Ion chromatography method for determining netilmicin sulfate related substances
The rapid separation of netilmicin sulfate-related substances by ion chromatography solves the problems of low sensitivity and poor separation ability in existing technologies, achieving efficient and accurate drug quality control and ensuring the quality standards of netilmicin sulfate.
Patent Information
- Application Number
- CN202511991538.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-02-03
AI Technical Summary
In the existing technology, the determination methods for related substances of netilmicin sulfate have low sensitivity, poor separation ability, and limited room for optimization of chromatographic conditions, which makes it difficult to control the quality of the drug.
Ion chromatography was employed using a Welch LP-C18 column. Mobile phase A consisted of 0.05% pentafluoropropionic acid solution-acetonitrile, and mobile phase B consisted of 0.2 mol/L trifluoroacetic acid solution-acetonitrile. Gradient elution was used, with a column temperature of 30-40℃. An integrating pulsed amperometric electrochemical detector was used. By combining the gradient elution program with specific solution preparation, rapid separation of netilmicin sulfate from other similar substances was achieved.
This technology enables rapid and accurate separation and determination of related substances in netilmicin sulfate, improving the accuracy and efficiency of drug quality control and ensuring medication safety.
Smart Images
Figure CN121453973A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical analysis technology, and more specifically to an ion chromatography method for determining related substances of netilmicin sulfate. Background Technology
[0002] Netilmicin sulfate is a semi-synthetic aminoglycoside antibiotic. During its production, the presence of multiple amino and hydroxyl groups makes it prone to impurities. This antibiotic is stable against aminoglycoside acetyltransferases and is particularly sensitive to strains that produce aminoglycoside acetyltransferases and are resistant to other aminoglycoside antibiotics. Netilmicin sulfate is a less common and less nephrotoxic aminoglycoside, with lower ototoxicity compared to gentamicin, tobramycin, and amikacin, and superior clinical efficacy. Due to its greater safety and efficacy, it has become the first-line drug for treating serious infections caused by susceptible Gram-negative bacilli, including Pseudomonas aeruginosa.
[0003] To ensure the research and production quality of netilmicin sulfate, it is particularly urgent for pharmaceutical manufacturers to develop an accurate and rapid analytical method for determining the content of related substances in netilmicin sulfate. Currently, the 2025 edition of the Chinese Pharmacopoeia (ChP2025) still uses ELSD to detect its related substances. This method suffers from low sensitivity, poor separation ability, and limited optimization space for chromatographic conditions. EP10.0 carries the PED method, which has higher sensitivity, but the mobile phase contains a large amount of buffer salts, resulting in long column equilibration time, shortened column life, and poor separation. A review of domestic and foreign literature and patents reveals that current methods for determining impurities in netilmicin sulfate are relatively limited, hindering companies' control over product quality.
[0004] Therefore, there is an urgent need for an accurate and efficient analytical method for determining related substances of netilmicin sulfate. Summary of the Invention
[0005] This invention provides an ion chromatography method for determining related substances of netilmicin sulfate, which can rapidly separate netilmicin sulfate from other similar substances. The method is low in cost, has good separation effect, and strong specificity, which can make up for the shortcomings of existing technologies, ensure the quality control of netilmicin sulfate, continuously improve the quality standards of this type of drug, thereby improving drug quality and further protecting the safety of medication for the general public.
[0006] Specifically, this invention discloses an ion chromatography method for determining related substances of netilmicin sulfate. The method employs ion chromatography with a Welch LP-C18 column or other equivalent columns. The mobile phase A is 0.05% pentafluoropropionic acid solution-acetonitrile, and the mobile phase B is 0.2 mol / L trifluoroacetic acid solution-acetonitrile, using gradient elution. A base is added after the column. The detector is an integrating pulsed amperometric electrochemical detector. The column temperature is 30-40℃, the flow rate is 0.6-1.0 mL / min, and the injection volume is 20-30 μL.
[0007] Preferably, the ratio of 0.05% pentafluoropropionic acid solution to acetonitrile in mobile phase A is 97:3.
