Method for detecting taurine and tyrosine content in pediatric compound amino acid injection

Abstract

The application provides a method for detecting the contents of taurine and tyrosine in pediatric compound amino acid injection, and belongs to the technical field of drug analysis, wherein the method comprises: detecting the sample to be measured by using high performance liquid chromatography; and in the detection process, an electric mist detector is used, and gradient elution is performed by using 0.1%vol-0.5%vol nonafluorovaleric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B. The application realizes the complete baseline separation of taurine, tyrosine and the remaining amino acids in the pediatric compound amino acid injection by using high performance liquid chromatography and the electric mist detector, and gradient elution is performed by using nonafluorovaleric acid aqueous solution-acetonitrile as the mobile phase, so that the technical problem that taurine and tyrosine cannot be simultaneously determined in the same analysis method is solved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for detecting taurine and tyrosine in pediatric compound amino acid injections. Background Technology

[0002] Pediatric compound amino acid injection (19AA-I) is an intravenous parenteral nutrition infusion specially designed for children. It contains 19 amino acids, including taurine and tyrosine, as well as their excipients. Ensuring the accuracy of the content of each amino acid is crucial in clinical use.

[0003] However, in this compound preparation, taurine does not contain a conjugated chromophore and has no characteristic absorption in the 200-400 nm UV-Vis region, making it impossible to directly measure using a UV detector. Tyrosine, on the other hand, contains a phenolic hydroxyl group and a benzene ring structure, exhibiting characteristic UV absorption at 274 nm. The significant difference in UV absorption characteristics between taurine and tyrosine makes it difficult to simultaneously measure them using the same analytical method, and also presents a dilemma in detector selection. Summary of the Invention

[0004] In view of the technical problems existing in the background art, this application provides a method for detecting taurine and tyrosine content in pediatric compound amino acid injection, aiming to solve the technical problem that taurine and tyrosine cannot be determined simultaneously in the same analytical method.

[0005] This application provides a method for detecting taurine and tyrosine content in a pediatric compound amino acid injection, comprising: detecting the sample to be tested using high performance liquid chromatography; wherein the detector used is an electro-fogging detector, and gradient elution is performed using a 0.1% vol to 0.5% vol aqueous solution of nonafluorovalerate as mobile phase A and acetonitrile as mobile phase B.

[0006] Furthermore, the gradient elution procedure is as follows: Furthermore, the detector used in the method is an electro-fogging detector with a fogging temperature of 45~55℃.

[0007] Furthermore, the high-performance liquid chromatography method uses a chromatographic column with porous carbon graphite as the packing material. The specifications of the chromatographic column include: column length 150~250mm, inner diameter 3.0~4.6mm, and packing particle size 3~5μm.

[0008] Furthermore, the detection conditions of the high performance liquid chromatography method include: the volumetric flow rate of the mobile phase is 0.5~1.0 mL / min.

[0009] Furthermore, the detection conditions for the high-performance liquid chromatography method include: an injection volume of 9~15 μL.

[0010] Furthermore, the detection conditions for the high-performance liquid chromatography method include: a column temperature of 15~25℃.

[0011] Furthermore, the linear range for detecting taurine content by the method is 12.21560 μg / mL to 36.64680 μg / mL, and the linear range for detecting tyrosine content is 22.51746 μg / mL to 67.55238 μg / mL.

[0012] Furthermore, the test sample includes a test solution, a reference solution, and a system suitability solution; The preparation method of the test solution includes: measuring pediatric compound amino acid injection, diluting it with water, shaking it well, filtering it, and taking the filtrate. The method for preparing the reference solution includes: measuring the reference stock solution, diluting it with water to obtain the reference solution; the reference stock solution contains taurine reference standard and tyrosine reference standard; The method for preparing the system suitability solution includes: taking phenylalanine reference standard, adding the reference standard stock solution, and diluting with water.

[0013] Furthermore, the system suitability requirements of the high performance liquid chromatography method include: when detecting the system suitability solution, the theoretical plate number of taurine and tyrosine peaks is not less than 2000, the tailing factor of taurine and tyrosine is not greater than 1.5, and the resolution between taurine and tyrosine and adjacent peaks is not less than 1.5.

