Method for detecting impurity I in amikacin sulfate injection and application

Abstract

The invention discloses a method for detecting an impurity I in an amikacin sulfate injection, which adopts a sulfonic strong ion exchange chromatographic column and a specific buffer solution for gradient elution, and can realize high-sensitivity detection without an ampere detector. Wherein a chromatographic column adopts a sulfonyl strong cation sodium system exchange column, gradient elution is performed by using a mobile phase, the mobile phase consists of a first buffer solution, a second buffer solution and a regeneration solution, and the first buffer solution is a citrate buffer solution I; the second buffer solution is a citrate buffer solution II; the regeneration liquid is an ethylenediaminetetraacetic acid sodium hydroxide solution. By screening and optimizing chromatographic conditions such as flow velocity, temperature, wavelength and the like, accurate quantitative detection of the impurity is realized. According to systematic methodology verification, the method has the advantages of high specificity, high sensitivity, good precision, high accuracy and the like, and the impurity I in the amikacin sulfate injection can be quantitatively or qualitatively detected, so that the safety of clinical medication of the amikacin sulfate injection is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and in particular to a method for detecting impurity I in amikacin sulfate injection. Background Art

[0002] Amikacin sulfate injection is an aminoglycoside antibiotic with an antibacterial spectrum and activity similar to kanamycin. Its mechanism of action is the inhibition of bacterial protein synthesis, similar to other aminoglycoside antibiotics, which exhibit bactericidal properties. The active ingredient, amikacin, is unstable and easily degrades at high temperatures, producing impurities. This leads to unstable product quality and, in turn, affects patient safety. Therefore, research on amikacin's impurities is warranted. The primary degradation impurities are kanamycin, impurity I, and impurity J. Impurity I has the molecular formula (C4H9NO3) and the structural formula is as follows:

[0003]

[0004] Molecular weight 119.12

[0005] 2(S)-4-amino-2-hydroxybutyric acid

[0006] The Chinese Pharmacopoeia standard for kanamycin uses thin-layer chromatography. Impurity J can be detected using the related substance method, but the specificity of impurity I does not meet the requirements. The EP standard's related substance method can detect impurity I, but this method requires an ammeter for analysis, and the sensitivity of impurity I is poor. Therefore, a separate method for detecting impurity I is necessary to effectively determine its content in amikacin sulfate injection. Summary of the Invention

[0007] The present invention aims to solve the problems of high cost and poor sensitivity in the prior art for detecting impurity I in amikacin sulfate injection, and aims to provide a method for detecting impurity I in amikacin sulfate injection and its application. The method has the advantages of strong specificity, high sensitivity, good precision, high accuracy, etc., and can qualitatively or quantitatively detect impurity I in amikacin sulfate injection, thereby improving the safety of clinical use of amikacin sulfate injection.

[0008] The present invention is achieved through the following technical solutions:

[0009] The first object of the present invention is to provide a method for detecting impurity I in amikacin sulfate injection, which uses high performance liquid chromatography to detect impurity I in amikacin sulfate injection. The instrument is an amino acid analyzer, the chromatographic column is a sulfonic acid strong cationic sodium system exchange column, and gradient elution is performed using buffer A (11.8g of trisodium citrate dihydrate and 6.5g of citric acid monohydrate are dissolved in 500ml of water, 70ml of methanol and 5.6ml of hydrochloric acid are added, diluted to 1000ml with water, mixed, and the pH is adjusted to 3.45 with a saturated sodium hydroxide solution) and buffer B (19.6g of trisodium citrate dihydrate, 3.1g of sodium hydroxide, and 5.0g of boric acid are dissolved in water and diluted to 1000ml, mixed, and the pH is adjusted to 10.85 with a saturated sodium hydroxide solution). Sodium hydroxide solution (22g of sodium hydroxide and 0.2g of ethylenediaminetetraacetic acid are dissolved in water and diluted to 1000ml, mixed) is used as regeneration solution D.

[0010] Furthermore, the chromatographic conditions are as follows: a detection wavelength of 570 nm, a buffer pump flow rate of 0.43 ml / ml to 0.47 ml / min, a derivatization pump flow rate of 0.24 ml / min to 0.26 ml / min, and an injection volume of 20 μl to 100 μl.

