Analysis method and application of tobramycin sulfate injection

Abstract

The application discloses an analysis method of tobramycin sulfate injection and application thereof, and the analysis method comprises the following steps: performing a pre-column derivatization reaction on a sample containing tobramycin and impurities by using a derivatization reagent, and then performing high performance liquid chromatography analysis on the sample after derivatization. The analysis is performed by gradient elution of a mobile phase A and a mobile phase B composed of water, acetonitrile and phosphoric acid, wherein the content of acetonitrile in the mobile phase B is higher than that in the mobile phase A, and effective separation of tobramycin and related impurities is realized by multi-stage gradient elution. The method can simultaneously separate and detect process impurities, degradation impurities and unknown impurities, has good separation degree and stability indicating capacity, and is suitable for quality control and drug stability research of tobramycin sulfate injection.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to an analytical method and application of tobramycin sulfate injection. Background Technology

[0002] Tobramycin sulfate injection is a widely used aminoglycoside antibiotic preparation for clinical anti-infective treatment. Its active ingredient, tobramycin, is composed of multiple amino sugars and deoxystreptamine linked by glycosidic bonds. During the production and storage of tobramycin sulfate injection, various process impurities and degradation impurities are generated. Currently, the ChP2025 and USP standards for injections have limited coverage of specific impurities under the injection category. Furthermore, due to the lack of conjugated systems in its molecular structure, it exhibits almost no absorption in the ultraviolet region, posing a significant challenge to conventional high-performance liquid chromatography-ultraviolet detection (HPLC-UV) methods.

[0003] In existing technologies, the analysis of related substances in tobramycin raw materials and injections largely relies on pharmacopoeia methods. However, different pharmacopoeias vary in their inclusion of impurities. For example, some pharmacopoeias do not explicitly specify certain impurities, while others only list a few known impurities and lack systematic analytical methods for these known impurities. Therefore, in actual quality control, the problem of not being able to comprehensively detect all relevant impurities remains.

[0004] Furthermore, tobramycin is prone to generating various process impurities and degradation impurities during preparation and storage, such as kanamycin B, niberamamide, neomycinamide, and other structural analogs. These impurities may undergo further transformation under different conditions (such as high temperature, acid and alkali, light, and oxidation), increasing the complexity of analysis. Moreover, since most tobramycin-related impurities belong to the aminoglycoside class, their polarity, molecular weight, and ionization behavior are very similar, making it difficult for traditional liquid chromatography methods to simultaneously and effectively separate multiple process impurities and degradation impurities.

[0005] Therefore, solving the problem of "difficulty in simultaneously separating multiple structurally similar aminoglycoside impurities" and developing a high-resolution, high-performance liquid chromatography method that can simultaneously monitor multiple specific impurities is crucial for ensuring the safety of clinical drug use. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides an analytical method and application for tobramycin sulfate injection. The analytical method is based on a combination of pre-column derivatization reaction and high-performance liquid chromatography analysis, which can detect related substances in tobramycin sulfate injection.

[0007] According to the present invention, an analytical method for tobramycin sulfate injection is provided, comprising the following steps: Step S01: Derivatize the tobramycin sulfate injection sample with a derivatizing reagent; Step S02: Perform high performance liquid chromatography analysis on the derivatized tobramycin sulfate injection sample, using mobile phase A and mobile phase B for gradient elution. Both mobile phase A and mobile phase B are solution systems composed of water, acetonitrile and phosphoric acid, and the acetonitrile content in mobile phase B is higher than that in mobile phase A. The gradient elution includes: In the first stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is increased from an initial percentage to an intermediate percentage; In the second phase, the aforementioned intermediate percentage remains unchanged; In the third stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is increased to the elution percentage; In the fourth stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is restored to the initial percentage.

[0008] In some embodiments of the invention, in the first stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is increased from the initial percentage and reaches the intermediate percentage after 20 to 30 minutes (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 20, or 30 minutes); in the second stage, the intermediate percentage is maintained for 30 to 40 minutes (e.g., 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 minutes); in the third stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is increased to the elution percentage, and elution is performed for 2 to 7 minutes (e.g., 2, 3, 4, 5, 6, or 7 minutes); in the fourth stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is reduced to the initial percentage and maintained for 5 to 9 minutes (e.g., 5, 6, 7, 8, or 9 minutes).

[0009] In some embodiments of the invention, the initial percentage is 19%-23% (e.g., 19%, 20%, 21%, 22%, or 23%), the intermediate percentage is 67%-73% (e.g., 67%, 68%, 69%, 70%, 71%, 72%, or 73%), and the elution percentage is 92%-98% (e.g., 92%, 93%, 94%, 95%, 96%, 97%, or 98%).

[0010] In some embodiments of the invention, during the first stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B increases from the initial percentage and reaches 32%-36% (e.g., 33%, 34%, 35% or 36%) after 12 to 16 minutes (e.g., 13, 14, 15 or 16 minutes).

[0011] In some embodiments of the invention, the third stage involves increasing the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B to the elution percentage within 10 seconds, and eluting for 2 to 7 minutes (e.g., 2, 3, 4, 5, 6, or 7 minutes); the fourth stage involves decreasing the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B to the initial percentage within 10 seconds, and continuing for 5 to 9 minutes (e.g., 5, 6, 7, 8, or 9 minutes).

[0012] In some embodiments of the present invention, the chromatographic column used for the high-performance liquid chromatography analysis is a phenylsilane-bonded silica column.

[0013] In some embodiments of the present invention, the phenylsilane-bonded silica gel column has an inner diameter of 2-5 mm (e.g., 2 mm, 4.6 mm or 5 mm), a length of 100-250 mm, and a packing particle size of 2-5 μm (e.g., 3 μm or 5 μm).

