A method for detecting related substances in N-Fmoc-L-phenylalanine

Abstract

The invention provides a method for detecting related substances in N-fluorenylmethoxycarbonyl-L-phenylalanine, including HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH and Fmoc-Phe-Phe-OH; the detection method is a high performance liquid chromatography method, including: (1) chromatographic conditions: a. chromatographic column: using octadecylsilane bonded silica gel as a filler; b. mobile phase: using phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B; c. elution program: using a gradient elution program; d. detector: the detector is a photodiode array detector with a detection wavelength of 220nm; (2) preparing a mixed standard solution and a test solution; (3) determination method: taking a diluent, a mixed standard solution and a test solution, respectively injecting them into a high performance liquid chromatograph, and obtaining the retention time t of each component after injection. R This method has the characteristics of simplicity, strong specificity, and high detection sensitivity, and plays a positive role in the quality control of Fmoc-protected amino acids and peptide APIs in industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and relates to a method for detecting related substances in N-fluorenylmethoxycarbonyl-L-phenylalanine. Background Art

[0002] Most of the peptide drugs currently on the market can be prepared by chemical synthesis. Peptide drugs are mainly composed of amino acids or amino acid derivatives. They can be connected step by step by amino acids or condensed by multiple peptide chain fragments, and then the final product can be obtained through subsequent cleavage and purification. The mainstream of chemical synthesis is solid phase synthesis, in which the Fmoc method uses alkali-removable Fmoc as an α-amino protecting group, and the side chain protection uses trifluoroacetic acid-removable tert-butyloxycarbonyl. The related substances of the Fmoc-protected amino acids used in the synthesis directly affect the final quality and yield of the product. If there is a set of detection methods with high accuracy, it can prevent the impurities in the protected amino acids from being brought into the product, reduce the difficulty of the subsequent purification process, and at the same time improve the yield, and ultimately reduce the production cost.

[0003] N-Fmoc-Phe-OH, abbreviated as Fmoc-Phe-OH, is an Fmoc-protected amino acid and is a frequently used product in the solid phase synthesis of peptides. From the process of protecting amino acids, it can be seen that N-9-Fmoc-Osu, the starting material for preparing Fmoc-protected amino acids, will bring in unreacted Fmoc-Osu, as well as other side reaction impurities. When preparing Fmoc-Phe-OH, according to deduction, the following impurities may be produced: 1. Residual impurities in the process: Fmoc-Osu (9-fluorenylmethyl-N-succinimidyl carbonate); 2. Side reaction impurities in the process: Fmoc-OH (9-fluorenylmethanol), HOSU (N-hydroxysuccinimide), Fmoc-β-Ala-OH (N-fluorenylmethoxycarbonyl-β-alanine), Fmoc-β-Ala-Phe-OH (N-fluorenylmethoxycarbonyl-β-alanine-phenylalanine), Fmoc-Phe-Phe-OH (N-fluorenylmethoxycarbonyl-phenylalanine-phenylalanine). The presence of these impurities will seriously affect the quality of Fmoc-Phe-OH, thereby affecting the final product of solid phase synthesis, making it difficult to meet quality control standards and increasing production costs. Therefore, if an analytical method that can effectively detect various potential impurities is developed, it will have important guiding significance for the production and purification of protected amino acids, and establishing strict quality control standards will have positive significance for reducing the difficulty of final product purification and improving product quality.

[0004] The existing technical solutions either use methanol as the mobile phase, or the mobile phase contains triethylamine, phosphoric acid or buffer salts. Methanol has ultraviolet absorption at low wavelengths. If methanol is used as the mobile phase, the sensitivity of the analytical method will be reduced, and the system pressure will be high. The presence of triethylamine in the mobile phase will cause irreversible damage to the chromatographic column and shorten the service life of the chromatographic column; during gradient operation, phosphoric acid in the mobile phase will cause serious baseline drift; the presence of buffer salts in the mobile phase will make the system baseline unstable, and there will be more interfering peaks in the blank test, which will reduce the sensitivity of the method, and the buffer salts are easy to precipitate, which will cause system pipeline blockage. In addition, the existing technical solution detection has a situation where the liquid phase purity is far different from the result of the titration method to measure the content. One of the reasons for this situation may be that the method does not completely separate the main peak from the impurities during the detection process, and the main peak contains impurity peaks, so that the purity is high in the result. Such a method for analyzing related substances is defective, and the results obtained are inaccurate, and there is no substantial guiding role in production, research and development, and purification.