[0014] The beneficial effects of this application are as follows: This application provides a method for detecting taurine and tyrosine in pediatric compound amino acid injections. The method utilizes high-performance liquid chromatography (HPLC) and an electrospray detector, employing a nonafluoropentanoic acid aqueous solution-acetonitrile mobile phase with gradient elution. This method only requires dilution and filtration of the solution before direct injection analysis. Based on the direct determination principle of solute particle charge using a CAD detector, it completely eliminates the cumbersome steps of traditional amino acid detection, such as derivatization reagent preparation, derivatization reaction temperature control, and derivatization product stabilization. The sample pretreatment time is reduced from 30-60 minutes in traditional methods to less than 2 minutes, significantly reducing the risk of errors introduced by human operation and significantly improving the detection capabilities for batch samples. This application uses porous graphite carbon as a packing material, utilizing its surface polar selectivity and planar molecular adsorption characteristics to solve the problems of weak taurine retention and easy efflux with dead volume in conventional reversed-phase systems. Furthermore, through an optimized gradient elution program, complete baseline separation of taurine, tyrosine, and other amino acids in the pediatric compound amino acid injection is achieved. This application eliminates the strong background noise problem in CAD detection by using nonafluorovalerate aqueous solution as the mobile phase. Simultaneously, the low-volatile acid mobile phase system reduces corrosion of the chromatographic column and instrument tubing, extending the column's lifespan and lowering consumable costs for long-term testing. The detection method proposed in this application has a lower operational threshold, making it more suitable for application in scenarios such as routine quality inspection in pharmaceutical companies and batch sample analysis by third-party testing institutions. It can also be extended to the determination of various compound amino acid injections.

[0015] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of this application, the accompanying drawings used in this application will be briefly described below. Obviously, the drawings described below are merely some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort.

[0017] Figure 1 This is the chromatogram of the reference solution in Example 1 of this application; Figure 2 This is a chromatogram of the system suitability solution in Example 1 of this application; Figure 3 This is the chromatogram of the test solution in Example 1 of this application; Figure 4 The linear fitting curve of taurine in Example 1 of this application; Figure 5This is the linear fitting curve of tyrosine in Example 1 of this application. Detailed Implementation

[0018] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0020] In the description of the embodiments of this application, technical terms such as "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly indicating the number, specific order, or primary and secondary relationship of the indicated technical features.

[0021] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0022] In the description of the embodiments in this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0023] In the description of the embodiments of this application, the term "multiple" refers to two or more (including two), similarly, "multiple sets" refers to two or more (including two sets), and "multiple pieces" refers to two or more (including two pieces).

[0024] In the detection of taurine and tyrosine, taurine has no characteristic absorption in the ultraviolet-visible region of 200-400 nm, while tyrosine contains phenolic hydroxyl groups and benzene ring structures and has characteristic ultraviolet absorption at 274 nm. The significant difference in their ultraviolet absorption characteristics leads to a contradiction in the selection of detectors when they are measured simultaneously in the same analytical method.

[0025] To address the technical challenge of simultaneously detecting taurine and tyrosine in pediatric compound amino acid injections, this application provides a method for detecting taurine and tyrosine in such injections. The method utilizes high-performance liquid chromatography (HPLC) and an electrospray detector, employing a nonafluorovalerate aqueous solution-acetonitrile mobile phase with gradient elution. This method allows for direct injection analysis after solution dilution and filtration, significantly reducing the risk of errors introduced by human intervention. It also overcomes the drawbacks of weak taurine retention and easy efflux with dead volume in conventional reversed-phase systems, achieving complete baseline separation of taurine and tyrosine from other amino acids in pediatric compound amino acid injections.

[0026] This application provides a method for detecting taurine and tyrosine content in a pediatric compound amino acid injection, comprising: detecting the sample to be tested using high performance liquid chromatography; wherein the detector used is an electro-fogging detector, and gradient elution is performed using a 0.1% vol to 0.5% vol aqueous solution of nonafluorovalerate as mobile phase A and acetonitrile as mobile phase B.

