Method for detecting glycerol, acetic acid and sodium hydrogen sulfite in compound amino acid injection

Abstract

The invention belongs to the field of medicine quality detection, and particularly relates to a method for detecting glycerol, acetic acid and sodium hydrogen sulfite in a compound amino acid injection, which is used for simultaneously carrying out quantitative detection on glycerol, acetic acid and sodium hydrogen sulfite in the compound amino acid injection. An ion exclusion chromatography technology is adopted; the used chromatographic conditions are as follows: a chromatographic column is an Xtimate Sugar-H column (300 mm * 7.8 mm, 5 [mu] m); the mobile phase is 7.5 * 10 <-5 > mol.L <-1 > sulfuric acid solution, the flow velocity is 0.5 mL.min <-1 >, the column temperature is 65 DEG C, the detection wavelength is 200 nm, and the sample size is 10 microliters. The method for simultaneously measuring the content of glycerol, acetic acid and sodium hydrogen sulfite in the compound amino acid injection (14AA) is established, interference of 14 kinds of high-concentration amino acids can be eliminated, special instruments are not needed, specificity is high, and universality is high.

Description

Technical Field

[0001] This invention belongs to the field of drug quality testing, specifically a method for detecting glycerol, acetic acid and sodium bisulfite in compound amino acid injection. Background Technology

[0002] Compound amino acid injection, as a balanced parenteral nutrition preparation, is used to improve the nutritional status of patients before and after surgery, improve protein digestion and absorption disorders, and solve mild malnutrition caused by insufficient protein intake or excessive consumption. Glycerol, as a source of energy required by human physiology, provides a weakly acidic environment. When infused together with amino acids, it significantly improves amino acid metabolism and promotes the full utilization of amino acids. Acetic acid mainly comes from lysine acetate in the formulation, and may also be introduced during pH adjustment in the formulation process. Its content affects the pH value of the injection, which may in turn affect amino acid degradation, polymerization, clarity, and osmotic pressure. Sodium bisulfite is a commonly used antioxidant used to inhibit the production of impurities from easily oxidized amino acids in the formulation. Sulfites can induce hypersensitivity reactions in the body, affecting medication safety. Currently, compound amino acid injection (14AA) is not included in the pharmacopoeias of various countries. The current standard follows the national drug standard WS1-(XG-005)-2020, which does not include quality control for the above-mentioned glycerol, acetic acid, and sodium bisulfite components. In the 2025 edition of the Chinese Pharmacopoeia, Part II, glycerol is determined by chemical titration (sodium periodate oxidation method). Acetic acid and sodium bisulfite are listed as pharmaceutical excipients in Part IV of the Chinese Pharmacopoeia and are detected by oxidation titration and acid-base titration methods, respectively. However, the titration method is cumbersome, has low sensitivity, and has certain limitations.

[0003] Existing reports on the detection methods for glycerol, acetic acid, and sodium bisulfite mainly include gas chromatography (GC) and ion chromatography (IGC). GC requires sample pretreatment, quantification with an internal standard, and calibration with known concentration standards. IGC has significant advantages in analyzing thermally unstable components such as PAD / conductivity suppression, but complex sample matrices can affect the retention time, peak shape, and detection sensitivity of the target analyte, and it requires specific equipment. Considering the numerous components in compound amino acid injections, other components can easily interfere with the analysis of the analytes. In 2024, the National Pharmacopoeia Commission revised the drug standard for compound amino acid injection (14AA) and published a draft standard for this product, adding inspection items for these three components. Glycerol is determined by potentiometric titration, sodium bisulfite by UV-Vis spectrophotometry after reaction with basic fuchsin, and acetic acid is detected by liquid chromatography-UV end absorption. These three methods involve numerous manual steps, multiple instrument types, and low sensitivity, and cannot simultaneously determine all three components, making them unsuitable for the release inspection of large batches of compound amino acid injection samples.

[0004] According to the "Technical Requirements for Consistency Evaluation of Marketed Generic Chemical Drugs (Injectables) (Draft for Comments)" issued by the National Center for Drug Evaluation, the concentration and dosage of excipients (antibacterial agents, buffers, antioxidants, etc.) in generic injectable drugs should meet relevant limit requirements. Establishing a scientific and feasible content determination method is of great significance for product quality control and formulation analysis. Therefore, it is urgent to develop a rapid and effective analytical method for the quantitative detection of compound amino acid injections containing glycerol, acetic acid, and sodium bisulfite. This will help to comprehensively examine the stability and safety of the injection and provide a research basis for the quality control methods of compound amino acid injections. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, at least one technical problem raised in the background art is solved.

