Detection method of kanamycin in amikacin sulfate injection

Abstract

The present invention relates to the technical field of drug quality control, and in particular to a method for detecting kanamycin in amikacin sulfate injection. The detection method comprises the following steps: S1 dilutes the amikacin sulfate injection with water, and obtains a test solution by derivatization treatment with an acetonitrile solution containing phenyl isothiocyanate and triethylamine; S2 prepares linear solutions of kanamycin sulfate of different concentrations, and obtains a reference solution by derivatization treatment; S3 elutes the test solution and the reference solution by HPLC, and then enters UV detection and detection to calculate the kanamycin content; the mobile phase used for the elution is 0.08-0.12 mol / L sodium acetate solution (pH value is 6.5±0.2)-methanol, and the volume ratio of the two is 65-75:25-35. The detection method of the present invention has low cost, can effectively separate the components to be tested, has high method sensitivity, good specificity, high precision, good accuracy, and accurate and reliable test results.

Description

Technical Field

[0001] The invention relates to the technical field of drug quality control, and in particular to a method for detecting kanamycin in amikacin sulfate injection. Background Art

[0002] The chemical name of Amikacin Sulfate is O-3-amino-3-deoxy-α-D-glucopyranosyl-(1→4)-O-[6-amino-6-deoxy-α-D-glucopyranosyl-(l→6)]-N3-(4-amino-2-hydroxy-1-oxobutyl)-2-deoxy-L-streptamine sulfate. The structural formula of Amikacin Sulfate is as follows:

[0003] .

[0004] Amikacin sulfate is synthesized from the fermentation product kanamycin. Its structural feature is that the aminohydroxybutyryl chain is bound to the streptamine part of the kanamycin A molecule. Its physical, chemical, pharmacological and pharmacokinetic characteristics are similar to those of other aminoglycoside antibiotics. Its action site is the 30S subunit of the bacterial ribosome, which inhibits the synthesis of bacterial proteins and produces a bactericidal effect. Amikacin sulfate belongs to the aminoglycoside antibiotics, which is characterized by its stability to the aminoglycoside inactivating enzymes produced by many intestinal gram-negative bacteria. Such enzymes are inactivated and then lose their antibacterial activity. Few resistant strains are produced, and strains resistant to other aminoglycoside antibiotics are still sensitive to this product.

[0005] The toxic and side effects of aminoglycosides are mainly manifested in nephrotoxicity and ototoxicity, and the incidence of damage to both by kanamycin is higher than that of amikacin. In addition, clinical use of kanamycin can also produce toxic reactions in the hematopoietic system, causing leukopenia. Therefore, it is very necessary to control the content of kanamycin in amikacin sulfate injection. Since the content of kanamycin in amikacin sulfate injection is very low, there is a large amount of interference from excipients, and kanamycin lacks a chromophore that can be detected by ultraviolet light, it brings certain difficulties to the detection of kanamycin. The sources of kanamycin: one is the starting material for raw material synthesis; the other is the degradation product generated by the degradation reaction of amikacin sulfate injection. The structural formula of kanamycin is as follows:

[0006] .

[0007] The European Pharmacopoeia uses HPLC electrochemical detector to detect amikacin sulfate-related substances (including kanamycin). There are also reports on the use of evaporative light scattering to detect kanamycin, but both of these detection methods require the purchase of additional instruments for detection. Therefore, other methods still need to be developed to detect kanamycin. The Chinese Pharmacopoeia uses thin layer chromatography to detect kanamycin, but thin layer chromatography is cumbersome to operate, has low sensitivity, and can only be qualitative but not quantitative.

[0008] Kanamycin in amikacin sulfate does not contain a double-bond side chain structure and cannot absorb ultraviolet light. Therefore, it needs to be derivatized like amino acids to absorb ultraviolet light for ultraviolet detection.

[0009] Chinese patent publication number CN103512965A discloses a method of using HPLC pre-column derivatization to quantify kanamycin in amikacin sulfate injection. However, the 2,4,6-trinitrobenzenesulfonic acid used in the method is a controlled reagent and is expensive. The price of 10mL of 5% 2,4,6-trinitrobenzenesulfonic acid aqueous solution is as high as 3,000 yuan.

[0010] Chinese patent publication number CN114236022A discloses the use of another derivatization reagent, phenyl isothiocyanate, to derivatize kanamycin. This reagent is cheap and easily available. However, after reproducing the method, it was found that the separation between the kanamycin peak and the derivatization reagent peak was poor, and the recovery rate exceeded the range of 80%-115%, which did not meet the requirements.

[0011] Therefore, it is very necessary to develop a method for detecting kanamycin in amikacin sulfate injection that can solve the above technical problems. Summary of the invention

[0012] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for detecting kanamycin in amikacin sulfate injection, which has low cost, can effectively separate the components to be tested, has high method sensitivity, good specificity, high precision, good accuracy, and accurate and reliable detection results.