[0008] Preferably, the ratio of 0.2 mol / L trifluoroacetic acid solution to acetonitrile in mobile phase B is 96:4. Preferably, the 0.2 mol / L trifluoroacetic acid solution contains 0.1 mol / L sodium hydroxide solution and 0.05% W / V anhydrous sodium sulfate.
[0009] Preferably, the alkaline solution added after the column is a 50% sodium hydroxide solution containing 0.005 mol / L disodium ethylenediaminetetraacetate solution, with a flow rate of 0.2-0.5 ml / min.
[0010] Preferably, the mobile phase flow rate is 0.8 ml / min, the column temperature is 30℃, and the injection volume is 25 μL.
[0011] Preferably, the reference electrode of the integrating pulse amperometric electrochemical detector is an Ag / AgCl composite electrode with a titanium alloy counter electrode; the detection electrode is a gold electrode with a diameter of 3 mm, and the four-potential detection waveform is as follows:
[0012] Preferably, the gradient elution program is as follows:
[0013] Preferably, the method further includes solution preparation: Preparation of test solution: Take 42 mg netilmicin sulfate, place it in a 50 ml volumetric flask, add mobile phase A to dissolve and dilute to the mark, and shake well; Impurity A stock solution: Accurately weigh 18 mg of impurity A sulfate reference standard, place it in a 10 ml volumetric flask, add acetonitrile-water 1:1 to dissolve and dilute to the mark, and shake well; Impurity B stock solution: Weigh 18 mg of impurity B sulfate reference standard accurately, place it in a 10 ml volumetric flask, add acetonitrile-water 1:1 to dissolve and dilute to the mark, and shake well. Netilmicin stock solution: Weigh 14 mg netilmicin sulfate reference standard accurately, place it in a 10 ml volumetric flask, add acetonitrile-water at a 1:1 ratio to dissolve and dilute to the mark, and shake well; Preparation of control solution: Accurately measure 0.5 ml of impurity A stock solution, 1.0 ml of impurity B stock solution and 0.6 ml of netilmicin stock solution, place them in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well; Blank solvent: diluent; The diluent is mobile phase A.
[0014] Preferably, impurity A is sisomicin sulfate and impurity B is 1-N-ethylgaloglycosamine sulfate.
[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention discloses an ion chromatography method for determining netilmicin sulfate-related substances that is convenient, environmentally friendly, and accurate. This method has good precision, high accuracy, good repeatability, and strong specificity, which can make up for the shortcomings of the prior art and strictly control the related substances of netilmicin sulfate. Attached Figure Description
[0016] Figure 1 For the localization chromatograms of each component; Figure 2 This represents the linear range of netilmicin sulfate; Figure 3 The linear range of impurity A; Figure 4 This represents the linear range of impurity B. Detailed Implementation
[0017] The present invention will now be described in detail with reference to various embodiments. However, it should be noted that these embodiments are not intended to limit the present invention. Equivalent changes or substitutions in function, method, or structure made by those skilled in the art based on these embodiments are all within the protection scope of the present invention.
[0018] In this invention, the term "blank solution" refers to a solution prepared under conditions identical to those used when measuring the sample in high-performance liquid chromatography (HPLC) analysis to eliminate interference. The measured result is called the "blank value," which should be subtracted from the sample measurement result to improve the accuracy of the determination.
[0019] In this invention, the term "test solution" refers to the solution of the sample to be tested in an ion chromatography detection and analysis method.
[0020] In this invention, the term "reference solution" refers to a solution of a standard sample in an ion chromatography detection and analysis method.
[0021] In this invention, the term "mobile phase" refers to the substance that carries the analyte forward during ion chromatography.
[0022] This invention discloses an ion chromatography method for determining related substances of netilmicin sulfate, including the main instruments and chromatographic conditions involved: Dionex-ICS6000 ion chromatograph (dual pump, eluent generator, conductivity detector, AS-AP autosampler, Chameleon chromatography workstation).
[0023] The chromatographic column used was a Welch LP-C18 250mm×4.6mm 5μm.
