Method for detecting cysteine in compound amino acid injection and application thereof

Abstract

The application provides a detection method and application of cysteine in a compound amino acid injection, and belongs to the technical field of analytical chemistry. The detection method comprises the following steps: adopting high performance liquid chromatography, and the chromatographic conditions comprise the following steps: adopting a chromatographic column filled with octadecylsilane bonded silica gel, gradient elution is carried out with a mobile phase, and an ultraviolet detector is adopted for detection. The detection method of cysteine provided by the application effectively improves the peak shape and separation degree of cysteine by screening and optimizing the chromatographic conditions such as the proportion and pH of the mobile phase, flow rate, column temperature and wavelength, and accurate quantitative detection of the substance is realized. The detection method is verified by a systematic methodology, has the advantages of strong specificity, good linear relationship, good accuracy, strong durability, good stability and the like, and can quantitatively detect the cysteine in the compound amino acid injection, so that the effectiveness and safety of the relevant compound amino acid injection for clinical medication are ensured.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, specifically to a method for detecting cysteine ​​in compound amino acid injections and its application. Background Technology

[0002] Compound amino acid injections are a common type of parenteral nutrition injection with a wide range of clinical applications. Currently, there are many types of amino acids on the market, such as compound amino acid injections (18AA-VII) and compound amino acid injections (18AA-IX), which contain cysteine. However, cysteine ​​is easily oxidized and degraded, leading to unstable product quality and consequently affecting patient safety and drug efficacy. Therefore, accurate quantitative research on the cysteine ​​content is necessary.

[0003] According to existing literature, various derivatizing reagents are used to derivatize samples before quantitative detection using high-precision instruments such as amino acid analyzers and high-performance liquid chromatographs. However, derivatization with derivatizing agents is cumbersome, and the derivatized samples are unstable, greatly affected by reaction time and concentration, leading to unstable measurement results. Existing technologies also report the use of colorimetric methods for detection, using chromogenic agents to induce a colorimetric reaction in cysteine, followed by detection using UV-Vis spectrophotometry. This method also suffers from cumbersome operation, colorimetric effects being greatly affected by reaction time and temperature, and the accuracy of UV-based results is unsatisfactory. Summary of the Invention

[0004] The technical problem to be solved by this invention is the many shortcomings of existing methods for detecting cysteine ​​in compound amino acid injections. The purpose is to provide a method for detecting cysteine ​​in compound amino acid injections and its application. This method is easy to operate and can accurately and quantitatively detect the content of cysteine ​​in compound amino acid injections, thereby ensuring the quality of compound amino acid injections and improving the effectiveness and safety of clinical medication.

[0005] This invention is achieved through the following technical solution:

[0006] A method for detecting cysteine ​​in a compound amino acid injection includes: high performance liquid chromatography (HPLC), with chromatographic conditions including: using an octadecylsilane-bonded silica gel column, gradient elution with a mobile phase, detection with an ultraviolet detector at a wavelength of 195 nm-205 nm, a mobile phase flow rate of 0.9 ml / min to 1.1 ml / min, an injection volume of 10 μl to 30 μl, and a column temperature of 25 °C to 35 °C.

[0007] The mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is a mixed solution of methanol and sodium octane sulfonate buffer solution; mobile phase B is an aqueous solution of acetonitrile.

[0008] Further, in a preferred embodiment of the present invention, the mobile phase A is a mixed solution of methanol and 3-6 mmol / L sodium octane sulfonate buffer solution; wherein the volume ratio of sodium octane sulfonate buffer solution to methanol is 97:3-99:1.

[0009] Furthermore, in a preferred embodiment of the present invention, the pH of the mobile phase A is 1.9 to 2.1.

[0010] Furthermore, in a preferred embodiment of the present invention, the mobile phase A is adjusted with phosphoric acid.

[0011] Furthermore, in a preferred embodiment of the present invention, the volume ratio of acetonitrile to water in the mobile phase B is 70:30 to 90:10.

