Method for simultaneously determining tryptophan and phenylalanine related impurities in compound amino acid injection

Abstract

The application provides a method for simultaneously determining tryptophan and phenylalanine related impurities in a compound amino acid injection, and belongs to the technical field of chemical analysis. The method comprises the following steps: respectively preparing a test sample solution, a control sample solution and a mixed control sample solution, then respectively injecting the solutions into a high performance liquid chromatograph for detection, recording a chromatogram, and calculating the content of each impurity; wherein the chromatographic conditions of the high performance liquid chromatograph comprise: using ammonium formate buffer-methanol as mobile phase A, and using acetonitrile-mobile phase A as mobile phase B for gradient elution. The application develops a new mobile phase system, which can ensure good separation of each impurity, and uses four different detection wavelengths, which is beneficial to obtaining a better peak shape and lower background interference, so as to realize simultaneous determination of tryptophan and phenylalanine related impurities (8 known impurities and unknown impurities) in the compound amino acid injection.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis technology, specifically to a method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injections. Background Technology

[0002] Compound amino acid injections are widely used parenteral nutrition drugs in clinical practice, containing 9 to 20 components such as tryptophan and phenylalanine. During the production and storage of amino acids, tryptophan and phenylalanine are prone to oxidation and high-temperature degradation, producing various related impurities. Known impurities of tryptophan mainly include 5-hydroxytryptamine, 2-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, kynurenine, and formylkynurenine, totaling seven. Other unknown impurities may also be present. The main impurity of phenylalanine is phenylethylamine.

[0003] If the aforementioned impurities exceed the threshold values ​​specified in ICH Q3B(R2): Impurities in New Drug Formulations, they may possess toxicity, thereby affecting the safety and efficacy of the drug. Therefore, it is necessary to detect and control the relevant impurities in amino acid injections.

[0004] Currently, high-performance liquid chromatography-mass spectrometry (LC-MS) is mostly used to simultaneously determine multiple impurities in compound amino acid injections. This is mainly because compound amino acid injections typically consist of 9 to 20 components, resulting in a very complex matrix. Furthermore, the structure of amino acids themselves means that most components have no UV absorption, and their degradation products also lack absorption. This makes conventional analytical instruments such as HPLC, UV, or time-of-flight detectors unsuitable for the required detection. However, while LC-MS offers high sensitivity and good selectivity, its expensive equipment, high operating costs, and complex method development make it unsuitable as a routine quality control method.

[0005] High-performance liquid chromatography (HPLC) is a commonly used method for impurity detection, and it has been applied to the detection of impurities in compound amino acid injections. However, current HPLC methods for detecting impurities in compound amino acid injections mostly target one or a few similar impurities. For impurities related to tryptophan and phenylalanine in compound amino acid injections, existing HPLC methods are insufficient for the simultaneous determination of these impurities.

[0006] In view of this, it is necessary to develop a simple, economical, accurate method based on high performance liquid chromatography that can simultaneously determine tryptophan and phenylalanine-related impurities in compound amino acid injections. Summary of the Invention

[0007] In view of the technical problems existing in the background art, this application provides a method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injection. By developing a new mobile phase system, adopting a multi-wavelength detection strategy, and establishing a wavelength correction method for co-eluting impurities, the method can achieve the simultaneous determination of tryptophan and phenylalanine-related impurities in compound amino acid injection in a simple, economical and accurate manner.

[0008] This application provides a method for simultaneously determining tryptophan and phenylalanine-related impurities in a compound amino acid injection, comprising the following steps: Take the compound amino acid injection to be tested, dilute it with water and make up to volume to obtain the test solution; Take tryptophan reference standard, dissolve it in water and dilute it quantitatively to obtain the reference standard solution; Take the impurity reference standard stock solution, dilute it quantitatively with water to obtain a mixed reference standard solution; the impurity reference standard stock solution contains formylkynurenine, 5-hydroxytryptamine, 2-hydroxytryptamine, 5-hydroxytryptamine, 3-phenylaminoalanine, dioxindolealanine, kynurenine, and phenylethylamine. The test solution, the reference solution, and the mixed reference solution were injected into a high-performance liquid chromatograph for detection, the chromatograms were recorded, and the content of each impurity was calculated. The chromatographic conditions of the high-performance liquid chromatograph include: Gradient elution was performed using ammonium formate buffer-methanol as mobile phase A and acetonitrile-mobile phase A as mobile phase B.

[0009] In some embodiments, the chromatographic conditions of the high-performance liquid chromatograph further include: Using a DAD as the detector, the detection wavelengths include 210nm, 250nm, 270nm, and 324nm.

[0010] In some embodiments, when the detection wavelength is 250 nm, in the chromatogram of the mixed reference solution, dioxindole alanine has two chromatographic peaks, formylkynurenine and 2-hydroxytryptophan co-elute, and the resolution of adjacent chromatographic peaks is not less than 1.5.

[0011] In some embodiments, the external standard method is used to calculate the content of each impurity based on the peak area; The peak area of ​​2-hydroxytryptophan among the impurities was calculated using the wavelength correction method.

[0012] In some embodiments, the peak area of ​​2-hydroxytryptophan is calculated using the following formula: In the formula, A C，250nmThis indicates the peak area of ​​2-hydroxytryptophan at a detection wavelength of 250 nm; A A+C,250nm This represents the peak area of ​​the co-elution peak of 2-hydroxytryptophan and formicylkynurenine at a detection wavelength of 250 nm; A A，324nm This represents the peak area of ​​formylkynurenine when the detection wavelength is 324 nm; f is the wavelength correction factor, with a value range of 2.60~2.65.

