Method for determining content of related substances in 4-boron [10B]-L-phenylalanine

Abstract

The invention provides a method for determining the content of related substances in 4-boron [10B]-L-phenylalanine, and relates to the field of pharmaceutical analysis. The method for determining the content of the related substances in the 4-boron [10B]-L-phenylalanine comprises the following steps: respectively injecting a reference solution and a test solution into a high performance liquid chromatograph for analysis, and carrying out gradient elution by using a mobile phase A and a mobile phase B to obtain the content of the related substances in the 4-boron [10B]-L-phenylalanine, the total integral number of methanol and acetonitrile in the mobile phase B is higher than the total integral number of methanol and acetonitrile in the mobile phase A; and calculating the content of each component in the test solution based on the relationship between the concentration of each component in the reference solution and the chromatographic peak area and the chromatographic peak area of each component in the test solution. According to the method disclosed by the invention, L-tyrosine and L-phenylalanine in 4-boron [10B]-L-phenylalanine can be effectively eluted, separated and quantitatively detected.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical analysis, and more particularly to a method for determining 4-boron[ 10 Methods for determining the content of related substances in β-L-phenylalanine. Background Technology

[0002] For the treatment of unresectable, locally advanced, or locally recurrent head and neck cancers, an increasing number of medical institutions and scholars are focusing on neutron capture therapy, which destroys cancer cells through the reaction of radionuclides and neutrons. Boron neutron capture therapy (BNCT) is particularly effective due to its neutron dispersion cross-sectional area being approximately 2000 times larger than other biological components. 4-boron[ 10 β-L-phenylalanine, as the active ingredient in BNCT drugs, can provide the raw material for developing drugs of different dosage forms, thereby realizing the possibility of clinical treatment with BNCT drugs. Meanwhile, 4-boron[ 10 During the production, storage, and transportation of β-L-phenylalanine, byproducts or other impurities may be generated. Therefore, the determination of the content of related substances is extremely important for the control of drug quality.

[0003] 4-Boron[ 10 B]-L-phenylalanine is used as the raw material for BNCT drugs, and its main impurities include at least one of L-tyrosine and L-phenylalanine.

[0004] 4-Boron[ 10 The structural formula of β-L-phenylalanine is: The structural formula of L-tyrosine is: The structural formula of L-phenylalanine is: 4-Boron[ 10 [B]-L-phenylalanine is structurally highly similar to impurities, making the development of an HPLC method capable of accurately determining both its main component and major impurity components challenging. Currently, no method exists that can simultaneously determine 4-boron[ 10 HPLC methods for β-L-phenylalanine and major impurities are reported. Summary of the Invention

[0005] The purpose of this application is to provide a method for determining 4-boron[ 10 Methods for determining the content of relevant substances in β-L-phenylalanine are proposed to address the aforementioned issues.

[0006] To achieve the above objectives, this application adopts the following technical solution: A method for determining 4-boron[ 10 A method for determining the content of related substances in L-Tyrosine, wherein the related substances include at least one of L-tyrosine and L-phenylalanine; The method includes: A reference solution and a test solution were prepared using an aqueous phosphoric acid solution as a diluent. The reference solution contained a known concentration of 4-boron[…]. 10 [B]-L-phenylalanine, L-tyrosine and L-phenylalanine, the test solution contains 4-boron[ 10 [B]-L-phenylalanine; The reference solution and the test solution were injected into a high-performance liquid chromatograph for analysis. The detection conditions of the high-performance liquid chromatograph included: an octadecylsilane-bonded silica column, a detection wavelength of 205-254 nm, and mobile phases including mobile phase A and mobile phase B, with gradient elution between mobile phase A and mobile phase B. Both mobile phase A and mobile phase B included potassium dihydrogen phosphate buffer solution, methanol, and acetonitrile. The total volume fraction of methanol and acetonitrile in mobile phase B was higher than that in mobile phase A. Based on the relationship between the concentration and chromatographic peak area of ​​each component in the reference solution, and the chromatographic peak area of ​​each component in the test solution, the contents of L-tyrosine and L-phenylalanine in the test solution are calculated.

[0007] According to an embodiment of this application, the concentration of the phosphoric acid aqueous solution is 0.1-5%.

[0008] According to an embodiment of this application, the pH of the potassium dihydrogen phosphate buffer solution is adjusted by phosphoric acid, and the pH of the potassium dihydrogen phosphate buffer solution is 2.5~5.0; And / or, the concentration of the potassium dihydrogen phosphate buffer solution is 0.005~0.05 mol / L.