[0027] Among them, high performance liquid chromatography (HPLC) has high separation efficiency, wide application range, fast analysis speed and good reproducibility. The response of the electro-fogging detector does not depend on the optical properties of the analyte and has the characteristics of good repeatability and high sensitivity. Using nonafluorovaleric acid aqueous solution as mobile phase A, relying on ion pair interaction and gradient elution, it can fully separate the two target substances from other amino acids in pediatric compound amino acid injection, eliminate matrix interference, and at the same time, eliminate the strong background noise problem generated in CAD detection from the root. The mobile phase system with low volatile acid can reduce the corrosion of chromatographic column and instrument tubing, extend the service life of chromatographic column, and reduce the consumable cost of long-term detection.

[0028] It can be noted that, in some embodiments, the pediatric compound amino acid injection specifically refers to pediatric compound amino acid injection (19AA-I). By using the above method for detection, complete baseline separation of taurine, tyrosine, and the remaining 17 amino acids in pediatric compound amino acid injection (19AA-I) can be achieved.

[0029] Furthermore, in some embodiments, the gradient elution procedure is as follows: In the technical solution of this application embodiment, a nonafluorovalerate aqueous solution with a concentration of 0.1% vol to 0.5% vol is used as mobile phase A, and acetonitrile is used as mobile phase B. This allows the highly polar taurine to be fully retained on the reversed-phase chromatography column, while improving the chromatographic peak shape of the moderately polar tyrosine, reducing tailing and adsorption, and improving the separation of taurine, tyrosine and other components. In the initial stage of the elution program, 100% mobile phase A is maintained to ensure effective retention and enrichment of the analyte at the column head. The proportion of acetonitrile is gradually increased, and the taurine and tyrosine are separated sequentially by increasing the elution intensity. Then, the column is washed with a high proportion of mobile phase B to effectively remove the strongly retained matrix and impurities. Finally, the initial mobile phase is restored to ensure injection reproducibility and test stability.

[0030] Furthermore, in some embodiments, the detector used is an electro-fogging detector with a fogging temperature of 45~55°C.

[0031] In the technical solution of this application embodiment, the electro-fogging detector is a general-purpose quality detector whose response does not depend on the optical properties of the analyte. Theoretically, it can respond to all non-volatile and semi-volatile compounds. The temperature of the electro-fogging detector is 45~55℃, which can ensure that the mobile phase is fully evaporated, minimize baseline noise, and avoid the volatilization loss of taurine and tyrosine at this temperature, thus ensuring detection sensitivity and quantitative accuracy.

[0032] Furthermore, in some embodiments, the chromatographic column used in the high-performance liquid chromatography method is a porous carbon graphite as the packing material; the specifications of the chromatographic column include: column length 150~250mm, inner diameter 3.0~4.6mm, and packing particle size 3~5μm.

[0033] In the technical solution of this application embodiment, porous graphite carbon is used as a filler to replace conventional C18 and amino acid-specific silica gel columns. By utilizing its surface polar selectivity and planar molecular adsorption characteristics, the retention capacity of highly polar taurine is greatly enhanced. This solves the problem of weak taurine retention and easy outflow with dead volume in conventional reversed-phase systems. It can simultaneously ensure the stable retention, peak symmetry and effective separation of taurine and tyrosine from matrix impurities, and achieve simultaneous and accurate quantification of both without batch detection.

[0034] Furthermore, in some embodiments, the volumetric flow rate of the mobile phase used in the high-performance liquid chromatography is 0.5~1.0 mL / min.

[0035] In the technical solution of this application embodiment, a suitable volumetric flow rate can ensure that taurine and tyrosine are fully separated, and under the premise of good peak shape, a suitable column pressure is maintained to protect the column. At the same time, the flow rate can match the temperature of the electro-atomization detector to ensure that the mobile phase is fully and uniformly atomized.

[0036] Furthermore, in some embodiments, the detection conditions for high-performance liquid chromatography include an injection volume of 9-15 μL.

[0037] In the technical solution of this application embodiment, the injection volume range of 9~15μL can avoid the problems of peak tailing and decreased resolution caused by sample injection overload. It can ensure that taurine and tyrosine are well separated while the peak shape is sharp and symmetrical. At the same time, this injection range is within the high-precision range (8~15μL) of the autosampler, which can reduce the injection volume error and ensure the repeatability of the detection.