[0012] Furthermore, in a preferred embodiment of the present invention, the gradient elution conditions are as follows:

[0013] Time: 0 min, 95-100% mobile phase A, 0-5% mobile phase B;

[0014] Time: 18 min, 95-100% mobile phase A, 0-5% mobile phase B;

[0015] Time: 18.5 min, 40-60% mobile phase A, 40-60% mobile phase B;

[0016] Time: 21.5 min, 40-60% mobile phase A, 40-60% mobile phase B;

[0017] Time: 22 min, 95-100% mobile phase A, 0-5% mobile phase B;

[0018] Time: 40 min, 95-100% mobile phase A, 0-5% mobile phase B.

[0019] Furthermore, in a preferred embodiment of the present invention, the wavelength is 200 nm.

[0020] Furthermore, in a preferred embodiment of the present invention, the flow rate of the mobile phase is 1.0 ml / min.

[0021] Furthermore, in a preferred embodiment of the present invention, the column temperature is 30°C.

[0022] The present invention also provides the application of the method for detecting cysteine ​​in the compound amino acid injection in the detection of cysteine ​​in the compound amino acid injection.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] This invention effectively improves the peak shape and resolution of cysteine ​​by screening and optimizing chromatographic conditions such as mobile phase ratio, pH, flow rate, column temperature, and wavelength, and achieves accurate quantitative detection of this substance. The detection method has undergone systematic methodological validation and possesses advantages such as high specificity, good linearity, high accuracy, strong robustness, and good stability. It can quantitatively detect cysteine ​​in compound amino acid injections, thereby ensuring the efficacy and safety of related compound amino acid injections in clinical use. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0026] Figure 1 This is an HPLC schematic diagram of a cysteine ​​reference solution.

[0027] Figure 2 This is an HPLC schematic diagram of a solution suitable for the cysteine ​​system. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The illustrative embodiments and descriptions of this invention are for explanation only and are not intended to limit the invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0029] The technical solution of the specific embodiments of the present invention is as follows:

[0030] This embodiment provides a method for detecting cysteine ​​in a compound amino acid injection, comprising: high-performance liquid chromatography (HPLC), wherein the chromatographic conditions include: using an octadecylsilane-bonded silica gel column, gradient elution with a mobile phase, detection with a UV detector at a wavelength of 195 nm-205 nm, and further, at a wavelength of 200 nm; a mobile phase flow rate of 0.9 ml / min to 1.1 ml / min, and further, at 1.0 ml / min; an injection volume of 10 μl to 30 μl, and further, 15 μl to 25 μl, and even further, 20 μl; and a column temperature of 25°C to 35°C, and further, at 30°C.

[0031] The mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is a mixed solution of methanol and sodium octane sulfonate buffer solution. Specifically, mobile phase A is a mixture of methanol and 3–6 mmol / L sodium octane sulfonate buffer solution, with a volume ratio of sodium octane sulfonate buffer solution to methanol of 97:3–99:1, and more specifically, a volume ratio of sodium octane sulfonate buffer solution to methanol of 98:2. The pH of mobile phase A is 1.9–2.1, and more specifically, the pH of mobile phase A is 2.0. The pH of mobile phase A is adjusted using phosphoric acid.

[0032] Mobile phase B: An aqueous solution of acetonitrile. The volume ratio of acetonitrile to water in mobile phase B is 70:30 to 90:10, and more specifically, the volume ratio of acetonitrile to water in mobile phase B is 80:20.

[0033] The conditions for gradient elution are as follows:

[0034] Time: 0 min, 95-100% mobile phase A, 0-5% mobile phase B;

[0035] Time: 18 min, 95-100% mobile phase A, 0-5% mobile phase B;

[0036] Time: 18.5 min, 40-60% mobile phase A, 40-60% mobile phase B;

[0037] Time: 21.5 min, 40-60% mobile phase A, 40-60% mobile phase B;

[0038] Time: 22 min, 95-100% mobile phase A, 0-5% mobile phase B;

[0039] Time: 40 min, 95-100% mobile phase A, 0-5% mobile phase B.