[0013] The present invention is achieved through the following technical solutions:

[0014] A method for detecting kanamycin in amikacin sulfate injection comprises the following steps:

[0015] S1 dilutes amikacin sulfate with water and obtains a test solution after derivatization;

[0016] S2 prepares linear solutions of kanamycin sulfate of different concentrations, and obtains reference solutions after derivatization treatment;

[0017] S3: The test solution and the reference solution are eluted by HPLC, and then enter into a UV detector for detection, and the kanamycin content is calculated;

[0018] In step S1 or S2, the derivatization reagent used in the derivatization treatment is an acetonitrile solution containing phenyl isothiocyanate and triethylamine; after the derivatization is completed, sulfuric acid solution is added to terminate the derivatization;

[0019] In step S3, the mobile phase used for elution is 0.08-0.12 mol / L sodium acetate solution-methanol, the volume ratio of the two is 65-75:25-35, and the pH value of the sodium acetate solution is 6.5±0.2.

[0020] The mechanism of the derivatization treatment is that phenyl isothiocyanate (derivation reagent) reacts with kanamycin under alkaline conditions (addition of triethylamine) to generate a kanamycin derivative having ultraviolet absorption and can be used for ultraviolet detection. The specific reaction is as follows:

[0021] .

[0022] In some embodiments of the present invention, the concentration of the sodium acetate solution is 0.1 mol / L.

[0023] In some embodiments of the present invention, the preparation of the test solution in step S1 includes:

[0024] S11 dilutes amikacin sulfate with water to obtain solution A;

[0025] S12 adds an acetonitrile solution containing phenyl isothiocyanate and triethylamine to carry out derivatization to obtain solution B;

[0026] S13 is added with sulfuric acid solution, and then fixed to volume with acetonitrile solution to obtain.

[0027] In some preferred embodiments of the present invention, in step S11, each 1 mL of solution A contains 4.0 mg-5.0 mg of amikacin.

[0028] In some preferred embodiments of the present invention, in step S12, the mass ratio of phenyl isothiocyanate to triethylamine is 1:1.

[0029] In some preferred embodiments of the present invention, in step S12, the concentration of phenyl isothiocyanate in the acetonitrile solution containing phenyl isothiocyanate and triethylamine is 2.5 mg / mL.

[0030] The acetonitrile solution containing phenyl isothiocyanate and triethylamine in the present invention can be prepared by simultaneously adding 2.5 mg of phenyl isothiocyanate and 2.5 mg of triethylamine to 1 mL of acetonitrile; or 5 mg / mL of phenyl isothiocyanate acetonitrile solution and 5 mg / mL of triethylamine acetonitrile solution are prepared separately, and then mixed in equal volumes to prepare the solution. The latter preparation method is preferred.

[0031] In some preferred embodiments of the present invention, in step S12, the derivatization conditions are: temperature 60-80°C, preferably 70°C; time 10-30 min, preferably 20 min.

[0032] In some preferred embodiments of the present invention, the volume ratio of solution A to the acetonitrile solution containing phenyl isothiocyanate and triethylamine is 1:4.

[0033] In some preferred embodiments of the present invention, in step S13, the concentration of the sulfuric acid solution is 0.1 mol / L-0.2 mol / L.

[0034] In some preferred embodiments of the present invention, the volume ratio of solution A to sulfuric acid solution is 1:0.4-0.6, preferably 1:0.5.

[0035] In some preferred embodiments of the present invention, in step S13, the volume concentration of the acetonitrile solution is 50%.

[0036] In some embodiments of the present invention, the derivatization steps of step S2 and step S1 are the same.

[0037] In some embodiments of the present invention, step S2 comprises the following steps:

[0038] Accurately weigh an appropriate amount of kanamycin sulfate, dissolve it in purified water and dilute it to make a solution containing 20µg of kanamycin per 1mL, accurately pipette 0.08mL, 0.2mL, 1mL, 2mL and 6mL of 20.0µg / mL kanamycin solution into a 20mL volumetric flask, respectively, add 2mL of 5mg / mL phenyl isothiocyanate acetonitrile solution and 2mL of 5mg / mL triethylamine acetonitrile solution, shake well, react in a 60-80℃ water bath for 10-30min, take it out, add 0.5mL of 0.1mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the scale to obtain a reference solution; the reference solutions of different concentrations correspond to kanamycin linear solutions containing 0.08µg / mL, 0.2µg / mL, 1µg / mL, 2µg / mL and 6µg / mL, respectively, based on the kanamycin concentration.

[0039] In some embodiments of the present invention, the detection wavelength of the HPLC method in step S3 is 242 nm.

[0040] In some embodiments of the present invention, in step S3, a 0.1 mol / L-0.2 mol / L sulfuric acid solution is used to adjust the pH of the sodium acetate solution to 6.5±0.2, preferably a 0.1 mol / L sulfuric acid solution, and the pH value is preferably 6.5.

[0041] In some embodiments of the present invention, the flow rate of the mobile phase in step S3 is 0.8-1.2 mL / min, preferably 1.0 mL / min.

[0042] In some embodiments of the present invention, the column temperature of the HPLC method in step S3 is 25-35°C, preferably 30°C.

[0043] In some embodiments of the present invention, the injection volume of the HPLC method in step S3 is 10 μL.

[0044] In some embodiments of the present invention, the chromatographic column used in the HPLC method in step S3 is an octadecylsilane bonded silica gel chromatographic column.

[0045] In some embodiments of the present invention, the elution method in step S3 is isocratic elution, and the elution time is 60 minutes.