[0011] Furthermore, the gradient elution conditions are:

[0012] Time 0 min, 98% to 100% buffer A, 0% to 2% buffer B;

[0013] Time 3.5 min, 98%-100% buffer A, 0%-2% buffer B;

[0014] Time 11.0 min, 83%-87% buffer A, 13%-17% buffer B;

[0015] Time 17.0 min, 78%-82% buffer A, 18%-22% buffer B;

[0016] Time 23.0 min, 73%-77% buffer A, 23%-27% buffer B;

[0017] Time 33.0 min, 13%-17% buffer A, 83%-87% buffer B;

[0018] Time 41.0 min, 13%-17% buffer A, 83%-87% buffer B;

[0019] Time 41.1 min, 0% to 2% buffer A, 98% to 100% buffer B;

[0020] Time 48.0 min, 0% to 2% buffer A, 98% to 100% buffer B;

[0021] Time 48.1 min, 100% regeneration solution D;

[0022] Time 52.0 min, 100% regeneration solution D;

[0023] Time 52.1 min, 98% to 100% buffer A, 0% to 2% buffer B;

[0024] Time 65.1 min, 98% to 100% buffer A, 0% to 2% buffer B;

[0025] Furthermore, the temperature program is:

[0026] Time 0.0min, 52~62℃;

[0027] Time 21.0min, 52~62℃;

[0028] Time: 25.0 min, 74-78°C;

[0029] Time 50.0min, 74-78℃;

[0030] Time 55.0min, 52~62℃;

[0031] Time 65.1min, 52~62℃;

[0032] The second purpose of the present invention is to provide the application of the above method in detecting impurity I in amikacin sulfate injection.

[0033] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0034] The present invention achieves accurate quantitative detection of this impurity by screening and optimizing chromatographic conditions such as flow rate, temperature, and wavelength. This method, which has been systematically validated, exhibits strong specificity, high sensitivity, good precision, and high accuracy. It can quantitatively or qualitatively detect impurity I in amikacin sulfate injection, thereby improving the safety of amikacin sulfate injection in clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of impurity I reference solution;

[0036] Figure 2 Schematic diagram of the impurity I sensitivity solution;

[0037] Figure 3 This is a linear relationship diagram of impurity I in Example 2. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with examples and drawings. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0039] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0040] The technical solutions of the specific embodiments of the present invention are as follows:

[0041] A method for detecting impurity I in amikacin sulfate injection comprises detecting impurity I in amikacin sulfate injection with an amino acid analyzer, using a sulfonic acid strong cationic sodium system exchange column as a chromatographic column, performing gradient elution with buffer A (taking 11.8 g of trisodium citrate dihydrate and 6.5 g of citric acid monohydrate, adding 500 ml of water to dissolve, adding 70 ml of methanol and 5.6 ml of hydrochloric acid, diluting to 1000 ml with water, mixing, and adjusting the pH to 3.45 with a saturated sodium hydroxide solution) and buffer B (taking 19.6 g of trisodium citrate dihydrate, 3.1 g of sodium hydroxide, and 5.0 g of boric acid, dissolving in water and diluting to 1000 ml, mixing, and adjusting the pH to 10.85 with a saturated sodium hydroxide solution), and using a sodium hydroxide solution (taking 22 g of sodium hydroxide and 0.2 g of ethylenediaminetetraacetic acid, dissolving in water and diluting to 1000 ml, mixing) as a regeneration solution D.

[0042] The detection wavelength is 570 nm;

[0043] The buffer pump flow rate is 0.43 ml / ml to 0.47 ml / min, preferably 0.45 ml / min;

[0044] The flow rate of the derivatization pump is 0.24 ml / min to 0.26 ml / min, preferably 0.25 ml / min;

[0045] The injection volume is 20 μl to 100 μl, preferably 50 μl;

[0046] The gradient elution conditions are:

[0047] Time 0 min, 98% to 100% buffer A, 0% to 2% buffer B;

[0048] Time 3.5 min, 98%-100% buffer A, 0%-2% buffer B;

[0049] Time 11.0 min, 83%-87% buffer A, 13%-17% buffer B;

[0050] Time 17.0 min, 78%-82% buffer A, 18%-22% buffer B;

[0051] Time 23.0 min, 73%-77% buffer A, 23%-27% buffer B;