[0040] Furthermore, this embodiment provides a method for detecting cysteine ​​in a compound amino acid injection, comprising: using high performance liquid chromatography, wherein the chromatographic conditions include: using a chromatographic column packed with octadecylsilane-bonded silica gel, gradient elution with a mobile phase, detection with an ultraviolet detector at a detection wavelength of 200 nm, a mobile phase flow rate of 1.0 ml / min, an injection volume of 20 μl, and a column temperature of 30 °C.

[0041] The mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is a mixed solution of methanol and sodium octane sulfonate buffer solution. Specifically, mobile phase A is a mixture of methanol and 4 mmol / L sodium octane sulfonate buffer solution, with a volume ratio of sodium octane sulfonate buffer solution to methanol of 98:2. The pH of mobile phase A is 2.0 (adjusted using phosphoric acid).

[0042] Mobile phase B: An aqueous solution of acetonitrile, with a volume ratio of acetonitrile to water of 80:20.

[0043] The conditions for gradient elution are as follows:

[0044] Time: 0 min, 100% mobile phase A, 0% mobile phase B;

[0045] Time: 18 min, 100% mobile phase A, 0% mobile phase B;

[0046] Time: 18.5 min, 50% mobile phase A, 50% mobile phase B;

[0047] Time: 21.5 min, 50% mobile phase A, 50% mobile phase B;

[0048] Time: 22 min, 100% mobile phase A, 0% mobile phase B;

[0049] Time: 40 min, 100% mobile phase A, 0% mobile phase B.

[0050] The specific implementation scheme of the present invention is as follows: wherein,

[0051] Compound Amino Acid Injection (18AA-Ⅶ), provided by Sichuan Kelun Pharmaceutical Co., Ltd.

[0052] Negative sample: Compound Amino Acid Injection (18AA-VII) (excluding cysteine), provided by Sichuan Kelun Pharmaceutical Co., Ltd.

[0053] Example 1: Specificity Experiment

[0054] A method for detecting cysteine ​​in a compound amino acid injection, comprising:

[0055] Reference stock solution: Accurately weigh an appropriate amount of cysteine ​​hydrochloride reference standard, dilute to the mark with mobile phase A, and prepare a solution containing approximately 0.35 mg / ml of cysteine.

[0056] Reference solution: Accurately measure 1.0 ml each of cysteine ​​negative sample and reference stock solution, place them in the same 10 ml volumetric flask, dilute to the mark with mobile phase A, and shake well to obtain (0.035 mg / ml).

[0057] Test solution: Accurately measure 1.0 ml of the test sample and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and shake well to obtain the solution.

[0058] Chromatographic conditions:

[0059] Chromatographic column: Shiseido MGⅡC18, 250mm×4.6mm, 5μm;

[0060] Mobile phase A: 4 mmol / L sodium octane sulfonate buffer (pH adjusted to 2.0 with phosphoric acid): methanol = (98:2);

[0061] Mobile phase B: Acetonitrile-water (80:20);

[0062] Column temperature: 30℃;

[0063] Flow rate: 1.0 ml / min;

[0064] Wavelength: 200nm;

[0065] Injection volume: 20 μl;

[0066] The gradient elution conditions are as follows:

[0067] Time: 0 min, 100% mobile phase A, 0% mobile phase B;

[0068] Time: 18 min, 100% mobile phase A, 0% mobile phase B;

[0069] Time: 18.5 min, 50% mobile phase A, 50% mobile phase B;

[0070] Time: 21.5 min, 50% mobile phase A, 50% mobile phase B;

[0071] Time: 22 min, 100% mobile phase A, 0% mobile phase B;

[0072] Time: 40 min, 100% mobile phase A, 0% mobile phase B.