[0046] In the present invention, 5 mg / mL phenyl isothiocyanate acetonitrile solution and 5 mg / mL triethylamine acetonitrile solution are preferably used to derive kanamycin in amikacin sulfate injection to generate kanamycin derivatives with ultraviolet absorption. After the derivatization is completed, 0.5 mL of 0.1 mol / L sulfuric acid solution is quickly added to adjust the derivatization solution from alkaline to acidic, and the derivatization reaction is terminated. A chromatographic column with octadecyl bonded silica gel as the stationary phase, methanol and 0.1 mol / L sodium acetate buffer solution are used as the mobile phase for isocratic elution, and the kanamycin in amikacin sulfate injection can be detected quickly and efficiently, so that the kanamycin detection method of this product has strong specificity, high precision, good accuracy, convenient operation, high cost performance, and can effectively control product quality.

[0047] The present invention optimizes the chromatographic conditions of Chinese Patent Publication No. CN114236022A, and studies the optimized method. The specificity meets the requirements, and the separation between the kanamycin derivative peak and the amikacin derivative peak and the derivatization reagent peak all meet the requirements. However, when the accuracy is investigated, it is found that the recovery rate of the solution exceeds the range of 80%-115%, which does not meet the requirements. Subsequently, the derivatization conditions are optimized, and the method after the chromatographic conditions and the derivatization conditions are optimized is used to perform methodological verification. The accuracy, precision, specificity, quantitative limit, detection limit, linearity and range, and durability all meet the requirements. The optimized method can accurately quantify the content of kanamycin in amikacin sulfate injection.

[0048] The beneficial effects of the present invention are:

[0049] The present invention optimizes the chromatographic conditions so that the separation between the kanamycin derivative peak and the amikacin derivative peak and the derivatization reagent peak meets the requirements, and optimizes the derivatization conditions so that the accuracy of the method meets the requirements; the HPLC pre-column derivatization method provided by the present invention has high cost performance, is safe and environmentally friendly, can effectively separate and determine kanamycin in amikacin sulfate injection, and has high sensitivity, strong specificity, and good precision and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a diagram of the Kanamycin Detection-Blank Solution of Amikacin Sulfate Injection in Example 1.

[0051] Figure 2 for Figure 1 The vertical axis enlargement diagram.

[0052] Figure 3 This is a diagram of the Kanamycin Detection-Blank Excipient Solution of Amikacin Sulfate Injection in Example 1.

[0053] Figure 4 for Figure 3 The vertical axis enlargement diagram.

[0054] Figure 5 This is a diagram of the Kanamycin Detection-reference solution of Amikacin Sulfate Injection in Example 1.

[0055] Figure 6 for Figure 5 The vertical axis enlargement diagram.

[0056] Figure 7 This is a diagram of the test solution of amikacin sulfate injection kanamycin detection in Example 1.

[0057] Figure 8 This is the kanamycin detection-linear relationship diagram of Example 1. DETAILED DESCRIPTION

[0058] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0059] Example 1

[0060] The reagents, equipment, and process parameters used in this example are as follows:

[0061] (1) Amikacin sulfate injection was obtained from Zhongfu Pharmaceutical Co., Ltd., batch number: C30220401, specification: 2 mL: 0.2 g;

[0062] (2) The source of kanamycin reference substance is China Food and Drug Inspection Institute, batch number is 130556-201502, and the content is 67.9%;

[0063] Instrument: Liquid chromatograph Thermo U3000;

[0064] Chromatographic column: Octadecylsilane bonded silica gel as filler (Phenomemex Hydro-12p80 column, 4.6mm×150mm, 4µm or chromatographic column with equivalent performance);

[0065] Mobile phase: 0.1 mol / L sodium acetate solution (adjust the pH value to 6.5 with 0.1 mol / L sulfuric acid solution)-methanol (70:30) as the mobile phase;

[0066] Detector: Ultraviolet absorption detector (UV);

[0067] Detection wavelength: 242nm;

[0068] Flow rate: 1.0 mL / min;

[0069] Column temperature: 30°C;

[0070] Injection volume: 10 μL;

[0071] Chromatography workstation: Chameleon.

[0072] Preparation of derivatization reagents:

[0073] 5 mg / mL phenyl isothiocyanate acetonitrile solution: Accurately weigh 500 mg of phenyl isothiocyanate, place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and the solution is ready.

[0074] 5 mg / mL triethylamine acetonitrile solution: Accurately weigh 500 mg of triethylamine, place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0075] Test sample stock solution: Take an appropriate amount of Amikacin Sulfate Injection, dissolve it in purified water and dilute it to make a solution containing approximately 4.0 mg of amikacin per 1 mL.

[0076] Preparation of test solution: accurately measure 1 mL of the test stock solution and place it in a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70 °C water bath for 20 min, take out and immediately add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and let cool, then dilute with 50% acetonitrile and make up to the mark;

[0077] Preparation of blank solution: accurately measure 1 mL of water and place it in a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and immediately add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the scale.

[0078] Preparation of reference solutions (linear solution 1-linear solution 5):

[0079] Reference substance stock solution: Accurately weigh an appropriate amount of kanamycin (kanamycin sulfate), dissolve it in purified water and dilute it to make a solution containing 20 µg of kanamycin per 1 mL.

[0080] Linear solution 1: Accurately pipette 0.08 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and let cool, then dilute with 50% acetonitrile and make up to the mark; prepare a solution containing 0.08 µg / mL kanamycin (calculated as kanamycin concentration).

