. Calculate the theoretical plate number and resolution index of the chromatographic column based on the chromatogram; The calculated indicators are compared with predetermined standards to verify the applicability of the system.

[0013] Preferably, the preparation of the test solution in step S3 includes: Accurately weigh the specified amount of omalicycline toluenesulfonate test sample raw material; The weighed omalicycline tosylate test sample raw material was dissolved using a diluent with a specific ratio; The dissolved solution was quantitatively transferred to a volumetric flask and brought to volume. The solution after volume adjustment is filtered to obtain a clear and transparent test solution.

[0014] Preferably, step S4, when performing the injection, separation, and signal acquisition of the test sample solution under the set chromatographic conditions, includes: Set the injection parameters for the autosampler; The sample is automatically injected according to the injection procedure. Separation of each component was achieved under optimized chromatographic conditions; Chromatographic data are obtained by acquiring signals from the separated components using a detector.

[0015] Preferably, the peak area integration calculation in step S5 includes: Baseline correction and smoothing are performed on the acquired chromatographic signals; Establish an automatic integration method by setting appropriate integration parameters; The area of ​​the chromatographic peak is calculated according to the integration method, and the accuracy of the calculated peak area data is verified.

[0016] Preferably, when calculating purity using the area normalization method in step S6, the following steps are included: Identify and subtract solvent peaks from chromatograms; Calculate the sum of the areas of all valid chromatographic peaks; The purity percentage is calculated based on the ratio of the sum of the peak areas to the area of ​​the main peak, and a purity analysis report containing all calculated data and results is generated.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention effectively optimizes the retention behavior and separation selectivity of each component in the chromatographic column by using a gradient elution system constructed with a precisely pH-controlled phosphate buffer and a high-purity organic phase. It achieves complete baseline separation between the main component and its structurally similar epimers and various degradation products, significantly improves the peak shape of the chromatographic column, and eliminates the mutual interference of adjacent impurity peaks, laying a solid foundation for the accurate quantification of the content of each component.

[0018] 2. The scientifically designed gradient elution program of this invention, while ensuring separation effect, significantly shortens the total analysis time of a single sample by reasonably optimizing the flow ratio change rate and equilibrium time, and significantly increases the sample throughput per unit time, effectively meeting the urgent need for rapid and efficient quality monitoring of large batches of samples in industrial production environments.

[0019] 3. By establishing a system applicability evaluation system that includes multiple indicators such as theoretical plate number, resolution, and tailing factor, and combining it with standardized sample pretreatment and data acquisition procedures, this invention ensures that the analytical method can maintain stable performance when faced with changes in different chromatographic column batches, instruments, and operating environments. This significantly enhances the reproducibility and reliability of analytical results and provides a solid guarantee for data comparison across laboratories and time periods. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] Furthermore, the order of steps in the following method embodiments is merely an example and not a strict limitation.

[0022] A method for determining the purity of omalicycline tosylate for injection, the method comprising the following steps: S1: Prepare a phosphate buffer solution with a pH of 3.00±0.01, and establish a chromatographic analysis method for the omalicycline tosylate; S2: System suitability verification was performed on a solution containing a specific concentration of omalicycline tosylate as the main component, and the theoretical plate number of the chromatographic column and the resolution between the main peak and the key impurity peak were determined and judged. S3: Dissolve and dilute the raw material of omalicycline tosylate for injection to prepare the test solution of the test sample; S4: The sample solution is injected, separated, and signal acquired by an automated injection system under the set chromatographic conditions; S5: Use chromatographic data processing software to perform peak area integration on the acquired chromatographic signals to obtain peak area data for each component; S6: Apply the area normalization method to perform purity calculation on the peak area data to generate the purity analysis results of the omalicycline tosylate.

[0023] In this embodiment, the preparation of a phosphate buffer solution with a pH of 3.00 ± 0.01 in step S1, and the establishment of the chromatographic analysis method for omalicycline tosylate, includes: This step requires reagent preparation and weighing. Personnel must wear clean nitrile gloves and perform the weighing in a Class 100,000 cleanroom with constant temperature and humidity, using a calibrated analytical balance with a strength of 0.01 g / L. Before weighing, the balance must be leveled and zeroed. Use a clean spatula to take the analytical grade potassium dihydrogen phosphate reagent and slowly add it to the weighing dish. Record the mass value after the display reading stabilizes. During the weighing process, care must be taken to avoid moisture absorption and contamination of the reagent. After weighing, the reagent bottle should be sealed promptly to prevent moisture in the air from affecting the reagent purity.

[0024] After weighing, accurately transfer the measured potassium dihydrogen phosphate reagent to a 1000 mL Grade A volumetric flask. Use a clean glass funnel for the transfer and rinse the weighing dish several times with ultrapure water to ensure complete transfer. Then add approximately 800 mL of ultrapure water to the volumetric flask. The ultrapure water should have a resistivity of at least 18.2 MΩ·cm and be filtered through a 0.22 μm aqueous filter membrane before use. Place the volumetric flask in an ultrasonic cleaner, set the water temperature to 25 ± 2 °C, the ultrasonic power to 300 W, and sonicate for 15 minutes until the reagent is completely dissolved and the solution is clear and transparent.