[0073] Detection method: The reference solution and the test solution were accurately measured and injected into the high performance liquid chromatograph for detection. The results are shown in Table 1.

[0074] Table 1 Results of cysteine ​​specificity investigation

[0075]

[0076] As shown in Table 1, the retention times of cysteine ​​in the reference solution and the test solution were basically the same. In the test solution, the resolution between cysteine ​​and adjacent peaks was greater than 1.5, indicating good separation effect. This demonstrates that the chromatographic conditions of the present invention can effectively separate and detect cysteine.

[0077] Example 2: Linear Experiment

[0078] A method for detecting cysteine ​​in a compound amino acid injection, comprising:

[0079] Reference stock solution: Accurately weigh an appropriate amount of cysteine ​​hydrochloride reference standard, dilute to the mark with mobile phase A, and prepare a solution containing approximately 0.35 mg / ml of cysteine.

[0080] Linear solutions with concentrations of 70.5200 μg / ml, 52.8900 μg / ml, 35.2600 μg / ml, 17.6300 μg / ml, 7.0520 μg / ml, and 0.1410 μg / ml were prepared from the control stock solution.

[0081] Chromatographic conditions:

[0082] Chromatographic column: Shiseido MGⅡC18, 250mm×4.6mm, 5μm;

[0083] Mobile phase A: 4 mmol / L sodium octane sulfonate buffer (pH adjusted to 2.0 with phosphoric acid): methanol = (98:2);

[0084] Mobile phase B: Acetonitrile-water (80:20);

[0085] Column temperature: 30℃;

[0086] Flow rate: 1.0 ml / min;

[0087] Wavelength: 200nm;

[0088] Injection volume: 20 μl.

[0089] The gradient elution conditions are as follows:

[0090] Time: 0 min, 100% mobile phase A, 0% mobile phase B;

[0091] Time: 18 min, 100% mobile phase A, 0% mobile phase B;

[0092] Time: 18.5 min, 50% mobile phase A, 50% mobile phase B;

[0093] Time: 21.5 min, 50% mobile phase A, 50% mobile phase B;

[0094] Time: 22 min, 100% mobile phase A, 0% mobile phase B;

[0095] Time: 40 min, 100% mobile phase A, 0% mobile phase B.

[0096] Detection method: Six linear solutions, reference solutions and 0.1410 μg / ml limit of quantitation solutions of different concentrations were accurately measured and injected into the high performance liquid chromatograph for detection. The results are shown in Table 2.

[0097] Table 2. Results of linearity study of cysteine.

[0098]

[0099] As shown in Table 2, the correlation coefficient r = 1 for the linear equation, indicating that the linear relationship is good under the chromatographic conditions of this invention.

[0100] Example 3: Accuracy Experiment

[0101] A method for detecting cysteine ​​in a compound amino acid injection, comprising:

[0102] Reference stock solution: Accurately weigh an appropriate amount of cysteine ​​hydrochloride reference standard, dilute to the mark with mobile phase A, and prepare a solution containing approximately 0.35 mg / ml of cysteine.

[0103] Reference solution: Accurately measure 1.0 ml each of cysteine ​​negative sample and reference stock solution, place them in the same 10 ml volumetric flask, dilute to the mark with mobile phase A, and shake well to obtain (0.035 mg / ml).

[0104] Negative sample solution: Accurately measure 1.0 ml of cysteine-deficient negative sample, place it in a 10 ml volumetric flask, dilute to the mark with mobile phase A, and shake well to obtain the solution.

[0105] Unspecified test solution: Accurately measure 0.5 ml of the test sample and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and shake well.

[0106] 70% recovery solution: Accurately measure 0.5 ml of the test sample and place it in a 10 ml volumetric flask. Accurately add 0.2 ml of the reference stock solution, dilute to the mark with mobile phase A, and shake well to obtain the solution.