[0013] In some embodiments, the ammonium formate buffer includes an ammonium formate solution and a pH adjuster; The pH value of the ammonium formate buffer solution is 3.1~4.5; The concentration of ammonium formate in the formate solution is 18~22 mmol / L; The pH adjuster includes one of phosphoric acid, hydrochloric acid, and formic acid.

[0014] In some embodiments, in mobile phase A, the volume ratio of ammonium formate buffer to methanol is 96:4 to 98:2; in mobile phase B, the volume ratio of acetonitrile to mobile phase A is 25:75 to 35:65.

[0015] In some embodiments, the gradient elution is performed using a linear gradient as shown in the table below: In the table above, the time increments sequentially from top to bottom; at the same time, the sum of mobile phase A and mobile phase B is 100%.

[0016] In some embodiments, the chromatographic conditions of the high-performance liquid chromatograph further include: Use a C18 column or a column with equivalent performance; The column temperature is 38~52℃; The temperature of the injection plate is 3~10℃; The injection volume is 10~50μL; The elution flow rate was 0.9~1.1 mL / min.

[0017] In some embodiments, the concentration of tryptophan in the reference solution is 5 μg / mL; In the mixed reference solution, the concentrations of formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 5-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, and kynurenine are 2 μg / mL, and the concentration of phenethylamine is 1 μg / mL.

[0018] The beneficial effects of this application include: This application provides a method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injections. By developing a new mobile phase system, it achieves better peak shape and lower background interference while ensuring good separation of each impurity. Based on this, this application sets four detection wavelengths according to the ultraviolet absorption characteristics of each impurity, enabling comprehensive detection of tryptophan and phenylalanine-related impurities in compound amino acid injections. Furthermore, for impurities with co-eluting peaks, this application establishes a wavelength correction method, effectively solving the technical problem of difficulty in quantitatively determining different impurities under co-eluting conditions and improving the reliability of the measurement results.

[0019] Through the above methods, this application achieves the simultaneous determination of seven known impurities of tryptophan (5-hydroxytryptamine, 2-hydroxytryptophan, 5-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, kynurenine, and formylkynurenine), unknown impurities of tryptophan, and phenylethylamine, an impurity of phenylalanine, in compound amino acid injections using the same HPLC method. Furthermore, the method provided in this application has low requirements for instruments and equipment, low operating costs, high sensitivity, and is not easily affected by interference. It can be applied to various compound amino acid injections containing tryptophan and phenylalanine and has good prospects for practical application.

[0020] 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

[0021] 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.

[0022] Figure 1 This is the chromatogram of the mixed reference standard in Example 1 of this application at 250 nm; Figure 2 This is the chromatogram of the mixed reference standard in Example 1 of this application at 324 nm; Figure 3 This is the chromatogram of the mixed reference standard in Example 1 of this application at 210 nm; Figure 4 This is the chromatogram of the reference standard in Example 1 of this application at 270 nm; Figure 5 This is the chromatogram of the undamaged test sample at 270 nm in Example 1 of this application. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] 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 specifying the number, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this application, "multiple" means two or more (including two), unless otherwise explicitly defined.

[0026] 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.

[0027] Among existing impurity detection methods, LC-MS instruments are expensive, have high operating costs, and are complex to develop. HPLC is relatively simpler and more economical, but it is difficult to simultaneously determine tryptophan and phenylalanine-related impurities in compound amino acid injections. To simultaneously determine tryptophan and phenylalanine-related impurities in compound amino acid injections in a simple, economical, and accurate manner, this application provides a method for the simultaneous determination of tryptophan and phenylalanine-related impurities in compound amino acid injections. By developing a new mobile phase system, employing a multi-wavelength detection strategy, and establishing a wavelength correction method for co-eluting impurities, the simultaneous determination of tryptophan and phenylalanine-related impurities in compound amino acid injections is achieved simply, economically, and accurately.

[0028] This application provides a method for simultaneously determining tryptophan and phenylalanine-related impurities in a compound amino acid injection, comprising the following steps: Take the compound amino acid injection to be tested, dilute it with water and make up to volume to obtain the test solution; Take tryptophan reference standard, dissolve it in water and dilute it quantitatively to obtain the reference standard solution; Take the impurity reference standard stock solution, dilute it quantitatively with water to obtain a mixed reference standard solution; the impurity reference standard stock solution contains formylkynurenine, 5-hydroxytryptamine, 2-hydroxytryptamine, 5-hydroxytryptamine, 3-phenylaminoalanine, dioxindolealanine, kynurenine, and phenylethylamine. The test solution, reference solution, and mixed reference solution were injected into a high-performance liquid chromatograph for detection. The chromatograms were recorded, and the content of each impurity was calculated. The chromatographic conditions for high performance liquid chromatography include: Gradient elution was performed using ammonium formate buffer-methanol as mobile phase A and acetonitrile-mobile phase A as mobile phase B.

[0029] In the technical solution of this application embodiment, by developing a new mobile phase system, while ensuring good separation of various impurities, it is beneficial to obtain better peak shape and lower background interference, so as to realize the simultaneous determination of tryptophan and phenylalanine-related impurities in compound amino acid injection.

[0030] It should be noted that the compound amino acid injection in this application can be any type of compound amino acid injection containing tryptophan and phenylalanine, and this application does not limit its specific type. For example only, the compound amino acid injection can be compound amino acid injection (18AA), compound amino acid injection (15-HBC), compound amino acid injection (9AA), compound amino acid injection (14AA), compound amino acid injection (19AA-I), compound amino acid injection (18AA-VII), compound amino acid injection (17AA), compound amino acid injection (15AA), compound amino acid injection (20AA), compound amino acid injection (18AA-V), compound amino acid injection (18AA-IX), compound amino acid injection (18AA-VI), compound amino acid injection (20AA), etc.