[0025] Next, adjust the pH. Transfer the completely dissolved solution to a clean glass beaker and insert the electrode of a precision pH meter calibrated with a standard buffer solution. Turn on the magnetic stirrer and set the speed to [speed value missing]. Keep the solution under moderate stirring. Slowly add 0.1 mol / L dilute phosphoric acid solution dropwise using a micropipette, waiting 30 seconds after each addition to allow the pH value to stabilize before taking the reading. During the adjustment process, strictly control the temperature at 25 ± 0.5℃, using a constant temperature water bath to maintain a constant solution temperature. When the pH value approaches the target value, switch to a more precise micro-addition method until the pH value is accurately reached at 3.00 ± 0.01.

[0026] After pH adjustment, transfer the solution back to the volumetric flask and dilute to the mark with ultrapure water. When diluting, ensure the concave portion of the liquid level is tangent to the mark. After capping, invert the flask 10 times to mix thoroughly, ensuring the solution is homogeneous. Then, filter the solution using a 0.45μm nylon microporous membrane and a vacuum filtration apparatus. Discard the initial 10mL of filtrate and collect the remaining filtrate in a clean glass container.

[0027] Finally, the solution is degassed. The filtered buffer solution is transferred to an ultrasonic cleaner, with the water temperature set to 25°C and the ultrasonic power to 250W, and the ultrasonic treatment lasting 20 minutes. During the treatment, the process is paused every 5 minutes, and the container is gently shaken to allow air bubbles to escape completely. After degassed, the buffer solution is immediately transferred to a dedicated storage bottle, which has been pre-washed with ultrapure water and purged with helium. The storage bottle is then filled with high-purity helium to a pressure of 0.1 MPa, sealed, and stored to prevent dissolved carbon dioxide from affecting the pH value.

[0028] Following the rigorous procedures outlined above, the final phosphate buffer solution fully met the requirements for chromatographic analysis: pH 3.00 ± 0.01, free of solid particles, and dissolved oxygen content below 0.5 mg / L. This satisfies the high standards for mobile phases in high-performance liquid chromatography (HPLC), ensuring the accuracy and reproducibility of the analytical results. The entire preparation process was conducted in a clean environment, with all operations performed according to predetermined procedures and detailed records kept of all parameters to guarantee the traceability of the experimental process.

[0029] When implementing time-series adjustments to control the ratio of aqueous to organic phases to form a gradient program, systematic gradient optimization experiments are required. An Agilent 1260 Infinity II high-performance liquid chromatography system, equipped with a quaternary gradient pump, autosampler, column oven, and diode array detector, was used. Before optimization, based on the chemical properties of omalicycline tosylate and preliminary experimental results, the initial gradient conditions were determined: a pH 3.0 phosphate buffer ratio of 50%–90%, an acetonitrile ratio of 10%–50%, and a total run time of 30–60 minutes. Freshly prepared mobile phase was used before each experiment, and the column was ensured to be fully equilibrated.

[0030] The gradient optimization process employed a multivariate experimental design, conducting single-factor experiments with other conditions fixed to examine the effects of different initial organic phase ratios (10%, 15%, and 20%) on compound retention behavior. Subsequently, the effect of gradient slope variations was investigated, setting different gradient change rates: 0.5%, 1.0%, and 1.5% organic phase / min. Simultaneously, the gradient shape was optimized, comparing the separation effects of linear, convex, and concave gradients. Each condition was repeated at least three times, using a system suitability solution containing the main components and major known impurities.

[0031] During the data acquisition and analysis phase, all chromatograms were recorded and analyzed using the OpenLab CDS chromatographic data processing system. The following key parameters were examined: resolution between the main peak and nearest-neighbor impurity peak (≥1.5), resolution between all impurity peaks, total run time, peak symmetry factor (0.8-1.2), baseline stability, and solvent consumption. The optimal mobile phase ratio for each time point was determined by overlaying and comparing chromatograms under different conditions. Particular attention was paid to the separation of diastereomers within the 8–12 minute timeframe and the elution of strongly retained impurities within the 20–25 minute timeframe.

[0032] Based on extensive experimental data, a detailed gradient program table was developed, containing the following key parameters: the initial ratio was set at 85% aqueous phase and 15% organic phase, held for 5 minutes; then, within 20 minutes, it was linearly changed to 70% aqueous phase and 30% organic phase; after another 10 minutes, it was linearly changed to 50% aqueous phase and 50% organic phase, held for 5 minutes; finally, within 5 minutes, it was rapidly returned to the initial ratio and held for 10 minutes for column equilibration. The flow rate was maintained at 1.0 mL / min for each time period, and the column temperature was controlled at 30℃. The gradient program table specifies the accurate ratios at 21 time points, with time accuracy controlled to 0.1 minutes and ratio accuracy controlled to 0.1%.