[0014] In some embodiments of the present invention, the conditions for the phenylsilane-bonded silica gel column are set as follows: flow rate of 1.0-1.5 ml / min (e.g., 1.1 ml / min, 1.2 ml / min, 1.3 ml / min or 1.4 ml / min), column temperature of 20-35 ℃ (e.g., 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃ or 35 ℃), and detection wavelength of 360-375 nm.

[0015] In some embodiments of the present invention, the derivatizing reagent is 2,4-dinitrofluorobenzene; the mobile phase A comprises water, acetonitrile and phosphoric acid in a volume ratio of (925-975):(45-55):(0.7-0.9); the mobile phase B comprises water, acetonitrile and phosphoric acid in a volume ratio of (260-300):(700-740):(0.7-0.9).

[0016] The present invention also relates to the application of the analytical method described above in the separation and detection of process impurities and degradation impurities in tobramycin sulfate injection, wherein the process impurities include impurity D, impurity F, kanamycin B, niberamamide and neomycin, and the degradation impurities include apramycin, wherein impurity D is 6-O-(3-amino-3-deoxy-α-D-deoxyglucopyranosyl)-2-deoxy-streptomycin, and impurity F is 3-N-carboxytobramycin.

[0017] Beneficial effects: Because tobramycin and its related impurities are all aminoglycoside compounds, lacking ultraviolet (UV) absorbing groups, they cannot be effectively detected directly using a UV detector, making it difficult to simultaneously separate multiple structurally similar aminoglycoside impurities. This invention provides an analytical method for tobramycin sulfate injection, employing a pre-column derivatization reaction and a gradient elution program. The reaction uses 2,4-dinitrofluorobenzene as the derivatizing reagent. Utilizing the primary amino group in the molecular structure of tobramycin and its impurities, a nucleophilic substitution reaction occurs between the derivatizing reagent and the primary amino group in the sample under an alkaline buffer environment, introducing a functional group with UV absorption characteristics, thereby enabling strong UV absorption at a specific wavelength. Analysis is then performed using high-performance liquid chromatography (HPLC). A gradient elution program is constructed using a water-acetonitrile-phosphoric acid system, systematically designed based on the polarity differences of tobramycin and its related impurities, their retention behavior after derivatization, and the elution characteristics of unknown degradation products. By adjusting the proportion of acetonitrile in the mobile phase, impurities of different polarities and structures are separated within a reasonable retention time, ensuring good separation between the main peak and adjacent impurity peaks. In this gradient elution method, a lower slope of acetonitrile is used in the initial stage to improve the separation of highly polar process impurities. A stable acetonitrile ratio is maintained in the middle stage to stably separate the tobramycin main peak and its adjacent degradation impurities. The acetonitrile ratio is further increased in the final stage to remove strongly retained impurities and reduce the impact of residual peaks on subsequent analyses. Therefore, this analytical method not only effectively separates various structurally similar aminoglycoside impurities and solves the separation problem between multi-component impurities (such as niberamide and kanamycin B, which have very similar polarities), but also effectively separates various impurities of similar polarity. It achieves effective separation of tobramycin from various process impurities and degradation impurities, including neomycin, niberamide, kanamycin B, and apramycin, overcoming the detection challenge of tobramycin's lack of UV absorption, and simultaneously detecting multiple known and unknown impurities. Furthermore, it improves the stability, repeatability, and coverage of unknown impurities. Attached Figure Description

[0018] Some embodiments of the invention have been described herein by way of example only, with reference to the accompanying drawings. It is important to emphasize, in particular, that the details shown are by way of example and for the purpose of illustrative discussion of embodiments of the invention. In this regard, the description taken in conjunction with the drawings will make it clear to those skilled in the art how to practice embodiments of the invention.

[0019] In the attached diagram: Figure 1 This is a schematic diagram of the analytical method for tobramycin sulfate injection of the present invention.

[0020] Figure 2 This is a schematic diagram of typical peak values ​​of various impurities generated by the analytical method for tobramycin sulfate injection of the present invention.

[0021] Figure 3 This is a schematic diagram of typical peak spectra of various impurities generated by the USP method for detecting impurities. Detailed Implementation

[0022] The following describes specific embodiments of the analytical method for tobramycin sulfate injection of the present invention with reference to the accompanying drawings. It should be noted that the implementation of the present invention is not limited to the following embodiments. Those skilled in the art can understand the advantages and effects of the present invention from the content disclosed in this specification. The present invention can be implemented or applied through other different specific embodiments, and various details in this specification can also be modified and changed based on different viewpoints and applications without departing from the concept of the present invention.

[0023] The active ingredient in tobramycin sulfate injection is tobramycin, and its relevant information is as follows: Chemical structural formula:

[0024] Name: Tobramycin Chemical name: O -3-Amino-3-deoxy- α - O -glucopyranosyl-(1→6)- O -[2,6-diamino-2,3,6-trideoxy- α [-D-ribopyranosyl-(1→4)]-2-deoxy-D-streptamine English name: Tobramycin Molecular formula: C 18 H 37 N5O9 Molecular weight: 467.52 CAS No.: 32986-56-4 Tobramycin has multiple amino groups and glycoside structures. Due to the lack of conjugated systems in its molecular structure, it has almost no absorption in the ultraviolet region, which poses a significant inconvenience for conventional high-performance liquid chromatography-ultraviolet detection (HPLC-UV) methods.

[0025] This invention provides an analytical method for tobramycin sulfate injection. The method utilizes the primary amino groups contained in tobramycin and its impurities to react with a derivatizing reagent under alkaline conditions, introducing functional groups with strong ultraviolet absorption, thereby significantly improving its response under an ultraviolet detector. Furthermore, by optimizing the composition of the mobile phase and gradient elution conditions, various impurities with similar polarities can be effectively separated.

