A method for determining the content of isopamicin sulfate

Abstract

The invention provides a high performance liquid chromatography method for determining the content of isopamicin sulfate, wherein the method uses an ultraviolet detector, an octadecylsilane bonded silica gel chromatographic column, and an ion pair buffered salt solution as a mobile phase. The method of the invention is simple to operate, has good durability, stable results, high accuracy of the detection result, good repeatability, and low detection cost, and is suitable for determining the content of isopamicin sulfate and its preparations.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for determining the content of isepamicin sulfate. Background Art

[0002] Isopamicin is an aminoglycoside antibiotic formed by the combination of the amino group of gentamicin B and isoserine. It inhibits protein synthesis by binding to bacterial ribosomes, thereby exerting its antibacterial effect. It is used to treat a variety of infectious diseases, such as sepsis, secondary infections caused by trauma, burns and surgical trauma, pneumonia, secondary infections caused by chronic respiratory tract diseases, cystitis, pyelonephritis, and peritonitis.

[0003]

[0004] As shown in Formula I, the clinical use of isopamicin is in the form of sulfate, in which the main structure of isopamicin is three amino sugar rings, containing only one carbonyl group and no conjugated structure. According to relevant information, isopamicin sulfate and its injection are included in the Chinese Pharmacopoeia and the Japanese Pharmacopoeia. The content determination in the Japanese Pharmacopoeia API and injection quality standards adopts the method of ion pair chromatography-post-column derivatization fluorescence detector, and the o-phthalaldehyde derivatizer reacts with isopamicin to generate a fluorescent compound, and the fluorescence detection is used for quantitative analysis; the content determination in the Chinese Pharmacopoeia API and injection quality standards adopts reversed phase chromatography-evaporative light scattering detection method, and the universal detection is used for quantitative analysis. The two determination methods use different principles. The Japanese Pharmacopoeia post-column derivatization fluorescence detection method is complicated to operate, and it is necessary to prepare the mobile phase and derivatization agent at the same time, build the derivatization device and pipeline, and the derivatization agent needs to be stored at low temperature during the entire use process. Relatively speaking, the Chinese Pharmacopoeia evaporative light scattering detection method is more convenient to operate. However, with the long-term use of the evaporative light scattering detection method, we found that the sugar ring contained in the isopamicin structure may remain in the evaporative light scattering detector, resulting in the need for special flushing and maintenance of the instrument after the test to ensure the performance of the detector. This method has poor durability. Based on the above reasons, it is of great significance to develop a chromatographic method with simple operation, good durability and stable results for the determination of the content of isopamicin sulfate. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the existing method of complex operation and poor durability, and provide a method for determining the content of isopamicin sulfate using a high performance liquid chromatography-ultraviolet detector, selecting an ultraviolet detector, using an octadecylsilane bonded silica gel chromatographic column with an ion pair mobile phase system, to achieve the content detection of isopamicin sulfate. The method has obvious advantages, such as simple method operation, good durability, stable results, high accuracy of the test results, good repeatability, low detection cost, and is suitable for the content determination of isopamicin sulfate and its preparations.

[0006] The present invention provides the following technical solutions.

[0007] A method for determining the content of isopamicin sulfate is a high performance liquid chromatography method, characterized in that an ultraviolet detector is selected, an octadecylsilane bonded silica gel chromatographic column is adopted, and an ion pair buffered salt solution is used as a mobile phase.

[0008] The detection wavelength of the ultraviolet detector described in the present method is 200-230 nm, preferably 210-230 nm, and more preferably 210 nm.

[0009] The chromatographic column of the present method is a YMC-Pack ODS-AM column, preferably a YMC-Pack ODS-AM, 150×4 mm, 5 μm.

[0010] The mobile phase of the method is an anhydrous sodium sulfate-sodium pentane sulfonate aqueous solution, preferably 2.84% anhydrous sodium sulfate-0.52% sodium pentane sulfonate, and the pH value is adjusted to 3.0-4.0 with phosphoric acid or glacial acetic acid, and the pH value is more preferably 3.5.

[0011] Furthermore, in the above method of the present invention, the flow rate is 0.8-1.2 ml / min, preferably 1.0 ml / min.

[0012] Furthermore, in the above method of the present invention, the column temperature is 20-30°C, preferably 23-27°C, and more preferably 25°C.

[0013] Furthermore, in the above method of the present invention, the injection volume is 5-50 μl, preferably 10 μl.

