Impurity detection method of compound amino acid injection

Abstract

The invention mainly relates to the technical field of drug analysis and detection, in particular to an impurity detection method of a compound amino acid injection. High performance liquid chromatography is adopted for detection, and chromatographic conditions are as follows: a chromatographic column adopts octadecylsilane chemically bonded silica as a filler; the wavelength of a UV detector is 210 to 225 nm; mobile phases: a mobile phase A: a sodium dihydrogen phosphate solution with a pH value of 4.0-6.5 and methanol; a mobile phase B: acetonitrile and water; the elution mode is gradient elution; the impurities comprise a tryptophan impurity B, a tryptophan impurity C, a tryptophan impurity D, a tryptophan impurity E, a tryptophan impurity H, a tryptophan impurity G and indole-3-formaldehyde. The detection method disclosed by the invention is good in specificity and high in accuracy; different detection methods do not need to be adopted for separate detection, multiple impurities in the preparation can be detected at the same time, and the detection frequency is reduced; the detection time is shortened and the detection cost is saved; a reliable method is provided for the preparation process of the compound amino acid injection and the quality control of the product, and the medication safety of a patient is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis and detection, and particularly relates to a method for detecting impurities in compound amino acid injection. BACKGROUND

[0002] Amino acids are the basic units of protein composition, and there are 20 kinds of amino acids in the composition of proteins in the body. Amino acids are divided into essential amino acids and non-essential amino acids. Essential amino acids cannot be synthesized in the body and must be taken in through food or supplements to meet the metabolic needs of the body. If the body does not intake enough amino acids, amino acid supplements need to be used for supplementation. Common amino acid supplements are divided into oral and injection types. Injection of amino acid supplements is to directly inject amino acid injection through intravenous injection, and the amino acid is dissolved in the body fluid and directly absorbed by the muscle or other tissues, which has a quick effect.

[0003] Compound amino acid injection is composed of multiple amino acids, and the components are relatively complex. As one of the main raw materials, tryptophan is unstable in physical and chemical properties and is easily oxidized during production, transportation and storage, thereby reducing the content of tryptophan in compound amino acid injection and producing various impurities. Some impurities can cause many adverse reactions in clinical practice. Therefore, it is necessary to study the tryptophan-related impurities in the preparation and establish a detection method with strong specificity, high accuracy and high efficiency. However, the existing methods for detecting tryptophan impurities in compound amino acid injection, such as the Chinese Pharmacopoeia (ChP), the European Pharmacopoeia (EP), the United States Pharmacopoeia (USP) and the Japanese Pharmacopoeia (JP), cannot simultaneously determine the contents of tryptophan impurities (B, C, D, E, H, G) and indole-3-methyl aldehyde in compound amino acid injection containing tryptophan, and different detection methods need to be used for separate detection. SUMMARY

[0004] The purpose of the present application is to avoid the shortcomings of the prior art and provide a method for detecting impurities in compound amino acid injection.

[0005] To achieve the above purpose, the present application provides the following technical solution: a method for detecting impurities in compound amino acid injection, which uses high performance liquid chromatography for detection, and the chromatographic conditions are as follows:

[0006] Chromatographic column: octadecylsilane bonded silica gel as filler;

[0007] UV detector: 210-225 nm;

[0008] Mobile phase: mobile phase A: pH = 4.0-6.5 sodium dihydrogen phosphate solution, methanol;

[0009] Mobile phase B: acetonitrile, water;

[0010] The elution mode is gradient elution.

[0011] The elution procedure of the gradient elution is as follows:

[0012] 0-60min, 95%-100%→60%-75% mobile phase A, 5%-0%→40%-25% mobile phase B;

[0013] 60-65min, 60%-75%→0%-5% mobile phase A, 40%-25%→100%-95% mobile phase B;

[0014] 65-69min, 0%-5%→95%-100% mobile phase A, 100%-95%→5%-0% mobile phase B;

[0015] 69-80min, 95%-100% mobile phase A, 5%-0% mobile phase B;

[0016] The impurities are tryptophan impurity B, tryptophan impurity C, tryptophan impurity D, tryptophan impurity E, tryptophan impurity H, tryptophan impurity G, indole-3-carboxaldehyde.

[0017] Preferably, the column specification: column length 250, inner diameter 4.6mm.