[0006] The technical solution adopted by the present invention to solve its technical problem is: the detection method of glycerol, acetic acid and sodium bisulfite in the compound amino acid injection of the present invention, which simultaneously performs quantitative detection of glycerol, acetic acid and sodium bisulfite in the compound amino acid injection; This detection method employs ion exclusion chromatography. The chromatographic conditions used in this ion exclusion chromatography technique were as follows: Ultimate Sugar-H column (300 mm × 7.8 mm, 5 μm); isocratic elution; mobile phase: 7.5 × 10⁻⁶ m. -5 mol·L -1 Sulfuric acid solution, flow rate: 0.5 mL / min -1 The column temperature was 65℃, the detection wavelength was 200 nm, and the injection volume was 10 μL.

[0007] As a further technical solution of the present invention, the detection method includes the following steps: Take blank solution, glycerol reference solution, acetic acid reference solution, sulfite reference solution, mixed reference solution and test solution, inject them into the chromatographic conditions described above and record the chromatograms; Under these chromatographic conditions, glycerol elutes at 15.8 min, acetic acid at 17.2 min, and sodium bisulfite at 18.2 min.

[0008] As a further technical solution of the present invention: the resolution between glycerol and adjacent peaks in the test sample is 0 at a column temperature of 30-50℃, 0-3 at a column temperature of 50-65℃, and 2.5-3 at a column temperature of 65-70℃.

[0009] As a further technical solution of the present invention: the separation degree between glycerol and adjacent peaks in the test sample decreases with the increase of mobile phase concentration, and the separation degree between glycerol and adjacent peaks in the test sample at mobile phase concentrations of 0.005-0.025 mol / L is 1-4.

[0010] As a further technical solution of the present invention: the resolution between acetic acid and adjacent peaks is 1.5-4 at a column temperature of 30-55℃ and 2.5-4 at a column temperature of 50-70℃.

[0011] As a further technical solution of the present invention: the separation degree between glycerol and adjacent peaks in the test sample increases with the increase of mobile phase concentration, and the separation degree between glycerol and adjacent peaks in the test sample at mobile phase concentrations of 0.005-0.025 mol / L is 0-6.5.

[0012] As a further technical solution of the present invention: the preparation of the glycerol reference solution includes the following steps: accurately weigh 300 mg of glycerol reference standard and place it in a 10 mL volumetric flask, dissolve and dilute it with water to the mark, shake well, and use it as the glycerol reference solution.

[0013] As a further technical solution of the present invention: the preparation of the acetic acid reference solution includes the following steps: accurately weigh 12.5 mg of anhydrous sodium acetate reference standard and place it in a 10 mL volumetric flask, dissolve and dilute it to the mark with water, shake well, and use it as the acetate reference solution.

[0014] As a further technical solution of the present invention, the preparation of the sulfite reference solution includes the following steps: accurately weigh 5.0 mg of sodium bisulfite reference standard and place it in a 10 mL volumetric flask, dissolve and dilute it to the mark with water, shake well, and use it as the sulfite reference solution.

[0015] As a further technical solution of the present invention: the preparation of the mixed reference solution includes the following steps: the preparation of the mixed reference solution includes the following steps: accurately weigh 600 mg of glycerol reference standard, 25.0 mg of anhydrous sodium acetate, and 10 mg of sodium bisulfite reference standard and place them in a 10 mL volumetric flask, dissolve and dilute with water to the mark, shake well, accurately transfer 5 mL, place it in a 10 mL volumetric flask, dilute with water to the mark, shake well, and use it as the mixed reference solution; The preparation of the test solution includes the following steps: accurately measure an appropriate amount of compound amino acid injection (14AA), filter, and take the filtrate as the test solution.