[0031] It should be noted that the concentrations of the test solution, reference solution, and mixed reference solution can be adjusted according to actual needs, and this application is not limited thereto. For example only, in some embodiments, 5 mL of the compound amino acid injection to be tested is taken, diluted with water, and brought to a final volume of 10 mL to obtain the test solution; in some embodiments, the concentration of tryptophan in the reference solution is 5 μg / mL; in some embodiments, the concentrations of formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 5-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, and kynurenine in the mixed reference solution are 2 μg / mL, and the concentration of phenethylamine is 1 μg / mL.

[0032] Furthermore, in some embodiments, the chromatographic conditions of the high-performance liquid chromatograph also include: Using a DAD as the detector, the detection wavelengths include 210nm, 250nm, 270nm, and 324nm.

[0033] In the embodiments of this application, by employing the HPLC-UV / DAD method and setting different detection wavelengths, different impurities can be detected. Specifically, 210 nm is specifically used for detecting phenethylamine, which exhibits strong absorption at 210 nm; 250 nm is mainly used for detecting impurities such as formylkynurenine, 5-hydroxytryptamine, 2-hydroxytryptamine, 3-phenylaminoalanine, dioxindolealanine, and kynurenine, all of which show good absorption at 250 nm; 270 nm is mainly used for detecting other unknown impurities related to tryptophan, ensuring comprehensive monitoring of unknown impurities; 324 nm is specifically used for detecting formylkynurenine, which exhibits characteristic absorption at 324 nm, significantly improving detection sensitivity and selectivity. Based on the four detection wavelengths set in the embodiments of this application, it is possible to simultaneously determine seven known impurities of tryptophan (5-hydroxytryptamine, 2-hydroxytryptophan, 5-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, kynurenine, and formylkynurenine), unknown impurities of tryptophan, and phenylethylamine, an impurity of phenylalanine, in compound amino acid injection.

[0034] Furthermore, in some embodiments, when the detection wavelength is 250 nm, in the chromatogram of the mixed reference solution, dioxindole alanine has two chromatographic peaks, formylkynurenine and 2-hydroxytryptophan co-elute, and the resolution of adjacent chromatographic peaks is not less than 1.5.

[0035] In the embodiments of this application, once the mixed reference solution meets the above conditions, it meets the system suitability requirements, and impurities such as formylkynurenine, 5-hydroxytryptamine, 2-hydroxytryptamine, 5-hydroxytryptamine, 3-phenylaminoalanine, dioxindolealanine, and kynurenine can be separated well.

[0036] Furthermore, in some embodiments, the external standard method is used to calculate the content of each impurity based on the peak area; The peak area of ​​2-hydroxytryptophan among the impurities was calculated using the wavelength correction method.

[0037] The external standard method is used to calculate the content of each impurity based on the peak area. The formula is as follows: C 杂质-供试品 =A 杂质-供试品 ×C 杂质-对照品 ÷A 杂质-对照品 ×n÷N×100% In the formula, C 杂质-供试品Indicates the concentration of the corresponding impurity in the test solution, A 杂质-供试品 C represents the peak area of ​​the corresponding impurity in the chromatogram of the test solution. 杂质-对照品 A represents the concentration of the corresponding impurity in the reference solution of that impurity. 杂质-对照品 The term "reference solution" refers to the peak area of ​​the corresponding impurity in the chromatogram of the reference solution. "n" represents the dilution factor of the test solution, and "N" represents the labeled amount of tryptophan or phenylalanine in the product. The reference solutions corresponding to the known impurities 5-hydroxytryptamine, 2-hydroxytryptamine, 5-hydroxytryptamine, 3-phenylaminoalanine, dioxindolealanine, kynurenine, formylkynurenine, and phenylethylamine constitute the mixed reference solution. The reference solution corresponding to the unknown impurity of tryptophan is the reference solution itself.

[0038] Since formylkynurenine and 2-hydroxytryptophan co-elute at 250 nm, the peak areas of these two impurities cannot be directly determined by the area of ​​the co-elution peak alone. Therefore, this application employs a multi-wavelength switching detection scheme. The peak area of ​​formylkynurenine is calculated using its characteristic absorption at 324 nm, and then wavelength correction is performed based on this calculation to determine the peak area of ​​2-hydroxytryptophan at 250 nm.

[0039] Specifically, in some embodiments, the peak area of ​​2-hydroxytryptophan is calculated using the following formula: In the formula, A C，250nm This indicates the peak area of ​​2-hydroxytryptophan at a detection wavelength of 250 nm; A A+C,250nm This represents the peak area of ​​the co-elution peak of 2-hydroxytryptophan and formicylkynurenine at a detection wavelength of 250 nm; A A，324nm This represents the peak area of ​​formylkynurenine when the detection wavelength is 324 nm; f is the wavelength correction factor, with a value range of 2.60~2.65.

[0040] In the embodiments of this application, for two impurities with co-eluting peaks, the wavelength correction method is established to calculate the content of the corresponding impurities by utilizing the difference in the absorption coefficients of each impurity at different wavelengths. This effectively solves the technical problem of difficulty in quantitatively measuring different impurities under co-eluting conditions and improves the reliability of the measurement results.