[0052] Time 33.0 min, 13%-17% buffer A, 83%-87% buffer B;

[0053] Time 41.0 min, 13%-17% buffer A, 83%-87% buffer B;

[0054] Time 41.1 min, 0% to 2% buffer A, 98% to 100% buffer B;

[0055] Time 48.0 min, 0% to 2% buffer A, 98% to 100% buffer B;

[0056] Time 48.1 min, 100% regeneration solution D;

[0057] Time 52.0 min, 100% regeneration solution D;

[0058] Time 52.1 min, 98% to 100% buffer A, 0% to 2% buffer B;

[0059] Time 65.1 min, 98% to 100% buffer A, 0% to 2% buffer B;

[0060] The temperature program is:

[0061] Time 0.0min, 52~62℃;

[0062] Time 21.0min, 52~62℃;

[0063] Time: 25.0 min, 74-78°C;

[0064] Time 50.0min, 74-78℃;

[0065] Time 55.0min, 52~62℃;

[0066] Time 65.1min, 52~62℃;

[0067] As a preferred embodiment, the present embodiment provides a method for detecting impurity I in amikacin sulfate injection, comprising: using an amino acid analyzer, chromatographic conditions including using a sulfonic acid strong cationic sodium system exchange column, using buffer A (take 11.8g of trisodium citrate dihydrate and 6.5g of citric acid monohydrate, add 500ml of water to dissolve, add 70ml of methanol and 5.6ml of hydrochloric acid, dilute to 1000ml with water, mix well, and adjust the pH to 3.45 with a saturated sodium hydroxide solution) and buffer B (take citric acid dihydrate and 5.6ml of hydrochloric acid, dilute to 1000ml, mix well, and adjust the pH to 3.45 with a saturated sodium hydroxide solution. 19.6 g of trisodium benzoate, 3.1 g of sodium hydroxide, and 5.0 g of boric acid were dissolved in water and diluted to 1000 ml, mixed, and the pH was adjusted to 10.85 with a saturated sodium hydroxide solution for gradient elution. Sodium hydroxide solution (22 g of sodium hydroxide and 0.2 g of ethylenediaminetetraacetic acid were dissolved in water and diluted to 1000 ml, mixed) was used as the regeneration solution D. The detection wavelength was 570 nm, the buffer pump flow rate was 0.45 ml / min, the derivatization pump flow rate was 0.25 ml / min, and the injection volume was 50 μl.

[0068] The gradient elution conditions are:

[0069] Time 0 min, 100% buffer A;

[0070] Time 3.5 min, 100% buffer A;

[0071] Time 11.0 min, 85% buffer A, 15% buffer B;

[0072] Time 17.0 min, 80% buffer A, 20% buffer B;

[0073] Time 23.0 min, 75% buffer A, 25% buffer B;

[0074] Time 33.0 min, 15% buffer A, 85% buffer B;

[0075] Time 41.0 min, 15% buffer A, 85% buffer B;

[0076] Time 41.1 min, 100% buffer B;

[0077] Time 48.0 min, 100% buffer B;

[0078] Time 48.1 min, 100% regeneration solution D;

[0079] Time 52.0 min, 100% regeneration solution D;

[0080] Time 52.1 min, 100% buffer A;

[0081] Time 65.1 min, 100% buffer A;

[0082] The temperature program is:

[0083] Time 0.0 min, 60 °C;

[0084] Time: 21.0 min, 60°C;

[0085] Time: 25.0 min, 76°C;

[0086] Time 50.0 min, 76°C;

[0087] Time 55.0min, 60℃;

[0088] Time: 65.1 min, 60°C;

[0089] The specific embodiments of the present invention are as follows:

[0090] This product refers to amikacin sulfate injection, provided by Hunan Kelun Pharmaceutical Co., Ltd.

[0091] Example 1: Specificity experiment

[0092] Reference substance stock solution: Accurately weigh about 5 mg of impurity I into a 10 ml volumetric flask, dissolve it in 0.02 mol / L hydrochloric acid solution and dilute to the mark, then shake well.

[0093] Reference substance solution: Accurately measure 1 ml of reference substance stock solution, place it in a 50 ml volumetric flask, add 0.02 mol / L hydrochloric acid solution to dilute to the scale, and shake well.