[0016] The beneficial effects of this invention are as follows: This invention employs ion exclusion chromatography, taking into account the structural characteristics of each component in the compound amino acid injection (14AA). The amino groups of amino acids exhibit strong ionization and no retention, while polyols such as glycerol and weak acids such as acetic acid and sulfite are repelled by ionization, resulting in chromatographic retention. Therefore, this method can eliminate the influence of high-concentration amino acids. A method for simultaneously determining the content of glycerol, acetic acid, and sodium bisulfite in the compound amino acid injection (14AA) has been established. This method can eliminate the interference of 14 high-concentration amino acids, does not require specialized instruments, and is highly specific and versatile. Attached Figure Description

[0017] The invention will now be further described with reference to the accompanying drawings.

[0018] Figure 1 This is a specificity test chromatogram of the detection method for glycerol, acetic acid and sodium bisulfite in the compound amino acid injection in Example 1; Figure 2 This is a chromatogram of the forced degradation test for the detection method of glycerol, acetic acid and sodium bisulfite in the compound amino acid injection in Example 1; Figure 3 Linear spectrum of glycerol in Example 1; Figure 4 Linear spectrum of acetic acid in Example 1; Figure 5 Linear spectrum of sodium bisulfite in Example 1; Figure 6 This is an optimized column temperature diagram of the detection method for glycerol, acetic acid and sodium bisulfite in the compound amino acid injection in Example 3; Figure 7 This is an optimized diagram of the mobile phase conditions for the detection method of glycerol, acetic acid and sodium bisulfite in the compound amino acid injection in Example 3; in, Figure 6 and Figure 7 R1 represents the resolution between glycerol and its adjacent peak; R2 represents the resolution between acetic acid and its adjacent peak. Detailed Implementation

[0019] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0020] like Figure 1-7As shown in the embodiments of the present invention, the detection method for glycerol, acetic acid and sodium bisulfite in the compound amino acid injection is described. The detection method of the present invention adopts ion exclusion chromatography. Ion exclusion chromatography usually uses a high-capacity cation exchange resin column with overall sulfonation. During the separation process, a semi-permeable membrane is formed on the surface of the stationary phase. Dissociated ions cannot pass through the membrane layer due to Donnan repulsion, while undissociated molecules can interact with the stationary phase through the membrane layer and have a certain retention effect, thereby separating the target analytes. The method for detecting glycerol, acetic acid, and sodium bisulfite in this compound amino acid injection includes the following steps: Provide a test solution and a reference solution, wherein the test solution is the compound amino acid injection to be tested; and the reference solution is an aqueous solution of glycerol reference standard, an aqueous solution of anhydrous sodium acetate reference standard, and an aqueous solution of sodium bisulfite reference standard. The test solution and the reference solution were respectively subjected to liquid chromatography detection. Based on the obtained peak areas of the test sample and the reference solution, as well as the concentration of the reference solution, the contents of glycerol, acetic acid and sodium bisulfite in the compound amino acid injection were obtained. The conditions for the liquid chromatography detection include: Chromatographic column: Strong cation hydrogen exchange column packed with sulfonated cross-linked styrene / divinylbenzene copolymer; Mobile phase: Acidic aqueous solution; Flow rate: 0.45-0.55 mL / min; Column temperature: 63-67℃; Detector: Ultraviolet detector.

[0021] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0022] In this invention, the compound amino acid injection to be tested preferably includes compound amino acid injection (14AA), and the prescription content of glycerol in the compound amino acid injection (14AA) is preferably 30g / 1000mL.

[0023] In this invention, the test solution is preferably obtained by filtering the compound amino acid injection (14AA) to be tested. The reference solution is preferably obtained by mixing glycerol, anhydrous sodium acetate and sodium bisulfite reference standards and water. This invention does not have special requirements for the method of mixing the glycerol, anhydrous sodium acetate and sodium bisulfite reference standards and water, such as accurately weighing the glycerol, anhydrous sodium acetate and sodium bisulfite reference standards, dissolving and quantitatively diluting them with water, and shaking well to obtain the reference solution.

[0024] In this invention, the concentrations of glycerol, acetic acid and sodium bisulfite in the test solution are 25-35 mg / mL, 0.72-1.0 mg / mL and 0.23-0.35 mg / mL, respectively, and more preferably 30 mg / mL, 0.90 mg / mL and 0.29 mg / mL.

[0025] In this invention, when the test solution and reference solution are subjected to liquid chromatography detection, it is preferable to further include: a detection blank solution, used to detect and eliminate interference signals present in the instrument itself or impurity peaks related to the solvent; the blank solution is preferably water.