[0081] Linear solution 2: Accurately pipette 0.2 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and let cool, then dilute with 50% acetonitrile and make up to the mark; prepare a solution containing 0.2 µg / mL kanamycin (measured in kanamycin concentration).

[0082] Linear solution 3: Accurately pipette 1 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and let cool, then dilute with 50% acetonitrile and make up to the mark; prepare linear solutions containing 1 µg / mL kanamycin (based on kanamycin concentration).

[0083] Linear solution 4: Accurately pipette 2 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and let cool, then dilute with 50% acetonitrile and make up to the mark; prepare a solution containing 2 µg / mL kanamycin (measured in kanamycin concentration).

[0084] Linear solution 5: Accurately pipette 6 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and let cool, then dilute with 50% acetonitrile and make up to the mark; prepare a solution containing 6 µg / mL of kanamycin (based on kanamycin concentration).

[0085] Accuracy test:

[0086] Accuracy-quantitation limit: accurately measure 1 mL of reference substance stock solution, place in a 25 mL volumetric flask, add water to dissolve and dilute to the mark, shake well, accurately measure 2 mL of the diluted reference substance stock solution, place in a 20 mL volumetric flask, accurately measure 1 mL of the test substance stock solution, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70 ° C water bath for 20 min, take out and add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the mark (prepare 3 copies in parallel);

[0087] Accuracy -75%: Take 1mL of the test sample stock solution and place it in a 20mL volumetric flask, accurately add 3mL of the reference sample stock solution, add 2mL of 5mg / mL phenyl isothiocyanate acetonitrile solution, 2mL of 5mg / mL triethylamine acetonitrile solution, shake well, react in a 70℃ water bath for 20min, take out and add 0.5mL of 0.1mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the scale (prepare 3 copies in parallel);

[0088] Accuracy -100%: Take 1mL of the test sample stock solution and place it in a 20mL volumetric flask, accurately add 4mL of the reference sample stock solution, add 2mL of 5mg / mL phenyl isothiocyanate acetonitrile solution, 2mL of 5mg / mL triethylamine acetonitrile solution, shake well, react in a 70℃ water bath for 20min, take out and add 0.5mL of 0.1mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the mark (prepare 3 copies in parallel);

[0089] Accuracy -125%: Take 1mL of the test sample stock solution and place it in a 20mL volumetric flask, accurately add 5mL of the reference sample stock solution, add 2mL of 5mg / mL phenyl isothiocyanate acetonitrile solution, 2mL of 5mg / mL triethylamine acetonitrile solution, shake well, react in a 70℃ water bath for 20min, take out and add 0.5mL of 0.1mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the mark (prepare 3 copies in parallel);

[0090] Take 10 μL of the corresponding test solution and reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, calculate the kanamycin content in the test solution based on the linear relationship generated by the reference solution, and calculate the accuracy. The results are shown in Table 1.

[0091] Table 1 Accuracy

[0092]

[0093] The results showed that the mean recovery rate at the quantitative limit concentration was 83.3%. In the range of 75%-120%, the relative standard deviation (RSD%) of the three recoveries was 4.34%, no more than 10%, which met the requirements; the mean recoveries at concentrations of 75%, 100% and 125% were 109.3%, 99.1% and 106.4% respectively, all within the range of 80%-115%. The relative standard deviation (RSD%) of the nine recovery data was 4.51%, no more than 5%, which met the requirements. The method has good accuracy.

[0094] Precision test:

[0095] Repeatability solution: Take 1 mL of the test sample stock solution and place it in a 20 mL volumetric flask, accurately add 4 mL of the reference sample stock solution, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution, and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the mark (prepare 6 copies in parallel).

[0096] Intermediate precision solution: Six samples of the test solution were prepared by different people on different days and analyzed on different instruments.

[0097] Take 10 μL of the corresponding test solution and reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, calculate the kanamycin content in the test solution based on the linear relationship generated by the reference solution, and calculate the repeatability and intermediate precision. The results are shown in Table 2.

[0098] Table 2 Repeatability and intermediate precision

[0099]

[0100] The results showed that the relative standard deviations (RSD%) of the kanamycin content in the six samples provided by the two analysts were 0.90% and 3.65%, respectively, which were no more than 5%, meeting the repeatability requirements; the difference in the mean values ​​of the kanamycin content in the six samples provided by the two experimenters was 0.20%, which was no more than 0.20%, meeting the requirements, and the precision of this method was good.

[0101] Limit of quantitation test:

[0102] Quantitation limit solution: Accurately measure 1 mL of the reference stock solution, place it in a 25 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, then accurately measure 2 mL, place it in a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70 ° C water bath for 20 min, take it out and add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the scale.

[0103] Take 10 μL of the quantitative limit solution and inject it into the liquid chromatograph respectively, record the chromatogram, and calculate the quantitative limit results. The results are shown in Table 3.

[0104] Table 3 Limit of quantitation

[0105]

[0106] The results showed that the intensity ratios of the kanamycin derivative peaks and noise peaks of the six quantitative limit samples were 12.7, 10.9, 13.6, 14.0, 18.9, and 13.2, all not less than 10; the RSD% of the peak areas of the six quantitative limit samples kanamycin derivatives was 5.97%, not more than 10%. The quantitative limit of this method is 0.04%.