[0024] Reagents and chemicals (Impurity A is sisomicin sulfate, impurity B is 1-N-ethylgaloglycosamine sulfate): Netilmicin sulfate reference standard, Netilmicin sulfate impurity A reference standard, Netilmicin sulfate impurity B reference standard.
[0025] All water used in the experiment was purified water prepared using milli-Q-Reference.
[0026] Preparation of the test solution: Take about 42 mg of this product, place it in a 50 ml volumetric flask, add mobile phase A to dissolve and dilute to the mark, and shake well.
[0027] Impurity A stock solution: Weigh approximately 18 mg of impurity A sulfate reference standard accurately, place it in a 10 ml volumetric flask, add acetonitrile-water (1:1) to dissolve and dilute to the mark, and shake well.
[0028] Impurity B stock solution: Weigh approximately 18 mg of impurity B sulfate reference standard accurately, place it in a 10 ml volumetric flask, add acetonitrile-water (1:1) to dissolve and dilute to the mark, and shake well.
[0029] Netilmicin stock solution: Weigh approximately 14 mg of netilmicin sulfate reference standard accurately, place it in a 10 ml volumetric flask, add acetonitrile-water (1:1) to dissolve and dilute to the mark, and shake well.
[0030] Preparation of control solution: Accurately measure 0.5 ml of impurity A stock solution, 1.0 ml of impurity B stock solution and 0.6 ml of netilmicin stock solution, place them in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well.
[0031] During the assay, the flow rate was 0.8 ml / min; the column temperature was 35℃; the injection volume was 25 μl; gradient elution was used; and the blank solvent, reference standard, and test sample were detected separately. The results are as follows: Figure 1 , Figure 2 , Figure 3 and Figure 4 As shown.
[0032] According to the formula: = Unknown impurity content % = in: Peak area of impurities in the test solution; Peak area of netilmicin in the reference solution; The dilution factor of the test solution; The dilution factor of the reference solution; Weigh netilmicin sulfate reference standard (mg). : Sample weight (mg); The content of the reference standard is retained to one decimal place; L 1: Volatile content of netilmicin sulfate reference standard; L 2: Moisture content of the test sample; 2M netilmicin molecular weight, 951.14; Molecular weight of netilmicin sulfate: 1441.54; = Known impurity content % = in: Peak areas of known impurities in the test solution; Peak areas of known impurities in the reference solution; The dilution factor of the test solution; The dilution factor of the reference solution; : Sample weight of impurity reference standard (mg); : Sample weight (mg); The content of impurity reference standards, retained to one decimal place; Moisture content of the test sample; Total impurities (%) = Known impurity content + 。
[0033] The following examples were performed under the chromatographic conditions described above: Example 1: Limit of Detection and Limit of Quantification Accurately measure each reference standard stock solution, dilute stepwise with diluent, and calculate the limit of detection with a signal-to-noise ratio (S / N) of not less than 3; inject three times consecutively and calculate the limit of quantitation with a signal-to-noise ratio of not less than 10.
[0034] The results showed that the signal-to-noise ratios (SNRs) of netilmicin sulfate, impurity A, and impurity B in the detection limit solution were all ≥3, and the concentrations of netilmicin sulfate, impurity A, and impurity B at 0.070 μg / ml, 0.060 μg / ml, and 0.062 μg / ml were guaranteed for detection. In the quantitation limit solution, the SNRs of netilmicin sulfate, impurity A, and impurity B were all ≥10, and the concentrations of netilmicin sulfate, impurity A, and impurity B at 0.174 μg / ml, 0.151 μg / ml, and 0.155 μg / ml were guaranteed for accurate quantification.
[0035] The results of the limit of quantitation are shown in Table 1.
[0036] Table 1: The detection limit results are shown in Table 2.