[0009] According to an embodiment of this application, in the mobile phase A, the volume ratio of the potassium dihydrogen phosphate buffer solution to methanol and acetonitrile is (85~98):(1~10):(1~5).

[0010] According to an embodiment of this application, in the mobile phase B, the volume ratio of the potassium dihydrogen phosphate buffer solution to methanol and acetonitrile is (15~30): (5~25): (45~80).

[0011] According to an embodiment of this application, the gradient elution includes: Maintain the volume fraction of mobile phase A at 100% for 0-8 minutes; Within 8–15 minutes, the volume fraction of mobile phase A was linearly reduced from 100% to 80%, while the volume fraction of mobile phase B was linearly increased from 0% to 20%. Within 15-20 minutes, the volume fraction of mobile phase A was linearly reduced from 80% to 0%, while the volume fraction of mobile phase B was linearly increased from 20% to 100%. For 20-30 minutes, maintain the volume fraction of mobile phase B at 100%. After 30-40 minutes, restore the mobile phase ratio to its initial state.

[0012] According to an embodiment of this application, the detection conditions of the high performance liquid chromatography include: the column length of the chromatographic column is 150~250mm, the inner diameter of the chromatographic column is 4.6mm, and the particle size of the packing material of the chromatographic column is 2.7~5μm.

[0013] According to an embodiment of this application, the detection conditions for the high-performance liquid chromatography include a column temperature of 20~45℃.

[0014] According to an embodiment of this application, the detection conditions for the high-performance liquid chromatography include: a flow rate of 0.7~1.2 mL / min; The injection volume is 5~25μl.

[0015] According to embodiments of this application, the reference solution contains 4-boron[ 10 The concentration of L-phenylalanine in the reference solution is 0.09~0.9 μg / ml, and the concentration of L-tyrosine in the reference solution is 0.15~3.0 μg / ml. And / or, the test solution contains 4-boron[ 10 The concentration of β-L-phenylalanine was 0.003~0.45 mg / ml.

[0016] Compared with the prior art, the beneficial effects of this application include: This application provides a rapid, simple, accurate, and efficient HPLC analytical method capable of analyzing 4-boron[ 10 This application provides an effective elution, separation, and quantitative detection of L-tyrosine and L-phenylalanine in 4-L-phenylalanine. The method achieves complete separation of impurity peaks from the main peak, as well as among impurity peaks themselves, resulting in ideal detection results. Furthermore, the method exhibits excellent overall performance: it is easy to operate, has a short run time, and possesses strong specificity, high sensitivity, and strong stability. In addition, the method demonstrates good linearity in the low concentration range, strong repeatability, and high accuracy, is less affected by personnel and instrumentation, and is stable and reliable. In summary, this application provides a method for the effective elution, separation, and quantitative detection of L-tyrosine and L-phenylalanine in 4-boron[ 10 β-L-phenylalanine provides the basis for research, development, and quality testing. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation on the scope of this application.

[0018] Figure 1 The chromatogram of the blank solution obtained using the method of Example 1; Figure 2 Chromatogram of the system suitability solution obtained using the method of Example 1; Figure 3 Chromatogram of the reference solution obtained using the method of Example 1; Figure 4 The chromatogram of the test solution obtained using the method of Example 1; Figure 5 The chromatogram of the system suitability solution obtained from Comparative Example 1; Figure 6 The chromatogram of the reference solution obtained in Comparative Example 2 is shown below. Figure 7 The chromatogram is of the system suitability solution obtained from Comparative Example 3. Detailed Implementation

[0019] As used in this article: "Prepared from" is synonymous with "comprising". The terms "comprising", "including", "having", "containing", or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0020] The conjunction "composed of..." excludes any unspecified elements, steps, or components. If used in a claim, this phrase makes the claim closed, excluding materials other than those described, except for associated conventional impurities. When the phrase "composed of..." appears in a clause of the body of a claim rather than immediately following it, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0021] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1–5” is disclosed, the described range should be interpreted as including ranges “1–4”, “1–3”, “1–2”, “1–2 and 4–5”, “1–3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0022] In these embodiments, unless otherwise specified, the portions and percentages are all by weight.

[0023] "Parts by mass" refers to the basic unit of measurement that expresses the mass ratio of multiple components. One part can represent any unit mass, such as 1g or 2.689g. If we say that component A has "a" parts by mass and component B has "b" parts by mass, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it can mean that the mass of component A is aK and the mass of component B is bK (where K is any number representing a multiplier). It is important to understand that, unlike parts by mass, the sum of the mass parts of all components is not limited to 100 parts.