[0038] Furthermore, in some embodiments, the detection conditions for high-performance liquid chromatography include: a column temperature of 15~25°C.

[0039] In the technical solution of this application embodiment, a suitable column temperature can stabilize the viscosity of the mobile phase, keep the column pressure within a safe range, reduce the damage of high pressure to the packing material, and at the same time, a mild and low temperature can inhibit the oxidation of tyrosine phenolic hydroxyl groups, avoiding interference from impurity peaks in quantitative analysis.

[0040] Furthermore, in some embodiments, the linear range for detecting taurine content includes 12.21560 μg / mL to 36.64680 μg / mL, and the linear range for detecting tyrosine content includes 22.51746 μg / mL to 67.55238 μg / mL.

[0041] In the technical solution of this application embodiment, the lower limit of the linear range is lower than the lowest concentration of the qualified test sample and the upper limit is higher than the highest concentration of the qualified test sample, which can completely cover all qualified sample diluents with contents ranging from 80% to 120% of the labeled amount, thus meeting the requirements for the determination range of taurine and tyrosine in pediatric compound amino acid injection.

[0042] Furthermore, in some embodiments, the test sample includes a test solution, a reference solution, and a system suitability solution; The preparation method of the test solution includes: measuring 0.5~1.5mL of pediatric compound amino acid injection, placing it in a 10mL volumetric flask, diluting it with water, shaking it well, filtering it, and taking the filtrate. The preparation method of the reference solution includes: measuring appropriate amounts of taurine reference standard and tyrosine reference standard, diluting with 0.1 mol / L hydrochloric acid solution to prepare reference standard stock solution I containing approximately 1-2 mg of taurine and 2-3 mg of tyrosine per 1 mL, and then diluting with water to prepare reference standard stock solution II containing approximately 60-65 μg of taurine and 105-115 μg of tyrosine per 1 mL. Take different volumes of reference standard stock solution II, dilute quantitatively with water, and shake well.

[0043] The method for preparing the system suitability solution includes: taking an appropriate amount of phenylalanine reference standard, adding the reference standard stock solution, and diluting with water to prepare a solution containing approximately 20-30 μg of taurine, 40-50 μg of tyrosine, and 0.40-0.55 mg of phenylalanine per 1 mL.

[0044] In the technical solution of this application embodiment, the test sample solution is diluted with water to reduce the high concentration of amino acids in the injection to the chromatographic linear range. Filtration removes particulate impurities to protect the chromatographic column and eliminate baseline interference, directly reflecting the true content of the analyte in the test sample. The reference standard is first prepared with hydrochloric acid to prepare reference standard stock solution I because the phenolic hydroxyl group of tyrosine is easily oxidized in a neutral aqueous environment. Adding dilute hydrochloric acid can inhibit this characteristic, thereby preparing a stable high-concentration mother liquor. Then, it is diluted with water to obtain reference standard stock solution II, and the solvent is unified to eliminate peak area system bias. The system suitability solution mixes phenylalanine with reference standard stock solution II, which can detect the resolution, theoretical plate number, and tailing factor of three groups of amino acids with similar retention behavior: taurine, tyrosine, and phenylalanine.

[0045] Furthermore, the system suitability requirements for high performance liquid chromatography include: when detecting system suitability solutions, the theoretical plate number of taurine and tyrosine peaks is not less than 2000, the tailing factor of taurine and tyrosine is not greater than 1.5, and the resolution between taurine and tyrosine peaks and adjacent peaks is not less than 1.5.

[0046] The technical solution of this application embodiment simultaneously meets the three system applicability requirements of theoretical plate number, tailing factor, and resolution, avoiding interference such as peak broadening, peak tailing, and peak overlap in the detection of amino acids in high performance liquid chromatography. The detection results are accurate and reliable, and the instrument is easy and efficient to operate. It can be applied to the determination of various compound amino acid injections and is more suitable for promotion and application in scenarios such as daily quality inspection of pharmaceutical companies and batch sample analysis of third-party testing institutions.