[0018] Preferably, the column packing particle size 5μm.

[0019] Preferably, the column flow rate is 0.4-0.6mL / min.

[0020] Preferably, the column temperature is 38-45℃.

[0021] Preferably, the injection volume is 5-20μL.

[0022] Preferably, the concentration of the sodium dihydrogen phosphate solution is 0.02-0.04mol / L.

[0023] Preferably, the sodium dihydrogen phosphate solution is adjusted to PH with sodium hydroxide and / or potassium hydroxide.

[0024] Preferably, the volume ratio of the sodium dihydrogen phosphate solution to methanol in the mobile phase A is 90:10-97:3.

[0025] Preferably, the volume ratio of acetonitrile to water in the mobile phase B is 90:10-70:30.

[0026] The compound amino acid injection impurity detection method has the following beneficial effects:

[0027] The chromatographic column is filled with octadecylsilane bonded silica, and the mobile phase is 0.02-0.04 mol / L sodium dihydrogen phosphate, methanol, water and acetonitrile, and the separation and detection of various tryptophan impurities (B, C, D, E, H, G) and indole-3-carbinol are realized by gradient elution through a high performance liquid chromatograph. The detection method has high specificity and accuracy, and can simultaneously detect tryptophan impurities (B, C, D, E, H, G) and indole-3-carbinol. Compared with the prior art, the method can simultaneously detect multiple impurities in the preparation without using different detection methods, thereby reducing the detection times, shortening the detection time and saving the detection cost, and is a kind of impurity detection method for compound amino acid injection with high specificity, high accuracy, high efficiency and low cost. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Chromatogram of the blank solution in Example 1;

[0029] Figure 2 Chromatogram of the control solution in Example 1;

[0030] Figure 3 Chromatogram of the system suitability solution in Example 1;

[0031] Figure 4 Chromatogram of the test solution in Example 1; DETAILED DESCRIPTION

[0032] Preferred embodiments of the present application will be described in more detail with reference to the drawings. Although the preferred embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0033] Preparation of the solution:

[0034] Blank solution: 10% acetonitrile.

[0035] Control solution: accurately weigh tryptophan impurity B control, tryptophan impurity C control, tryptophan impurity D control, tryptophan impurity E control, tryptophan impurity H control, tryptophan impurity G control, and indole-3-carbinol control, dissolve them in 10% acetonitrile to prepare a mixed solution containing 5-15 μg of each of tryptophan impurities (B, E, G), 1.5-3.5 μg of each of tryptophan impurities (C, D, H), and 2-5 μg of indole-3-carbinol per 1 ml, which is the control solution.

[0036] Test solution: dilute the compound amino acid injection sample with 10% acetonitrile, shake well, and obtain the test solution.

[0037] System suitability solution: Take appropriate amounts of the test solution and the reference solution, mix them well, and prepare a system suitability solution.

[0038] Chromatographic conditions

[0039] Equipment: High-performance liquid chromatograph;

[0040] UV detector: wavelength 210-225nm;

[0041] Chromatographic column: Octadecylsilane-bonded silica gel as packing material; specifications: column length 250 mm × inner diameter 4.6 mm, packing particle size 5 μm;

[0042] Flow rate: 0.4-0.6 mL / min;

[0043] Column temperature: 38-45℃.

[0044] Gradient elution table:

[0045] Table 1 Gradient Elution Table

[0046]

[0047]

[0048] Mobile phase A: 0.02-0.04 mol / L sodium dihydrogen phosphate solution (pH adjusted to 4.0-6.5 with sodium hydroxide and / or potassium hydroxide): methanol = 90:10-97:3;

[0049] Mobile phase B: Acetonitrile: Water = 90:10-70:30.

[0050] Take blank solution, reference solution, system suitability solution, and test solution, and inject them into the HPLC system according to the above-described conditions. The injection volume is 10 μL, and the chromatograms are recorded. Analyze the chromatograms of the blank solution, reference solution, system suitability solution, and test solution, and use resolution (R) to evaluate the degree of separation between adjacent chromatographic peaks. Specifically, the higher the resolution (R) between adjacent chromatographic peaks, the better the separation effect between adjacent components in the chromatographic system, and the stronger the reliability of the analytical results.