[0005] In order to develop an analytical method that can effectively detect various potential impurities, technicians have been conducting research and experiments for many years, but there has been no obvious progress. As an analytical technology with certain technological breakthroughs in recent years, technicians have developed a protected amino acid test method using acetonitrile, ultrapure water, and trifluoroacetic acid as mobile phases. However, when this test method is actually used, in order to ensure the accuracy of the test results, the concentration of the mobile phase (trifluoroacetic acid) must reach more than 2%. If the concentration of the mobile phase is too high, the mobile phase will be too acidic, and the sample will be partially decomposed in the mobile phase, and other impurities will be generated, which will not only lead to inaccurate test results, but also the mobile phase is highly corrosive and causes great damage to the instrument. If the concentration is lower than this, the impurity separation effect is poor and the detection accuracy will decrease. Therefore, the accuracy and practicality of this test method are not high. Summary of the invention

[0006] In order to better control product quality during peptide synthesis, impurities must be accurately analyzed. To solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides a method for detecting related substances in N-fluorenylmethoxycarbonyl-L-phenylalanine, wherein the related substances are HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, and Fmoc-Phe-Phe-OH;

[0008] Furthermore, the detection method comprises:

[0009] (1) Chromatographic conditions:

[0010] a. Chromatographic column: octadecylsilane bonded silica gel is used as filler;

[0011] b. Mobile phase: Mobile phase A is purified water, and 85% v / v phosphoric acid is added to adjust the pH to 2.1-2.5;

[0012] Mobile phase B was acetonitrile;

[0013] c. Elution program: The elution time is 50 min, using the gradient elution program shown in Table 1:

[0014] Table 1

[0015] Time / min Mobile phase A%, v / v Mobile phase B%, v / v 0 75 25 50 20 80

[0016] d. Detector: The detector is a photodiode array (i.e., DAD) detector;

[0017] e. Column temperature: 30℃~40℃;

[0018] (2) Mix the standard solution and the test solution:

[0019] a. Prepare standard solution: accurately weigh appropriate amount of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Phe-Phe-OH, and Fmoc-Phe-OH reference substances, dissolve them in diluent and quantitatively dilute them to prepare a mixed standard solution containing 0.001 mg of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, and Fmoc-Phe-Phe-OH per ml, and 0.5 mg of Fmoc-Phe-OH;

[0020] b. Prepare the test solution: accurately weigh an appropriate amount of the test sample, dissolve it in a diluent and quantitatively dilute it to make a solution containing 0.5 mg / mL of the test sample per 1 ml;

[0021] (3) Determination method: Take the diluent, the mixed standard solution and the test solution and inject them into the high performance liquid chromatograph respectively. After injection, the retention time t of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Phe-Phe-OH and Fmoc-Phe-OH are obtained respectively. R ;

[0022] (4) Content calculation results:

[0023] The area normalization method is used to calculate the content of each impurity in the test solution. The calculation formula is:

[0024]

[0025] A 杂质峰 : impurity peak area;

[0026] A 总峰 : The sum of all peak areas in the test sample;

[0027] Further, the detection wavelength of the DAD detector is 220 nm;

[0028] Further, the flow rate of the mobile phase is 0.8 to 1.2 ml / min; preferably, the flow rate of the mobile phase is 1.0 ml / min;

[0029] Further, the injection volume is 5 to 20 μl; preferably, the injection volume is 10 μl;

[0030] Further, the diluent is acetonitrile;