[0033] Input the optimized gradient program into the chromatography workstation control system. Create a new method file in the OpenLab CDS software, and sequentially input the flow ratios for each time point on the "Gradient Program" page. Set the gradient curve type to linear, enable the automatic mixing function, and set the maximum system pressure limit to 400 bar and the minimum pressure limit to 0 bar. After inputting the data, perform system validation tests by running blank gradients and standard solutions to confirm that the actual gradient executed matches the set values ​​with a deviation of <0.5%. Simultaneously verify the column oven temperature control accuracy (±0.5℃) and flow rate accuracy (±0.05 mL / min).

[0034] Document a reproducible gradient elution method, including a complete gradient procedure table, system configuration requirements, validation data, and acceptance criteria. Establish a method maintenance procedure, specifying monthly system suitability tests and fine-tuning procedures when column batches are changed. All relevant parameters are stored in the controlled methods folder on the chromatography workstation and backed up to the Laboratory Information Management System (LIMS) to ensure reproducibility and reliability when the method is transferred between different instruments and operators.

[0035] In this embodiment, S2 involves system suitability verification of a solution containing a specific concentration of omalicycline tosylate as the main component. This verification includes determining and judging the theoretical plate number of the chromatographic column and the resolution between the main peak and key impurity peaks. When calculating the theoretical plate number for column efficiency, the necessary instruments and materials for system suitability testing are required. The experiment uses a high-performance liquid chromatography (HPLC) system equipped with a diode array detector. The instrument's condition has been fully validated: pump flow rate accuracy is controlled within ±0.5%, detector wavelength accuracy is within ±1 nm, and column oven temperature control accuracy is ±0.5℃. A certified system suitability solution containing a specific concentration of omalicycline tosylate and its main related substances is used. The solution is used immediately after preparation and stored under specified conditions. The chromatographic column is a designated brand and model, with a valid column efficiency certification. The column temperature is controlled at 30±0.5℃.

[0036] Before chromatographic acquisition, equilibrate the chromatographic system with the initial mobile phase conditions for at least 30 minutes until the baseline stabilizes. Baseline noise should be less than 0.1 mAU and drift less than 0.5 mAU / h. Flush the injection needle six times with system suitability solution before injection to remove air bubbles and ensure sample representativeness. Set the injection volume to 10 μL, the flow rate to 1.0 mL / min, and the detection wavelength to 280 nm. After each injection, allow sufficient time for complete elution of all components to ensure the chromatogram fully records all peak information. Perform six consecutive injections to assess system reproducibility; the relative standard deviation of retention time should not exceed 1.0%.

[0037] When selecting the principal component chromatographic peak as the calculation object, the peak must first be accurately identified. During the principal component peak confirmation process, it is necessary to ensure that its retention time matches the expected value, the peak shape is symmetrical, and there are no shoulders or bifurcations. Using the peak identification function of the chromatography workstation, appropriate peak width parameters and slope thresholds are set to ensure that the principal peak is correctly identified. Simultaneously, peak purity is checked, and multi-wavelength spectra acquired using a diode array detector are used to verify whether the principal peak is a single compound, ruling out the possibility of co-elution.

[0038] When calculating the theoretical plate number using the built-in formula of the chromatography workstation, the system automatically performs the calculation according to the USP or EP standard method. The calculation formula is N = 16 × (tR / W)², where tR is the retention time of the main peak and W is the peak width. The chromatography workstation first accurately measures the retention time with an accuracy of 0.01 minutes; then it determines the peak initiation and endpoint and measures the peak width using the tangent method. The calculation process is completed automatically, and all intermediate parameters are recorded simultaneously. The system also performs automatic verification to ensure that the measurement points are selected correctly and to avoid measurement errors caused by baseline noise or interfering peaks.

[0039] When verifying the calculation results against preset quality standards, strict quality standards need to be established in advance. Based on pharmacopoeia requirements and internal quality standards, the minimum theoretical plate count is set at 15,000. The system automatically compares the calculation results with the preset standards and generates a pass / fail conclusion. Simultaneously, the results of six consecutive injections are examined, and the relative standard deviation is calculated, requiring it to not exceed 2.0%. All data are automatically recorded in the system suitability test report, including chromatograms, calculation process, final results, and conclusions.

[0040] A complete system suitability validation report is generated, including instrument information, chromatographic conditions, solution information, raw data, calculation results, and conclusions. All electronic data is securely stored to ensure data integrity and traceability. If test results do not meet requirements, the system will automatically initiate an investigation procedure, indicating possible causes and suggested corrective actions. Only after the system suitability test is passed can formal sample analysis begin, ensuring the quality control of the entire analytical process.

[0041] When measuring the resolution between the main peak and impurity peaks to obtain resolution data between the main peak and adjacent impurity peaks, it is essential to ensure that the chromatographic system is in a stable state. The experiment used validated chromatographic conditions: a Waters XBridge BEH C18 column (4.6 × 250 mm, 5 μm), a column temperature maintained at 30.0 ± 0.5℃, a mobile phase of pH 3.0 phosphate buffer-acetonitrile system, run according to a predetermined gradient program, a flow rate of 1.0 mL / min, and a detection wavelength of 280 nm. The system suitability solution was prepared according to quality standards, containing the specified concentration of omalicycline tosylate as the main component and its main related substances. Before use, the solution was filtered through a 0.45 μm microporous membrane and kept at a constant temperature of 10℃ in an autosampler.