[0051] The reagent peaks in the blank solution were determined by analyzing the chromatogram of the blank solution.

[0052] The chromatogram of the reference solution serves as a standard reference for the target analyte. It must have clear, complete peaks, high resolution, and be unaffected by interference from the blank solution reagent peaks, ensuring accurate identification and measurement of all peaks. The retention time and compound name of the main peak in the chromatogram are labeled for comparison with the detection peaks of the target analyte in the test sample.

[0053] The system suitability solution is a mixed solution of the control sample solution and the test sample solution. The separation degree and sensitivity and other parameters reflected in the chromatogram of the system suitability solution can verify whether the analysis method of the application can accurately separate and detect the components in the sample. The separation degree between each impurity peak in the system suitability solution and other peaks in the test sample solution determines whether the other peaks in the test sample interfere with each other. Specifically, if the separation degree of each impurity peak is large, and the other peaks in the test sample do not interfere with each other, it indicates that the detection method of the application has good specificity and high accuracy, and can detect the impurities in the test sample.

[0054] The chromatogram of the test sample solution is the chromatogram of the sample to be measured. By comparing with the chromatogram of the control sample and the chromatogram of the system suitability solution, the impurities detected in the test sample are determined.

[0055] Example 1: Impurity detection method of compound amino acid injection

[0056] Preparation of the solution:

[0057] Blank solution: 10% acetonitrile.

[0058] Control sample solution: accurately weigh tryptophan impurity B control sample, tryptophan impurity C control sample, tryptophan impurity D control sample, tryptophan impurity E control sample, tryptophan impurity H control sample, tryptophan impurity G control sample, and indole-3-carboxaldehyde control sample, dissolve them in 10% acetonitrile to prepare a mixed solution containing 5-15 μg of each of tryptophan impurities (B, E, G), 1.5-3.5 μg of each of tryptophan impurities (C, D, H), and 2-5 μg of indole-3-carboxaldehyde per 1 ml, which is the control sample solution.

[0059] Test sample solution: an appropriate amount of compound amino acid injection sample is diluted with 10% acetonitrile and shaken to obtain the test sample solution.

[0060] System suitability solution: mix the test sample solution and the control sample solution to prepare the system suitability solution.

[0061] Chromatographic conditions

[0062] Equipment: Shimadzu LC-2030

[0063] UV detector: wavelength 215 nm

[0064] Chromatographic column: octadecylsilane bonded silica gel as filler; specifications: column length 250 x inner diameter 4.6 mm, filler particle size 5 μm.

[0065] Flow rate: 0.5 mL / min

[0066] Column temperature: 40°C

[0067] Gradient elution table:

[0068] Table 2 Gradient Elution Table of Example 1

[0069] Time (min) Mobile phase A (%) Mobile phase (B) 0 100 0 3 95 5 60 65 35 65 2 98 67 0 100 69 95 5 80 100 0

[0070] Mobile phase A: 0.03 mol / L sodium dihydrogen phosphate solution (pH adjusted to 5.2 with sodium hydroxide): methanol = 92:8;

[0071] Mobile phase B: Acetonitrile: Water = 85:15.

[0072] Take the blank solution, system suitability solution, reference solution, and test solution, and inject them into the system according to the above-described high-performance liquid chromatography (HPLC) conditions. The injection volume is 10 μL, and the chromatograms are recorded. The chromatogram of the blank solution is shown below. Figure 1 As shown; the chromatogram of the reference solution is as follows. Figure 2 As shown; the chromatogram of the system suitability solution is as follows. Figure 3 As shown; the chromatogram of the test solution is as follows. Figure 4 As shown.

[0073] like Figure 1 As shown in the chromatogram of the blank solution, the baseline of the spectrum is relatively stable when the retention time is 0-65 min, and multiple consecutive short peaks appear when the retention time is 65 min-74 min. Therefore, the chromatographic peak at the retention time of 65 min-74 min is the reagent peak of the blank solution.