[0041] Through the synergistic effect of the novel mobile phase system, multi-wavelength detection strategy, and wavelength correction method established for co-eluting impurities in the above embodiments, the method provided in this application can simultaneously determine tryptophan and phenylalanine-related impurities in compound amino acid injections. Moreover, the method provided in this application has low requirements for instruments and equipment, low operating costs, high sensitivity, and is not easily affected by interference. It can be applied to various compound amino acid injections containing tryptophan and phenylalanine and has good prospects for practical application.

[0042] Furthermore, in some embodiments, the ammonium formate buffer comprises an ammonium formate solution and a pH adjuster; wherein the pH value of the ammonium formate buffer is 3.1 to 4.5; the concentration of ammonium formate in the ammonium formate solution is 18 to 22 mmol / L; and the pH adjuster comprises one of phosphoric acid, hydrochloric acid, and formic acid.

[0043] In the embodiments of this application, the ammonium formate buffer solution formed by the above method is used as the main component of mobile phase A, which is beneficial to obtaining better separation effect so as to simultaneously determine multiple impurities.

[0044] Furthermore, in some embodiments, in mobile phase A, the volume ratio of ammonium formate buffer to methanol is 96:4 to 98:2; in mobile phase B, the volume ratio of acetonitrile to mobile phase A is 25:75 to 35:65.

[0045] Furthermore, in some embodiments, gradient elution is performed using a linear gradient as shown in the table below: In the table above, the time increments sequentially from top to bottom; at the same time, the sum of mobile phase A and mobile phase B is 100%.

[0046] In the above embodiments, by adjusting the composition of each mobile phase and the elution gradient, different impurities can be eluted efficiently, so as to achieve simultaneous detection of multiple impurities.

[0047] Furthermore, in some embodiments, the chromatographic conditions of the high-performance liquid chromatograph also include: Use a C18 column or other equivalent column; column temperature: 38-52℃; injection plate temperature: 3-10℃; injection volume: 10-50μL; elution flow rate: 0.9-1.1mL / min.

[0048] The C18 column is a chromatographic column using octadecylsilane-bonded silica gel as the packing material. The specific manufacturer and size can be selected according to actual needs, and this application does not limit them. For example only, in some embodiments, the C18 column is Spectrum Red AQ-C18 (inner diameter 4.6 mm × column length 250 mm, packing particle size 3 μm).

[0049] It can be noted that the method provided in this application has good applicability, and the above parameters can be appropriately adjusted within the corresponding range to achieve the required technical effect and meet the application needs of different situations.

[0050] 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.

[0051] Example 1 This embodiment provides a method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injections, including the following steps: S1. Prepare test solutions (undamaged test solutions, oxidatively degraded test solutions, and high-temperature degraded test solutions). Accurately measure 5 mL of compound amino acid injection (18AA), place it in a 10 mL volumetric flask, dilute with water and bring to volume, shake well to obtain an intact test solution.

[0052] Accurately measure 10 mL of compound amino acid injection (18AA) and place it in a 20 mL volumetric flask. Add 2 mL of 3% hydrogen peroxide solution and let it stand at room temperature for 4 h. Dilute with water to the mark, shake well, filter with a PES filter membrane, and take the filtrate to obtain the oxidative degradation test solution.

[0053] Take 25 mL of compound amino acid injection (18AA), place it in a screw-top bottle, place it in an oven at 105℃ for 2 hours, remove it, cool it, shake it well, transfer it to a 50 mL volumetric flask, dilute it with water to the mark, shake it well, filter it with a PES filter membrane, and take the filtrate to obtain the high-temperature degradation test solution.

[0054] S2. Preparation of reference solution Take the tryptophan reference standard, accurately weigh it, dissolve it in water, and dilute it quantitatively to obtain the reference solution; the concentration of tryptophan in the reference solution is 5 μg / mL.

[0055] S3. Preparation of mixed reference solution Accurately weigh the reference standards of formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 5-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, kynurenine, and phenethylamine, dissolve them separately in 50% methanol solution, and quantitatively dilute them to obtain impurity reference standard stock solutions. In these stock solutions, the concentrations of formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 5-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, and kynurenine are 100 μg / mL, and the concentration of phenethylamine is 50 μg / mL. Accurately measure this impurity reference standard stock solution and quantitatively dilute it with water to obtain a mixed reference standard solution. In this mixed reference standard solution, the concentrations of formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 5-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, and kynurenine are 2.0 μg / mL, and the concentration of phenethylamine is 1.0 μg / mL.

[0056] S4. Inject the three test solutions, reference solutions, and mixed reference solutions into the high-performance liquid chromatograph for detection, record the chromatograms, and calculate the concentration of each impurity.

[0057] The chromatographic conditions for high performance liquid chromatography include: Chromatographic column: Mid-Spectrum Red AQ-C18 (inner diameter 4.6 mm × column length 250 mm, packing particle size 3 μm); Gradient elution was performed using ammonium formate buffer-methanol as mobile phase A and acetonitrile-mobile phase A as mobile phase B, as shown in Table 1. The ammonium formate buffer consisted of 20 mmol / L ammonium formate solution and phosphate as a pH adjuster, with a pH of 3.70. The volume ratio of ammonium formate buffer to methanol was 98:2, and the volume ratio of acetonitrile to mobile phase A was 30:70. The elution flow rate was 1 mL / min; the column temperature was 50 °C; the injection plate temperature was 4 °C; and the injection volume was 10 μL. Using a DAD detector, the detection wavelengths for 5-hydroxytryptamine, 2-hydroxytryptamine, 3-phenylaminoalanine, dioxindolealanine, and kynurenine are 250 nm; the detection wavelength for formylkynurenine is 324 nm; the detection wavelength for phenylethylamine is 210 nm; and the detection wavelength for other impurities of tryptophan is 270 nm.