[0042] During chromatographic acquisition, the system was first equilibrated with the initial mobile phase until baseline stability was achieved, with baseline noise <0.1 mAU and drift <0.5 mAU / h. Then, 10 μL of system suitability solution was precisely pipetted and injected for analysis. After all components were completely eluted, complete chromatographic data were acquired. Six consecutive injections were performed to assess system reproducibility, with the relative standard deviation of the main peak retention time not exceeding 1.0%. All chromatographic data were automatically saved to the chromatography workstation and processed and analyzed using OpenLab CDS 2.4 software.

[0043] When identifying the main peak and its nearest adjacent impurity peak in a chromatogram, the main peak's identity is confirmed through retention time comparison and UV spectral analysis. The retention time of the main peak should be consistent with that of the reference solution, and its UV spectrum should have a similarity index greater than 980 with the reference solution in the 200-400 nm range. Subsequently, the nearest adjacent impurity peak is identified. This impurity peak is usually located before or after the main peak, and its separation from the main peak is a key focus. Using the peak identification function of the chromatographic working peak, appropriate peak detection parameters are set: peak width 0.1 min, slope threshold 50 μV / min, and minimum peak area 100 μV·s, ensuring that all relevant peaks are correctly identified.

[0044] When measuring the retention times and peak widths of the two peaks, the chromatography workstation automatically performs precise measurements. The retention time tR is measured with an accuracy of 0.01 min, determined by the elution time corresponding to the peak apex. The peak width W is measured using the tangent method specified by USP: the target chromatographic peak is selected in the workstation, and the system automatically draws tangents at the inflection points of the rising and falling edges of the peak. The distance between the intersection points of the two tangents and the baseline is the peak width value, with a measurement accuracy of 0.001 min. Simultaneously, the retention times of the main peak tR1 and the impurity peak tR2, as well as the respective peak widths W1 and W2, are recorded. All measurements are automatically recorded in a data table, and the average value and relative standard deviation are calculated.

[0045] The resolution value was automatically calculated by chromatographic data processing software according to the resolution calculation formula specified in the pharmacopoeia. The resolution Rs calculation formula specified in the USP General Rules was adopted: Where tR2 is the retention time of the later eluting peak, tR1 is the retention time of the earlier eluting peak, and W1 and W2 are the peak widths of the two peaks, respectively. The chromatography workstation automatically retrieves the measured retention time and peak width data, substitutes them into the formula for calculation, and retains the result to two decimal places. The software also performs data validity verification to ensure that the measurement points are selected correctly and to eliminate measurement errors caused by baseline fluctuations or peak overlap.

[0046] This data is recorded as a key indicator for system suitability evaluation. The resolution calculation result is automatically entered into the system suitability test report, and all raw data used in the calculation process are also recorded. According to quality standards, the resolution between the main peak and the nearest adjacent impurity peak should be no less than 1.5. The system automatically compares the calculation results with preset standards and generates a pass / fail conclusion. All data and conclusions are automatically saved to the Laboratory Information Management System (LIMS) to ensure data integrity and traceability. If the results do not meet the requirements, the system will automatically initiate a deviation investigation procedure, indicating possible causes and suggesting corrective measures.

[0047] In this embodiment, step S3 involves dissolving and adjusting the volume of the omalicycline tosylate test sample raw material to prepare the test solution, including: When using precision weighing techniques to dissolve and dilute the test sample, precise weighing is required. The experiment is conducted in a GMP-compliant Class 100,000 cleanroom, with the ambient temperature controlled at 20±2℃ and relative humidity at 45±5%. A Sartorius CPA225D analytical balance (0.0001g) calibrated by a legally recognized metrology institution is used. This balance is equipped with a windproof cover and anti-static device, and the calibration certificate is valid. Daily calibration is performed using standard weights before each use. Before weighing, personnel must wear powder-free nitrile gloves and wipe the balance surface and weighing area with a lint-free cloth soaked in 75% ethanol. The raw material for the injectable omalicycline tosylate test sample is taken. The raw material is stored at 2-8℃ and equilibrated to room temperature in a desiccator before use. Using a clean spatula, an appropriate amount of the test sample is taken and weighed using the weight difference method, accurately weighing 50.0mg and recording the actual weight value to four decimal places. The ambient temperature and humidity are monitored in real time during the weighing process to ensure that the weighing accuracy meets the requirements.

[0048] After weighing, perform quantitative transfer and preliminary dissolution of the sample. Carefully transfer the accurately weighed sample to a 50 mL Class A volumetric flask using clean, dust-free weighing paper. Rinse the weighing dish and weighing paper multiple times, v / v, using approximately 30 mL of diluent (water:acetonitrile = 80:20) to ensure complete transfer of the sample into the flask. Place the volumetric flask in a KQ-500DE CNC ultrasonic cleaner, set the ultrasonic power to 300 W, and the water temperature to 20 ± 2 °C for 20 minutes. Pause every 5 minutes during ultrasonication and gently shake the flask to ensure the sample on the flask wall is fully dissolved. Visually inspect to ensure the sample is completely dissolved, the solution is clear and transparent, and there are no visible insoluble matter or suspended particles.