[0074] like Figure 2 As shown, the chromatogram of the reference solution displays the elution times of each impurity. The chromatographic peaks of tryptophan impurities (B1, B2, D, C, E, G, H) and indole-3-carboxaldehyde are clearly visible. The peaks of each tryptophan impurity do not overlap, indicating high resolution. In this embodiment, all eight impurities can be detected in a single test, demonstrating the high resolution and efficiency of the detection method of this invention. The experimental results of the reference solution in this embodiment are as follows:

[0075] Table 3. Test results of reference solution

[0076] Name Retention time Tryptophan impurity B1 12.416 Tryptophan impurity B2 14.142 Tryptophan impurity D 16.016 Tryptophan impurity C 18.030 Tryptophan impurity E 20.440 Tryptophan impurity G 21.386 Tryptophan impurity H 33.715 Indole-3-carbinol 59.058

[0077] like Figure 3 As shown in the chromatogram of the system suitability solution, there are more peaks and better resolution. The detection results are as follows:

[0078] Table 4. System Suitability Solution Test Results

[0079]

[0080]

[0081] Through and Figure 2 The comparison shows that after adding the test solution to the reference solution, all substances in the reference solution were analyzed and detected. Furthermore, the resolution between each impurity peak in both the system suitability solution and the test solution and other peaks in the test solution was greater than 1.4, indicating no interference between the impurity peaks and other peaks. This demonstrates that the detection method of this invention has good specificity, high accuracy, and high detection efficiency, and is suitable for the detection of tryptophan impurities (B, D, C, E, G, H) and indole-3-carboxaldehyde in compound amino acid injections.

[0082] like Figure 4 As shown, the test results of the compound amino acid injection sample in this embodiment are as follows:

[0083] Table 5. Test Results of the Samples

[0084] Peak number Compound name Retention time Theoretical plates Separation 3 Tryptophan impurity B1 12.458 11537 1.7 4 Tryptophan impurity B2 14.187 16378 3.8 6 Tryptophan impurity C 18.061 32255 9.1 8 Tryptophan impurity G 21.405 10302 3.3 9 Tryptophan impurity H 33.742 66244 18.0

[0085] Therefore, it can be seen that the compound amino acid injection tested in this embodiment contains tryptophan impurities (B1, B2, C, G, H). Since the test sample does not contain impurities D, E and indole-3-carboxaldehyde, they were not detected. The detection method of the present invention can detect multiple tryptophan impurities at the same time, reduce the number of tests, shorten the detection time, and save detection costs. It is a highly specific, accurate, efficient, and low-cost method for detecting impurities in amino acid injections.

[0086] Example 2: Impurity Detection Method for Compound Amino Acid Injection

[0087] Solution preparation:

[0088] Blank solution: 10% acetonitrile.

[0089] Reference solution: Accurately weigh the reference standards for tryptophan impurity B, C, D, E, H, G, and indole-3-carboxaldehyde, and dissolve them in 10% acetonitrile to prepare a mixed solution containing 5-15 μg each of tryptophan impurities (B, E, G), 1.5-3.5 μg each of tryptophan impurities (C, D, H), and 2-5 μg of indole-3-carboxaldehyde per 1 ml.

[0090] Test solution: Take an appropriate amount of compound amino acid injection sample, dilute with 10% acetonitrile, and shake well to prepare the test solution.

[0091] System suitability solution: Take appropriate amounts of the test solution and the reference solution, mix them well, and prepare a system suitability solution.

[0092] Chromatographic conditions

[0093] Equipment: Shimadzu LC-2030

[0094] UV detector: wavelength 225nm

[0095] Chromatographic column: Octadecylsilane-bonded silica gel as packing material; specifications: column length 250 × inner diameter 4.6 mm, packing particle size 5 μm.

[0096] Flow rate: 0.6 mL / min

[0097] Column temperature: 45℃

[0098] Gradient elution table:

[0099] Table 6 Gradient Elution Table of Example 2

[0100] Time (min) Mobile phase A (%) Mobile phase (B) 0 100 0 3 95 5 60 70 30 65 5 95 67 3 97 69 100 0 80 100 0

[0101] Mobile phase A: 0.04 mol / L sodium dihydrogen phosphate solution (pH adjusted to 6.5 with potassium hydroxide): methanol = 97:3;

[0102] Mobile phase B: Acetonitrile: Water = 90:10.

[0103] Take blank solution, system suitability solution, reference solution and test solution, and inject them for detection according to the above high performance liquid chromatography conditions, with an injection volume of 10 μL.