[0024] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0025] A method for determining 4-boron[ 10 A method for determining the content of related substances in L-Tyrosine, wherein the related substances include at least one of L-tyrosine and L-phenylalanine; The method includes: Diluent screening: based on 4-boron[ 10 [B]-L-phenylalanine solubility screening diluent, 4-boron[ 10 [B]-L-phenylalanine is insoluble in water but soluble in acidic solutions. Therefore, aqueous solutions of phosphoric acid, formic acid, acetic acid, and hydrochloric acid were screened. The results showed that aqueous solutions of formic acid and acetic acid interfered with the elution of the impurity L-tyrosine and were unsuitable. Moreover, the pH of hydrochloric acid solution was approximately 0.5, while the recommended pH range for the chromatographic column is 2-8. To ensure column life, hydrochloric acid solution was not suitable as a diluent. Phosphoric acid solution, on the other hand, had a suitable pH and did not interfere with the detection targets. Therefore, aqueous solution of phosphoric acid was selected as the diluent.

[0026] A reference solution and a test solution were prepared using an aqueous phosphoric acid solution as a diluent. The reference solution contained a known concentration of 4-boron[…]. 10 [B]-L-phenylalanine, L-tyrosine and L-phenylalanine, the test solution contains 4-boron[ 10 [B]-L-phenylalanine; The reference solution and the test solution were separately injected into a high-performance liquid chromatograph (HPLC) for analysis. The HPLC detection conditions included: an octadecylsilane-bonded silica column; a detection wavelength of 205–254 nm (e.g., 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, 254 nm, or any value between 205–254 nm); and mobile phases A and B, with gradient elution using mobile phases A and B. Both mobile phases A and B comprise potassium dihydrogen phosphate buffer, methanol, and acetonitrile. The total volume fraction of methanol and acetonitrile in mobile phase B was higher than that in mobile phase A. Using potassium dihydrogen phosphate buffer as part of the mobile phase stabilized the retention times of each substance. Gradually increasing the ratio of methanol to acetonitrile through gradient elution improved the separation between substances and avoided mutual interference.

[0027] Based on the relationship between the concentration and chromatographic peak area of ​​each component in the reference solution, and the chromatographic peak area of ​​each component in the test solution, the contents of L-tyrosine and L-phenylalanine in the test solution are calculated.

[0028] According to embodiments of this application, the concentration of the phosphoric acid aqueous solution is 0.1% to 5%; if the concentration of the phosphoric acid aqueous solution is too low, the acidity is insufficient, making it difficult to dissolve 4-boron[ 10 [B]-L-phenylalanine; if the concentration of phosphoric acid aqueous solution is too high and the acidity is too strong, it will affect the life of the chromatographic column.

[0029] For example, the concentration of the phosphoric acid aqueous solution is 0.1%, 1%, 2%, 3%, 4%, 5%, or any value between 0.1% and 5%.

[0030] According to an embodiment of this application, the pH of the potassium dihydrogen phosphate buffer solution is adjusted by phosphoric acid, and the pH of the potassium dihydrogen phosphate buffer solution is 2.5 to 5.0; for example, the pH of the potassium dihydrogen phosphate buffer solution is 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or any value between 2.5 and 5.0.

[0031] The concentration of the potassium dihydrogen phosphate buffer solution is 0.005~0.05 mol / L. For example, the concentration of the potassium dihydrogen phosphate buffer solution is 0.005 mol / L, 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, or any value between 0.005 and 0.05 mol / L.

[0032] According to an embodiment of this application, in the mobile phase A, the volume ratio of the potassium dihydrogen phosphate buffer solution to methanol and acetonitrile is (85~98):(1~10):(1~5). Adding methanol and acetonitrile to mobile phase A with a relatively low proportion of organic phase within the above-mentioned content range helps to accelerate the production of 4-boron[…]. 10 The retention time of β-L-phenylalanine and L-tyrosine, and the retention time of 4-boron[ 10 B]-L-phenylalanine is not affected by baseline fluctuations caused by diluents, ensuring the accuracy of each component detection.

[0033] For example, in mobile phase A, the volume ratio of the potassium dihydrogen phosphate buffer solution to methanol and acetonitrile is any value between 85:1:1, 92:7:1, 98:10:5, or (85~98):(1~10):(1~5).