[0107] 100% recovery solution: Accurately measure 0.5 ml of the test sample and place it in a 10 ml volumetric flask. Accurately add 0.5 ml of the reference stock solution, dilute to the mark with mobile phase A, and shake well to obtain the solution.

[0108] 130% recovery solution: Accurately measure 0.5 ml of the test sample and place it in a 10 ml volumetric flask. Accurately add 0.8 ml of the reference stock solution, dilute to the mark with mobile phase A, and shake well to obtain the solution.

[0109] Chromatographic conditions:

[0110] Chromatographic column: Shiseido MGⅡC18, 250mm×4.6mm, 5μm;

[0111] Mobile phase A: 4 mmol / L sodium octane sulfonate buffer (pH adjusted to 2.0 with phosphoric acid): methanol = (98:2);

[0112] Mobile phase B: Acetonitrile-water (80:20);

[0113] Column temperature: 30℃;

[0114] Flow rate: 1.0 ml / min;

[0115] Wavelength: 200nm;

[0116] Injection volume: 20 μl.

[0117] The gradient elution conditions are as follows:

[0118] Time: 0 min, 100% mobile phase A, 0% mobile phase B;

[0119] Time: 18 min, 100% mobile phase A, 0% mobile phase B;

[0120] Time: 18.5 min, 50% mobile phase A, 50% mobile phase B;

[0121] Time: 21.5 min, 50% mobile phase A, 50% mobile phase B;

[0122] Time: 22 min, 100% mobile phase A, 0% mobile phase B;

[0123] Time: 40 min, 100% mobile phase A, 0% mobile phase B.

[0124] Detection method: The reference solution, negative sample solution, unspecified test solution, 70% recovery solution, 100% recovery solution and 130% recovery solution were accurately injected into the high performance liquid chromatograph for detection. The results are shown in Table 3.

[0125] Table 3. Results of the accuracy assessment of cysteine.

[0126]

[0127] As shown in Table 3, the recoveries of the nine spiked solutions were all between 99.72% and 101.94%, with an average recovery of 101.32% and an RSD of 0.71%. This indicates that the accuracy of cysteine ​​is good under the chromatographic conditions of this invention.

[0128] Example 4: Durability Test

[0129] A method for detecting cysteine ​​in a compound amino acid injection, comprising:

[0130] Reference stock solution: Accurately weigh an appropriate amount of cysteine ​​hydrochloride reference standard, dilute to the mark with mobile phase A, and prepare a solution containing approximately 0.35 mg / ml of cysteine.

[0131] Reference solution: Accurately measure 1.0 ml each of cysteine ​​negative sample and reference stock solution, place them in the same 10 ml volumetric flask, dilute to the mark with mobile phase A, and shake well to obtain (0.035 mg / ml).

[0132] Test solution: Accurately measure 1.0 ml of the test sample and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and shake well to obtain the solution.

[0133] Chromatographic conditions:

[0134] Chromatographic column: Shiseido MGⅡC18, 250mm×4.6mm, 5μm;

[0135] Mobile phase A: 4 mmol / L sodium octane sulfonate buffer (pH adjusted with phosphoric acid) and methanol;

[0136] Mobile phase B: Acetonitrile-water (80:20);

[0137] Injection volume: 20 μl;

[0138] The gradient elution conditions are as follows:

[0139] Time: 0 min, 100% mobile phase A, 0% mobile phase B;

[0140] Time: 18 min, 100% mobile phase A, 0% mobile phase B;

[0141] Time: 18.5 min, 50% mobile phase A, 50% mobile phase B;

[0142] Time: 21.5 min, 50% mobile phase A, 50% mobile phase B;

[0143] Time: 22 min, 100% mobile phase A, 0% mobile phase B;

[0144] Time: 40 min, 100% mobile phase A, 0% mobile phase B.

[0145] Cysteine ​​robustness was investigated by varying column temperature, flow rate, wavelength, and pH. The column temperature, flow rate, wavelength, and pH settings are shown in Table 4 below.