[0026] Specifically, the present invention is achieved through the following technical solutions: Please see Figure 1 , Figure 1 This is a schematic flowchart of the analytical method for tobramycin sulfate injection according to the present invention. The present invention provides an analytical method for tobramycin sulfate injection, comprising the following steps: Step S01: Derivatize the sample of tobramycin sulfate injection containing tobramycin and impurities using a derivatization reagent; Step S02: Perform high performance liquid chromatography analysis on the derivatized tobramycin sulfate injection sample, using mobile phase A and mobile phase B for gradient elution. Both mobile phase A and mobile phase B are solution systems composed of water, acetonitrile and phosphoric acid, and the acetonitrile content in mobile phase B is higher than that in mobile phase A. The gradient elution includes: In the first stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is increased from an initial percentage to an intermediate percentage; In the second phase, the aforementioned intermediate percentage remains unchanged; In the third stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is increased to the elution percentage; In the fourth stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is restored to the initial percentage.

[0027] In some embodiments of the present invention, in the first stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is increased from the initial percentage, reaching the intermediate percentage after 20 to 30 minutes; in the second stage, the intermediate percentage is maintained for 30 to 40 minutes; in the third stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is increased to the elution percentage, and elution is performed for 2 to 7 minutes; in the fourth stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is decreased to the initial percentage, and this is maintained for 5 to 9 minutes.

[0028] The derivatization reaction involves mixing the test solution with a derivatizing reagent and an alkaline buffer solution, allowing the derivatizing reagent to react with tobramycin and its impurities in the test solution. Pre-column derivatization is performed under heating conditions to convert tobramycin and its impurities into derivatives with UV absorption. Gradient elution is performed using a system of water, acetonitrile, and phosphoric acid as the mobile phase, and separation and detection are performed using a high-performance liquid chromatography (HPLC) column as the stationary phase.

[0029] In some embodiments, the derivatizing reagent is 2,4-dinitrofluorobenzene, and the alkaline buffer solution is a tris(hydroxymethyl)aminomethane solution. The core principle of the analytical method in this application is that tobramycin and its related impurities belong to the aminoglycoside class of compounds, containing multiple primary amino groups in their molecular structure, but lacking conjugated chromophores suitable for UV detection. Therefore, they exhibit almost no significant absorption signal under typical UV detection conditions. To improve detection sensitivity, this invention uses 2,4-dinitrofluorobenzene as a pre-column derivatizing reagent, causing it to undergo a nucleophilic substitution reaction with the primary amino groups in tobramycin and various impurity molecules, forming derivatives with a nitrobenzene ring structure. Because the formed derivatized products have strong n→π* electron transition absorption, they can be effectively detected by a UV detector at a wavelength of approximately 360-375 nm.

[0030] In some embodiments, the derivatization reaction is carried out under heating conditions in the temperature range of 50-70 °C for 30-60 minutes. In a preferred embodiment, the derivatization reaction is carried out at approximately 60 °C for 30-60 minutes because moderately increasing the reaction temperature can effectively increase the reaction rate between the primary amino group and 2,4-dinitrofluorobenzene, making the derivatization reaction more complete, thereby improving the detection sensitivity and signal stability of the relevant substances. If the reaction temperature is too low or the reaction time is insufficient, some amino compounds may be incompletely derivatized, resulting in a lower peak area or a decrease in the detection ability of unknown impurities; conversely, if the reaction temperature is too high or the time is too long, some derivatized products may be further degraded, or even additional by-products may be generated, affecting the chromatographic separation effect and the accuracy of the analytical results.

[0031] In some embodiments, the mobile phase includes mobile phase A and mobile phase B. Mobile phase A comprises water, acetonitrile, and phosphoric acid in a volume ratio of (925-975):(45-55):(0.7-0.9), and mobile phase B comprises water, acetonitrile, and phosphoric acid in a volume ratio of (260-300):(700-740):(0.7-0.9). Because tobramycin-related impurities have high structural similarity and similar polarities, this invention further incorporates a gradient elution method. By gradually increasing the proportion of organic phase in the mobile phase, the distribution behavior and hydrophobic interactions between each derivatized product and the stationary phase are altered. This allows different process impurities and degradation impurities to be gradually eluted within an appropriate retention time, thereby improving the separation of adjacent peaks and avoiding peak overlap.

[0032] In some embodiments, the chromatographic column used in the high-performance liquid chromatography (HPLC) analysis is a phenylsilane-bonded silica column. The phenylsilane-bonded silica column has an inner diameter of 2-5 mm, a length of 100-250 mm, and a packing particle size of 2-5 μm. The conditions for the phenylsilane-bonded silica column are set as follows: flow rate of 1.0-1.5 ml / min, column temperature of 20-35 ℃, and detection wavelength of 360-375 nm. Preferably, the detection wavelength is 360-365 nm, and more preferably, the detection wavelength is 365 nm. This invention uses a phenylsilane-bonded silica column, which, in addition to providing the hydrophobic effect of general reversed-phase chromatography, may also generate π-π interactions between the phenyl stationary phase and the nitro aromatic rings generated after derivatization, further improving the separation effect between structurally similar impurities. Therefore, the method of this invention can not only effectively detect known process impurities but also detect a variety of unknown degradation impurities under forced degradation conditions, demonstrating good impurity coverage.