[0094] Test solution: Accurately measure 1 ml of the product and place it in a 50 ml volumetric flask. Add 0.02 mol / L hydrochloric acid solution to dilute to the scale and shake well.

[0095] 100% recovery solution: Accurately measure 1 ml of this product and place it in a 50 ml volumetric flask. Accurately add 1 ml of the reference substance stock solution, dilute to the scale with 0.02 mol / L hydrochloric acid solution, and shake well.

[0096] Chromatographic conditions:

[0097] Chromatographic column: LCAK06 / Na (4.6×150 mm, 7 μm);

[0098] Buffer A: Take 11.8g of trisodium citrate dihydrate and 6.5g of citric acid monohydrate, add 500ml of water to dissolve, add 70ml of methanol and 5.6ml of hydrochloric acid, dilute to 1000ml with water, mix well, and adjust the pH to 3.45 with saturated sodium hydroxide solution.

[0099] Buffer solution B: Dissolve 19.6 g of trisodium citrate dihydrate, 3.1 g of sodium hydroxide, and 5.0 g of boric acid in water and dilute to 1000 ml. Mix well and adjust the pH to 10.85 with a saturated sodium hydroxide solution.

[0100] Regeneration solution D: Take 22g of sodium hydroxide and 0.2g of EDTA, dissolve in water and dilute to 1000ml, mix well;

[0101] The detection wavelength is 570 nm;

[0102] The buffer pump flow rate is 0.45 ml / min;

[0103] The flow rate of the derivatization pump was 0.25 ml / min;

[0104] The injection volume was 50 μl;

[0105] The gradient elution conditions are:

[0106] Time 0 min, 100% buffer A;

[0107] Time 3.5 min, 100% buffer A;

[0108] Time 11.0 min, 85% buffer A, 15% buffer B;

[0109] Time 17.0 min, 80% buffer A, 20% buffer B;

[0110] Time 23.0 min, 75% buffer A, 25% buffer B;

[0111] Time 33.0 min, 15% buffer A, 85% buffer B;

[0112] Time 41.0 min, 15% buffer A, 85% buffer B;

[0113] Time 41.1 min, 100% buffer B;

[0114] Time 48.0 min, 100% buffer B;

[0115] Time 48.1 min, 100% regeneration solution D;

[0116] Time 52.0 min, 100% regeneration solution D;

[0117] Time 52.1 min, 100% buffer A;

[0118] Time 65.1 min, 100% buffer A;

[0119] The temperature program is:

[0120] Time 0.0 min, 60 °C;

[0121] Time: 21.0 min, 60°C;

[0122] Time: 25.0 min, 76°C;

[0123] Time 50.0 min, 76°C;

[0124] Time 55.0min, 60℃;

[0125] Time: 65.1 min, 60°C;

[0126] Detection method: Accurately measure the reference solution, test solution and test sample spiked solution and inject them into the high performance liquid chromatography instrument for detection. The results are shown in Table 1.

[0127]

[0128]

[0129] Table 1 Specificity test results

[0130] As shown in Table 1, the retention time of impurity I in the reference solution and the test solution is consistent, and there is no chromatographic peak at the retention time of impurity I in the test solution, indicating that the method has good specificity.

[0131] Example 2: Linearity and sensitivity test

[0132] A method for detecting impurity I in amikacin sulfate injection, comprising:

[0133] Reference substance stock solution: Accurately weigh about 5 mg of impurity I into a 10 ml volumetric flask, dissolve it in 0.02 mol / L hydrochloric acid solution and dilute to the mark, then shake well.

[0134] Impurity I linear solution: Take the reference substance stock solution and dilute it with 0.02mol / L hydrochloric acid solution to prepare solutions containing impurity I at 20μg / ml, 15μg / ml, 10μg / ml, 5μg / ml, 3μg / ml, 1μg / ml, and 0.5μg / ml, respectively.

[0135] Chromatographic conditions:

[0136] Chromatographic column: LCAK06 / Na (4.6×150 mm, 7 μm);

[0137] Buffer A: Take 11.8g of trisodium citrate dihydrate and 6.5g of citric acid monohydrate, add 500ml of water to dissolve, add 70ml of methanol and 5.6ml of hydrochloric acid, dilute to 1000ml with water, mix well, and adjust the pH to 3.45 with saturated sodium hydroxide solution.