[0026] In this invention, the preferred chromatographic column is the Yuexu Ultimate Sugar-H column (7.8mm × 300mm, 5μm). The chromatographic column described in this invention enables the separation of amino acids and impurities from glycerol, acetic acid, and sodium bisulfite in the test solution without interfering with detection.

[0027] The acidic aqueous solution preferably includes an aqueous sulfuric acid solution; the concentration of the aqueous sulfuric acid solution is preferably 0.007-0.008 mol / L, more preferably 0.0075 mol / L; the use of an acidic aqueous solution as the mobile phase in this invention is beneficial for the regeneration of the chromatographic column.

[0028] The preferred flow rate is 0.5 mL / min. At the flow rate described in this invention, the peak shapes for glycerol, acetic acid, and sodium bisulfite are good.

[0029] The column temperature is preferably 63-67℃, and more preferably 65℃.

[0030] The detector used in liquid chromatography is preferably an ultraviolet detector, and the detection wavelength of the ultraviolet detector is preferably 190-210 nm, more preferably 200 nm. Compared with differential refractive index detectors, the ultraviolet detector used in this invention has a stable baseline, low noise, and high sensitivity.

[0031] The preferred injection volume for liquid chromatography detection is 10 μL.

[0032] In this invention, the contents of glycerol, acetic acid, and sodium bisulfite in the compound amino acid injection are preferably obtained by multiplying the concentrations of glycerol, acetic acid, and sodium bisulfite in the test solution by a dilution factor. The dilution factor is a multiple of the volume of the test solution to be tested compound amino acid injection. The formulas for calculating the concentrations of glycerol, acetic acid, and sodium bisulfite in the test solution are as follows: C 甘油、醋酸及亚硫酸氢钠 =A 供试品 / A 对照品 ×C 对照品 ×P 对照品 In the formula, A 供试品 A represents the peak areas of glycerol, acetic acid, and sodium bisulfite in the test solution. 对照品 C represents the peak areas of glycerol, acetate, and sulfite in the reference solution. 对照品 The concentration of the reference solution is mg / mL; P 对照品 The purity of the reference standard is %; C甘油、醋酸及亚硫酸氢钠 The concentrations of glycerol, acetic acid, and sodium bisulfite in the test solution are expressed in mg / mL.

[0033] This invention uses high-performance liquid chromatography (HPLC) with strong cation exchange to detect the content of glycerol, acetic acid, and sodium bisulfite. It eliminates interference from amino acids and other components in the injection or solution, allows for direct determination, provides accurate results, and is simple, rapid, and widely applicable.

[0034] To further illustrate the present invention, the method for detecting the content of glycerol, acetic acid and sodium bisulfite in the compound amino acid injection provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0035] In this embodiment of the invention, the information on the reagents, medicines, and instruments used includes: 1) Reagents and chemicals: Glycerin reference standard (Bailingwei, batch number: LF70Y162, purity: 99.7%), anhydrous sodium acetate reference standard (ThermoFisher, batch number: 059326, purity: 99.9%), sodium bisulfite reference standard (SIGMA, batch number: MKCP4836, purity: 64.4%).

[0036] Compound Amino Acid Injection (14AA) (Batch No.: K25041301, commercially available product); Sulfuric acid (batch number: 20200605, Beijing Tongguang Fine Chemical Co., Ltd.); ultrapure water (Milli-Q, laboratory-made, greater than 18.2 MΩ·cm).

[0037] Compound Amino Acid Injection (14AA) is a sterile aqueous solution prepared from 14 amino acids and glycerol, containing 30g of glycerol per 1000mL. The prescription components are shown in Table 1. Table 1. Prescription components of compound amino acid injection (14AA)

[0038] 2) Instruments and equipment: Thermo U-3000 chromatograph (equipped with Chromeleon version 7.2 network data system, Thermo Fisher), IQ7000 pure water system (Milli-Q), XP205 electronic balance (METTLER).

[0039] The Thermo U-3000 chromatograph mainly includes the following sensors: Leakage sensor: Used to detect system leaks and ensure experimental safety.

[0040] Temperature sensor: integrated into the column oven, with a temperature control accuracy of ±0.1℃.

[0041] Pressure sensor: Monitors the pressure of the mobile phase and supports back pressure up to 100 MPa.

[0042] Ultraviolet detector sensor: includes a deuterium lamp and a dual-wavelength detection cell for spectral analysis.