[0033] In some embodiments of the present invention, the volume percentage of phosphoric acid in the mobile phase is 0.07% to 0.09%. Adding a specific proportion of phosphoric acid to the mobile phase can regulate the acid-base environment of the mobile phase, maintaining a more stable ionization state for tobramycin and its derivatized products, thereby improving chromatographic peak shape and enhancing analytical reproducibility. Simultaneously, an appropriate acidic environment also helps reduce secondary interactions between the analyte and residual silanol groups on the column, avoiding peak tailing caused by ion adsorption. Furthermore, when the phosphoric acid content is controlled within the range of 0.07% to 0.09%, both the retention capacity of the main peak and the separation effect of various related impurities can be considered. When the proportion of phosphoric acid is too low, the acidity of the mobile phase is insufficient, and some amino compounds may have poor peak shapes due to unstable ionization, or even a decrease in the separation degree of adjacent impurities; while when the proportion of phosphoric acid is too high, the retention time of some impurities may be excessively shortened, causing the chromatographic peaks to become too concentrated, thus affecting the separation ability of unknown impurities. Therefore, by controlling the phosphate content in the mobile phase within the aforementioned specific range, the chromatographic separation effect, peak shape symmetry, and method stability of tobramycin and its related impurities can be effectively improved, thereby enhancing the overall reliability of related substances analysis.

[0034] It should be further noted that the present invention also relates to the application of the analytical method described above in the separation and detection of process impurities and degradation impurities in tobramycin sulfate injection, wherein the process impurities include impurity D, impurity F, kanamycin B, niberamamide and neomycin, and the degradation impurities include apramycin, wherein impurity D is 6-O-(3-amino-3-deoxy-α-D-deoxyglucopyranosyl)-2-deoxy-streptomycin, and impurity F is 3-N-carboxytobramycin.

[0035] The process impurities, degradation impurities, and unknown impurities in tobramycin sulfate injection are mostly aminoglycoside structures that are highly similar to tobramycin itself. The only differences between them are in the position of glycosyl substitution, the number of hydroxyl groups, the state of amino substitution, or the degree of local oxidation. Therefore, the components are very similar in terms of polarity, ionization behavior, and chromatographic retention characteristics, which makes it easy for traditional liquid chromatography methods to have overlapping retention times, insufficient peak separation, or ineffective detection of unknown impurities.

[0036] The analytical method of this invention introduces tobramycin and related impurity molecules into nitro aromatic structures with strong UV absorption capabilities through pre-column derivatization reactions. This not only significantly improves the detection sensitivity of each component, but also, due to the differences in the number of reactive amine groups, steric hindrance, and derivatization efficiency among different impurity molecules, the derivatized products exhibit further differences in hydrophobicity and chromatographic retention behavior, which is beneficial for improving the separation effect between structurally similar impurities.

[0037] Furthermore, this invention combines a phenyl-bonded silica stationary phase with a gradient elution procedure, utilizing the differences in hydrophobic interactions, hydrogen bonding, and π-π interactions between the derivatized product and the stationary phase. This allows different process impurities and degradation impurities to be eluted sequentially at different retention times, thereby avoiding peak overlap caused by the simultaneous elution of multiple impurities with similar polarities.

[0038] Especially for apramycin, niberlaxamine, and some unknown degradation impurities, due to their high structural similarity to the main component of tobramycin, it is often difficult to obtain sufficient separation under traditional reversed-phase liquid chromatography conditions. This invention, by adjusting the acetonitrile ratio, phosphoric acid concentration, and gradient change rate in the mobile phase, can effectively alter the distribution balance of each derivatized product between the stationary and mobile phases, thereby improving the separation ability between adjacent peaks and maintaining a high degree of separation between the main peak and adjacent impurity peaks.

[0039] On the other hand, this invention, through forced degradation experiments, discovered that tobramycin may undergo reactions such as glycosidic bond breakage, amino oxidation, hydrolysis, and molecular rearrangement under high temperature, acid, alkali, oxidation, and light conditions, thereby forming a variety of known and unknown degradation products. Because these degradation products exhibit significant differences in polarity and structure, the analytical method must not only possess excellent separation capabilities but also sufficient impurity coverage and stability indication capabilities.

[0040] The analytical method described herein can effectively detect newly added degradation peaks under different degradation conditions while maintaining good purity of the main peak. This demonstrates that the method can effectively distinguish between the main component, process impurities, and degradation impurities, avoiding co-elution of degradation products with the main peak and thus preventing quantification accuracy. Therefore, this invention is not only applicable to routine quality control of tobramycin sulfate injection, but also to formulation stability studies, production process optimization, degradation pathway analysis, and product release testing, which is of great significance for improving drug quality consistency and safety.

[0041] The following examples illustrate the present invention in more detail. However, the present invention is not limited to the following examples and may be implemented by appropriate modifications within the scope of the present invention, and any of them are included within the scope of the present invention.

[0042] Example 1: Chromatographic Detection Samples and Instruments Samples: Test solutions: Tobramycin sulfate injection from Shanghai Hefeng Pharmaceutical Co., Ltd., and Tobramycin sulfate injection from Flynn Pharma Ltd. Accurately measure an appropriate amount of the injection solution and dilute quantitatively with water to prepare a solution containing approximately 0.2 mg per ml.

[0043] Instrument: High-performance liquid chromatograph equipped with an ultraviolet (UV) detector or a diode array detector (DAD).

[0044] Sample preparation Derivatization reagent: Take an appropriate amount of 2,4-dinitrofluorobenzene, dissolve it in anhydrous ethanol and dilute it quantitatively to prepare a solution containing about 10 mg per 1 ml.

[0045] Alkaline buffer solution: Take about 300 mg of tris(hydroxymethyl)aminomethane, place it in a 100 ml volumetric flask, add 20 ml of water to dissolve it, dilute with dimethyl sulfoxide to the mark, shake well, and cool to room temperature.

[0046] Accurately measure 15 ml of the test solution and place it in a 50 ml volumetric flask. Add 10 ml each of the derivatization reagent and the alkaline buffer solution. Heat in a water bath at 60 ℃ ± 2 ℃ for 50 minutes. Dilute to the mark with acetonitrile and shake well. Let stand for 16 hours.