[0138] Buffer solution B: Dissolve 19.6 g of trisodium citrate dihydrate, 3.1 g of sodium hydroxide, and 5.0 g of boric acid in water and dilute to 1000 ml. Mix well and adjust the pH to 10.85 with a saturated sodium hydroxide solution.

[0139] Regeneration solution D: Take 22g of sodium hydroxide and 0.2g of EDTA, dissolve in water and dilute to 1000ml, mix well;

[0140] The detection wavelength is 570 nm;

[0141] The buffer pump flow rate is 0.45 ml / min;

[0142] The flow rate of the derivatization pump was 0.25 ml / min;

[0143] The injection volume was 50 μl;

[0144] The gradient elution conditions are:

[0145] Time 0 min, 100% buffer A;

[0146] Time 3.5 min, 100% buffer A;

[0147] Time 11.0 min, 85% buffer A, 15% buffer B;

[0148] Time 17.0 min, 80% buffer A, 20% buffer B;

[0149] Time 23.0 min, 75% buffer A, 25% buffer B;

[0150] Time 33.0 min, 15% buffer A, 85% buffer B;

[0151] Time 41.0 min, 15% buffer A, 85% buffer B;

[0152] Time 41.1 min, 100% buffer B;

[0153] Time 48.0 min, 100% buffer B;

[0154] Time 48.1 min, 100% regeneration solution D;

[0155] Time 52.0 min, 100% regeneration solution D;

[0156] Time 52.1 min, 100% buffer A;

[0157] Time 65.1 min, 100% buffer A;

[0158] The temperature program is:

[0159] Time 0.0 min, 60 °C;

[0160] Time: 21.0 min, 60°C;

[0161] Time: 25.0 min, 76°C;

[0162] Time 50.0 min, 76°C;

[0163] Time 55.0min, 60℃;

[0164] Time: 65.1 min, 60°C;

[0165] Detection method: Seven linear solutions of different concentrations were accurately measured and injected into the amino acid analyzer for detection. The results are shown in Table 2 and the linear relationship diagram is shown in Figure 3 shown.

[0166]

[0167]

[0168] Table 2 Linearity and sensitivity investigation results

[0169] As shown in Table 2, the linear equation correlation coefficient r reaches 1.0000, indicating a good linear relationship. At the quantitative limit concentration of impurity I, the signal-to-noise ratio is greater than 10, indicating that the chromatographic conditions of the present invention have a high sensitivity for impurity I.

[0170] Example 3: Accuracy Test

[0171] Reference substance stock solution: Weigh about 10 mg of impurity I into a 20 ml volumetric flask, dissolve it in 0.02 mol / L hydrochloric acid solution and dilute to the mark, then shake well.

[0172] Reference substance solution: Accurately measure 1 ml of reference substance stock solution, place it in a 50 ml volumetric flask, add 0.02 mol / L hydrochloric acid solution to dilute to the scale, and shake well.

[0173] Test solution: Accurately weigh 1 ml of the product and place it in a 50 ml volumetric flask. Dissolve it in 0.02 mol / L hydrochloric acid solution and dilute to the mark. Shake well.

[0174] 30%, 100%, and 150% recovery solutions: Accurately weigh 1 ml of this product into a 50 ml volumetric flask. Accurately add 0.3 ml, 1.0 ml, and 1.5 ml of the reference substance stock solution, respectively. Dilute to the mark with 0.02 mol / L hydrochloric acid solution and shake well. Prepare three replicates for each concentration.

[0175] Chromatographic conditions:

[0176] Chromatographic column: LCAK06 / Na (4.6×150 mm, 7 μm);

[0177] Buffer A: Take 11.8g of trisodium citrate dihydrate and 6.5g of citric acid monohydrate, add 500ml of water to dissolve, add 70ml of methanol and 5.6ml of hydrochloric acid, dilute to 1000ml with water, mix well, and adjust the pH to 3.45 with saturated sodium hydroxide solution.

[0178] Buffer solution B: Dissolve 19.6 g of trisodium citrate dihydrate, 3.1 g of sodium hydroxide, and 5.0 g of boric acid in water and dilute to 1000 ml. Mix well and adjust the pH to 10.85 with a saturated sodium hydroxide solution.