[0014] Furthermore, in the above method of the present invention, the concentration of the injection solution is 0.5-2.0 mg / ml, preferably 0.5-1.5 mg / ml, and more preferably 1.0 mg / ml.

[0015] Furthermore, the method of the present invention comprises the following steps: (1) Preparation of reference solution: Take an appropriate amount of isopamicin reference substance, dissolve and dilute with water to prepare a reference solution; (2) Preparation of test solution: Take an appropriate amount of isopamicin sulfate injection and dilute it with water to prepare the test solution; Alternatively, take an appropriate amount of isopamicin sulfate API, dissolve and dilute with water to prepare a test solution; Alternatively, take an appropriate amount of other isopamicin sulfate preparations except isopamicin sulfate injection to prepare the test solution; (3) Take the reference solution and the test solution and inject them into the high performance liquid chromatography for detection.

[0016] In summary, the present invention is designed to determine the content of isepamicin sulfate using a high performance liquid chromatography-ultraviolet detector, which has obvious advantages over existing technical methods, such as post-column derivatization fluorescence detection method and evaporative light scattering detection method, simple operation, good durability, stable results, high detection result accuracy, good repeatability, low detection cost, and is suitable for the content determination of isepamicin sulfate and its preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the chromatogram of the test solution 1 at a wavelength of 200 nm in Example 1; Figure 2 This is the chromatogram of the test solution 1 at a wavelength of 210 nm in Example 1; Figure 3 This is the chromatogram of the test solution 1 at a wavelength of 230 nm in Example 1; Figure 4 It is the chromatogram of blank auxiliary material solution in Example 2; Figure 5 This is the chromatogram of the test solution spiked with impurities in Example 2; Figure 6 Overlapping chromatograms of linear solutions 1 to 5 in Example 3; Figure 7 The chromatograms are overlapped for the high concentration test solution, the medium concentration test solution, and the low concentration test solution in Example 4. DETAILED DESCRIPTION

[0018] The following examples are used to further illustrate and understand the spirit of the present invention, but are not intended to limit the scope of protection of the present invention in any way.

[0019] Example 1 Investigation of detection wavelength Detector and chromatographic conditions: HPLC instrument: Shimadzu, LC-20AD XR ,PDA detector; Detection wavelength: 190nm, 200nm, 210nm, 230nm, 240nm; Mobile phase: 2.84% anhydrous sodium sulfate-0.52% sodium pentane sulfonate-glacial acetic acid aqueous solution (pH 3.5); Chromatographic column: YMC-Pack ODS-AM, 150×4.0 mm, 5 μm; column temperature 25°C; flow rate 1.0 ml / min; injection volume: 10 μl.

[0020] (1) Sample preparation: Reference solution: Take an appropriate amount of isopamicin reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing approximately 1 mg / ml per 1 ml.

[0021] Test solution: Take an appropriate amount of isopamicin sulfate injection and dilute it with water to make a solution containing about 1 mg / ml per 1 ml. Prepare 6 copies in parallel.

[0022] (2) Detection: Take the reference solution and the test solution and inject them into the HPLC instrument, and record the chromatogram. Calculate the measurement results at different wavelengths. The typical chromatogram is shown in Figure 1-Figure 3 , the results are shown in Table 1.

[0023] Table 1 Detection wavelength investigation test results

[0024] When the detection wavelength is 190nm, the baseline noise is high. When the detection wavelength is 240nm, the peak area of ​​isopamicin is low and the response is small. Both situations may lead to large fluctuations in the peak area, affecting the determination results. The range of the contents of the six test solutions at 190nm and 240nm wavelengths is 6.8% and 5.2%, respectively, and the RSD is 2.7% and 1.9%, respectively. It can be seen that the determination results are unstable at this wavelength. When the detection wavelength is 200nm, 210nm, and 230nm, isopamicin has a good ultraviolet response. The average values ​​of the determination results of the six test solutions are 101.2%, 101.4%, and 101.4%, respectively, with a range of 1.6%, 0.4%, and 0.7%, and the RSD of the detected amount is 0.7%, 0.2%, and 0.3%, respectively. The results are parallel, indicating that this method is applicable to a wide wavelength range, and the results are stable and reproducible in the wavelength range of 200nm~230nm.