[0034] According to an embodiment of this application, in the mobile phase B, the volume ratio of the potassium dihydrogen phosphate buffer solution to methanol and acetonitrile is (15~30):(5~25):(45~80). Adding methanol and acetonitrile to mobile phase B, with a higher proportion of organic phase within the above-mentioned range, helps to advance the retention time of L-phenylalanine and elute contaminants that easily adhere to the chromatographic column without affecting subsequent detection solutions, thus ensuring the accuracy of results between different test solutions.

[0035] For example, in mobile phase B, the volume ratio of potassium dihydrogen phosphate buffer solution to methanol and acetonitrile is any value between 15:5:45, 20:16:64, 30:25:80, or (15~30): (5~25): (45~80).

[0036] According to an embodiment of this application, the gradient elution includes: Maintain the volume fraction of mobile phase A at 100% for 0-8 minutes; Within 8–15 minutes, the volume fraction of mobile phase A was linearly reduced from 100% to 80%, while the volume fraction of mobile phase B was linearly increased from 0% to 20%. Within 15-20 minutes, the volume fraction of mobile phase A was linearly reduced from 80% to 0%, while the volume fraction of mobile phase B was linearly increased from 20% to 100%. For 20-30 minutes, maintain the volume fraction of mobile phase B at 100%. After 30-40 minutes, the mobile phase ratio is restored to its initial state. By using a gradient elution of mobile phase A and mobile phase B in a certain ratio, 4-boron[ 10 L-phenylalanine, L-tyrosine, and L-phenylalanine elute at different retention times without interfering with each other, ensuring separation between them, improving method specificity, and effectively shortening the time required for all components to be eluted, thus improving detection efficiency.

[0037] According to an embodiment of this application, the detection conditions of the high performance liquid chromatography include: the column length of the chromatographic column is 150~250mm, the inner diameter of the chromatographic column is 4.6mm, and the particle size of the packing material of the chromatographic column is 2.7~5μm.

[0038] For example, the column length of the chromatographic column is 150 mm, 160 mm, 170 mm, 180 mm, 190 mm, 200 mm, 210 mm, 220 mm, 230 mm, 240 mm, 250 mm or any value between 150 and 250 mm; the particle size of the packing material of the chromatographic column is 2.7 μm, 3 μm, 4 μm, 5 μm or any value between 2.7 and 5 μm.

[0039] According to embodiments of this application, the detection conditions for the high-performance liquid chromatography include a column temperature of 20–45°C. For example, the column temperature can be any value between 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or 20–45°C.

[0040] According to an embodiment of this application, the detection conditions for the high-performance liquid chromatography include a flow rate of 0.7 to 1.2 mL / min; for example, a flow rate of 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min or any value between 0.7 and 1.2 mL / min.

[0041] The injection volume is 5 to 25 μl. For example, the injection volume can be 5 μl, 10 μl, 15 μl, 20 μl, 25 μl, or any value between 5 and 25 μl.

[0042] According to embodiments of this application, the reference solution contains 4-boron[ 10 The concentration of L-phenylalanine in the reference solution is 0.09~0.9 μg / ml, and the concentration of L-tyrosine in the reference solution is 0.15~3.0 μg / ml.

[0043] For example, 4-boron in the reference solution [ 10 The concentrations of β-L-phenylalanine were 0.09 μg / ml, 0.1 μg / ml, 0.15 μg / ml, 0.2 μg / ml, 0.25 μg / ml, 0.3 μg / ml, 0.35 μg / ml, 0.4 μg / ml, 0.45 μg / ml, 0.5 μg / ml, 0.55 μg / ml, 0.6 μg / ml, 0.65 μg / ml, 0.7 μg / ml, 0.75 μg / ml, 0.8 μg / ml, 0.85 μg / ml, 0.9 μg / ml, or 0.09~0.9 μg / ml. The concentration of L-tyrosine in the reference solution is any value between 0.15 μg / ml, 0.5 μg / ml, 1 μg / ml, 1.5 μg / ml, 2 μg / ml, 2.5 μg / ml, 3.0 μg / ml, or any value between 0.15 and 3.0 μg / ml; the concentration of L-phenylalanine in the reference solution is any value between 0.15 μg / ml, 0.5 μg / ml, 1 μg / ml, 1.5 μg / ml, 2 μg / ml, 2.5 μg / ml, 3.0 μg / ml, or any value between 0.15 and 3.0 μg / ml.