[0043] Example 1 The method for detecting the content of glycerol, acetic acid and sodium bisulfite in compound amino acid injection (14AA) includes the following steps: Chromatographic conditions: Ultimate Sugar-H column (300 mm × 7.8 mm, 5 μm); isocratic elution; mobile phase: 7.5 × 10⁻⁶ m. -5 mol·L -1 Sulfuric acid solution, flow rate: 0.5 mL / min -1 The column temperature was 65℃, the detection wavelength was 200 nm, and the injection volume was 10 μl.

[0044] Chromatograms of blank solution (water), reference solution (glycerol concentration 30 mg / mL, sodium acetate concentration 1.25 mg / mL, and sodium bisulfite concentration 0.5 mg / mL), and test solution (obtained by filtering compound amino acid injection (14AA) and collecting the filtrate) are shown in the figure. Figure 1 From top to bottom, the solutions are blank solution, reference solution, and test solution. And the following related experiments were conducted: (1) Specificity test: Blank solvent, reference solution, and test solution were injected and analyzed under the prescribed chromatographic conditions. The results are shown in Figure 1. Under these chromatographic conditions, glycerol eluted at 15.8 min, acetic acid at 17.2 min, and sulfite at 18.2 min. The theoretical plate number based on the glycerol peak was 26195, and the separation from other peaks was good. Other components in the blank solution and test solution did not interfere with the determination of glycerol, acetic acid, and sulfite, indicating that this method has good specificity.

[0045] (2) Forced degradation test Take 2 mL of each compound amino acid injection (14AA) and place them in a 10 mL volumetric flask, and perform strong acid degradation (1 mol·L⁻¹) separately. -1 1 mL of hydrochloric acid solution destroys the degradation product over 2 hours; strong alkali degradation (1 mol·L⁻¹) -1The degradation methods included: sodium hydroxide solution (1 mL, 2 h for degradation), oxidative degradation (1 mL 3% hydrogen peroxide solution, 1 h for degradation), high-temperature degradation (boiling water bath for 2 h), and photodegradation (365 nm UV irradiation for 48 h). Results showed that glycerol and acetic acid were relatively stable under various degradation conditions, while sulfite was extremely unstable under oxidative and photodegradation conditions, readily oxidizing to sulfate. The degradation impurities did not interfere with the determination of glycerol, acetic acid, and sulfite. The results are as follows: Figure 2 As shown.

[0046] (3) Linear Accurately weigh appropriate amounts of glycerol, anhydrous sodium acetate, and sodium bisulfite reference standards, dissolve and dilute them in ultrapure water to a concentration equivalent to 15.0-60.0 mg / mL of glycerol. -1 The acetic acid concentration is 0.450-1.80 mg·mL. -1 The sulfite concentration is 0.150-0.600 mg·mL. -1 A mixed reference solution was prepared. Linear regression was performed on the peak area (Y) against the concentration (X) to obtain the regression curve, correlation coefficient, and linear range. The results are shown in Table 2.

[0047] Table 2. Linear ranges and regression equations for glycerol, acetic acid, and sulfite.

[0048] (4) Limit of quantitation and limit of detection The reference solution was diluted, and the concentration measured when S / N≥10 was taken as the limit of quantitation, and the concentration measured when S / N≥3 was taken as the limit of detection. The results are shown in Table 3.

[0049] Table 3 Limits of Quantification and Limits of Detection for Glycerol, Acetic Acid, and Sulfite

[0050] (5) Precision and repeatability tests The reference solution was measured six times consecutively under the proposed chromatographic conditions. The RSD of the glycerol peak area was 0.31%, the RSD of the acetic acid peak area was 0.20%, and the RSD of the sulfite peak area was 0.42%, indicating that the instrument has good precision.

[0051] Accurately measure and filter the compound amino acid injection (14AA). Prepare six parallel aliquots of the filtrate. Inject and analyze according to the planned chromatographic conditions. The RSDs for the glycerol peak content determination, acetic acid content determination, and sulfite content determination, calculated using the external standard method, were 0.54%, 0.70%, and 1.19%, respectively, indicating good repeatability of the method.