[0025] Example 2 Specificity Investigation Detector and chromatographic conditions: HPLC instrument: Shimadzu, LC-20AD XR , VWD / PDA detector; Detection wavelength: 210nm; mobile phase: 2.84% anhydrous sodium sulfate-0.52% sodium pentane sulfonate (pH adjusted to 3.5 with phosphoric acid or glacial acetic acid); chromatographic column: YMC-Pack ODS-AM, 150×4.0mm, 5μm; column temperature 25℃; flow rate 1.0ml / min; injection volume: 10μl.

[0026] (1) Sample preparation: Blank excipient solution: Take appropriate amount of sodium chloride and sodium bisulfite, dissolve them in water according to the prescribed amount and quantitatively dilute them to form a blank excipient solution.

[0027] Impurity spiked test solution: Take appropriate amount of D-isopamicin reference substance, HAPA gentamicin B reference substance, 3''-N-Demethyl -1,3''-N-di-HAPA gentamicin B reference substance, 1-N-HAPA-HAPA B reference substance, isoserine reference substance, and gentamicin B reference substance and prepare a solution containing about 1 mg / ml of isopamicin and about 10 μg / ml of each impurity per 1 ml with isopamicin sulfate injection.

[0028] (2) Detection: Take the above blank excipient solution and the impurity spiked test solution and inject them into the high performance liquid chromatograph and record the chromatogram. The typical chromatogram is shown in Figure 4-Figure 5 , the results are shown in Table 2.

[0029] Table 2 Specificity test results

[0030] Blank excipient solutions and impurity-rich spiked test solutions were prepared to investigate the specificity of this method. In order to increase the impurity response, the impurity spiked test solution was determined using a high-sensitivity detector. The separation of the main peak and the adjacent impurity peaks (D-isopamicin and 1-N-HAPA-HAPA B) and the peak purity of the main peak were used as evaluation indicators. The results showed that the main peak of isopamicin and the adjacent impurity peaks were well separated, with separations of 2.2 and 1.8, respectively, and the peak purity of the main peak met the requirements. The blank excipients and isopamicin-related impurities did not interfere with the determination of isopamicin content, and this method had good specificity.

[0031] Example 3 Linearity Investigation Detector and chromatographic conditions: HPLC instrument: Shimadzu, LC-20AD XR , VWD / PDA detector; Detection wavelength: 210nm; mobile phase: 2.84% anhydrous sodium sulfate-0.52% sodium pentane sulfonate-glacial acetic acid aqueous solution (pH 3.5); chromatographic column: YMC-Pack ODS-AM, 150×4.0mm, 5μm; column temperature 25℃; flow rate 1.0ml / min; detection wavelength: 210nm; injection volume: 10μl.

[0032] (1) Sample preparation: Linear solutions 1-5: Take an appropriate amount of isopamicin reference substance, weigh accurately, dissolve in water and quantitatively dilute to make solutions containing approximately 0.5, 0.8, 1.0, 1.2, and 1.5 mg / ml per 1 ml.

[0033] (2) Detection: Take each linear solution and inject it into the liquid chromatograph, and record the chromatogram. The typical chromatogram is shown in Figure 6 .

[0034] (3) Calculation: With the concentration of isopamicin (mg / ml) as the abscissa (X) and the peak area of ​​the main peak as the ordinate (Y), the least square method was used for linear regression analysis to obtain the linear regression equation and linear correlation coefficient r. The results are shown in Table 3.

[0035] Table 3 Linearity test results

[0036] The results showed that the method had good linearity, the linear correlation coefficient was 1.000, and the linear correlation concentration range was at least 0.5 mg / ml~1.5 mg / ml.

[0037] Example 4 Precision and Accuracy Investigation The detector and chromatographic conditions are the same as in Example 3.

[0038] (1) Sample preparation: Reference solution: same as in Example 1.

[0039] Blank excipient solution: same as Example 2.

[0040] Blank test solution: Take an appropriate amount of isopamicin sulfate API, dissolve it in water and quantitatively dilute it to make a solution containing about 1.0 mg / ml per 1 ml. Prepare 3 copies in parallel.

[0041] Low concentration test solution: Take an appropriate amount of isopamicin sulfate API, dissolve it in blank excipient solution and quantitatively dilute it to make a solution containing about 0.8 mg / ml per 1 ml. Prepare 3 copies in parallel.

[0042] Medium concentration test solution: Take an appropriate amount of isopamicin sulfate API, dissolve it in blank excipient solution and quantitatively dilute it to make a solution containing about 1.0 mg / ml per 1 ml. Prepare 3 copies in parallel.