[0146] Table 4 Durability Condition Parameters

[0147] condition Column temperature ℃ Flow rate (ml / min) wavelength nm pH value mobile phase A ratio Example 4-1a 25 1.0 200 2.0 98：2 Example 4-1b 35 1.0 200 2.0 98：2 Example 4-2a 30 0.9 200 2.0 98：2 Example 4-2b 30 1.1 200 2.0 98：2 Example 4-3a 30 1.0 195 2.0 98：2 Example 4-3b 30 1.0 205 2.0 98：2 Example 4-4a 30 1.0 200 1.9 98：2 Example 4-4b 30 1.0 200 2.1 98：2 Examples 4-5a 30 1.0 200 2.0 97：3 Example 4-5b 30 1.0 200 2.0 99：1

[0148] Detection method: Set the robustness condition parameters according to Table 4, accurately measure the test solution and perform detection in a high performance liquid chromatograph. The results are shown in Table 5.

[0149] Table 5 Results of Cysteine ​​Durability Study

[0150]

[0151] As shown in Table 5, after fine-tuning the chromatographic conditions, the cysteine ​​content of the test samples ranged from 89.64% to 92.32%, with an average content of 90.68% and an RSD of 0.93%. This indicates that the cysteine ​​method exhibits good robustness under the chromatographic conditions of this invention.

[0152] Example 5 Stability Experiment:

[0153] A method for detecting cysteine ​​in a compound amino acid injection, comprising:

[0154] Reference stock solution: Accurately weigh an appropriate amount of cysteine ​​hydrochloride reference standard, dilute to the mark with mobile phase A, and prepare a solution containing approximately 0.35 mg / ml of cysteine.

[0155] Reference solution: Accurately measure 1.0 ml each of cysteine ​​negative sample and reference stock solution, place them in the same 10 ml volumetric flask, dilute to the mark with mobile phase A, and shake well to obtain (0.035 mg / ml).

[0156] Test solution: Accurately measure 1.0 ml of the test sample and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and shake well to obtain the solution.

[0157] Chromatographic conditions:

[0158] Chromatographic column: Shiseido MGⅡC18, 250mm×4.6mm, 5μm;

[0159] Mobile phase A: 4 mmol / L sodium octane sulfonate buffer (pH adjusted to 2.0 with phosphoric acid): methanol = (98:2);

[0160] Mobile phase B: Acetonitrile-water (80:20);

[0161] Column temperature: 30℃;

[0162] Flow rate: 1.0 ml / min;

[0163] Wavelength: 200nm;

[0164] Injection volume: 20 μl;

[0165] The gradient elution conditions are as follows:

[0166] Time: 0 min, 100% mobile phase A, 0% mobile phase B;

[0167] Time: 18 min, 100% mobile phase A, 0% mobile phase B;

[0168] Time: 18.5 min, 50% mobile phase A, 50% mobile phase B;

[0169] Time: 21.5 min, 50% mobile phase A, 50% mobile phase B;

[0170] Time: 22 min, 100% mobile phase A, 0% mobile phase B;

[0171] Time: 40 min, 100% mobile phase A, 0% mobile phase B.

[0172] Detection method: The reference solution and the test solution were accurately measured and detected by high performance liquid chromatography. The results are shown in Table 6.

[0173] Table 6 Results of the cysteine ​​stability study

[0174] Time point Reference peak area Sample content 0h 394.587 93.60％ 2h 396.184 93.57％ 4h 395.631 93.62％ 8h 395.923 93.65％ 12h 394.107 94.06％ 16h 395.106 93.57％ 24h 395.209 93.81％ RSD 0.19％ 0.19％

[0175] As shown in Table 6, the RSD of both the control peak area and the sample content was less than 1% within 24 hours. This indicates that cysteine ​​has good stability under the chromatographic conditions of this invention.