[0107] Detection limit test:

[0108] Detection limit solution: Accurately measure 3 mL of the reference stock solution, place it in a 10 mL volumetric flask, dilute to the mark with water, and shake well; accurately measure 1 mL, place it in a 25 mL volumetric flask, dissolve it in water and dilute to the mark, shake well, then accurately measure 2 mL, place it in a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70 ° C water bath for 20 min, take it out, add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the mark.

[0109] Take 10 μL of the detection limit solution and inject it into the liquid chromatograph respectively, record the chromatogram, and calculate the quantitative limit results. The results are shown in Table 4.

[0110] Table 4 Detection limits

[0111]

[0112] The results showed that when the sample concentration was 200ug / mL, the detection limit of kanamycin was 0.01%, and the signal-to-noise ratio was 4.4, not less than 3. That is, when the kanamycin content in the test sample reached 0.01%, it could be detected, and this method had a high detection sensitivity.

[0113] Preparation of specific solution:

[0114] Blank excipient solution: Take 1 mL of blank excipient solution without amikacin, place it in a 25 mL volumetric flask, add water to dilute to the scale, and shake well; accurately measure 1 mL and place it in a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70 ° C water bath for 20 min, take it out and immediately add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the scale.

[0115] Take 10 μL of blank solution, blank excipient solution, linear solution 4, and test solution, respectively, and inject them into the liquid chromatograph to record the chromatograms. The detection results of kanamycin and amikacin sulfate are shown in Table 5; the chromatograms of blank solution, blank excipient solution, reference solution, and test solution are shown in Figure 1-Figure 7 As shown, wherein "KAN" represents kanamycin.

[0116] Table 5 Specificity test results

[0117]

[0118] The results showed that the blank solution and blank excipient solution in the specific solution had no interference with the detection of kanamycin.

[0119] Linearity test:

[0120] Take 10 μL of each of linear solution 1, linear solution 2, linear solution 3, linear solution 4, and linear solution 5, inject them into the liquid chromatograph, and record the chromatogram. With kanamycin concentration as the horizontal axis and kanamycin derivative peak area as the vertical axis, a linear regression equation is drawn. The results are shown in Table 6; the linear relationship diagram is shown in Figure 8 .

[0121] Table 6 Linearity and range

[0122]

[0123] The results showed that when the kanamycin concentration was 0.0821μg / mL-6.1579μg / mL, the linear regression equation was y = 18.2164x - 0.4916; the correlation coefficient (r) was 0.9991, not less than 0.99. In the above concentration range, there was a good linear relationship between the kanamycin concentration and the peak area of ​​kanamycin derivatives.

[0124] Durability test:

[0125] (1) Linear solution 4, accuracy -100% solution was placed at room temperature for 0, 8, 35, and 51 hours, 10 μL was taken and injected into the liquid chromatograph, and the chromatogram was recorded. The kanamycin content in the test solution was calculated based on the linear relationship generated by the reference solution, and the solution stability results were calculated. The results are shown in Table 7.

[0126] Table 7 Durability-Solution Stability

[0127]

[0128] The results showed that the ratio of the peak area of ​​the reference solution at each time point to the peak area at 0h was in the range of 95%-105%, and the solution was stable within 51 hours. The kanamycin content of the test solution (with sample added) at each time point was compared with that at 0h, and the difference in kanamycin content was no more than 0.20%, and the solution was stable within 36 hours, indicating that this method had good durability.

[0129] (2) Take 10 μL of blank solution, reference solution (linear solution 1-linear solution 5), and accuracy-100% solution, respectively, and inject them into the liquid chromatograph under different chromatographic conditions, and record the chromatograms. Calculate the kanamycin content in the test solution based on the linear relationship generated by the reference solution, and calculate the durability results of different chromatographic conditions. The results are shown in Table 8.

[0130] Table 8 Durability - Chromatographic Conditions

[0131]

[0132] The results showed that when the mobile phase pH was 6.5±0.2 and the proportion of methanol in the mobile phase was 30%±5%, the linear correlation coefficient (R) was greater than 0.995, the peak area of ​​the response reference solution accounted for the peak area of ​​the reference solution in the range of 95%-105%, and the system applicability of the method met the requirements. The kanamycin difference under each detection condition was less than 0.20%, so the durability of the method was good under each chromatographic condition.

[0133] Sample testing:

[0134] Take samples of the test sample at 0 day, high temperature (60℃) for 5 days, high temperature (60℃) for 10 days, high temperature (60℃) for 30 days, accelerated (40℃, 75%±5%RH) for 3 months, and accelerated (40℃, 75%±5%RH) for 6 months, and prepare them according to the above test sample solution preparation method. The linear solution is the same as the above linear solution 1 to linear solution 5.

[0135] Take 10 μL of each of the above solutions and inject them into the liquid chromatograph to record the chromatogram. Calculate the kanamycin content in the test solution based on the linear relationship generated by the reference solution, and calculate the solution stability results. The results are shown in Table 9.

[0136] Table 9 Stability sample test results

[0137]

[0138] The results showed that this method can accurately quantify the content of kanamycin in amikacin sulfate injection and monitor the change trend of kanamycin during the stability study, which is conducive to better monitoring the quality and safety of amikacin sulfate injection products.