[0044] The test solution contains 4-boron[ 10 The concentration of β-L-phenylalanine is 0.003~0.45 mg / ml. For example, the test solution contains 4-boron[ 10 The concentration of β-L-phenylalanine is 0.003 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.25 mg / ml, 0.3 mg / ml, 0.35 mg / ml, 0.4 mg / ml, 0.45 mg / ml, or any value between 0.003 and 0.45 mg / ml.

[0045] The implementation schemes of this application will be described in detail below with reference to specific embodiments. However, those skilled in the art will understand that the following embodiments are only for illustrating this application and should not be regarded as limiting the scope of this application. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased commercially.

[0046] Example 1: Specificity Test Prepare blank solutions and 4-boron[ 10 [B]-L-phenylalanine positioning solution, L-tyrosine positioning solution, L-phenylalanine positioning solution, system suitability solution, and test solution were injected and determined according to the above chromatographic method: (1) Blank solution (diluent): 1% phosphoric acid aqueous solution.

[0047] (2) Reference solution: Take 4-boron[ 10 A suitable amount of β-L-phenylalanine, L-tyrosine, and L-phenylalanine reference standards were dissolved in diluent and diluted to the mark to prepare a solution containing approximately 4-boron. 10 A solution of L-phenylalanine 0.45 μg / ml, L-tyrosine 1.5 μg / ml, and L-phenylalanine 1.5 μg / ml.

[0048] (3) 4-Boron[ 10 [B]-L-phenylalanine (main component) positioning solution: Take 4-boron[ 10 A suitable amount of β-L-phenylalanine reference standard was dissolved in diluent and diluted to the mark to prepare a solution containing approximately 4-boron[ 10 A solution of 0.3 mg / ml of β-L-phenylalanine.

[0049] (4) L-tyrosine positioning solution: Take an appropriate amount of L-tyrosine reference standard, add diluent to dissolve and dilute to the mark to prepare a solution containing about 1.5 μg / ml of L-tyrosine.

[0050] (5) L-phenylalanine positioning solution: Take an appropriate amount of L-phenylalanine reference standard, add diluent to dissolve and dilute to the mark to prepare a solution containing about 1.5 μg / ml of L-phenylalanine.

[0051] (6) System suitability solution: Take 4-boron[ 10 A suitable amount of β-L-phenylalanine, L-tyrosine, and L-phenylalanine reference standards were dissolved in diluent and diluted to the mark to prepare a solution containing approximately 4-boron. 10 A solution of L-phenylalanine 0.3 mg / ml, L-tyrosine 1.5 μg / ml, and L-phenylalanine 1.5 μg / ml.

[0052] (7) Test solution: Prepare a solution containing approximately 4-boron using an appropriate amount of the test sample and a diluent. 10 A solution of 0.3 mg / ml of β-L-phenylalanine.

[0053] (8) Chromatographic conditions: The chromatographic column is an Agilent 5 TC-C18(2) (4.6×250mm, 5μm) column with octadecylsilane-bonded silica gel as the stationary phase. Detection wavelength: 210nm; Column temperature: 30℃; Injector temperature: 25℃; Injection volume: 20 μl; Flow rate: 1.0 ml / min; Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate buffer solution (pH 3.0) - methanol - acetonitrile (volume ratio 92:7:1); Mobile phase B: 0.01 mol / L potassium dihydrogen phosphate buffer solution (pH 3.0) - methanol - acetonitrile (volume ratio 20:16:64); The elution gradients are shown in Table 1.

[0054] Table 1 Elution gradient table for Example 1

[0055] The test results are shown in Tables 2 and 3.

[0056] Table 2 Resolution Results

[0057] Table 3 System suitability and solution repeatability results

[0058] Figure 1 This is the chromatogram of the blank solution obtained using the method of Example 1 in this application. Figure 1 It can be seen that the blank solution in 4-boron[ 10 The peak positions of β-L-phenylalanine, L-tyrosine, and L-phenylalanine were all empty, indicating that the diluent did not interfere with the determination.

[0059] According to Table 2 and Figure 2 , Figure 4 It can be seen that 4-boron[ 10 4-boron[B]-L-phenylalanine and various impurities can be effectively detected. In the chromatograms of the system suitability solution and the test solution, 4-boron[ 10 The retention times of β-L-phenylalanine and its impurities are consistent with their retention times in their corresponding positioning solutions.