[0043] High concentration test solution: Take an appropriate amount of isopamicin sulfate API, dissolve it in blank excipient solution and quantitatively dilute it to make a solution containing about 1.2 mg / ml per 1 ml. Prepare 3 copies in parallel.

[0044] (2) Detection: Take the reference solution and the test solution and inject them into the liquid chromatograph, and record the chromatogram. The typical chromatogram is shown in Figure 7 .

[0045] (3) Calculation: Calculate the recovery rate and RSD of the recovery rate of 9 test sample solutions at each concentration using the external standard method.

[0046] Table 4 Precision and accuracy results

[0047] The results showed that the recovery rates of the 9 test solutions were 99.7%~100.3%, the average recovery rate was 100.0%, and the RSD of the recovery rate was 0.3%, indicating that this method is accurate, reliable and has good precision.

[0048] Example 5 Study on the durability of chromatographic conditions Detector and chromatographic conditions HPLC instrument 1: Shimadzu, LC-20AD XR , VWD / PDA detector; HPLC instrument 2: Waters, e2695, PDA detector; Detection wavelength: 210nm; mobile phase: 2.84% anhydrous sodium sulfate-0.52% sodium pentane sulfonate (pH adjusted to 3.5 with phosphoric acid or glacial acetic acid); chromatographic column: YMC-Pack ODS-AM, 150×4.0mm, 5μm; injection volume: 10μl, flow rate 1.0ml / min, column temperature 25℃.

[0049] Change condition 1: flow rate 0.9ml / min, 1.1ml / min.

[0050] Change condition 2: mobile phase pH 3.4, 3.6.

[0051] Variation condition 3: column temperature 23°C, column temperature 27°C.

[0052] (1) Sample preparation: The solutions used are the test solution and the reference solution, which are the same as those in Example 1.

[0053] (2) Detection: Inject the test sample solution and the reference sample solution into a liquid chromatograph, record the chromatogram, and calculate the ratio to normal conditions.

[0054] Table 5 Column durability test results

[0055] The results showed that by changing the flow rate, mobile phase pH value, and column temperature, the retention time of the main peak was between 7.3 and 8.9 min, the peak elution was stable, and the detection could be completed within 10 minutes. The detection results were consistent with those under normal conditions. This method has good durability, simplicity, and low detection cost.

Claims

1. A method for determining the content of isopamicin sulfate, the method being a high performance liquid chromatography method, characterized in that: A UV detector was used, an octadecylsilane bonded silica gel chromatographic column was used, an ion pair buffered salt solution was used as the mobile phase, and the isopamicin sulfate was a compound shown in Formula 1 。 2. The method according to claim 1, wherein the ultraviolet detector has a detection wavelength of 200 to 230 nm, preferably 210 to 230 nm.

3. The method according to claim 1, wherein the chromatographic column is a YMC-Pack ODS-AM column.

4. The method according to claim 1, wherein the mobile phase is an anhydrous sodium sulfate-sodium pentane sulfonate aqueous solution.

5. The method according to claim 1 or 4, wherein the mobile phase is a 2.84% anhydrous sodium sulfate-0.52% sodium pentane sulfonate aqueous solution, and phosphoric acid or glacial acetic acid is used to adjust the pH value to 3.0-4.0, preferably the pH value is 3.

5.

6. The method according to claim 1, wherein the flow rate is 0.8-1.2 ml / min, preferably 1.0 ml / min.

7. The method according to claim 1, wherein the column temperature is 20-30°C, preferably 23-27°C.

8. The method according to claim 1, wherein the injection volume is 5-50 μl, preferably 10 μl.

9. The method according to claim 1, wherein the concentration of the injection solution is 0.5-2 mg / ml, preferably 0.5-1.5 mg / ml.

10. The method according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: (1) Preparation of reference solution: Take an appropriate amount of isopamicin reference substance, dissolve and dilute with water to prepare a reference solution; (2) Preparation of test solution: Take an appropriate amount of isopamicin sulfate injection and dilute it with water to prepare the test solution; Alternatively, take an appropriate amount of isopamicin sulfate API, dissolve and dilute with water to prepare a test solution; Alternatively, take an appropriate amount of other isopamicin sulfate preparations except isopamicin sulfate injection to prepare the test solution; (3) Take the reference solution and the test solution and inject them into the high performance liquid chromatography for detection.

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