[0047] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0048] I. Method Development Example 1 This embodiment provides a method for detecting taurine and tyrosine content in a pediatric compound amino acid injection (19AA-I), including: 1. Instruments and reagents Vanquish Core high-performance liquid chromatograph with electro-fogging detector (Thermo Fisher Scientific, USA); XPR6UD5 analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.).

[0049] Nonafluorovalerate (batch number I2306046, Shanghai Aladdin Biochemical Technology Co., Ltd.); Acetonitrile (batch number F22M7J201, Fisher Chemical).

[0050] Taurine (batch number 111616-201605, National Institutes for Food and Drug Control); Tyrosine (batch number 140609-202215, National Institutes for Food and Drug Control); Phenylalanine (batch number 140676-201706, National Institutes for Food and Drug Control). Pediatric Compound Amino Acid Injection (19AA-I) (Batch No. P230902SC-01-2, Wuhan Jiu'an Pharmaceutical Research Institute Co., Ltd.).

[0051] 2. Testing Methods 2.1 Chromatographic conditions Column: Hypercarb (4.6 mm × 150 mm, 5 μm) or equivalent performance column Mobile phase: 0.3% vol nonafluoropentanoic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B. Flow rate: 0.7 mL / min Column temperature: 20℃ CAD atomizer temperature: 50℃ Injection volume: 10 μL Elution method: gradient elution 2.2 Solution Preparation Preparation of test solution: Accurately measure 1 mL of pediatric compound amino acid injection (19AA-I), place it in a 10 mL volumetric flask, dilute with water to the mark, shake well, filter, and collect the filtrate.

[0052] Preparation of reference solutions: Accurately weigh appropriate amounts of taurine and tyrosine reference standards, dissolve them in 0.1 mol / L hydrochloric acid solution, and quantitatively dilute to prepare a solution containing approximately 1.25 mg taurine and 2.2 mg tyrosine per mL, as reference solution I. Accurately measure an appropriate amount of reference solution I and dilute it with water to prepare a solution containing approximately 62.5 μg taurine and 110 μg tyrosine per mL, as reference solution II. Accurately measure 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL of reference solution II into 10 mL volumetric flasks, dilute to the mark with water, and shake well.

[0053] System suitability solution: Take an appropriate amount of phenylalanine reference standard, add an appropriate amount of reference standard stock solution II, and dilute with water to prepare a solution containing approximately 25 μg taurine, 44 μg tyrosine, and 0.48 mg phenylalanine per 1 mL.

[0054] Blank solvent: water Blank preparation solution: Accurately measure 10 mL of blank preparation (pediatric compound amino acid injection without taurine and tyrosine (19AA-I)), place it in a 100 mL volumetric flask, dilute with water to the mark, and shake well.

[0055] Taurine positioning solution: Weigh about 2.5 mg of taurine raw material, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the mark, shake well, and the solution is ready.

[0056] Tyrosine positioning solution: Weigh approximately 4.4 mg of tyrosine raw material, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the mark, shake well, and the solution is ready.

[0057] Phenylalanine positioning solution: Weigh approximately 4.8 mg of phenylalanine raw material, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the mark, shake well, and the solution is ready.

[0058] 2.3 System Applicability Requirements In the system suitability solution, the theoretical plate number of taurine and tyrosine peaks should not be less than 2000, the tailing factor of taurine and tyrosine should not be greater than 1.5, and the resolution between taurine and tyrosine peaks and adjacent peaks should not be less than 1.5.

[0059] 2.4 Measurement and Quantification Methods Inject the reference solution into the liquid chromatograph and record the chromatogram. Calculate the linear regression equation using the logarithm of the reference solution concentration and the logarithm of the corresponding peak area. Separately, determine the test sample solution using the same method and calculate the content of each amino acid in the test sample using the linear regression equation.

[0060] Example 2 This embodiment provides a method for detecting taurine and tyrosine content in pediatric compound amino acid injection (19AA-I). Compared with Example 1, the only difference is that the concentration of mobile phase A, nonafluorovalerate aqueous solution, is 0.1% vol, the elution times from the top right to the bottom in the gradient elution table are adjusted to 0 min, 5 min, 19 min, 25 min, 30 min, 32 min, and 40 min, respectively, the flow rate is adjusted to 0.5 mL / min, and the column temperature is adjusted to 15℃. Other experimental parameters and conditions are basically the same as in Example 1, and will not be repeated here.