[0058] Table 1 Gradient elution program After detection according to the method of this embodiment, the chromatograms of the mixed reference solution at 250 nm, 324 nm, and 210 nm are as follows: Figure 1 , Figure 2 , Figure 3As shown, the chromatogram of the reference solution at 270 nm is as follows. Figure 4 As shown, the chromatogram of the undamaged test solution at 270 nm is as follows. Figure 5 As shown.

[0059] The measurement results are as follows: ① The blank solvent does not interfere with the determination of known impurities of tryptophan and phenylalanine; ② At a wavelength of 250 nm, dioxindole alanine 1, dioxindole alanine 2, 5-hydroxytryptophan, kynurenine, 5-hydroxytryptophan, 3-phenylaminoalanine, and 2-hydroxytryptophan eluted sequentially in the mixed reference solution, with formylkynurenine and 2-hydroxytryptophan co-eluting. The resolution between each known impurity and adjacent peaks was greater than 3.3, and the system suitability met the requirements. ③ In the undamaged test sample solution, the minimum separation degree of each impurity is 5.6; in the oxidatively degraded test sample solution, the minimum separation degree of each impurity is 3.7; in the high-temperature degraded test sample solution, the minimum separation degree of each impurity is 3.0.

[0060] In this embodiment, the contents of each impurity in the undamaged test sample solution, the oxidatively degraded test sample solution, and the high-temperature degraded test sample solution are shown in Table 2.

[0061] Table 2. Detection results of impurity content The following sections describe the testing methods provided in this application, including limit of quantitation, linearity, accuracy, and robustness tests. The specific methods and results are as follows: (1) Limit of Quantification Gradually dilute the mixed reference solution with water, and inject the reference solution into the high-performance liquid chromatograph under the chromatographic conditions described above until the signal-to-noise ratio of each impurity is greater than 10. At this point, the concentration of each impurity is the limit of quantitation.

[0062] The concentration limits of each impurity were determined and are shown in Table 3.

[0063] Table 3 Detection results of the limit of quantitation The results showed that the quantitation limits of each impurity were all lower than 0.04% of the concentration of the test sample solution, and the signal-to-noise ratio of each impurity was greater than 10, indicating that the method provided in this embodiment has good sensitivity.

[0064] (2) Linearity test Take appropriate amounts of formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 5-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, kynurenine, phenylethylamine, and tryptophan reference standards, accurately weigh them, and dilute them with water to prepare linear concentration solutions as shown in Table 4.

[0065] Table 4 Linear Concentration Solutions The above-mentioned linear solutions were taken and measured according to the chromatographic conditions of this embodiment. Linearity was performed by comparing the concentration of each compound with the peak area response value. The linear results of each compound are shown in Table 5. Among them, the wavelength correction factor of formylkynurenine at a wavelength of 250 nm is 2.63.

[0066] Table 5 Linear Results The results showed that all impurities and tryptophan exhibited good linearity between the method's report limit and 200% of the limit.

[0067] (3) Accuracy test Take appropriate amounts of formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, kynurenine, and phenylethylamine, and prepare a reference stock solution with water. Accurately measure an appropriate amount of the stock solution into a 20 mL volumetric flask, accurately add appropriate amounts of each impurity stock solution and 10 mL of sample, and dilute with water to the mark to prepare recovery solutions at 20%, 100%, and 150% levels, respectively. Perform the determination according to the chromatographic conditions of this embodiment, record the chromatogram, and calculate the recovery rate of each impurity. The results are shown in Table 6.

[0068] Table 6 Impurity Recovery Results The results showed that the recovery rates of each impurity were between 95% and 120%, indicating that the method provided in this embodiment had good accuracy.

[0069] (4) Durability test The chromatographic parameters in this embodiment were fine-tuned to examine the robustness of the method. The parameter adjustments are shown in Table 7.

[0070] Table 7 Parameter Adjustment Status The impurity content of the samples was determined under different modified parameter conditions.

[0071] The results showed that under different chromatographic conditions, the blank solvent did not interfere with the determination of each impurity, and the minimum separation between each impurity was 1.6. Compared with the original chromatographic conditions, the changes in the determination results of each impurity content did not exceed 0.03%, while the changes in unknown impurities all exceeded 0.05%. The results indicate that the method provided in this embodiment has good robustness.

[0072] Example 2 This embodiment provides a method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injection. Compared with Example 1, the only difference is that the composition of the test solution and ammonium formate buffer, the column temperature, and the gradient elution procedure are changed. The remaining steps are the same as in Example 1 and will not be repeated here.

[0073] In this embodiment, the preparation steps of the test solution are as follows: Accurately measure 5 mL of compound amino acid injection (15-HBC), place it in a 10 mL volumetric flask, dilute with water and bring to volume, shake well to obtain an intact test solution.

[0074] Accurately measure 10 mL of compound amino acid injection (15-HBC) and place it in a 20 mL volumetric flask. Add 3 mL of 0.1% hydrogen peroxide solution, let it stand at room temperature for 1 h, dilute with water to the mark, and shake well to obtain the oxidized and degraded compound amino acid injection. Accurately measure 5 mL of the oxidized and degraded compound amino acid injection and place it in a 10 mL volumetric flask. Dilute with water and make up to volume. Shake well and filter through a filter membrane. Take the filtrate to obtain the oxidized and degraded test solution.