[0060] According to Table 2, Table 3 and Figure 2 , Figure 3 , Figure 4 It can be seen that in the chromatogram of the system suitability solution, the elution order is L-tyrosine, 4-boron […]. 10 The resolution between the main component peak and adjacent peaks of B-L-phenylalanine and L-phenylalanine is greater than 1.5, indicating that the method of this application can completely separate the relevant substances. The reference solution was tested 5 times under the same conditions. The test results showed that the RSD of the main component and impurities in the reference solution was ≤2.0%, indicating that the method of this application has high accuracy.

[0061] Example 2: Limit of Detection and Limit of Quantification Test The chromatographic detection conditions in Example 2 (such as chromatographic column, mobile phase, detection wavelength, column temperature, injector temperature, injection volume, flow rate, elution gradient, etc.) are the same as in Example 1.

[0062] (1) Limit of quantitation solution: Take 4-boron[ 10 Appropriate amounts of L-BPA, L-tyrosine, and L-phenylalanine reference standards were dissolved in diluent and gradually diluted before being measured on an instrument until the signal-to-noise ratio of the L-BPA peak, L-phenylalanine peak, and L-tyrosine peak was ≥10. The solutions at this concentration were used as the corresponding limits of quantitation solutions.

[0063] (2) Limit of detection solution: Take each limit of quantitation solution and dilute it step by step, then measure it on the instrument until the signal-to-noise ratio of L-BPA peak, L-phenylalanine peak and L-tyrosine peak is ≥3; use the solution at this concentration as the corresponding limit of detection solution.

[0064] The test results are shown in Tables 4 and 5 below.

[0065] Table 4 Detection Limit Results

[0066] Table 5 Results of Limit of Quantification

[0067] As shown in Tables 4 and 5, each known component has a low limit of detection and limit of quantitation, indicating that this method has high detection sensitivity.

[0068] Example 3: Linearity and Range Test The chromatographic detection conditions in Example 3 (such as chromatographic column, mobile phase, detection wavelength, column temperature, injector temperature, injection volume, flow rate, elution gradient, etc.) are the same as in Example 1.

[0069] (1) 4-Boron[ 10 [B]-L-phenylalanine series linear solutions: Take 4-boron[ 10 An appropriate amount of β-L-phenylalanine reference standard was prepared into solutions with concentrations of approximately 0.09 μg / ml, 0.225 μg / ml, 0.45 μg / ml, 0.675 μg / ml, 0.90 μg / ml, 3.0 μg / ml, 30 μg / ml, 150 μg / ml, 300 μg / ml, and 450 μg / ml, respectively, and then analyzed by an instrument.

[0070] (2) Linear solutions of L-tyrosine series: Take an appropriate amount of L-tyrosine reference standard and prepare solutions with concentrations of approximately 0.15 μg / ml, 0.75 μg / ml, 1.5 μg / ml, 2.25 μg / ml and 3.0 μg / ml respectively, and perform the determination on the instrument.

[0071] (3) Linear solutions of L-phenylalanine series: Take an appropriate amount of L-phenylalanine reference standard and prepare solutions with concentrations of approximately 0.15 μg / ml, 0.75 μg / ml, 1.5 μg / ml, 2.25 μg / ml and 3.0 μg / ml respectively, and perform the determination on the instrument.

[0072] Each of the test substances (4-boron[ 10 Using the concentrations of [B]-L-phenylalanine, L-tyrosine, and L-phenylalanine as the x-axis and their peak areas as the y-axis, a linear fit was performed to obtain the linear equations for each analyte. The results are shown in Table 6.

[0073] Table 6. Linearity and Range Results

[0074] As shown in Table 6, the linear relationships of the components under this method are good, and the linear range is wide.

[0075] Example 4: Precision Test The chromatographic detection conditions in Example 4 (such as chromatographic column, mobile phase, detection wavelength, column temperature, injector temperature, injection volume, flow rate, elution gradient, etc.) are the same as in Example 1.

[0076] To assess the precision of the method in this application, repeatability and intermediate precision tests were conducted. The acceptable criteria were as follows: for components with a content <0.05%, since the result is below the limit of quantitation and therefore inaccurate, only the result is recorded, and no RSD needs to be calculated; for components with a content ≥0.05%, the RSD should be ≤10%.

[0077] Repeatability test: Take the same batch of test solution and test it 6 times in parallel. The test is performed by the same person on the same instrument and the contents of L-tyrosine and L-phenylalanine are recorded.

[0078] Intermediate precision test: Experimenter 2 measured the contents of the same batch of test solutions prepared in parallel on different dates and with different instruments, and recorded the contents of L-tyrosine and L-phenylalanine.

[0079] The RSD of the 12 test results was calculated, and the results are shown in Table 7.