[0049] After the sample is completely dissolved, cool and dilute the solution to the required volume. Remove the volumetric flask from the ultrasonic water bath and place it on a clean bench in the laboratory. Allow it to cool at 20±2℃ for 15 minutes to allow the solution temperature to equilibrate with room temperature. After cooling, slowly add diluent to about 1 cm below the graduation mark on the volumetric flask. Then, use a dropper to add the diluent drop by drop until the lowest point of the meniscus is tangent to the graduation mark. During the volume adjustment process, keep your line of sight level with the graduation mark to ensure accurate readings. Cap the flask and mix it repeatedly 15 times using an alternating inverted and upright method to ensure the solution is thoroughly and evenly mixed. Avoid vigorous shaking that may generate air bubbles throughout the process.

[0050] After the solution was thoroughly mixed, it was filtered. A 0.45 μm microporous membrane made of Millipore Millex-HN nylon was used, and a 50 mL glass syringe was employed for filtration. First, the membrane was moistened with a small amount of the thoroughly mixed solution, and the filtration apparatus was rinsed. The first 5 mL of filtrate was discarded. The remaining filtrate was collected in a clean 2 mL HPLC sample vial, which had been pre-washed with ultrapure water and sonicated with methanol. After filtration, the vial was immediately capped and sealed, and the sample information, including sample name, batch number, preparation time, and expiration date, was clearly labeled.

[0051] The obtained test solution must undergo quality inspection. A suitable amount of filtrate should be visually inspected; the solution should be clear and transparent, free of suspended matter, precipitates, fibers, or other visible foreign matter. Simultaneously, the absorbance value should be measured at 280 nm using a UV-Vis spectrophotometer and compared with the reference solution to ensure the concentration is within the linear range of the standard curve. The prepared test solution should be placed in an autosampler, stored at 10°C, and analyzed within 24 hours to ensure sample stability. All preparation processes should be meticulously recorded in the experimental logbook, including weighing values, dissolution time, final volume, filtration conditions, and other parameters, ensuring the traceability of the experimental process.

[0052] In this embodiment, when the test sample solution is injected, separated, and signal acquired by the automatic injection system under the set chromatographic conditions in step S4, it includes: When performing liquid chromatography separation and signal acquisition of the test solution using an automated sample injection system, the system must first be prepared and initialized. The experiment used an Agilent 1260 Infinity II high-performance liquid chromatography system equipped with a G7129A high-performance autosampler. Before sample analysis, the autosampler was prepared as follows: the outer wall of the injection needle and the injection port were rinsed three times v / v using a dedicated cleaning solution, including water:methanol = 50:50, with each rinse volume being 500 μL; the inner wall of the injection needle and the sample loop were rinsed five times v / v using a diluent, with each rinse volume being 100 μL; the number of uses of the injection needle gasket was checked to ensure it was within its lifespan; and the waste bottle was confirmed to have sufficient capacity, and the sample tray temperature was controlled at 10 ± 1 °C. After completing the system preparation, the autosampler's self-test procedure was executed, including pressure testing, leak detection, and injection accuracy verification, to ensure the system was in optimal working condition.

[0053] When placing the prepared test solution into the autosampler sample tray, strict adherence to standard operating procedures is required. Use certified 2mL clear glass vials equipped with PTFE / silicone septa and pre-opened aluminum caps. Clearly label each vial with the sample number, batch number, and preparation time. When placing the vials, ensure they are upright to prevent tilting, which could hinder accurate needle positioning. Maintain a constant temperature of 4°C in the sample tray to prevent sample degradation during the waiting period. Simultaneously, establish a sequence of positions within the sample tray, including the test sample, quality control sample, blank solution, and system suitability test solution, ensuring the analytical order meets quality control requirements. Create a corresponding sample sequence list in the software, accurately entering the relevant information and analytical method for each sample.

[0054] The injection volume and injection sequence were set using OpenLab CDS control software. The injection volume was set to 10.0 μL, using a partial-fill injection mode, with a sample loop volume of 20 μL. The injection sequence was set according to quality control requirements: first, a blank solution was injected once, followed by a system suitability test solution. After confirming system suitability, the sequence of samples to be tested was injected. The sample sequence used a random distribution design, with one quality control sample inserted every 10 samples and one system suitability check sample inserted after every 20 samples. Injection parameters were set in the software: needle aspiration rate of 100 μL / min, injection rate of 500 μL / min, needle depth 0.5 mm from the bottom of the vial, and a needle cleaning time in the mobile phase after injection of 5 seconds. An injection interval was also set to ensure sufficient analysis time for each sample.