[0176] Screening experiment of chromatographic conditions as described in Example 1

[0177] A method for detecting cysteine ​​in a compound amino acid injection, comprising:

[0178] Reference stock solution: Accurately weigh an appropriate amount of cysteine ​​hydrochloride reference standard, dilute to the mark with mobile phase A, and prepare a solution containing approximately 0.35 mg / ml of cysteine.

[0179] Reference solution: Accurately measure 1.0 ml each of cysteine ​​negative sample and reference stock solution, place them in the same 10 ml volumetric flask, dilute to the mark with mobile phase A, and shake well to obtain (0.035 mg / ml).

[0180] Test solution: Accurately measure 1.0 ml of the test sample and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and shake well to obtain the solution.

[0181] Chromatographic conditions:

[0182] Chromatographic column: Shiseido MGⅡC18, 250mm×4.6mm, 5μm;

[0183] Mobile phase A: 4 mmol / L sodium octane sulfonate buffer (pH adjusted with phosphoric acid) and methanol;

[0184] Mobile phase B: Acetonitrile-water (80:20);

[0185] Injection volume: 20 μl;

[0186] The gradient elution conditions are as follows:

[0187] Time: 0 min, 100% mobile phase A, 0% mobile phase B;

[0188] Time: 18 min, 100% mobile phase A, 0% mobile phase B;

[0189] Time: 18.5 min, 50% mobile phase A, 50% mobile phase B;

[0190] Time: 21.5 min, 50% mobile phase A, 50% mobile phase B;

[0191] Time: 22 min, 100% mobile phase A, 0% mobile phase B;

[0192] Time: 40 min, 100% mobile phase A, 0% mobile phase B.

[0193] A control example with chromatographic conditions set for column temperature, flow rate, wavelength, and pH is shown in Table 7 below:

[0194] Table 7 Comparison Example Parameters and Conditions

[0195] condition Column temperature ℃ Flow rate (ml / min) wavelength nm pH value mobile phase A ratio Compare with Example 1-1a 20 1.0 200 2.0 98：2 Compare with Example 1-1b 40 1.0 200 2.0 98：2 Compare with Example 1-2a 30 0.8 200 2.0 98：2 Compare with Example 1-2b 30 1.2 200 2.0 98：2 Compare with Example 1-3a 30 1.0 190 2.0 98：2 Compare with Example 1-3b 30 1.0 210 2.0 98：2 Compare with Example 1-4a 30 1.0 200 1.8 98：2 Compare with Example 1-4b 30 1.0 200 2.2 98：2 Compare with Example 1-5a 30 1.0 200 2.0 96：4

[0196] Detection method: The conditions and parameters of the control example were set according to Table 7. The test solution was accurately measured and detected by high performance liquid chromatography. The results are shown in Table 8.

[0197] Table 8 Results of the Cysteine ​​Control Study

[0198]

[0199] In the column temperature investigation, the content results were normal at column temperatures of 20℃ and 40℃. Changing the column temperature affected the retention time, but had no significant impact on the resolution. Under normal conditions, the content results were normal at 30℃. Considering the commonly used column temperatures and column temperature durability screening results, a column temperature range of 25–35℃ was selected.

[0200] In the flow rate studies, the concentration results were normal at flow rates of 0.8 ml / min and 1.2 ml / min. At 0.8 ml / min, the retention time of the target peak was prolonged, but the separation effect was good. At 1.2 ml / min, the retention time was premature, posing a risk of poor separation, while the concentration results were normal under normal conditions at 1.0 ml / min. Considering the comprehensive results of the flow rate robustness screening, a flow rate range of 0.9 ml / min to 1.1 ml / min was selected.

[0201] In the wavelength evaluation, the content results at 190nm and 210nm were normal, with a higher response at 190nm than at 210nm, but this had no significant impact on actual detection. Under normal conditions, the content results at 200nm were also normal. Considering both the overall wavelength durability evaluation results and the selection criteria, the wavelength range of 195nm to 205nm was chosen.