[0052] (6) Stability test The reference solution and the test solution were placed at room temperature for 0, 2, 4, 6, 8, 12, 18, and 24 hours for determination. The results showed that after 24 hours of storage, the RSD of the peak area for glycerol in the test solution was 0.64%, for acetic acid it was 0.67%, and for sulfite it was 2.22%. For the reference solution, after 24 hours of storage at room temperature, the RSD of the peak area for glycerol was 0.67% and for acetic acid it was 0.49%. This indicates that the stability of glycerol and acetic acid in both the reference solution and the test solution was good at room temperature for 24 hours. However, the RSD of the peak area for sulfite was 2.88% after 8 hours and 6.65% after 24 hours, indicating that the stability of sulfite within 8 hours was basically satisfactory. However, after further storage at room temperature, sulfite will be further oxidized to sulfate; therefore, the solution preparation and usage time should be strictly controlled.

[0053] (7) Recovery rate test Accurately weigh 1500 mg of glycerol, 62.50 mg of anhydrous sodium acetate, and 23.43 mg of sodium bisulfite reference standard into a 25 mL volumetric flask. Dissolve and dilute to the mark with water, and shake well to obtain the reference standard stock solution. Accurately measure 2.5 mL of the reference standard stock solution into a 5 mL volumetric flask. Dissolve and dilute to the mark with water, and shake well to obtain the reference standard solution. Accurately measure 5 mL of compound amino acid injection (14AA) into a 10 mL volumetric flask. Add 1.5 mL, 2.5 mL, and 3.5 mL of the reference standard stock solution, respectively. Dilute to the mark with the mobile phase, shake well, filter, and collect the filtrate. Prepare three aliquots for each of the low, medium, and high concentrations. The results showed that, within the range of 80% to 120% of the labeled concentration, the average recovery rate of glycerol (n=9) was 100.0%, with an RSD of 0.77%; the average recovery rate of acetic acid (n=9) was 100.0%, with an RSD of 0.68%; and the average recovery rate of sulfite (n=9) was 101.4%, with an RSD of 1.10%, indicating that the method has good accuracy.

[0054] Table 4. Results of glycerol recovery (n=9)

[0055] Table 5 Results of Acetic Acid Recovery (n=9)

[0056] Table 6 Results of sulfite recovery (n=9)

[0057] (8) Durability The effects of flow rate, column temperature, and mobile phase concentration on the determination of glycerol, acetic acid, and sulfite content were investigated. When the flow rate, column temperature, and mobile phase concentration were slightly varied, the RSD% for glycerol content determination was 0.94% (n=7), for acetic acid content determination it was 0.42% (n=7), and for sulfite content determination it was 0.40% (n=7). The results showed that the changes in these conditions had almost no effect on the determination of glycerol, acetic acid, and sulfite content.

[0058] Example 2 (1) Selection of chromatographic column This method selects a certain degree of cross-linking of all-sulfonated H. ＋ High-capacity ion exchange resins are used as chromatographic packing materials. To accelerate Donnan membrane equilibration and achieve efficient separation, the resin should have a large pore size and an appropriate degree of cross-linking. Commonly used stationary phases are fully sulfonated cross-linked polystyrene cation exchange resins with high exchange capacity. The Ultimate Sugar-H column used in this method has a large pore size and approximately 8% cross-linking, achieving excellent separation. This column effectively distinguishes analytes from high-concentration amino acids through an ion repulsion mechanism, making it particularly suitable for the detection of weak acids and alcohols in compound amino acid injections.

[0059] (2) Investigation of chromatographic conditions In this method, dilute sulfuric acid was used as the mobile phase to investigate the separation of analytes under different column temperatures (30 ℃, 40 ℃, 50 ℃, 60 ℃, 65 ℃, 70 ℃). The results showed that the separation of glycerol and acetic acid from adjacent peaks was optimal at a column temperature of 65 ℃. The method also investigated the separation under different sulfuric acid concentrations (0.0050 mol·L⁻¹, 0.0075 mol·L⁻¹, 0.01 mol·L⁻¹, 0.015 mol·L⁻¹, 0.02 mol·L⁻¹, 0.025 mol·L⁻¹). The results showed that the separation of glycerol and acetic acid from adjacent peaks was optimal at a sulfuric acid concentration of 0.0075 mol·L⁻¹.