[0104] Example 3: Impurity Detection Method for Compound Amino Acid Injection

[0105] Solution preparation:

[0106] Blank solution: 10% acetonitrile.

[0107] Reference solution: Accurately weigh the reference standards for tryptophan impurity B, C, D, E, H, G, and indole-3-carboxaldehyde, and dissolve them in 10% acetonitrile to prepare a mixed solution containing 5-15 μg each of tryptophan impurities (B, E, G), 1.5-3.5 μg each of tryptophan impurities (C, D, H), and 2-5 μg of indole-3-carboxaldehyde per 1 ml.

[0108] Test solution: Take an appropriate amount of compound amino acid injection sample, dilute with 10% acetonitrile, and shake well to prepare the test solution.

[0109] System suitability solution: Take appropriate amounts of the test solution and the reference solution, mix them well, and prepare a system suitability solution.

[0110] Chromatographic conditions

[0111] Equipment: Shimadzu LC-2030

[0112] UV detector: wavelength 215nm

[0113] Chromatographic column: Octadecylsilane-bonded silica gel as packing material; specifications: column length 250 × inner diameter 4.6 mm, packing particle size 5 μm.

[0114] Flow rate: 0.4 mL / min

[0115] Column temperature: 38℃

[0116] Gradient elution table:

[0117] Table 7 Gradient Elution Table for Example 3

[0118] Time (min) Mobile phase A (%) Mobile phase (B) 0 100 0 3 95 5 60 65 35 65 5 95 67 5 95 69 95 5 80 100 0

[0119] Mobile phase A: 0.02 mol / L sodium dihydrogen phosphate solution (pH adjusted to 4.0 with sodium hydroxide): methanol / water = 90:10;

[0120] Mobile phase B: Acetonitrile: Water = 80:20.

[0121] Take blank solution, system suitability solution, reference solution and test solution, and inject them for detection according to the above high performance liquid chromatography conditions, with an injection volume of 10 μL.

[0122] It is understood that the above embodiments only illustrate preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can freely combine the above technical features without departing from the concept of the present invention, and can also make several modifications and improvements, all of which fall within the protection scope of the present invention. Therefore, all equivalent transformations and modifications made with respect to the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims

1. A method for detecting impurities in a compound amino acid injection, characterized in that, High-performance liquid chromatography (HPLC) was used for detection, and the chromatographic conditions were as follows: Column: Octadecylsilane-bonded silica gel as the packing material; UV detector: 210-225nm; Mobile phase: Mobile phase A: Sodium dihydrogen phosphate solution with pH = 4.0-6.5, methanol; Mobile phase B: Acetonitrile, water; The elution method is gradient elution; The gradient elution procedure is as follows: 0-60 min, 95%-100%→60%-75% mobile phase A, 5%-0%→40%-25% mobile phase B; 60-65 min, 60%-75% → 0%-5% mobile phase A, 40%-25% → 100%-95% mobile phase B; 65-69 min, 0%-5% → 95%-100% mobile phase A, 100%-95% → 5%-0% mobile phase B; 69-80 min, 95%-100% mobile phase A, 5%-0% mobile phase B; The impurities are tryptophan impurity B, tryptophan impurity C, tryptophan impurity D, tryptophan impurity E, tryptophan impurity H, tryptophan impurity G, and indole-3-carboxaldehyde.

2. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that, The chromatographic column specifications are: column length 250mm, inner diameter 4.6mm.

3. The method for detecting impurities in the compound amino acid injection according to claim 2, characterized in that, The chromatographic column packing material has a particle size of 5 μm.

4. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that, The flow rate of the chromatographic column is 0.4-0.6 mL / min.

5. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that, The column temperature of the chromatographic column is 38-45℃.

6. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that, The chromatographic injection volume is 5-20 μL.

7. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that, The concentration of the sodium dihydrogen phosphate solution is 0.02-0.04 mol / L.

8. The method for detecting impurities in the compound amino acid injection according to claim 7, characterized in that, The pH of the sodium dihydrogen phosphate solution is adjusted with sodium hydroxide and / or potassium hydroxide.

9. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that, The volume ratio of sodium dihydrogen phosphate solution to methanol in the mobile phase A is 90:10-97:

3.

10. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that, The volume ratio of acetonitrile to water in the mobile phase B is 90:10-70:30.

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