[0139] Example 2

[0140] Instrument: Liquid chromatograph Thermo U3000;

[0141] Chromatographic column: Octadecylsilane bonded silica gel was used as filler (Phenomemex Hydro-12p80 column, 4.6 mm × 150 mm, 4 µm);

[0142] Mobile phase: 0.1 mol / L sodium acetate (adjust the pH to 6.5 with 0.2 mol / L sulfuric acid solution)-methanol (70:30) as the mobile phase;

[0143] Detector: Ultraviolet absorption detector (UV);

[0144] Detection wavelength: 242nm;

[0145] Flow rate: 1.0 mL / min;

[0146] Column temperature: 30°C;

[0147] Injection volume: 10 μL;

[0148] Chromatography workstation: Chameleon.

[0149] 5 mg / mL phenyl isothiocyanate acetonitrile solution: Accurately weigh 500 mg of phenyl isothiocyanate, place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and the solution is ready.

[0150] 5 mg / mL triethylamine acetonitrile solution: Accurately weigh 500 mg of triethylamine, place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0151] Stock solution of test sample: Take an appropriate amount of amikacin sulfate injection and dilute it with purified water to make a solution containing approximately 4.0 mg of amikacin per 1 mL.

[0152] Preparation of test solution: Take 1 mL of the test stock solution and place it in a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 60 °C water bath for 10 min, take out and add 0.5 mL of 0.2 mol / L sulfuric acid, mix well and let cool, then dilute with 50% acetonitrile and make up to the mark;

[0153] Preparation of blank solution: accurately measure 1 mL of water and place it in a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 60°C water bath for 10 min, take out and add 0.5 mL of 0.2 mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the scale.

[0154] Preparation of reference solutions (linear solution 1-linear solution 5):

[0155] Reference stock solution: Accurately weigh an appropriate amount of kanamycin (kanamycin sulfate), dissolve and dilute with purified water to make a solution containing 20 µg of kanamycin per 1 mL

[0156] Linear solution 1: Accurately pipette 0.08 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 60°C water bath for 10 min, take out and add 0.5 mL of 0.2 mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the mark; prepare a solution containing 0.08 µg / mL kanamycin.

[0157] Linear solution 2: Accurately pipette 0.2 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 60°C water bath for 10 min, take out and add 0.5 mL of 0.2 mol / L sulfuric acid, mix well and let cool, then dilute with 50% acetonitrile and make up to the mark; prepare a solution containing 0.2 µg / mL kanamycin.

[0158] Linear solution 3: Accurately pipette 1 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 60°C water bath for 10 min, take out and add 0.5 mL of 0.2 mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the mark; prepare linear solutions containing 1 µg / mL kanamycin respectively.

[0159] Linear solution 4: Accurately pipette 2 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 60°C water bath for 10 min, take out and add 0.5 mL of 0.2 mol / L sulfuric acid, mix well and let cool, then dilute with 50% acetonitrile and make up to the mark; prepare a solution containing 2 µg / mL kanamycin.

[0160] Linear solution 5: Accurately pipette 6 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 60°C water bath for 10 min, take out and add 0.5 mL of 0.2 mol / L sulfuric acid, mix well and let cool, then dilute with 50% acetonitrile and make up to the mark; prepare a kanamycin solution containing 6 µg / mL.

[0161] Accuracy solution preparation:

[0162] Accuracy -75%: Take 1mL of the test sample stock solution and place it in a 20mL volumetric flask, accurately add 3mL of the reference sample stock solution, add 2mL of 5mg / mL phenyl isothiocyanate acetonitrile solution, 2mL of 5mg / mL triethylamine acetonitrile solution, shake well, react in a 60℃ water bath for 10min, take out and add 0.5mL of 0.2mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the mark (prepare 3 copies in parallel);

[0163] Accuracy -100%: Take 1mL of the test sample stock solution and place it in a 20mL volumetric flask, accurately add 4mL of the reference sample stock solution, add 2mL of 5mg / mL phenyl isothiocyanate acetonitrile solution, 2mL of 5mg / mL triethylamine acetonitrile solution, shake well, react in a 60℃ water bath for 10min, take out and add 0.5mL of 0.2mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the scale (prepare 3 copies in parallel);

[0164] Accuracy -125%: Take 1mL of the test sample stock solution and place it in a 20mL volumetric flask, accurately add 5mL of the reference sample stock solution, add 2mL of 5mg / mL phenyl isothiocyanate acetonitrile solution, 2mL of 5mg / mL triethylamine acetonitrile solution, shake well, react in a 60℃ water bath for 10min, take out and add 0.5mL of 0.2mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the mark (prepare 3 copies in parallel);

[0165] Take 10 μL of the corresponding test solution and reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, calculate the kanamycin content in the test solution based on the linear relationship generated by the reference solution, and calculate the accuracy. The results are shown in Table 10.

[0166] Table 10 Accuracy

[0167]

[0168] The results showed that the mean recoveries at concentrations of 75%, 100%, and 125% were 101.3%, 97.7%, and 102.5%, respectively, all within the range of 80%-115%. The relative standard deviation (RSD%) of the 9 recovery data was 3.92%, no more than 5%, which did not meet the requirements. This method had good accuracy.