[0179] Regeneration solution D: Take 22g of sodium hydroxide and 0.2g of EDTA, dissolve in water and dilute to 1000ml, mix well;

[0180] The detection wavelength is 570 nm;

[0181] The buffer pump flow rate is 0.45 ml / min;

[0182] The flow rate of the derivatization pump was 0.25 ml / min;

[0183] The injection volume was 50 μl;

[0184] The gradient elution conditions are:

[0185] Time 0 min, 100% buffer A;

[0186] Time 3.5 min, 100% buffer A;

[0187] Time 11.0 min, 85% buffer A, 15% buffer B;

[0188] Time 17.0 min, 80% buffer A, 20% buffer B;

[0189] Time 23.0 min, 75% buffer A, 25% buffer B;

[0190] Time 33.0 min, 15% buffer A, 85% buffer B;

[0191] Time 41.0 min, 15% buffer A, 85% buffer B;

[0192] Time 41.1 min, 100% buffer B;

[0193] Time 48.0 min, 100% buffer B;

[0194] Time 48.1 min, 100% regeneration solution D;

[0195] Time 52.0 min, 100% regeneration solution D;

[0196] Time 52.1 min, 100% buffer A;

[0197] Time 65.1 min, 100% buffer A;

[0198] The temperature program is:

[0199] Time 0.0 min, 60 °C;

[0200] Time: 21.0 min, 60°C;

[0201] Time: 25.0 min, 76°C;

[0202] Time 50.0 min, 76°C;

[0203] Time 55.0min, 60℃;

[0204] Time: 65.1 min, 60°C;

[0205] Detection method: Accurately measure the reference solution, test solution and 30%, 100%, and 150% recovery solutions and inject them into the amino acid analyzer for detection. The results are shown in Table 3.

[0206]

[0207]

[0208] Table 3 Accuracy inspection results

[0209] As shown in Table 3, the average recovery rate of impurity I in 9 recovered solutions was 102.06%, with a relative standard deviation (RSD) of 0.8%, indicating good accuracy of the method, indicating good accuracy of impurity I under the chromatographic conditions of the present invention.

[0210] Example 4: Durability Test

[0211] Reference substance stock solution: Weigh about 10 mg of impurity I into a 20 ml volumetric flask, dissolve it in 0.02 mol / L hydrochloric acid solution and dilute to the mark, then shake well.

[0212] Reference substance solution: Accurately measure 1 ml of reference substance stock solution, place it in a 50 ml volumetric flask, add 0.02 mol / L hydrochloric acid solution to dilute to the scale, and shake well.

[0213] Test solution: Accurately weigh 1 ml of the product and place it in a 50 ml volumetric flask. Dissolve it in 0.02 mol / L hydrochloric acid solution and dilute to the mark. Shake well.

[0214] Test sample spike solution: Accurately weigh 1 ml of the product and place it in a 50 ml volumetric flask. Accurately add 1.0 ml of the reference sample stock solution, then dilute to the scale with 0.02 mol / L hydrochloric acid solution and shake well.

[0215] Chromatographic conditions:

[0216] Chromatographic column: LCAK06 / Na (4.6×150 mm, 7 μm);

[0217] Buffer A: Take 11.8g of trisodium citrate dihydrate and 6.5g of citric acid monohydrate, add 500ml of water to dissolve, add 70ml of methanol and 5.6ml of hydrochloric acid, dilute to 1000ml with water, mix well, and adjust the pH to 3.45 with saturated sodium hydroxide solution.

[0218] Buffer solution B: Dissolve 19.6 g of trisodium citrate dihydrate, 3.1 g of sodium hydroxide, and 5.0 g of boric acid in water and dilute to 1000 ml. Mix well and adjust the pH to 10.85 with a saturated sodium hydroxide solution.

[0219] Regeneration solution D: Take 22g of sodium hydroxide and 0.2g of EDTA, dissolve in water and dilute to 1000ml, mix well;

[0220] The detection wavelength is 570 nm;

[0221] The injection volume was 50 μl;

[0222] The durability of impurity I was investigated by changing the flow rate, elution gradient, and temperature gradient. The conditions are set as shown in Table 4.