[0075] Take an appropriate amount of compound amino acid injection (15-HBC), transfer it to a suitable glass bottle, and place it under high-pressure steam at 121℃ for 30 minutes to destroy it. After removing it, let it cool and shake it well to obtain the high-temperature degraded compound amino acid injection. Accurately measure 10 mL of the high-temperature degraded compound amino acid injection, place it in a 20 mL volumetric flask, dilute it with water to the mark, shake it well, filter it with a filter membrane, and take the filtrate to obtain the high-temperature degraded test solution.

[0076] In this embodiment, the ammonium formate buffer includes an ammonium formate solution with a concentration of 20 mmol / L and hydrochloric acid as a pH adjuster. After adjusting the pH value with phosphoric acid, the pH value of the ammonium formate buffer is 3.15. In this embodiment, the column temperature is 47°C, and the gradient elution program is shown in Table 8.

[0077] Table 8 Gradient elution program After testing according to the method of this embodiment, the measurement results are as follows: ① The blank solvent (water) does not interfere with the determination of known impurities of tryptophan and phenylalanine; ② At a wavelength of 250 nm, dioxindole alanine 1, dioxindole alanine 2, 5-hydroxytryptophan, kynurenine, 5-hydroxytryptophan, 3-phenylaminoalanine, and 2-hydroxytryptophan eluted sequentially in the mixed reference solution, with formylkynurenine and 2-hydroxytryptophan co-eluting. The resolution between each known impurity and adjacent peaks was greater than 2.6; the system suitability met the requirements. ③ In the undamaged test sample solution, the minimum separation degree of each impurity is 4.3; in the oxidatively degraded test sample solution, the minimum separation degree of each impurity is 2.5; in the high-temperature degraded test sample solution, the minimum separation degree of each impurity is 2.7.

[0078] In this embodiment, the contents of each impurity in the undamaged test sample solution, the oxidatively degraded test sample solution, and the high-temperature degraded test sample solution are shown in Table 9.

[0079] Table 9. Detection results of impurity content The following sections describe the testing methods provided in this application, including limit of quantitation, linearity, accuracy, and robustness tests. The specific methods and results are as follows: (1) Limit of Quantification Gradually dilute the mixed reference solution with water, and inject the reference solution into the high-performance liquid chromatograph under the chromatographic conditions described above until the signal-to-noise ratio of each impurity is greater than 10. At this point, the concentration of each impurity is the limit of quantitation.

[0080] The concentration limits of each impurity were determined and are shown in Table 10.

[0081] Table 10 Detection results of limit of quantitation The results showed that the quantitation limits of each impurity were all below 0.05% of the concentration of the test sample solution, and the signal-to-noise ratio of each impurity was greater than 10, indicating that the method provided in this embodiment has good sensitivity.

[0082] (2) Linearity test Take appropriate amounts of formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 5-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, kynurenine, phenylethylamine, and tryptophan reference standards, accurately weigh them, and dilute them with water to prepare linear concentration solutions as shown in Table 11.

[0083] Table 11 Linear Concentration Solutions The above linear solutions were taken and measured according to the chromatographic conditions of this embodiment. Linearity was performed by comparing the concentration of each compound with the peak area response value. The linearity results of each compound are shown in Table 12, where the wavelength correction factor for formylkynurenine at 250 nm is 2.62.

[0084] Table 12 Linear Results The results showed that all impurities and tryptophan exhibited good linearity between the method's report limit and 200% of the limit.

[0085] (3) Accuracy test Take appropriate amounts of formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, kynurenine, and phenylethylamine, and prepare a reference stock solution with water. Accurately measure an appropriate amount of the stock solution into a 20 mL volumetric flask, accurately add appropriate amounts of each impurity stock solution and 10 mL of sample, and dilute with water to the mark to prepare recovery solutions at 25%, 100%, and 150% levels, respectively. Perform the determination according to the chromatographic conditions of this embodiment, record the chromatogram, and calculate the recovery rate of each impurity. The results are shown in Table 13.

[0086] Table 13 Impurity Recovery Results The results showed that the recovery rates of each impurity were between 95% and 120%, indicating that the method provided in this embodiment had good accuracy.

[0087] (4) Durability test The chromatographic parameters in this embodiment were fine-tuned to examine the robustness of the method. The parameter adjustments are shown in Table 14.

[0088] Table 14 Parameter Adjustment Status The impurity content of the samples was determined under different modified parameter conditions.

[0089] The results showed that under different chromatographic conditions, the blank solvent did not interfere with the determination of each impurity, and the minimum separation between each impurity was 1.6. Compared with the original chromatographic conditions, the changes in the determination results of each impurity content did not exceed 0.05%, while the changes in unknown impurities all exceeded 0.02%. The results indicate that the method provided in this embodiment has good robustness.

[0090] Example 3 This embodiment provides a method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injection. Compared with Example 1, the only difference is that the composition of the test solution and mobile phase A, the column temperature, and the gradient elution procedure are changed. The remaining steps are the same as in Example 1 and will not be repeated here.

[0091] In this embodiment, the preparation steps of the test solution are as follows: Accurately measure 5 mL of compound amino acid injection (9AA), place it in a 10 mL volumetric flask, dilute with water and bring to volume, shake well to obtain an intact test solution.

[0092] Accurately measure 10 mL of compound amino acid injection (9AA), place it in a 10 mL volumetric flask, add 2 mL of 3% hydrogen peroxide solution, let stand at room temperature for 4 h, dilute with water to the mark, shake well, filter with a PES filter membrane, and take the filtrate to obtain the oxidative degradation test solution.

[0093] Take 25 mL of compound amino acid injection (9AA), place it in a 50 mL volumetric flask, place it in an oven at 105℃ for 2 h, remove it, cool it, dilute it with water to the mark, shake it well, filter it with a PES filter membrane, and take the filtrate to obtain the high-temperature degradation test solution.