[0169] Comparative Example 1

[0170] The difference from Example 1 is mainly in the mobile phase composition and elution mode, which are as follows:

[0171] Instrument: Liquid chromatograph Thermo U3000;

[0172] Chromatographic column: Octadecylsilane bonded silica gel was used as filler (Phenomemex Hydro-12p80 column, 4.6 mm × 150 mm, 4 µm);

[0173] Mobile phase A: 80% acetonitrile;

[0174] Mobile phase B: 0.1 mol / L sodium acetate solution (adjusted to pH 6.5 with glacial acetic acid)-acetonitrile (93:7);

[0175] Detector: Ultraviolet absorption detector (UV);

[0176] Detection wavelength: 254nm;

[0177] Flow rate: 1.0 mL / min;

[0178] Column temperature: 30°C;

[0179] Injection volume: 10 μL;

[0180] Chromatography workstation: Chameleon.

[0181] The gradient elution program is shown in Table 11.

[0182] Table 11 Gradient elution program

[0183]

[0184] Preparation of derivatization reagents:

[0185] 5 mg / mL phenyl isothiocyanate acetonitrile solution: Accurately weigh 500 mg of phenyl isothiocyanate, place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and the solution is ready.

[0186] 5 mg / mL triethylamine acetonitrile solution: Accurately weigh 500 mg of triethylamine, place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0187] Preparation of test solution: Take an appropriate amount of amikacin sulfate injection, dissolve it in purified water and dilute it to make a solution containing about 4.0 mg of amikacin per 1 mL, take 1 mL and place it in a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70 ° C water bath for 20 min, take it out and immediately add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and let it cool, then dilute it with 50% acetonitrile and make up to the scale;

[0188] Preparation of blank solution: accurately measure 1 mL of water and place it in a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and immediately add 0.5 mL of 0.1 mol / L sulfuric acid, mix well and cool, then dilute with 50% acetonitrile and make up to the scale.

[0189] Reference solution: accurately weigh an appropriate amount of kanamycin (kanamycin sulfate), dissolve it in purified water and dilute it to make a solution containing 20µg of kanamycin per 1mL, accurately transfer 2mL to a 20mL volumetric flask, add 2mL of 5mg / mL phenyl isothiocyanate acetonitrile solution and 2mL of 5mg / mL triethylamine acetonitrile solution, shake well, react in a 70℃ water bath for 20min, take it out and add 0.5mL of 0.1mol / L sulfuric acid, mix well and let it cool, then dilute it with 50% acetonitrile and make up to the mark; make a solution containing 2µg / mL kanamycin.

[0190] Take 10 μL of blank solution, reference solution and test solution, inject them into liquid chromatograph respectively, record the chromatogram, and the results are shown in Table 12.

[0191] Table 12 Specificity test results

[0192]

[0193] The results showed that under this chromatographic condition, the peak of amikacin sulfate derivatives interfered with the detection of kanamycin derivatives peak, so the method specificity did not meet the requirements.

[0194] Comparative Example 2

[0195] The main difference from Example 1 is that no sulfuric acid solution is added to terminate the derivatization after the derivatization is completed, as follows:

[0196] Instrument: Liquid chromatograph Thermo U3000;

[0197] Chromatographic column: Octadecylsilane bonded silica gel was used as filler (Phenomemex Hydro-12p80 column, 4.6 mm × 150 mm, 4 µm);

[0198] Mobile phase: 0.1 mol / L sodium acetate (adjust the pH to 6.5 with 0.1 mol / L sulfuric acid solution)-methanol (70:30) as the mobile phase;

[0199] Detector: Ultraviolet absorption detector (UV);

[0200] Detection wavelength: 242nm;

[0201] Flow rate: 1.0 mL / min;

[0202] Column temperature: 30°C;

[0203] Injection volume: 10 μL;

[0204] Chromatography workstation: Chameleon.

[0205] 5 mg / mL phenyl isothiocyanate acetonitrile solution: Accurately weigh 500 mg of phenyl isothiocyanate, place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and the solution is ready.

[0206] 5 mg / mL triethylamine acetonitrile solution: Accurately weigh 500 mg of triethylamine, place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0207] Stock solution of test sample: Take an appropriate amount of amikacin sulfate injection and dilute it with purified water to make a solution containing approximately 4.0 mg of amikacin per 1 mL.

[0208] Preparation of test solution: Take 1 mL of the test stock solution and place it in a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70 °C water bath for 20 min, take out and cool, then dilute with 50% acetonitrile and make up to the mark;

[0209] Preparation of blank solution: Accurately measure 1 mL of water and place it in a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and cool, then dilute with 50% acetonitrile and make up to the scale.

[0210] Preparation of reference solutions (linear solution 1-linear solution 5):

[0211] Reference stock solution: Accurately weigh an appropriate amount of kanamycin (kanamycin sulfate), dissolve and dilute with purified water to make a solution containing 20 µg of kanamycin per 1 mL

[0212] Linear solution 1: Accurately pipette 0.08 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and cool, then dilute with 50% acetonitrile and make up to the mark; prepare a solution containing 0.08 µg / mL kanamycin.

[0213] Linear solution 2: Accurately pipette 0.2 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and cool, then dilute with 50% acetonitrile and make up to the mark; prepare a solution containing 0.2 µg / mL kanamycin.

[0214] Linear solution 3: Accurately pipette 1 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and cool, then dilute with 50% acetonitrile and make up to the mark; prepare linear solutions containing 1 µg / mL kanamycin respectively.

[0215] Linear solution 4: Accurately pipette 2 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and cool, then dilute with 50% acetonitrile and make up to the mark; prepare a solution containing 2 µg / mL kanamycin.