[0047] Chromatographic conditions and gradient elution A phenyl silica gel column was used as the stationary phase, and a water-acetonitrile-phosphoric acid mobile phase was employed with a gradient elution program. Phenylsilane-bonded silica gel (Alltima 4.6 mm × 250 mm, 5 μm or equivalent column) was used as the packing material. The resolution of each organic impurity was optimized by adjusting the proportion of organic phase (acetonitrile) in the mobile phase. Specific parameters are as follows: Mobile phase A: water-acetonitrile-phosphoric acid (950:50:0.8, v / v / v).

[0048] Mobile phase B: water-acetonitrile-phosphoric acid (280:720:0.8, v / v / v).

[0049] Perform gradient elution as shown in Table 1; control the flow rate to approximately 1.2 ml per minute; maintain the column temperature at 25 ℃; control the retention time of the tobramycin main peak in the elution program to between 50 and 60 minutes; set the detection wavelength to 365 nm; and the analyte injection volume to 45 μl. In Table 1, the data below mobile phase A represents the percentage of the volume of mobile phase A relative to the total volume of mobile phases A and B, also referred to as the volume percentage of mobile phase A; the data below mobile phase B represents the percentage of the volume of mobile phase B relative to the total volume of mobile phases A and B, also referred to as the volume percentage of mobile phase B.

[0050] Table 1: Gradient elution was performed using the following gradient table to ensure that the tobramycin main peak eluted within 50-60 minutes and maintained good separation (>1.5) from adjacent impurities:

[0051] The higher acetonitrile content in mobile phase B indicates that its elution capacity is stronger than that of mobile phase A. Since the chromatographic system used is a reversed-phase liquid chromatography system, the stationary phase has relatively strong hydrophobic interactions. Therefore, increasing the acetonitrile content in the mobile phase reduces the hydrophobic forces between the analyte and the stationary phase, allowing the analyte to gradually elute from the column. The gradient elution program of this invention includes multiple stages, each with different functions and separation objectives.

[0052] The first stage is the initial phase of gradient elution, where the volume percentage of mobile phase B increases from an initial percentage to an intermediate percentage. This stage lasts from 0 minutes to 20-30 minutes (preferably about 25 minutes). During this first stage, the proportion of mobile phase B slowly increases from about 21% to about 70%. Specifically, from about 0 to 14 minutes, the proportion of mobile phase B gradually increases from about 21% to about 34%, and further increases to about 70% from about 14 to 25 minutes. Since the acetonitrile content in mobile phase B is higher than that in mobile phase A, the acetonitrile proportion increases gradually at a lower slope, allowing highly polar and structurally similar process impurities, such as kanamycin B, niberamide, and neomycinamide, to be gradually separated over a longer period. The inventors have found that the aforementioned aminoglycoside impurities have similar polarities; if the acetonitrile proportion is rapidly increased in the initial stage of analysis, multiple impurity peaks can easily elute simultaneously, causing peak overlap. Therefore, this invention employs a gentler gradient change in the initial phase to improve the separation capability between process impurities in the initial phase. Furthermore, increasing the proportion of mobile phase B in the latter part of the first stage can enhance the overall elution capacity of the mobile phase, allowing the tobramycin main peak, which has strong retention capacity after derivatization, and some degradation impurities to be gradually eluted. Since the hydrophobicity of tobramycin is significantly increased after derivatization, it is necessary to increase the proportion of organic phase to avoid excessive retention of the main peak in the chromatographic column.

[0053] The second stage is a gradient elution plateau stage, which maintains the aforementioned intermediate percentage. This stage lasts 30 to 40 minutes, preferably approximately 35 minutes. In this embodiment, the time interval of the second stage corresponds to the gradient elution plateau stage in Table 1, starting from approximately 25 minutes and lasting approximately 60 minutes. During the second stage, the proportion of mobile phase B is maintained at approximately 70% to create a stable isocratic elution environment. Multiple structurally similar unknown degradation impurities often accompany the tobramycin peak. If the proportion of organic phase is continuously and rapidly increased, the peak and adjacent unknown impurities may elute simultaneously, thus reducing the separation efficiency. Therefore, by maintaining a fixed organic phase proportion in the later stages, the peak and unknown degradation impurities can be gradually separated under a more stable elution environment, thereby further improving the separation effect near the peak and enhancing the purity of the peak and the coverage of unknown impurities.

[0054] The third stage involves increasing the proportion of mobile phase B to the elution percentage, and this third stage lasts for 2 to 7 minutes, preferably about 5 minutes. In this embodiment, the time segment of the third stage corresponds to the gradient elution post-stage from about 60 minutes to 65 minutes in Table 1. In the third stage, the proportion of mobile phase B is increased to about 95% within about 0.1 minutes and maintained at the elution percentage for about 5 minutes to create high organic phase flushing conditions, which are used to remove highly retained impurities, oxidative degradation products, and some highly hydrophobic derivatization byproducts remaining in the chromatographic column. The inventors have found that if a high proportion of organic phase flushing is not performed, some highly retained substances may remain in the chromatographic column, leading to problems such as ghost peaks, baseline drift, or residual contamination in subsequent analyses. Therefore, by setting a high organic phase flushing stage, column contamination can be effectively reduced and the stability of continuous analysis can be improved.