[0080] Table 7 Precision Results

[0081] As shown in Table 7, the RSD of all 12 solutions measured for precision (repeatability and intermediate precision) was <10%, proving that the method is not affected by personnel and instruments and has good precision.

[0082] Example 5: Accuracy Test The chromatographic detection conditions in Example 5 (such as chromatographic column, mobile phase, detection wavelength, column temperature, injector temperature, injection volume, flow rate, elution gradient, etc.) are the same as in Example 1.

[0083] Nine portions of 4-boron[10B]-L-phenylalanine test solution with known content (the content of the main component is known, and the impurity content has been measured in Example 4) were taken and divided into three spiking groups, with three portions in each spiking group. Three reference solutions of low, medium, and high concentrations were precisely added to each sample, and diluent was added to prepare spiking test solutions equivalent to 50%, 100%, and 150% of the limit concentration of each component.

[0084] The samples were injected and determined according to the method in Example 1, and chromatograms were obtained. The spiked recoveries and RSDs of each component were calculated, and the results are shown in Table 8.

[0085] Table 8 Accuracy Results

[0086] As shown in Table 8, the recoveries of each component at 50%, 100%, and 150% of the limit concentration were all within the range of 80%–120%, and the RSDs were all <10%, which meets the requirements. This method has high accuracy and can accurately determine 4-boron. 10 Content of [B]-L-phenylalanine-related substances.

[0087] Example 6: Solution Stability Test The chromatographic detection conditions in Example 6 (such as chromatographic column, mobile phase, detection wavelength, column temperature, injector temperature, injection volume, flow rate, elution gradient, etc.) are the same as in Example 1.

[0088] The reference solution and the test solution were stored at room temperature (25℃) and 2~8℃ for a certain period of time, respectively, and then samples were taken. The content of each component in the solution was determined using the method of this application. The results are shown in Tables 9 and 10.

[0089] Table 9. Stability results of the reference solution

[0090] Table 10 Stability results of the test solution

[0091] According to Tables 9 and 10, both the reference solution and the test solution have high stability when placed at room temperature or 2-8℃, and the stability time is not less than 42h.

[0092] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the diluent used in Comparative Example 1 is different from that used in this application; the diluent used in Comparative Example 1 is a 1% formic acid aqueous solution. Everything else is the same as in Example 1.

[0093] Figure 5 The chromatogram of the system suitability solution obtained from Comparative Example 1 is shown below. Figure 5 It can be seen that using 1% formic acid aqueous solution as a diluent raises the baseline of the chromatogram, interfering with the elution of L-tyrosine, and adding an additional impurity peak next to it, affecting the accuracy of its content determination. The elution order is as follows: interference peak, L-tyrosine peak, 4-boron [ 10 [B]-L-phenylalanine peak, L-phenylalanine peak, and other unknown impurity peaks.

[0094] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the mobile phase is mobile phase A, and the mobile phase A is always kept at 100%.

[0095] Figure 6 The chromatogram of the reference solution obtained in Comparative Example 2 is shown below. Figure 6 It can be seen that when using only mobile phase A for elution, diluent baseline fluctuations interfere with 4-boron[ 10 [B]-L-phenylalanine elutes, as shown in the figure below, with a retention time of 6.901 min, leading to the 4-boron [ 10 The integral of β-L-phenylalanine was inaccurate; the RSD of the five tests of the reference solution was 2.6%, which is greater than 2.0%, failing to meet the requirements. However, in Example 1, 4-boron[ 10 The elution peak of β-L-phenylalanine was not interfered with, and the RSD was only 0.7%, indicating that Example 1 was better than Comparative Example 2. Meanwhile, the elution run time of Comparative Example 2 was longer than that of Example 1, and the detection efficiency was lower than that of Example 1.

[0096] Table 11 Comparison of test results between Example 1 and Comparative Example 2

[0097] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the mobile phase A is 0.01 mol / L potassium dihydrogen phosphate buffer solution (pH 3.0) - methanol (volume ratio 92:8). Everything else is the same as in Example 1.