[0060] (3) Method establishment and optimization This method, taking into account the structural characteristics of each component in compound amino acid injection (14AA), shows that the amino groups of amino acids exhibit strong ionization and no retention, while polyols such as glycerol and weak acids such as acetic acid and sulfite are repelled by ionization, resulting in chromatographic retention. Therefore, this method can eliminate the influence of high concentrations of amino acids. The established method utilizes ion exclusion chromatography with dilute sulfuric acid as the mobile phase to detect the content of multiple components in compound amino acid injection. It can effectively solve the problem of complex matrix interference in large compound preparations. The established ion exclusion chromatography method has high specificity and can well meet the quantitative requirements of excipients in compound amino acid injection.

[0061] Example 3 Based on the above-mentioned detection methods for glycerol, acetic acid, and sodium bisulfite in compound amino acid injection, this invention uses blank solution, glycerol reference solution, anhydrous sodium acetate reference solution, sulfite reference solution, mixed reference solution, and test solution, and injects them under specific chromatographic conditions for determination, recording the chromatograms (see Figure 1). Under these chromatographic conditions, glycerol elutes at 15.8 min, acetic acid at 17.2 min, and sodium bisulfite at 18.2 min; The resolution between glycerol and adjacent peaks in the test sample was 0 at column temperatures of 30-50℃, 0-3 at column temperatures of 50-65℃, and 2.5-3 at column temperatures of 65-70℃. The resolution between glycerol and adjacent peaks in the test sample decreases with increasing mobile phase concentration. The resolution between glycerol and adjacent peaks in the test sample at mobile phase concentrations of 0.005-0.025 mol / L is 1-4. The resolution between acetic acid and adjacent peaks was 1.5-4 at column temperatures of 30-55℃ and 2.5-4 at column temperatures of 50-70℃. The resolution between glycerol and adjacent peaks in the test sample increases with increasing mobile phase concentration. The resolution between glycerol and adjacent peaks in the test sample at mobile phase concentrations of 0.005-0.025 mol / L is 0-6.5. The preparation of blank solution, glycerol reference solution, acetic acid reference solution, sulfite reference solution, mixed reference solution, and test solution includes the following steps: The preparation of the blank solution includes the following steps: taking water as the blank solution and filtering it; The preparation of the glycerol reference solution includes the following steps: accurately weigh 300 mg of glycerol reference standard and place it in a 10 mL volumetric flask, dissolve and dilute with water to the mark, shake well, and this is the glycerol reference solution; The preparation of the acetic acid reference solution includes the following steps: accurately weigh 12.5 mg of anhydrous sodium acetate reference standard and place it in a 10 mL volumetric flask, dissolve and dilute it to the mark with water, shake well, and use it as the acetate reference solution; The preparation of the sulfite reference solution includes the following steps: accurately weigh 5.0 mg of sodium bisulfite reference standard and place it in a 10 mL volumetric flask, dissolve and dilute with water to the mark, shake well, and use it as the sulfite reference solution; The preparation of the mixed reference solution includes the following steps: accurately weigh 600 mg of glycerol reference standard, 25 mg of anhydrous sodium acetate reference standard, and 10 mg of sodium bisulfite reference standard into a 10 mL volumetric flask, dissolve and dilute with water to the mark, shake well, accurately transfer 5 mL into a 10 mL volumetric flask, add blank solvent to dilute and make up to volume, and use as the mixed reference solution. The preparation of the test solution includes the following steps: accurately measure an appropriate amount of compound amino acid injection (14AA), filter it, and use it as the test solution; It should be noted that the specific chromatographic conditions described above are: Rezex ROA-organic Acid H+ column (300 mm × 7.8 mm, 5 μm); isocratic elution; mobile phase: 7.5 × 10⁻⁶. -5 mol·L -1 Sulfuric acid solution, flow rate: 0.5 mL / min -1 The column temperature was 65℃, the detection wavelength was 200 nm, and the injection volume was 10 μL.

[0062] The working principle of this invention is as follows: This invention employs ion exclusion chromatography, combined with the structural characteristics of each component in the compound amino acid injection (14AA). The amino groups of amino acids exhibit strong ionization and no retention, while polyols such as glycerol and weak acids such as acetic acid and sulfite are repelled by ionization, resulting in chromatographic retention. Therefore, this method can eliminate the influence of high-concentration amino acids. A method for simultaneously determining the content of glycerol, acetic acid, and sodium bisulfite in compound amino acid injection (14AA) has been established. This method can eliminate the interference of 14 high-concentration amino acids, does not require specialized instruments, and has high specificity and versatility.