[0055] The fourth stage is the stage of restoring the proportion of mobile phase B to its initial percentage. This fourth stage lasts for 5 to 9 minutes, preferably about 7 minutes. In this embodiment, the time segment of the fourth stage corresponds to the column equilibration stage from about 65 minutes to 72 minutes in Table 1. In this fourth stage, the proportion of mobile phase B is reduced to the initial percentage within about 0.1 minutes and continues for about 7 minutes to restore mobile phase B to its initial conditions, allowing the chromatographic column to reach a stable equilibrium state again. The inventors have found that if column equilibration is not sufficiently performed, subsequent analyses are prone to problems such as retention time drift, peak shape changes, and decreased resolution. Therefore, by setting a column equilibration stage, this invention can effectively improve the reproducibility, stability, and batch-to-batch consistency of subsequent analyses.

[0056] This method can simultaneously detect process impurities and degradation impurities, including impurity D, impurity F, kanamycin B, niberlaxamine, neomycinamide, and other unknown impurities. The specific structures of these impurities are shown in Table 2 below. Table 2: Impurity Structure

[0057] The structural formulas of each impurity are as follows: Impurity D (BP injection impurity 2):

[0058] Impurity F:

[0059] Neomycin (BP feedstock impurity C):

[0060] Niberamamide (Acetamide) (BP feedstock impurity B):

[0061] Kanamycin B (BP raw material impurity A):

[0062] Apramycin (BP Injection Impurity 1):

[0063] Under the above conditions, the effects of changes in wavelength (365±2 nm), flow rate (1.2±0.05 ml / min), column temperature (25±5 ℃), and the proportion of phosphoric acid in the mobile phase (0.08%±0.01%) on the determination results were detected. The results showed that even after changes in the chromatographic conditions, the impurities could still be effectively separated and detected. Clear separation of the tobramycin main peak from multiple impurity peaks was observed, with the main peak retention time being approximately 50-60 minutes, and good separation between adjacent peaks. Figure 2 As shown in the figure, the upper part is a schematic diagram at a measurement range of 1600 mAU, where some impurity peaks are not clearly visible; therefore, please refer to the schematic diagram at a measurement range of 50 mAU in the lower part. Experimental results show that the described analytical method has good detection performance for impurities D, F, neomycin, niberamide, kanamycin B, and apramycin. The system exhibits good robustness under minor variations in wavelength, flow rate, column temperature, and phosphoric acid ratio, and the impurity peaks remain effectively separated.

[0064] Example 2: Strong Degradation Test To further verify the ability of the analytical method of the present invention to detect degradation products of tobramycin sulfate injection, forced degradation tests were conducted on the test samples under high temperature, acid, alkali, light and oxidative damage conditions, in order to simulate the extreme environmental conditions that the product may be subjected to during preparation, transportation, storage and use, and to observe the changes of the main components of tobramycin and its related impurities.

[0065] The high-temperature degradation test included two conditions: storage at 60 °C for 10 days and heating at 100 °C for 8-9 hours, to evaluate the thermal stability of tobramycin under long-term low-temperature storage and short-term high-temperature conditions. The acid degradation test involved adding 0.1 mol / L hydrochloric acid solution to the test sample and then evaporating it to dryness in a water bath at 60 °C to simulate the hydrolysis of glycosidic bonds under acidic conditions. The alkali degradation test used 1% sodium hydroxide solution to assess the molecular cleavage and degradation that may occur under alkaline conditions. The light degradation test followed the relevant guidelines for drug stability studies, with a total illuminance of not less than 1.2 × 10⁻⁶. 6 The product was continuously irradiated for 10 days under conditions of Lux·hr and near-ultraviolet energy of not less than 2000 W·hr / m² to simulate the impact of long-term light exposure on product quality; the oxidation damage test was conducted by treating the product with 3% hydrogen peroxide solution to assess the degradation trend under oxidative conditions.

[0066] Neither ChP2025 nor USP standards for injections include specific impurities, such as Figure 3 The diagram shows typical peak spectra of various impurities detected by the USP method. The upper part of the diagram is a schematic diagram at a range of 1800 mAU, where some impurity peaks are not obvious. Please refer to the lower part of the diagram at a range of 50 mAU for further information. The BP raw material includes impurities A (kanamycin B), B (niberlaxamine), and C (neomycin); the BP injection standard includes degradation impurities such as impurity 1 (ampramycin) and impurity 2 (impurity D). The forced degradation test results of the formulation samples show that the method can effectively detect all impurities, as shown in Table 3 below.

[0067] Table 3: Impurities detected in the strong degradation test

[0068] Note: High temperature damage 1 (conditions: 60 ℃, 10 days); High-temperature damage 2 (Conditions: 100 ℃, heating for 8~9 hours); Acid destruction (conditions: 2 drops of -0.1 mol / L HCl solution, evaporated to dryness in a 60 ℃ water bath); Alkali destruction (conditions: 2 drops of 1% NaOH solution, evaporate to dryness at 60 ℃); Light damage (conditions: total illuminance not less than 1.2 × 10⁶ Lux·hr, near-ultraviolet energy not less than 2000 W·hr / m², 10 days); Oxidative destruction (conditions: 3% H2O2 solution, 2 drops, evaporated to dryness in a 60 ℃ water bath).

[0069] The experimental results showed that under different degradation conditions, the main peak of tobramycin exhibited varying degrees of degradation, accompanied by the formation of various known and unknown impurities. Known impurities included niberamamide, kanamycin B, neomycin, impurity D, impurity F, and apramycin. Under oxidative, high-temperature, and acidic degradation conditions, several unknown degradation impurity peaks were detected, indicating that tobramycin exhibits a complex degradation pathway under extreme conditions.