[0061] Example 3 This embodiment provides a method for detecting taurine and tyrosine content in pediatric compound amino acid injection (19AA-I). Compared with Example 1, the only difference is that the concentration of mobile phase A, nonafluorovalerate aqueous solution, is 0.5% vol, the elution times from the top right to the bottom in the gradient elution table are adjusted to 0 min, 5 min, 16 min, 24 min, 30 min, 32 min, and 40 min, respectively, and the column temperature is adjusted to 22℃. Other experimental parameters and conditions are basically the same as in Example 1, and will not be repeated here.

[0062] Examples 4-5 Examples 4-5 provide a method for detecting taurine and tyrosine content in pediatric compound amino acid injection (19AA-I), which differs from Example 1 only in the flow rate of 0.6 mL / min and 0.8 mL / min, respectively.

[0063] Other experimental parameters and conditions are basically the same as in Example 1, and will not be repeated here.

[0064] Comparative Example 1 This comparative example provides a method for detecting taurine and tyrosine content in a pediatric compound amino acid injection (19AA-I). The only difference from Example 1 is that a 0.3% vol trifluoroacetic acid aqueous solution is used as mobile phase A for gradient elution. Other experimental parameters and conditions are basically the same as in Example 1 and will not be repeated here.

[0065] Comparative Example 2 This comparative example provides a method for detecting taurine and tyrosine content in pediatric compound amino acid injection (19AA-I). Compared with Example 1, the only difference is that the gradient elution procedure has been changed. Other experimental parameters and conditions are basically the same as those in Example 1, and will not be repeated here.

[0066] In this comparative example, the gradient elution procedure is as follows: II. Method Validation To fully verify the reliability, accuracy, and stability of the method for detecting taurine and tyrosine in pediatric compound amino acid injection (19AA-I) proposed in this invention for the simultaneous quantitative detection of taurine and tyrosine, three methodological verifications were carried out sequentially for the detection conditions of Example 1: linearity and range, repeatability, and accuracy. All verification experiments used the same instruments, reagents, reference standards, chromatographic conditions, determination, and quantification methods as the method development method. The specific verification process is as follows.

[0067] 1. Linearity and Range Validation Tyrosine reference stock solution: Weigh approximately 22 mg of tyrosine accurately, place it in a 10 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to dissolve it, and dilute to the mark. Shake well to obtain the solution.

[0068] Taurine reference stock solution: Weigh approximately 12.5 mg of taurine accurately, place it in a 10 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to dissolve it and dilute to the mark, shake well, and the solution is ready.

[0069] Mixed reference stock solution: Accurately measure 5 ml each of taurine reference stock solution and tyrosine reference stock solution, place them in a 100 ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0070] Preparation of linear solutions: Accurately measure the corresponding amount of mixed reference standard stock solution into the corresponding volumetric flask according to the table below, dilute with water to the mark, and shake well to obtain the solution.

[0071] 2. Repeatability verification The test solution, reference solution, and system suitability solution are prepared in the same manner as the method developed. Six parallel portions of the test solution are prepared.

[0072] 3. Accuracy Verification The test solution, reference solution, and system suitability solution are developed in accordance with the same method.

[0073] The linearity and range verification of the mixed reference stock solution were performed in the same way as in Method 2.

[0074] Accuracy blank solution: Accurately measure 10 ml of blank preparation (pediatric compound amino acid injection without taurine and tyrosine (19AA-I)), place it in a 100 ml volumetric flask, dilute with water to the mark, and shake well to obtain the blank solution.

[0075] Accuracy solution: Take 1 ml of blank preparation, add the mixed reference standard stock solution to an appropriate volumetric flask according to the table below, dilute with water to the mark, and shake well. Prepare 3 parallel aliquots for each concentration level.

[0076] III. Analysis of Test Results for Each Embodiment and Comparative Example The chromatograms of the reference solutions, system suitability solutions, and test solutions in Example 1 are as follows: Figure 1-3 As shown, where, Figure 1 The test is for the chromatogram of 4 mL of diluted reference solution "Reference Stock Solution II".