[0037] Table 2: The ion chromatography method described in this invention can efficiently separate related substances in netilmicin sulfate, and can also determine the content of related substances in the sample. This method exhibits a wide linear range (0.174-10.435 ug / ml for netilmicin sulfate, 0.151-22.622 ug / ml for impurity A, and 0.155-38.833 ug / ml for impurity B), with good linearity (r = 0.99992, 0.998, 0.999); high sensitivity (limits of quantitation for netilmicin sulfate, impurity A, and impurity B are 0.174 μg / ml, 0.151 μg / ml, and 0.155 μg / ml, respectively); and good repeatability (RSD for impurity B (n=6) is 1.2%, and the RSD for total impurities (n=6) is 1.3%). The method of this invention can rapidly and accurately determine related substances in netilmicin sulfate.
[0038] In summary, the method disclosed in this invention has good precision, high accuracy, good repeatability, and strong specificity, and can rapidly and accurately determine related substances in netilmicin sulfate.
[0039] The above descriptions are merely some embodiments of the present invention. It should be noted that those skilled in the art can make other modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.
Claims
1. An ion chromatography method for determining related substances of netilmicin sulfate, characterized in that, The method employs ion chromatography, using a Welch LP-C18 column or other equivalent columns. The mobile phase A is 0.05% pentafluoropropionic acid solution-acetonitrile, and the mobile phase B is 0.2 mol / L trifluoroacetic acid solution-acetonitrile, with gradient elution. Alkali is added post-column. The detector is an integrating pulsed amperometric electrochemical detector. The column temperature is 30-40℃, the flow rate is 0.6-1.0 mL / min, and the injection volume is 20-30 μL.
2. The method according to claim 1, characterized in that, The ratio of 0.05% pentafluoropropionic acid solution to acetonitrile in the mobile phase A is 97:
3.
3. The method according to claim 2, characterized in that, The ratio of 0.2 mol / L trifluoroacetic acid solution to acetonitrile in the mobile phase B is 96:
4.
4. The method according to claim 3, characterized in that, The 0.2 mol / L trifluoroacetic acid solution contains 0.1 mol / L sodium hydroxide solution and 0.05% W / V anhydrous sodium sulfate.
5. The method according to claim 4, characterized in that, The post-column alkali solution is a 50% sodium hydroxide solution containing 0.005 mol / L disodium ethylenediaminetetraacetate solution, with a flow rate of 0.2-0.5 ml / min.
6. The method according to claim 5, characterized in that, The mobile phase flow rate was 0.8 ml / min, the column temperature was 30℃, and the injection volume was 25 μL.
7. The method according to claim 6, characterized in that, The reference electrode of the integrated pulsed amperometric electrochemical detector is an Ag / AgCl composite electrode with a titanium alloy counter electrode; the detection electrode is a gold electrode with a diameter of 3 mm, and the four-potential detection waveform is as follows: 。 8. The method according to claim 7, characterized in that, The gradient elution procedure is as follows: 。 9. The method according to claim 8, characterized in that, The method also includes solution preparation: Preparation of test solution: Take 42 mg netilmicin sulfate, place it in a 50 ml volumetric flask, add mobile phase A to dissolve and dilute to the mark, and shake well; Impurity A stock solution: Accurately weigh 18 mg of impurity A sulfate reference standard, place it in a 10 ml volumetric flask, add acetonitrile-water 1:1 to dissolve and dilute to the mark, and shake well; Impurity B stock solution: Weigh 18 mg of impurity B sulfate reference standard accurately, place it in a 10 ml volumetric flask, add acetonitrile-water 1:1 to dissolve and dilute to the mark, and shake well. Netilmicin stock solution: Weigh 14 mg netilmicin sulfate reference standard accurately, place it in a 10 ml volumetric flask, add acetonitrile-water at a 1:1 ratio to dissolve and dilute to the mark, and shake well; Preparation of control solution: Accurately measure 0.5 ml of impurity A stock solution, 1.0 ml of impurity B stock solution and 0.6 ml of netilmicin stock solution, place them in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well; Blank solvent: diluent; The diluent is mobile phase A.
10. The method according to claim 9, characterized in that, Impurity A is sisomicin sulfate, and impurity B is 1-N-ethylgaloglycoamine sulfate.