[0223]

[0224] Table 4 Durability condition parameters

[0225] Detection method: Set the durability condition parameters according to Table 4, accurately measure the reference solution, test solution and test sample spiked solution and inject them into the amino acid analyzer for detection. The results are shown in Table 5.

[0226]

[0227] Table 5 Durability test results

[0228] As shown in Table 5, after fine-tuning the chromatographic conditions, the recovery rates of impurity I in the spiked solution of the test sample were between 100% and 105%, with a recovery rate variation of less than 2%. This indicates that the impurity I method has good robustness under the chromatographic conditions of the present invention.

[0229] In summary, the present invention achieves quantitative detection of impurity I by screening and optimizing chromatographic conditions such as the mobile phase elution program, column temperature program, buffer pump flow rate, and derivatization pump flow rate. This method has been systematically validated and has the advantages of strong specificity, high accuracy, high sensitivity, and strong durability. It can qualitatively or quantitatively detect impurity I in amikacin sulfate injection, thereby improving the safety of the product.

[0230] The above examples are only used to illustrate the present invention. In addition, there are many different implementation methods. These implementation methods are all conceivable by those skilled in the art after understanding the concept of the present invention. Therefore, they are not listed here one by one.

Claims

1. A method for detecting impurity I in amikacin sulfate injection, characterized in that: Impurity I in amikacin sulfate injection was detected by high performance liquid chromatography; the chromatographic column used a sulfonic acid strong cationic sodium system exchange column, and the mobile phase was used for gradient elution. The mobile phase consisted of a first buffer, a second buffer, and a regeneration solution. The first buffer was citrate buffer I; the second buffer was citrate buffer II; and the regeneration solution was ethylenediaminetetraacetic acid sodium hydroxide solution.

2. The detection method according to claim 1, wherein The preparation method of the citrate buffer solution I is as follows: 1) A solution prepared by adding 11.8 g of trisodium citrate dihydrate and 6.5 g of citric acid monohydrate to 500 ml of water; 2) Add 70 ml of methanol and 5.6 ml of hydrochloric acid to the solution, dilute with water to 1000 ml, and mix well; adjust the pH to 3.45 with a saturated sodium hydroxide solution.

3. The detection method according to claim 1, wherein The preparation method of the citrate buffer II is as follows: Dissolve 19.6 g of trisodium citrate dihydrate, 3.1 g of sodium hydroxide, and 5.0 g of boric acid in water and dilute to 1000 ml. Mix well and adjust the pH to 10.85 with a saturated sodium hydroxide solution.

4. The detection method according to claim 1, wherein The flow rate of the buffer pump is 0.43 ml / ml~0.47 ml / min, the flow rate of the derivatization pump is 0.24 ml / min~0.26 ml / min, and the injection volume is 20 μl~100 μl.

5. The detection method according to claim 1, wherein The mobile phase gradient of the gradient elution is: Time 0 min, 98%–100% buffer A, 0%–2% buffer B; Time 3.5 min, 98%-100% buffer A, 0%-2% buffer B; Time 11.0 min, 83%-87% buffer A, 13%-17% buffer B; Time 17.0 min, 78%-82% buffer A, 18%-22% buffer B; Time 23.0 min, 73%-77% buffer A, 23%-27% buffer B; Time 33.0 min, 13%-17% buffer A, 83%-87% buffer B; Time 41.0 min, 13%-17% buffer A, 83%-87% buffer B; Time 41.1 min, 0%–2% buffer A, 98%–100% buffer B; Time 48.0 min, 0%-2% buffer A, 98%-100% buffer B; Time 48.1 min, 100% regeneration solution D; Time 52.0 min, 100% regeneration solution D; Time 52.1 min, 98%–100% buffer A, 0%–2% buffer B; Time: 65.1 min, 98%~100% buffer A, 0%~2% buffer B.

6. The detection method according to claim 1, wherein The temperature gradient of gradient elution is: Time 0.0min, 52~62℃; Time: 21.0 min, 52-62°C; Time: 25.0 min, 74-78°C; Time 50.0min, 74~78℃; Time 55.0min, 52~62℃; Time: 65.1min, 52~62℃.

7. Use of the detection method according to any one of claims 1 to 6 in detecting impurity I in amikacin sulfate injection.

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