[0094] In this embodiment, the ammonium formate buffer includes an ammonium formate solution with a concentration of 20 mmol / L and a pH adjuster, phosphoric acid. After adjusting the pH value with phosphoric acid, the pH value of the ammonium formate buffer is 4.0. The volume ratio of ammonium formate buffer to methanol is 96:4. In this embodiment, the column temperature is 40°C, and the gradient elution program is shown in Table 15.

[0095] Table 15 Gradient elution program After testing according to the method of this embodiment, the measurement results are as follows: ① The blank solvent does not interfere with the determination of known impurities of tryptophan and phenylalanine; ② At a wavelength of 250 nm, dioxindole alanine 1, dioxindole alanine 2, 5-hydroxytryptophan, kynurenine, 5-hydroxytryptophan, 3-phenylaminoalanine, and 2-hydroxytryptophan eluted sequentially in the mixed reference solution, with formylkynurenine and 2-hydroxytryptophan co-eluting. The resolution between each known impurity and adjacent peaks was greater than 2.0. ③ In the undamaged test sample solution, the minimum separation degree of each impurity is 7.5; in the oxidatively degraded test sample solution, the minimum separation degree of each impurity is 2.3; in the high-temperature degraded test sample solution, the minimum separation degree of each impurity is 2.1.

[0096] In this embodiment, the contents of each impurity in the undamaged test sample solution, the oxidatively degraded test sample solution, and the high-temperature degraded test sample solution are shown in Table 16.

[0097] Table 16 Detection results of impurity content The following sections describe the testing methods provided in this application, including limit of quantitation, linearity, accuracy, and robustness tests. The specific methods and results are as follows: (1) Limit of Quantification Gradually dilute the mixed reference solution with water, and inject the reference solution into the high-performance liquid chromatograph under the chromatographic conditions described above until the signal-to-noise ratio of each impurity is greater than 10. At this point, the concentration of each impurity is the limit of quantitation.

[0098] The concentration limits of each impurity were determined and are shown in Table 17.

[0099] Table 17 Results of Limit of Quantitation Detection The results showed that the quantitation limits of each impurity were all lower than 0.03% of the concentration of the test sample solution, and the signal-to-noise ratio of each impurity was greater than 10, indicating that the method provided in this embodiment has good sensitivity.

[0100] (2) Linearity test Take appropriate amounts of formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 5-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, kynurenine, phenylethylamine, and tryptophan reference standards, accurately weigh them, and dilute them with water to prepare linear concentration solutions as shown in Table 18.

[0101] Table 18 Linear Concentration Solutions The above-mentioned linear solutions were taken and measured according to the chromatographic conditions of this embodiment. Linearity was performed by comparing the concentration of each compound with the peak area response value. The linearity results of each compound are shown in Table 19, where the wavelength correction factor for formylkynurenine at 250 nm is 2.64.

[0102] Table 19 Linear Results The results showed that all impurities and tryptophan exhibited good linearity between the method's report limit and 200% of the limit.

[0103] (3) Accuracy test Take appropriate amounts of formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, kynurenine, and phenylethylamine, and prepare a reference stock solution with water. Accurately measure an appropriate amount of the stock solution into a 20 mL volumetric flask, accurately add appropriate amounts of each impurity stock solution and 10 mL of sample, and dilute with water to the mark to prepare recovery solutions at 20%, 100%, and 150% levels, respectively. Perform the determination according to the chromatographic conditions of this embodiment, record the chromatogram, and calculate the recovery rate of each impurity. The results are shown in Table 20.

[0104] Table 20 Impurity Recovery Results The results showed that the recovery rates of each impurity were between 89% and 120%, indicating that the method provided in this embodiment had good accuracy.

[0105] (4) Durability test The chromatographic parameters in this embodiment were fine-tuned to examine the robustness of the method. The parameter adjustments are shown in Table 21.

[0106] Table 21 Parameter Adjustment Status The impurity content of the samples was determined under different modified parameter conditions.

[0107] The results showed that under different chromatographic conditions, the blank solvent did not interfere with the determination of each impurity, the minimum separation between each impurity was 1.3, and compared with the original chromatographic conditions, the changes in the determination results of each impurity content did not exceed 0.02%, and the content of unknown impurities did not change. The results indicate that the method provided in this embodiment has good robustness.

[0108] Comparative Example 1 This comparative example provides a method for determining tryptophan-related impurities in compound amino acid injections. Compared to Example 1, the difference lies in that this method cannot determine impurities in phenylalanine. The mobile phase system, chromatographic parameters, solution preparation, and method for calculating unknown impurities (self-comparison method) differ from Example 1. Therefore, this method cannot determine impurities in phenylalanine. The specific steps are as follows: The test sample solution is a compound amino acid injection stock solution (batch number: 925052511).

[0109] Accurately measure 1 ml of this product into a 100 ml volumetric flask, dilute with water to the mark, and shake well to obtain the reference solution.

[0110] For the reference solution, accurately weigh an appropriate amount of 2-hydroxytryptophan reference standard, dissolve it in water, and quantitatively dilute it to prepare a solution containing approximately 1 μg of 2-hydroxytryptophan per 1 ml.

[0111] For system suitability solution, take an appropriate amount of 2-hydroxytryptophan reference standard, add this product to dissolve and dilute to prepare a solution containing approximately 1 μg of 2-hydroxytryptophan per 1 ml.

[0112] The mixed reference solution is the same as in Example 1.

[0113] The system suitability solution, test solution, control solution, and mixed control solution were injected into the high-performance liquid chromatograph for detection. The chromatograms were recorded, and the contents of 2-hydroxytryptophan and unknown impurities were calculated.