[0216] Linear solution 5: Accurately pipette 6 mL of the reference stock solution into a 20 mL volumetric flask, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and cool, then dilute with 50% acetonitrile and make up to the mark; prepare a kanamycin solution containing 6 µg / mL.

[0217] Accuracy solution preparation:

[0218] Accuracy -75%: Take 1 mL of the test sample stock solution and place it in a 20 mL volumetric flask, accurately add 3 mL of the reference sample stock solution, add 2 mL of 5 mg / mL phenyl isothiocyanate acetonitrile solution, and 2 mL of 5 mg / mL triethylamine acetonitrile solution, shake well, react in a 70°C water bath for 20 min, take out and cool, then dilute with 50% acetonitrile and make up to the mark (prepare 3 copies in parallel);

[0219] Accuracy -100%: Take 1mL of the test sample stock solution and place it in a 20mL volumetric flask, accurately add 4mL of the reference sample stock solution, add 2mL of 5mg / mL phenyl isothiocyanate acetonitrile solution, 2mL of 5mg / mL triethylamine acetonitrile solution, shake well, react in a 70℃ water bath for 20min, take out and cool, then dilute with 50% acetonitrile and make up to the mark (prepare 3 copies in parallel);

[0220] Accuracy -125%: Take 1mL of the test sample stock solution and place it in a 20mL volumetric flask, accurately add 5mL of the reference sample stock solution, add 2mL of 5mg / mL phenyl isothiocyanate acetonitrile solution, and 2mL of 5mg / mL triethylamine acetonitrile solution, shake well, react in a 70℃ water bath for 20min, take out and cool, then dilute with 50% acetonitrile and make up to the mark (prepare 3 copies in parallel);

[0221] Take 10 μL of the corresponding test solution and reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, calculate the kanamycin content in the test solution based on the linear relationship generated by the reference solution, and calculate the accuracy. The results are shown in Table 13.

[0222] Table 13 Accuracy

[0223]

[0224] The results showed that the mean recoveries at concentrations of 75%, 100% and 125% were 74.8%, 92.5% and 127.6% respectively, which were above the range of 80%-115%. The relative standard deviation (RSD%) of the 9 recovery data was 24.5%, which was greater than 5% and did not meet the requirements. This derivatization method did not add sulfuric acid solution to adjust the pH value to acidic to terminate the derivatization, so the accuracy did not meet the requirements.

[0225] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for detecting kanamycin in amikacin sulfate injection, characterized in that: The steps include: S1 dilutes amikacin sulfate with water and obtains a test solution after derivatization; S2 prepares linear solutions of kanamycin sulfate of different concentrations, and obtains reference solutions after derivatization treatment; S3: The test solution and the reference solution are eluted by HPLC, and then enter into a UV detector for detection, and the kanamycin content is calculated; In step S1 or S2, the derivatization reagent used in the derivatization treatment is an acetonitrile solution containing phenyl isothiocyanate and triethylamine; the mass ratio of phenyl isothiocyanate to triethylamine is 1:1; after the derivatization is completed, sulfuric acid solution is added to terminate the derivatization; In step S3, the mobile phase used for elution is 0.08-0.12 mol / L sodium acetate solution-methanol, the volume ratio of the two is 65-75:25-35, and the pH of the sodium acetate solution is 6.5±0.

2.

2. The detection method according to claim 1, characterized in that: The chromatographic column used in the HPLC method in step S3 is an octadecylsilane bonded silica gel chromatographic column.

3. The detection method according to claim 1, characterized in that: The elution method in step S3 is isocratic elution, and the elution time is 60 min.

4. The detection method according to claim 1, characterized in that: The preparation of the test solution in step S1 includes: S11 dilutes amikacin sulfate with water to obtain solution A; S12 adds an acetonitrile solution containing phenyl isothiocyanate and triethylamine to carry out derivatization to obtain solution B; S13 is added with sulfuric acid solution, and then fixed to volume with acetonitrile solution to obtain.

5. The detection method according to claim 4, characterized in that: In step S11, each 1 mL of solution A contains 4.0 mg to 5.0 mg of amikacin.

6. The detection method according to claim 4, characterized in that: In step S12, the concentration of phenyl isothiocyanate in the acetonitrile solution containing phenyl isothiocyanate and triethylamine is 2.5 mg / mL; the derivatization conditions are: temperature 60-80° C., time 10-30 min.

7. The detection method according to claim 4, characterized in that: In step S13, the concentration of the sulfuric acid solution is 0.1 mol / L-0.2 mol / L; the volume concentration of the acetonitrile solution is 50%.

8. The detection method according to claim 4, characterized in that: The volume ratio of solution A to the acetonitrile solution containing phenyl isothiocyanate and triethylamine is 1:4; the volume ratio of solution A to the sulfuric acid solution is 1:0.4-0.

6.

9. The detection method according to any one of claims 1 to 8, characterized in that: The derivatization steps of step S2 are the same as those of step S1.

10. The detection method according to any one of claims 1 to 8, characterized in that: The detection wavelength of the HPLC method in step S3 is 242 nm; The pH value of the sodium acetate solution was adjusted to 6.5±0.2 using 0.1mol / L-0.2mol / L sulfuric acid solution; The flow rate of the mobile phase is 0.8-1.2 mL / min; Column temperature is 25-35°C; The injection volume was 10 μL.

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