[0202] In the mobile phase pH study, the content results were normal at mobile phase pH values ​​of 1.8 and 2.2. Different pH values ​​that are too high or too low may affect the actual peak elution and separation effect, posing a risk of poor separation. Under normal conditions, the content results were normal at pH value 2.0. Considering the comprehensive evaluation of the mobile phase pH durability screening results, a pH range of 1.9–2.1 was selected.

[0203] In the investigation of mobile phase A ratios, a 96:4 ratio showed normal content results, but the peak elution time was earlier, and the separation effect was not as good as under normal conditions. Under normal conditions, a 98:2 ratio resulted in normal resolution and content. Considering the comprehensive evaluation of the robustness screening results for mobile phase A ratios, the optimal range for the ratio of sodium octane sulfonate buffer to methanol in mobile phase A is 97:3 to 99:1.

[0204] This invention effectively improves the peak shape and resolution of cysteine ​​by screening and optimizing chromatographic conditions such as mobile phase ratio, pH, flow rate, column temperature, and wavelength, and achieves accurate quantitative detection of this substance. The detection method has undergone systematic methodological validation and possesses advantages such as high specificity, good linearity, high accuracy, strong robustness, and good stability. It can quantitatively detect cysteine ​​in compound amino acid injections, thereby ensuring the efficacy and safety of related compound amino acid injections in clinical use.

[0205] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting cysteine in a compound amino acid injection, characterized by, The compound amino acid injection is 18AA-VII, provided by Sichuan Kelun Pharmaceutical Co., Ltd., and includes The high performance liquid chromatography method is adopted, and the chromatographic conditions include: a chromatographic column adopting octadecylsilane bonded silica gel filler, gradient elution with a mobile phase, detection by an ultraviolet detector, a detection wavelength of 195nm-205nm, a flow rate of the mobile phase of 0.9mL / min-1.1mL / min, an injection amount of 15μL-25μL, and a column temperature of 25℃-35℃; The mobile phase is composed of a mobile phase A and a mobile phase B, the mobile phase A is a mixed solution of methanol and sodium octane sulfonate buffer salt solution, and the mobile phase B is an acetonitrile aqueous solution; The pH of the mobile phase A is 2-2.1; The mobile phase A is a mixed solution of methanol and 3-6mmol / L sodium octane sulfonate buffer solution; The pH of the mobile phase A is adjusted by phosphoric acid; The gradient elution conditions are as follows: Time: 0min, 95-100% of the mobile phase A and 0-5% of the mobile phase B; Time: 18min, 95-100% of the mobile phase A and 0-5% of the mobile phase B; Time: 18.5min, 40-60% of the mobile phase A and 40-60% of the mobile phase B; Time: 21.5min, 40-60% of the mobile phase A and 40-60% of the mobile phase B; Time: 22min, 95-100% of the mobile phase A and 0-5% of the mobile phase B; Time: 40min, 95-100% of the mobile phase A and 0-5% of the mobile phase B.

2. The method according to claim 1, wherein the cysteine is detected in the compound amino acid injection. The volume ratio of the sodium octane sulfonate buffer solution to methanol is 97:3-99:

1.

3. The method according to claim 1, wherein the cysteine is detected by the following method. The volume ratio of acetonitrile to water in the mobile phase B is 70:30-90:

10.

4. The method according to claim 1, wherein cysteine is detected in the compound amino acid injection. The wavelength is 200nm.

5. The method according to claim 1, wherein cysteine is detected in the complex amino acid injection. The flow rate of the mobile phase is 1.0mL / min.

6. The method according to claim 1, wherein cysteine is detected in the complex amino acid injection. The column temperature is 30℃.

7. Application of the method for detecting cysteine in the compound amino acid injection as claimed in any one of claims 1-6 in detecting cysteine in the compound amino acid injection.

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