[0063] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting glycerol, acetic acid, and sodium bisulfite in compound amino acid injection, characterized in that: This detection method can simultaneously perform quantitative detection of glycerol, acetic acid and sodium bisulfite in compound amino acid injection; This detection method employs ion exclusion chromatography. The chromatographic conditions used in this ion exclusion chromatography technique were as follows: Ultimate Sugar-H column (300 mm × 7.8 mm, 5 μm); isocratic elution; mobile phase: 7.5 × 10⁻⁶ m. -5 mol·L -1 Sulfuric acid solution, flow rate: 0.5 mL / min -1 The column temperature was 65℃, the detection wavelength was 200 nm, and the injection volume was 10 μL.

2. The method for detecting glycerol, acetic acid, and sodium bisulfite in the compound amino acid injection according to claim 1, characterized in that: The steps of this detection method include: Take blank solution, glycerol reference solution, acetic acid reference solution, sulfite reference solution, mixed reference solution and test solution, inject them into the chromatographic conditions described above and record the chromatograms; Under these chromatographic conditions, glycerol elutes at 15.8 min, acetic acid at 17.2 min, and sodium bisulfite at 18.2 min.

3. The method for detecting glycerol, acetic acid, and sodium bisulfite in the compound amino acid injection according to claim 2, characterized in that: The resolution between glycerol and adjacent peaks in the test sample was 0 at column temperatures of 30-50℃, 0-3 at column temperatures of 50-65℃, and 2.5-3 at column temperatures of 65-70℃.

4. The method for detecting glycerol, acetic acid, and sodium bisulfite in the compound amino acid injection according to claim 3, characterized in that: The resolution between glycerol and adjacent peaks in the test sample decreases with increasing mobile phase concentration. The resolution between glycerol and adjacent peaks in the test sample is 1-4 at mobile phase concentrations of 0.005-0.025 mol / L.

5. The method for detecting glycerol, acetic acid, and sodium bisulfite in the compound amino acid injection according to claim 4, characterized in that: The resolution between acetic acid and adjacent peaks was 1.5-4 at column temperatures of 30-55℃ and 2.5-4 at column temperatures of 50-70℃.

6. The method for detecting glycerol, acetic acid, and sodium bisulfite in the compound amino acid injection according to claim 5, characterized in that: The resolution between glycerol and adjacent peaks in the test sample increases with increasing mobile phase concentration. The resolution between glycerol and adjacent peaks in the test sample at mobile phase concentrations of 0.005-0.025 mol / L is 0-6.

5.

7. The method for detecting glycerol, acetic acid, and sodium bisulfite in the compound amino acid injection according to claim 1, characterized in that: The preparation of the glycerol reference solution includes the following steps: accurately weigh 300 mg of glycerol reference standard into a 10 mL volumetric flask, dissolve and dilute with water to the mark, and shake well to obtain the glycerol reference solution.

8. The method for detecting glycerol, acetic acid, and sodium bisulfite in the compound amino acid injection according to claim 7, characterized in that: The preparation of acetic acid reference solution includes the following steps: Accurately weigh 12.5 mg of anhydrous sodium acetate reference standard and place it in a 10 mL volumetric flask. Dissolve and dilute with water to the mark, and shake well to obtain the acetate reference standard solution.

9. The method for detecting glycerol, acetic acid, and sodium bisulfite in the compound amino acid injection according to claim 8, characterized in that: The preparation of the sulfite reference solution includes the following steps: accurately weigh 5.0 mg of sodium bisulfite reference standard and place it in a 10 mL volumetric flask, dissolve and dilute with water to the mark, shake well, and use it as the sulfite reference solution.

10. The method for detecting glycerol, acetic acid, and sodium bisulfite in the compound amino acid injection according to claim 9, characterized in that: The preparation of the mixed reference solution includes the following steps: Accurately weigh 600 mg of glycerol reference standard, 25.0 mg of anhydrous sodium acetate, and 10 mg of sodium bisulfite reference standard into a 10 mL volumetric flask. Dissolve and dilute with water to the mark, shake well, accurately transfer 5 mL into a 10 mL volumetric flask, dilute with water to the mark, shake well, and this is the mixed reference standard solution. The preparation of the test solution includes the following steps: accurately measure an appropriate amount of compound amino acid injection (14AA), filter, and take the filtrate as the test solution.

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