[0077] Depend on Figure 1-3It can be seen that the peaks of taurine and tyrosine in the reference solution are symmetrical and stable; in the system suitability solution, taurine, tyrosine and phenylalanine are baseline separated and the resolution meets the standard; in the test solution containing 19 amino acids, the other component peaks do not interfere with the two target peaks and the overall baseline is stable, indicating that this method can detect taurine and tyrosine at the same time, and the chromatographic system meets the requirements for quantitative detection.

[0078] The solutions in Examples 1-4 and Comparative Examples 1-2 were tested, and the results are shown in Table 1.

[0079] Table 1. Test results of each solution in Examples 1-4 and Comparative Examples 1-2. As shown in Table 1, the blank solution and the blank preparation solution did not interfere with the elution of taurine and tyrosine. Other chromatographic peaks in the test solution did not interfere with the determination of taurine and tyrosine. The retention times of taurine and tyrosine in the positioning solution, reference solution, and test solution were basically consistent, with a relative retention time of 1.00, which is within the acceptable range of 0.95 to 1.05. The resolutions of taurine and tyrosine with adjacent chromatographic peaks were 4.9 and 3.0, respectively, both higher than the limit of 1.5. The content of the two amino acids in the test sample was within the reasonable range of 98.0% to 102.0%, indicating that the method has a good separation effect and can accurately determine the content of taurine and tyrosine in pediatric compound amino acid injection (19AA-I). Examples 2-5 were tested using different concentrations of nonafluorovalerate, gradient programs, and flow rates. The results showed that the separation effect of taurine and tyrosine was basically the same as that of Example 1, and the effective separation of taurine, tyrosine, and the remaining 17 amino acids in the preparation was completely achieved. Comparative Example 1 used a 0.3% vol trifluoroacetic acid mobile phase, which had high baseline noise and matrix interference, affecting the accurate quantification of taurine and tyrosine. The elution program used in Comparative Example 2 did not achieve good separation of taurine and tyrosine, and it was also unable to separate the two target substances from the other amino acids in the pediatric compound amino acid injection (19AA-I), indicating matrix interference.

[0080] The linearity and range of taurine and tyrosine in Example 1 were verified, and the test results are as follows: Figure 4-5 As shown.

[0081] The linearity and range of taurine and tyrosine in Example 1 were verified, and the specific data are shown in Table 2.

[0082] Table 2. Linearity test results for taurine and tyrosine. Depend on Figure 4-5As shown in Table 2, this method performs linear regression after taking the logarithm of both peak area and concentration. The linear regression equation between the logarithm of taurine concentration and the logarithm of peak area is y = 0.8915x. The correlation coefficient r = 1.0000, with a value of -1.5069, shows excellent linear correlation within the concentration range of 12.21560 μg / mL to 36.64680 μg / mL. The linear regression equation for the logarithm of tyrosine concentration and the logarithm of peak area is y = 0.7686x - 0.6057, with a correlation coefficient r = 0.9993. This indicates good linear correlation within the range of 22.51746 μg / mL to 67.55238 μg / mL, with both correlation coefficients significantly higher than 0.998. This demonstrates a stable quantitative correspondence between the concentrations of the two target substances and the CAD detector response values ​​within the concentration range covering 50% to 150% of the labeled amount of the sample. The linear range completely covers the content fluctuation range of the pediatric compound amino acid injection (19AA-I) sample, meeting the requirements for accurate quantitative detection of taurine and tyrosine in this preparation.

[0083] The repeatability method of Example 1 was verified, and the test results are shown in Table 3.

[0084] Table 3. Repeatability verification results of Example 1 As shown in Table 3, the taurine content in the six test solutions ranged from 99.3% to 101.4%, with a calculated relative standard deviation (RSD) of 1.0%. The tyrosine content ranged from 100.7% to 101.0%, with an RSD of 0.1%. Both RSDs were less than 2.0%, indicating that the intra-day precision of this detection method is excellent. The fluctuations throughout the entire process of sample dilution and filtration, chromatographic separation, CAD detector acquisition and integration are minimal. Human operation and minor instrument errors have very little interference with the detection results. The results of multiple parallel determinations of sample content are highly consistent, demonstrating that the method is stable and reliable.