[0070] Further analysis revealed significant differences in the types and amounts of impurities generated under different degradation conditions. For example, acidic conditions more readily produce degradation products related to glycosidic bond cleavage, while oxidative conditions more readily form new, unknown oxidized impurities; high-temperature conditions may lead to rearrangement or hydrolysis of some aminoglycoside structures, generating multiple byproducts of similar polarity. These results demonstrate that the method of this invention can not only detect known process impurities but also possesses a broad detection capability for various degradation products. Specifically: Acid-base degradation: Under 0.1 mol / L HCl or 1% NaOH conditions, the tobramycin main peak showed significant degradation. Specifically, under 0.1 mol / L HCl, tobramycin readily underwent glycosidic bond hydrolysis and partial amino structure changes, generating multiple degradation products. Under 1% NaOH conditions, further sugar ring cleavage, molecular rearrangement, or desubstitution reactions may occur, resulting in a similarly significant main peak degradation. No significant co-elution was observed, indicating that the method of this invention has good separation and stability indication capabilities for both acid and alkaline degradation products. Furthermore, the inventors found significant differences in the types of degradation impurities formed under acid and alkaline degradation conditions. Acid degradation conditions more readily produced degradation products formed by glycosidic bond hydrolysis, while alkaline degradation conditions may further generate byproducts produced by sugar ring cleavage, molecular rearrangement, or desubstitution reactions. These results suggest that tobramycin may have different degradation pathways and mechanisms under different degradation environments. Therefore, the method of this invention not only needs to be able to detect multiple types of degradation products, but also needs to be able to effectively separate complex degradation impurity systems generated under different degradation conditions. Through the systematic design of gradient elution procedures and derivatization conditions, the separation between newly added degradation impurity peaks and the tobramycin main peak under different degradation conditions can be effectively achieved, thereby improving the applicability, stability indication ability, and coverage of unknown degradation impurities of the method of this invention for complex degradation systems. Although the main peak area of ​​the test sample decreased to varying degrees, the mass conservation rate of the system remained within a reasonable range of 98.0% to 105.0%. This result indicates that the amount of substance reduced due to the degradation of the main peak has been quantitatively and proportionally converted into various degradation impurities that can be effectively separated and detected by this method. There were no instances of missed detection of degradation products or strong retention or non-elution on the column, thus confirming that the method has a high quantitative detection capability for acid and alkali degradation products. Therefore, by systematically designing the gradient elution procedure and the ratio of organic phases, this invention can simultaneously take into account the detection and separation effects of degradation products of different polarities, thereby improving the applicability of the method to complex degradation systems and the ability to cover unknown degradation impurities.

[0071] Oxidative Degradation: Under 3% H2O2 conditions, the main peak purity factor still met the requirements, and multiple unknown degradation impurities were detected. The excellent detection capability exhibited by this method under oxidative stress testing is primarily due to its unique chemoselectivity based on pre-column derivatization. In the oxidative degradation pathway of H2O2, tobramycin molecules are prone to complex oxidative ring destruction or chemical bond breakage (amino groups in the molecular structure are easily oxidized to nitro groups or undergo chain scission). This method utilizes the reaction mechanism of specific binding between 2,4-dinitrofluorobenzene (DNFB) and primary amino groups to accurately detect ineffective degradation fragments that have lost their core active groups, and to directionally and focusedly detect key degradation products that still retain the aminoglycoside core skeleton (such as the clearly captured main unknown degradation impurities 1 and 2). This highly specific chemical filtration and detection mechanism enables the method of the present invention to more specifically monitor the oxidative stability of the core functional domain of tobramycin sulfate injection. Under oxidative degradation conditions, it can still maintain a good separation effect between the main peak and the adjacent degradation impurity peaks, and no obvious co-elution phenomenon was observed. This shows that the method of the present invention has good separation ability and stability indication ability for oxidative degradation products.

[0072] Separation: Under all destructive conditions, the separation between the main peak and the adjacent impurity peak was greater than 1.58, proving that the method can effectively distinguish between impurities generated during the process and degradation products generated during storage.

[0073] Furthermore, under all forced degradation conditions, the method of the present invention can maintain a good separation effect between the tobramycin main peak and the adjacent impurity peaks. The minimum resolution between the main peak and the adjacent chromatographic peaks is greater than 1.5, and the peaks of each chromatographic peak are intact without obvious tailing or co-elution, which shows that the method has excellent separation ability.

[0074] Meanwhile, peak purity analysis was performed using a diode array detector (DAD). The results showed that the purity of the tobramycin main peak remained at a high level under various degradation conditions, and no degradation impurities were observed to co-elute with the main peak. This further proves that the method of the present invention can effectively distinguish between the main component and degradation products and has good stability indication capability.

[0075] Furthermore, the main peak degradation rate and total impurity content measured under various forced degradation conditions by this invention both conform to the principle of mass conservation, indicating that this method has good recovery ability and detection accuracy for tobramycin and its degradation products, and can truly reflect the degradation of the test sample under different destructive conditions.

[0076] Therefore, the above results fully demonstrate that the analytical method established in this invention has good stability indication ability, impurity coverage ability and separation ability, and is suitable for the detection of related substances, quality control and stability study of tobramycin sulfate injection.

[0077] In summary, the method for analyzing related substances in tobramycin sulfate injection provided by this invention has at least the following technical effects and advantages: (1) The gradient elution procedure is not simply a change in the organic phase ratio, but a systematic design based on the polarity differences of tobramycin and its related impurities, their retention behavior after derivatization, and the elution characteristics of unknown degradation products. For example, in the initial stage of the gradient, a lower slope of organic phase is used to improve the separation effect between highly polar process impurities in the front stage; in the middle stage, a higher proportion of organic phase is maintained to stably separate the tobramycin main peak and its adjacent degradation impurities; in the later stage, the proportion of organic phase is further increased to remove strongly retained impurities and reduce the impact of residual peaks on subsequent analyses. Therefore, this invention can effectively separate and detect various structurally similar aminoglycoside impurities, while simultaneously ensuring the purity of the main peak, the coverage of unknown impurities, and the stability of the analytical method.