[0098] Figure 7 The chromatogram of the system suitability solution obtained from Comparative Example 3 is shown below. Figure 7 It can be seen that, using the mobile phase of Comparative Example 3, the diluent baseline fluctuation in the chromatogram interferes with 4-boron[ 10 The peak of β-L-phenylalanine affects the accuracy of the measurement. As shown in the figure below, the peak with a retention time of 6.720 min is affected by baseline lifting.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

[0100] Furthermore, those skilled in the art will understand that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are intended to be within the scope of this application and form different embodiments. For example, in the foregoing claims, any of the claimed embodiments can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of this application and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

Claims

1. A method for determining 4-boron[ 10 A method for determining the content of related substances in β-L-phenylalanine, characterized in that... The relevant substances include at least one of L-tyrosine and L-phenylalanine; The method includes: Using aqueous phosphoric acid as diluent, a control solution comprising known concentrations of 4-boro 10 n-L-phenylalanine, L-tyrosine and L-phenylalanine, and a test solution comprising 4-boro 10 n-L-phenylalanine is prepared. The reference solution and the test solution were respectively injected into a high-performance liquid chromatograph for analysis. The detection conditions of the high-performance liquid chromatograph included: an octadecylsilane-bonded silica column, a detection wavelength of 205-254 nm, and mobile phases including mobile phase A and mobile phase B, with gradient elution between mobile phase A and mobile phase B. Both mobile phase A and mobile phase B included potassium dihydrogen phosphate buffer solution, methanol, and acetonitrile. The total volume fraction of methanol and acetonitrile in mobile phase B was higher than that in mobile phase A. Based on the relationship between the concentration and chromatographic peak area of ​​each component in the reference solution, and the chromatographic peak area of ​​each component in the test solution, the contents of L-tyrosine and L-phenylalanine in the test solution are calculated.

2. The determination of 4-boron according to claim 1 10 A method for determining the content of related substances in β-L-phenylalanine, characterized in that... The concentration of the phosphoric acid aqueous solution is 0.1% to 5%.

3. The determination of 4-boron according to claim 1 10 A method for determining the content of related substances in β-L-phenylalanine, characterized in that... The potassium dihydrogen phosphate buffer solution has its pH adjusted by phosphoric acid, and the pH of the potassium dihydrogen phosphate buffer solution is 2.5~5.0; And / or, the concentration of the potassium dihydrogen phosphate buffer solution is 0.005~0.05 mol / L.

4. The determination of 4-boron according to claim 3. 10 A method for determining the content of related substances in β-L-phenylalanine, characterized in that... In the mobile phase A, the volume ratio of the potassium dihydrogen phosphate buffer solution to methanol and acetonitrile is (85~98):(1~10):(1~5).

5. The determination of 4-boron according to claim 3 [ 10 A method for determining the content of related substances in β-L-phenylalanine, characterized in that... In the mobile phase B, the volume ratio of the potassium dihydrogen phosphate buffer solution to methanol and acetonitrile is (15~30): (5~25): (45~80).

6. The determination of 4-boron according to claim 1 10 A method for determining the content of related substances in β-L-phenylalanine, characterized in that... The gradient elution includes: Maintain the volume fraction of mobile phase A at 100% for 0-8 minutes; Within 8–15 minutes, the volume fraction of mobile phase A was linearly reduced from 100% to 80%, while the volume fraction of mobile phase B was linearly increased from 0% to 20%. Within 15-20 minutes, the volume fraction of mobile phase A was linearly reduced from 80% to 0%, while the volume fraction of mobile phase B was linearly increased from 20% to 100%. For 20-30 minutes, maintain the volume fraction of mobile phase B at 100%. After 30-40 minutes, restore the mobile phase ratio to its initial state.

7. The determination of 4-boron according to claim 1 10 A method for determining the content of related substances in β-L-phenylalanine, characterized in that... The detection conditions for the high performance liquid chromatography include: the column length is 150~250mm, the inner diameter of the column is 4.6mm, and the particle size of the packing material is 2.7~5μm.

8. The determination of 4-boron according to claim 1 10 A method for determining the content of related substances in β-L-phenylalanine, characterized in that... The detection conditions for the high performance liquid chromatography include a column temperature of 20~45℃.

9. The determination of 4-boron according to claim 1 10 A method for determining the content of related substances in β-L-phenylalanine, characterized in that... The detection conditions for the high-performance liquid chromatography include: The flow rate is 0.7~1.2 mL / min; The injection volume is 5~25μl.

10. The determination of 4-boron according to any one of claims 1-9 10 A method for determining the content of related substances in β-L-phenylalanine, characterized in that... 4-boron in the reference solution 10 The concentration of L-phenylalanine in the reference solution is 0.09~0.9 μg / ml, and the concentration of L-tyrosine in the reference solution is 0.15~3.0 μg / ml. and / or, the concentration of 4-boron 10 B]-L-phenylalanine in the test solution is 0.003 to 0.45 mg / ml.

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