[0085] The accuracy method of Example 1 was verified, and the test results are shown in Table 4.

[0086] Table 4. Accuracy method verification of Example 1 As shown in Table 4, the recovery rates of taurine samples in each group ranged from 99.3% to 103.5%, with an RSD of 1.5% for the nine samples. The recovery rates of tyrosine samples in each group ranged from 95.1% to 98.5%, with an RSD of 1.1% for the nine samples. The relative standard deviations of taurine and tyrosine were both less than 2.0%, indicating that the method was minimally affected by the 17 amino acids and excipients in the pediatric compound amino acid injection (19AA-I). The entire process of sample dilution and filtration, chromatographic separation, and CAD detection resulted in no loss of target substances. The measured values ​​showed high consistency with the true values, demonstrating the method's accuracy and reliability. It can accurately quantify taurine and tyrosine in the preparation, meeting the accuracy requirements for drug quality control.

[0087] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for detecting taurine and tyrosine content in a pediatric compound amino acid injection, characterized in that, High performance liquid chromatography (HPLC) was used to detect the sample. The detector used was an electro-fogging detector, with a nonafluorovalerate aqueous solution of 0.1% vol to 0.5% vol as mobile phase A and acetonitrile as mobile phase B, for gradient elution.

2. The method for detecting taurine and tyrosine content in pediatric compound amino acid injection according to claim 1, characterized in that, The gradient elution procedure is as follows: At the same time, the sum of the volume ratios of mobile phase A and mobile phase B is 100%.

3. The method for detecting taurine and tyrosine content in pediatric compound amino acid injection according to claim 1, characterized in that, The atomization temperature of the electro-fogging detector is 45~55℃.

4. The method for detecting taurine and tyrosine content in pediatric compound amino acid injection according to claim 1, characterized in that, The high-performance liquid chromatography method uses a column packed with porous carbon graphite. The specifications of the chromatographic column include: column length 150~250mm, inner diameter 3.0~4.6mm, and packing particle size 3~5μm.

5. The method for detecting taurine and tyrosine content in pediatric compound amino acid injection according to claim 1, characterized in that, The detection conditions for the high-performance liquid chromatography method include: The volumetric flow rate of the mobile phase is 0.5~1.0 mL / min.

6. The method for detecting taurine and tyrosine content in pediatric compound amino acid injection according to any one of claims 1, characterized in that, The detection conditions for the high-performance liquid chromatography method include: an injection volume of 9~15 μL.

7. The method for detecting taurine and tyrosine content in pediatric compound amino acid injection according to any one of claims 1, characterized in that, The detection conditions for the high-performance liquid chromatography method include: a column temperature of 15~25℃.

8. The method for detecting taurine and tyrosine content in pediatric compound amino acid injection according to claim 1, characterized in that, The linear range for detecting taurine content by the method is 12.21560 μg / mL to 36.64680 μg / mL, and the linear range for detecting tyrosine content by the method is 22.51746 μg / mL to 67.55238 μg / mL.

9. The method for detecting taurine and tyrosine content in the pediatric compound amino acid injection according to claim 1, characterized in that, The test samples include test solution, reference solution and system suitability solution; The preparation method of the test solution includes: measuring pediatric compound amino acid injection, diluting it with water, shaking it well, filtering it, and taking the filtrate. The method for preparing the reference solution includes: measuring the reference stock solution, diluting it with water to obtain the reference solution; the reference stock solution contains taurine reference standard and tyrosine reference standard; The method for preparing the system suitability solution includes: taking phenylalanine reference standard, adding the reference standard stock solution, and diluting with water.

10. The method for detecting taurine and tyrosine content in the pediatric compound amino acid injection according to claim 9, characterized in that, The system suitability requirements for the high performance liquid chromatography method include: when detecting the system suitability solution, the theoretical plate number of taurine and tyrosine peaks is not less than 2000, the tailing factor of taurine and tyrosine is not greater than 1.5, and the resolution between taurine and tyrosine peaks and adjacent peaks is not less than 1.5.

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