[0055] The instrument automatically completes the sample aspiration and injection process, with the autosampler precisely executing a preset program. The injection needle first moves to the designated sample vial position, penetrates the septum, and descends to a predetermined depth, aspirating an appropriate amount of sample at a set speed. After aspiration, the injection needle rises and moves to the injection valve position, injecting the sample into the sample loop. The six-way valve switches positions, carrying the sample from the sample loop into the mobile phase flow path and into the chromatographic column. The entire injection process is performed at room temperature and completed within one minute to ensure sample stability during injection. After each injection, the injection needle automatically cleans its inner and outer walls, first rinsing the outer wall with cleaning solution and then rinsing the inner wall with diluent to prevent sample residue and cross-contamination.

[0056] During separation under the set chromatographic conditions, the chromatographic system operated according to an optimized gradient program. A binary gradient pump delivered the mobile phase at a flow rate of 1.0 mL / min, the column oven temperature was controlled at 30.0 ± 0.5 °C, and a WatersXBridge BEH C18 column (4.6 × 250 mm, 5 μm) was used. After the sample entered the column, separation was performed based on the retention characteristics of each component. Throughout the separation process, the system pressure was maintained within the range of 200-250 bar, with pressure fluctuations less than ± 2%. Column temperature, flow rate, and pressure were monitored in real time during separation to ensure stable chromatographic conditions. The separation time was controlled to be completed within 45 minutes to ensure complete elution of all target components.

[0057] When continuously acquiring chromatographic signals at a specific wavelength using a UV detector, an Agilent G7115A diode array detector was employed for signal acquisition. The detection wavelength was set to 280 nm, the bandwidth to 4 nm, and the reference wavelength was disabled. The acquisition frequency was 20 Hz, and the response time was set to 0.5 seconds. The detector underwent self-testing and wavelength calibration before analysis to ensure the accuracy of the optical system. Baseline noise and drift were monitored in real time during signal acquisition, with noise required to be less than 0.1 mAU and drift less than 0.5 mAU / h. The acquired chromatographic signals were transmitted to the data processing system in real time, saved in raw data format, and a real-time chromatogram was generated for monitoring and analysis. All acquired data was automatically timestamped and labeled with sample information to ensure data integrity and traceability. After analysis, the system automatically generated an injection sequence report, including the injection time, analysis status, and data volume for each sample.

[0058] In this embodiment, when performing peak area integration on the acquired chromatographic signal using chromatographic data processing software in step S5 to obtain peak area data for each component, the following steps are included: When performing peak area integration on the acquired chromatographic signals using chromatographic data processing software, the original chromatograms must first be preprocessed. The experiment used the Waters Empower 3 chromatographic data system. After opening the acquired raw chromatographic data file, baseline correction was performed first. Selecting the "Automatic Baseline Correction" function, the system first identified baseline drift in the chromatogram, fitted the baseline trajectory using the least squares method, set the correction window width to 1.0 minute, and the smoothing factor to 0.3, removing baseline shifts caused by changes in mobile phase composition, temperature fluctuations, etc. Subsequently, signal smoothing was performed using the Savitzky-Golay filtering algorithm, with a polynomial order of 2 and a window width of 13 data points, effectively eliminating high-frequency noise interference while maintaining the chromatographic peak shape characteristics. During processing, the comparison of the spectra before and after correction was displayed in real time, ensuring a flat baseline and a noise level reduced to below 20% of the original signal.

[0059] After preprocessing, the integration parameters are set and optimized. In the integration method editing interface, the peak width parameter is first set to 0.2 min, which is determined based on the average peak width of the earliest and latest peaks in the actual chromatogram. The slope threshold is set to 50 μV / min, calculated by analyzing the baseline noise level to ensure effective differentiation between signal and noise. The minimum peak area is set to 1000 μV·s, determined based on a signal-to-noise ratio of 3:1, which effectively filters out irrelevant small interference peaks. Peak detection parameters are also set: the peak start and end point recognition sensitivity is set to 5, the peak-to-valley ratio threshold is set to 2, and the shoulder peak detection sensitivity is set to 50. For complex peak shapes, the "peak purity angle" calculation function is enabled, and the minimum purity is set to 980 to ensure that each chromatographic peak can be correctly identified and resolved.

[0060] When the software automatically identifies and integrates each chromatographic peak, the system executes an automated processing flow according to the set parameters. First, peak detection is performed. The algorithm identifies the peak start and end points based on changes in the first derivative and determines the peak apex position using the second derivative. For each detected peak, the system automatically calculates 17 peak parameters, including retention time, peak height, peak area, symmetry factor, and resolution. For the resolution of overlapping peaks, the vertical segmentation method is used to handle severely overlapping peaks (resolution < 0.8), while the tangent-skim method is used to handle slightly overlapping peaks (resolution < 1.2). During integration, the system displays the integration baseline position in real time and marks the integration result for each peak, using different colors to distinguish between baseline-separated peaks, overlapping peaks, and shoulder peaks.

[0061] After automatic integration, the process moves to manual verification. Researchers first check the rationality of the integration baseline, confirming that the baseline location conforms to pharmacopoeia requirements, especially for complex baselines and overlapping peaks. They then verify the accuracy of each peak identification, focusing on major component peaks and key impurity peaks with retention times in the 5–15 minute range. For chromatographic peaks automatically marked as "requiring verification" by the system, manual integration tools are used for further processing: for leading peaks, an exponential correction function model is used for fitting; for tailing peaks, a Gaussian-Lorentz mixed model is used for analysis. Simultaneously, the integration event table is checked to ensure that all changes to integration parameters are recorded in detail.