[0114] The high-performance liquid chromatography (HPLC) conditions are as follows: Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Capcell PAK C18 AQ S3, 4.6 mm × 250 mm, 3 μm or equivalent column); 0.1% trifluoroacetic acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B, with gradient elution according to the table below; flow rate was 0.8 mL / min; column temperature was 20 °C; detector wavelength was 270 nm; injection volume was 10 μl.

[0115] Gradient elution table: After testing according to the method provided in this comparative example, the results are as follows: Comparing the above measurement results with those of Example 1, it can be seen that the comparative example can only quantitatively measure 2-hydroxytryptophan, a known impurity of tryptophan, and cannot simultaneously quantitatively measure formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 5-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, kynurenine, and phenylethylamine.

[0116] In summary, this application provides a method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injections, belonging to the field of chemical analysis technology. The method includes: preparing a test solution, a reference solution, and a mixed reference solution separately; then injecting each solution into a high-performance liquid chromatograph (HPLC) for detection, recording the chromatograms, and calculating the content of each impurity; wherein the chromatographic conditions of the HPLC include: using ammonium formate buffer-methanol as mobile phase A and acetonitrile-mobile phase A as mobile phase B, performing gradient elution. This application, by developing a new mobile phase system, ensures good separation of each impurity while employing four different detection wavelengths, which is beneficial for obtaining better peak shapes and lower background interference, thus enabling the simultaneous determination of tryptophan and phenylalanine-related impurities (eight known impurities and unknown impurities) in compound amino acid injections.

[0117] 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 simultaneously determining tryptophan and phenylalanine-related impurities in a compound amino acid injection, characterized in that, Includes the following steps: Take the compound amino acid injection to be tested, dilute it with water and make up to volume to obtain the test solution; Take tryptophan reference standard, dissolve it in water and dilute it quantitatively to obtain the reference standard solution; Take the impurity reference standard stock solution, dilute it quantitatively with water to obtain a mixed reference standard solution; the impurity reference standard stock solution contains formylkynurenine, 5-hydroxytryptamine, 2-hydroxytryptamine, 5-hydroxytryptamine, 3-phenylaminoalanine, dioxindolealanine, kynurenine, and phenylethylamine. The test solution, the reference solution, and the mixed reference solution were injected into a high-performance liquid chromatograph for detection, the chromatograms were recorded, and the content of each impurity was calculated. The chromatographic conditions of the high-performance liquid chromatograph include: Gradient elution was performed using ammonium formate buffer-methanol as mobile phase A and acetonitrile-mobile phase A as mobile phase B.

2. The method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injection according to claim 1, characterized in that, The chromatographic conditions of the high-performance liquid chromatograph also include: Using a DAD as the detector, the detection wavelengths include 210nm, 250nm, 270nm, and 324nm.

3. The method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injection according to claim 2, characterized in that, When the detection wavelength is 250 nm, in the chromatogram of the mixed reference solution, dioxindole alanine has two chromatographic peaks, formylkynurenine and 2-hydroxytryptophan co-elute, and the resolution of adjacent chromatographic peaks is not less than 1.

5.

4. The method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injection according to claim 3, characterized in that, The content of each impurity was calculated based on peak area using the external standard method. The peak area of ​​2-hydroxytryptophan among the impurities was calculated using the wavelength correction method.

5. The method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injection according to claim 4, characterized in that, The formula for calculating the peak area of ​​2-hydroxytryptophan is as follows: In the formula, A C，250nm This indicates the peak area of ​​2-hydroxytryptophan at a detection wavelength of 250 nm; A A+C,250nm This represents the peak area of ​​the co-elution peak of 2-hydroxytryptophan and formicylkynurenine at a detection wavelength of 250 nm; A A，324nm This represents the peak area of ​​formylkynurenine when the detection wavelength is 324 nm; f is the wavelength correction factor, with a value range of 2.60~2.

65.

6. The method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injection according to claim 1, characterized in that, The ammonium formate buffer solution includes an ammonium formate solution and a pH adjuster; The pH value of the ammonium formate buffer solution is 3.1~4.5; The concentration of ammonium formate in the formate solution is 18~22 mmol / L; The pH adjuster includes one of phosphoric acid, hydrochloric acid, and formic acid.

7. The method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injection according to claim 1, characterized in that, In mobile phase A, the volume ratio of ammonium formate buffer to methanol is 96:4 to 98:2; in mobile phase B, the volume ratio of acetonitrile to mobile phase A is 25:75 to 35:

65.

8. The method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injection according to claim 1, characterized in that, The gradient elution is performed using a linear gradient as shown in the table below: In the table above, the time increments sequentially from top to bottom; at the same time, the sum of mobile phase A and mobile phase B is 100%.

9. The method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injection according to claim 1, characterized in that, The chromatographic conditions of the high-performance liquid chromatograph also include: A C18 column was used. The column temperature is 38~52℃; The temperature of the injection plate is 3~10℃; The injection volume is 10~50μL; The elution flow rate was 0.9~1.1 mL / min.

10. The method for simultaneously determining tryptophan and phenylalanine-related impurities in compound amino acid injection according to claim 1, characterized in that, In the reference solution, the concentration of tryptophan is 5 μg / mL; In the mixed reference solution, the concentrations of formylkynurenine, 5-hydroxytryptophan, 2-hydroxytryptophan, 5-hydroxytryptophan, 3-phenylaminoalanine, dioxindolealanine, and kynurenine are 2 μg / mL, and the concentration of phenethylamine is 1 μg / mL.

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