[0078] (2) Using 2,4-dinitrofluorobenzene as a pre-column derivatization reagent, the primary amino group in tobramycin and its related impurities reacts with the derivatization reagent to introduce a nitrobenzene ring structure with strong ultraviolet absorption capacity. This effectively solves the technical problem that tobramycin and its aminoglycoside impurities cannot be directly detected by ultraviolet light due to the lack of conjugated chromogenic groups, and significantly improves detection sensitivity and detection capability.

[0079] (3) By systematically optimizing the derivatization conditions, mobile phase composition, gradient elution program and chromatographic separation conditions, the present invention can simultaneously achieve effective separation and detection of multiple process impurities and degradation impurities, including impurity D, impurity F, kanamycin B, niberlammamine, neomycin, apramycin and multiple unknown degradation impurities. Impurity D is 6-O-(3-amino-3-deoxy-α-D-deoxyglucosepyranosyl)-2-deoxy-streptomycin, and impurity F is 3-N-carboxytobramycin, which has good impurity coverage ability.

[0080] (4) The method of the present invention can maintain a good separation effect between the main peak and the adjacent impurity peak under forced degradation conditions such as high temperature, acid, alkali, light and oxidation. The minimum separation degree between the main peak and the adjacent chromatographic peak can reach more than 1.5, and the purity of the main peak is good, which shows that the method of the present invention has excellent separation ability.

[0081] (5) The present invention has been verified by durability tests. When the detection wavelength, flow rate, column temperature and mobile phase composition and other conditions change within a certain range, it can still stably detect and effectively separate various impurities. This shows that the method of the present invention has good robustness, reproducibility and operational stability, and is suitable for the detection, quality control and stability study of related substances in tobramycin sulfate injection.

[0082] (6) Compared with existing pharmacopoeia methods that can only monitor some known impurities, the method of the present invention can further detect all known impurities and a variety of unknown degradation impurities that may exist at present, and can simultaneously complete the detection of multiple types of related substances in a single analysis. Therefore, it is more conducive to comprehensively evaluating product quality and safety and has higher practical application value.

[0083] While the present invention has been disclosed above with reference to the preferred embodiments described above, it is not intended to limit the invention. Those skilled in the art can make modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of patent protection for this invention is determined by the claims appended to this specification. The present invention is not limited to any of the above-described embodiments or features. The present invention may include various additions or modifications to the described embodiments.

Claims

1. An analytical method for tobramycin sulfate injection, characterized in that, The analytical method includes the following steps: Step S01: Derivatize the sample of tobramycin sulfate injection containing tobramycin and impurities using a derivatization reagent; Step S02: Perform high performance liquid chromatography analysis on the derivatized tobramycin sulfate injection sample, using mobile phase A and mobile phase B for gradient elution. Both mobile phase A and mobile phase B are solution systems composed of water, acetonitrile and phosphoric acid, and the acetonitrile content in mobile phase B is higher than that in mobile phase A. The gradient elution includes: In the first stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is increased from an initial percentage to an intermediate percentage; In the second phase, the aforementioned intermediate percentage remains unchanged; In the third stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is increased from the intermediate percentage to the elution percentage; In the fourth stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is restored to the initial percentage.

2. The analytical method as described in claim 1, characterized in that, In the first stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B increases from the initial percentage and reaches the intermediate percentage after 20 to 30 minutes; In the second phase, maintain the intermediate percentage for 30 to 40 minutes; In the third stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is increased from the intermediate percentage to the elution percentage, and elution is performed for 2 to 7 minutes. In the fourth stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is reduced to the initial percentage and this process continues for 5 to 9 minutes.

3. The analytical method as described in claim 1, characterized in that, The initial percentage is 19%-23%, the intermediate percentage is 67%-73%, and the elution percentage is 92%-98%.

4. The analytical method as described in claim 2, characterized in that, In the first stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B increases from the initial percentage, reaching 32%-36% after 12 to 16 minutes.

5. The analytical method as described in claim 2, characterized in that, In the third stage, the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B is increased from the intermediate percentage to the elution percentage within 10 seconds, and elution is performed for 2 to 7 minutes. The fourth stage involves reducing the percentage of the volume of mobile phase B relative to the total volume of mobile phase A and mobile phase B to the initial percentage within 10 seconds, and continuing for 5 to 9 minutes.

6. The analytical method as described in claim 1, characterized in that, The chromatographic column used in the high-performance liquid chromatography analysis is a phenylsilane-bonded silica column.

7. The analytical method as described in claim 6, characterized in that, The phenylsilane-bonded silica gel column has an inner diameter of 2-5 mm, a length of 100-250 mm, and a packing particle size of 2-5 μm.

8. The analytical method as described in claim 6, characterized in that, The conditions for the phenylsilane-bonded silica gel column are set as follows: flow rate of 1.0-1.5 ml / min, column temperature of 20-35 ℃, and detection wavelength of 360-375 nm.

9. The analytical method as described in claim 1, characterized in that, The derivatizing reagent is 2,4-dinitrofluorobenzene; the mobile phase A comprises water, acetonitrile and phosphoric acid, with a volume ratio of (925-975):(45-55):(0.7-0.9); the mobile phase B comprises water, acetonitrile and phosphoric acid, with a volume ratio of (260-300):(700-740):(0.7-0.9).

10. The application of the analytical method according to any one of claims 1 to 9 in the separation and detection of process impurities and degradation impurities in tobramycin sulfate injection, characterized in that, The process impurities include impurity D, impurity F, kanamycin B, niberapamide, and neomycinamine, and the degradation impurities include apramycin. Impurity D is 6-O-(3-amino-3-deoxy-α-D-deoxyglucosepyranosyl)-2-deoxy-streptomycinamine, and impurity F is 3-N-carboxytobramycin.

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