[0062] A comparative analysis method was used to compare the current integration results with historical data, requiring that the relative standard deviation of the main peak area not exceed 2.0% and the relative standard deviation of the retention time not exceed 1.0%. For special peak shapes, peak purity analysis was used to verify the results using multi-wavelength spectral data acquired by a diode array detector, requiring that the purity angle of each peak be less than the threshold angle. After all verifications were completed, an integration report was generated, including integration parameter settings, detailed integration data for each peak, and records of manual interventions during the integration process. Finally, it was confirmed that all chromatographic peaks were correctly identified and integrated, and that the integration results met the data integrity requirements, satisfying the accuracy and reliability requirements for subsequent quantitative analysis. All integration methods and results were automatically saved to the database to ensure the traceability and reproducibility of the experimental process.

[0063] In this embodiment, when performing purity calculation on the peak area data using the area normalization method in step S6 to generate the purity analysis result of the omalicycline tosylate, the process includes: When performing purity calculations on peak area data using the area normalization method, peak area screening and processing are necessary. The experiment used the Waters Empower 3 chromatography data processing system. After opening the integrated chromatographic data file, solvent peaks were first identified and eliminated. The system automatically identified solvent peaks based on their retention time ranges, and confirmed this by combining the UV spectral characteristics acquired by the diode array detector: solvent peaks typically exhibit broad peaks with no characteristic UV absorption. Known invalid peaks were automatically identified and marked using the preset retention time windows in the method. The system established an exclusion list containing the retention time ranges and spectral characteristics of common invalid peaks, ensuring these peaks were not included in the calculation. All eliminated peaks were listed in detail in the final report, with the reasons for elimination noted.

[0064] After peak screening, the system automatically calculates the sum of the areas of all valid chromatographic peaks. The calculation uses double-precision floating-point arithmetic to ensure a precision of 0.0001 μV·s. First, the peak areas of each valid peak are summed to obtain the total peak area. Simultaneously, the system performs data validity verification: checking the integral quality factor (Q value) of each peak (requiring a Q value greater than 0.98); verifying peak symmetry (requiring a symmetry factor within the range of 0.8-1.2); and confirming the baseline noise level (requiring a signal-to-noise ratio greater than 10). For peaks that do not meet the quality requirements, the system automatically marks them and prompts for manual review. After calculation, a peak area summary table is generated, containing detailed information such as the retention time, peak area, and percentage of the total area for each peak.

[0065] Next, the purity of the principal components is calculated. The system automatically identifies the main peak and confirms it based on its retention time and UV spectral characteristics. The main peak area is taken, and the purity percentage is calculated using the area normalization method formula: Purity percentage = (Main peak area / Total peak area) × 100%. The calculation process uses a validated algorithm to ensure a calculation accuracy of 0.01%. The system also performs error analysis, considering factors such as integration error, baseline noise error, and injection volume error, to calculate the uncertainty of the final result. Typically, the expanded uncertainty is required to be no more than 0.2%.

[0066] The final analysis report is a complete document. The report uses a standard template and includes the following main components: sample information, chromatographic conditions, integration parameter settings, peak area summary table, purity calculation results, and data quality indicators. All numerical values ​​in the report are rounded according to significant figure rules, and purity results are retained to one decimal place. Chromatograms and an integration event table are also included to ensure the traceability of the results. The report is output in both PDF electronic and paper formats. The electronic report uses a digital signature to ensure data integrity, and the paper report is signed and confirmed by authorized personnel before being archived.

[0067] All calculations and results are automatically saved to the Laboratory Information Management System (LIMS), establishing a complete data traceability chain. The system records all parameters used in the calculation process, algorithm versions, operators, and other information. A data review process is also established: initial review by analysts, followed by verification by quality assurance personnel, and finally, approval and report issuance by authorized personnel. For abnormal results, the system automatically initiates an investigation procedure, requiring re-injection for verification or re-preparation of samples for analysis, ensuring the accuracy and reliability of the final results. The entire process complies with GMP data integrity requirements and meets the review standards of regulatory agencies.

[0068] Comparison table with existing methods Comparison Dimensions Existing liquid chromatography methods Method of the present invention Areas for improvement / advantages Aqueous phase treatment Generally, only ultrapure water or simple filtration is used; long-term storage can easily generate bubbles or dissolved oxygen, affecting the detection. Ultrasonic degassing combined with helium protection storage ensures long-term stability of the mobile phase. Improved detection baseline stability and reduced noise interference. organic phase system Common acetonitrile-water or methanol-water systems are not optimized for the target analyte. An acetonitrile-water system was selected, and 0.1% trifluoroacetic acid was added to improve peak shape and resolution. Improved separation selectivity and reduced peak tailing Testing conditions The detection wavelength is typically 254 nm or selected empirically. Specifically employing a 220nm detection wavelength to match the maximum absorption peak of the target compound. Higher sensitivity, lower limit of quantitation Verification content Some methods only perform linearity or recovery rate tests, resulting in unsystematic validation. The system validates a full set of indicators including linearity, precision, accuracy, and stability. The methodology is more comprehensive and conforms to ICH Q2 standards. applicability Unstable for detecting trace components in complex matrices It maintains high precision and recovery even in complex samples. It expands the scope of application and improves the reliability of detection. Compared with existing liquid chromatography detection methods, this invention systematically optimizes mobile phase preparation, detection condition selection, and methodological validation. By introducing ultrasonic degassing and helium-protected aqueous phase treatment, the long-term stability of the mobile phase is ensured. Combined with the optimization measure of adding trifluoroacetic acid to the acetonitrile-water system, the peak shape and resolution of the target compound are effectively improved. In terms of detection wavelength selection, the characteristic absorption peak of the target compound is determined to be at 220 nm, improving detection sensitivity. More importantly, the method of this invention has undergone a full set of validations conforming to international standards, ensuring its precision, accuracy, and stability. In summary, this invention not only solves the problems of detection instability and insufficient resolution in existing methods, but also has significant advantages in detection sensitivity and applicability, possessing high practical value and promising prospects for widespread application.

[0069] Therefore, the embodiments should be regarded as exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of the equivalents of the claims are intended to be included within the invention.

[0070] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0071] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the purity of omalicycline tosylate for injection, characterized in that, The method includes the following steps: S1: Prepare a phosphate buffer solution with a pH of 3.00±0.01, and establish a chromatographic analysis method for the omalicycline tosylate; S2: System suitability verification was performed on a solution containing a specific concentration of omalicycline tosylate as the main component, and the theoretical plate number of the chromatographic column and the resolution between the main peak and the key impurity peak were determined and judged. S3: Dissolve and dilute the raw material of omalicycline tosylate for injection to prepare a test solution of the raw material of omalicycline tosylate. S4: The sample solution is injected, separated, and signal acquired by an automated injection system under the set chromatographic conditions; S5: Use chromatographic data processing software to perform peak area integration on the acquired chromatographic signals to obtain peak area data for each component; S6: Apply the area normalization method to perform purity calculation on the peak area data to generate the purity analysis results of the omalicycline tosylate.

2. The method according to claim 1, characterized in that, The preparation of the phosphate buffer solution with a pH of 3.00 ± 0.01 in step S1 includes: The solution was prepared by precise weighing using superior grade potassium dihydrogen phosphate reagent. The pH value was precisely adjusted using a 0.1 mol / L dilute phosphoric acid solution under constant temperature conditions. The pH-adjusted solution is filtered through a microporous membrane to remove particulate matter; the filtered buffer solution is then subjected to ultrasonic degassing to obtain a phosphate buffer solution that meets the requirements for chromatographic analysis.

3. The method according to claim 1, characterized in that, The chromatographic analysis method established in S1 employs a gradient elution procedure, including: Configure a gradient scaling procedure that includes multiple time points; The flow parameters of the pump system are set according to the gradient program; The proportion of phosphate buffer solution is controlled by setting flow parameters; A chromatographic analysis method that optimizes the ratio variation conditions to obtain the best separation effect.

4. The method according to claim 1, characterized in that, When performing system suitability verification in S2, the following are included: Prepare system-suitable solutions containing specific concentrations of the main component and related impurities; Chromatograms were obtained by performing chromatographic analysis on the system suitability solution. Calculate the theoretical plate number and resolution index of the chromatographic column based on the chromatogram; The calculated indicators are compared with predetermined standards to verify the applicability of the system.

5. The method according to claim 1, characterized in that, The preparation of the test solution in step S3 includes: Accurately weigh the specified amount of omalicycline toluenesulfonate test sample raw material; The weighed omalicycline tosylate test sample raw material was dissolved using a diluent with a specific ratio; The dissolved solution was quantitatively transferred to a volumetric flask and brought to volume. The solution after volume adjustment is filtered to obtain a clear and transparent test solution.

6. The method according to claim 1, characterized in that, When performing the injection, separation, and signal acquisition of the test sample solution under the set chromatographic conditions in step S4, it includes: Program for setting injection parameters for the autosampler; The sample is automatically injected according to the injection procedure. Separation of each component was achieved under optimized chromatographic conditions; Chromatographic data are obtained by acquiring signals from the separated components using a detector.

7. The method according to claim 1, characterized in that, When performing peak area integration in S5, including Baseline correction and smoothing are performed on the acquired chromatographic signals; Establish an automatic integration method by setting appropriate integration parameters; The area of ​​the chromatographic peak is calculated according to the integration method, and the accuracy of the calculated peak area data is verified.

8. The method according to claim 1, characterized in that, When calculating purity using the area normalization method in S6, the following is included: Identify and subtract solvent peaks from chromatograms; Calculate the sum of the areas of all valid chromatographic peaks; The purity percentage is calculated based on the ratio of the sum of the peak areas to the area of ​​the main peak, and a purity analysis report containing all calculated data and results is generated.