[0031] Furthermore, the retention times t of HOSU, Fmoc-β-Ala-OH, Fmoc-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Osu, Fmoc-Phe-OH and Fmoc-Phe-Phe-OH are R They are 2.3-2.4min, 18.0-18.5min, 18.5-19.0min, 24.0-25.0min, 27.5-28.0min, 30.2-31.0min and 34.5-36.0min respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the HPLC chromatogram for simultaneous separation and detection of related substances in the Fmoc-Phe-OH sample solution in Example 1, wherein the peaks are A: HOSU; B: Fmoc-β-Ala-OH; C: Fmoc-OH; D: Fmoc-β-Ala-Phe-OH; E: Fmoc-Osu; F: Fmoc-Phe-OH; and G: Fmoc-Phe-Phe-OH.

[0033] Beneficial Effects

[0034] 1. The present invention adopts HPLC-UV detection method to achieve simultaneous detection of multiple related substances in N-Fmoc-L-phenylalanine:

[0035] 2. This method can effectively detect Fmoc-Phe-OH and 6 known impurities, and the separation degree is greater than 1.5, and the theoretical plate number of the main peak is greater than 100000; the present invention uses acetonitrile as the mobile phase B phase, because the acetonitrile cutoff wavelength is 190nm, which is lower than the cutoff wavelength of methanol 205nm, the absorption background is smaller than that of methanol, and the pressure is lower than that of methanol, and the impurity detection wavelength here is at the low wavelength of ultraviolet, so using acetonitrile as the mobile phase will minimize the influence of the mobile phase;

[0036] 3. The present invention uses appropriate sample concentration to obtain an appropriate signal-to-noise ratio (S / N). If the concentration is too low, it will affect the detection of impurities in the sample, and if the concentration is too high, the peak shape will be poor, affecting the accuracy of the detection;

[0037] 4. The present invention adopts an appropriate mobile phase flow rate. If the flow rate is too low, the peak will be obtained later and the theoretical plate number of the peak will be poor. If the flow rate is too high, the pressure will exceed the pressure that the instrument can withstand, causing certain damage to the instrument.

[0038] 5. This method has the characteristics of simple sample processing, strong specificity, high detection sensitivity, good stability of measurement results, strong method repeatability and durability. The method provided by the present invention plays a positive role in the quality control of Fmoc-protected amino acids and polypeptide raw materials in industrial production. DETAILED DESCRIPTION

[0039] In a specific implementation, it is obvious that a person skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

[0040] Embodiment 1:

[0041] A method for detecting and analyzing Fmoc-Phe-OH related substances, comprising:

[0042] (1) Chromatographic conditions:

[0043] a. Chromatographic column: Octadecylsilane bonded silica gel is used as filler. The specific chromatographic column adopts C 18 Chromatographic column, specification: 4.6*250mm, inner diameter 5μm;

[0044] b. Mobile phase: Mobile phase A is purified water, and 85% v / v phosphoric acid is added to adjust the pH to 2.1-2.5; mobile phase B is acetonitrile; the mobile phase flow rate is 1.0 ml / min;

[0045] c. Elution program: The elution time is 50 min, using the gradient elution program shown in Table 1:

[0046] Table 1

[0047] Time / min Mobile phase A%, v / v Mobile phase B%, v / v 0 75 25 50 20 80

[0048] d. Detector: The detector is a photodiode array (i.e., DAD) detector with a detection wavelength of 220 nm;

[0049] e. Column temperature: 35℃;

[0050] (2) Mix the standard solution and the test solution:

[0051] a. Prepare standard solution: accurately weigh appropriate amount of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Phe-Phe-OH, and Fmoc-Phe-OH reference substances, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed standard solution containing 0.001 mg of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, and Fmoc-Phe-Phe-OH per ml, and 0.5 mg of Fmoc-Phe-OH;

[0052] b. Prepare the test solution: accurately weigh about 25 mg of the test sample, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain;

[0053] (3) Determination method: Take the diluent acetonitrile, the mixed standard solution and the test solution, and inject them into the high performance liquid chromatograph respectively. The injection volume is 10 μl. After injection, the retention time t of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Phe-Phe-OH and Fmoc-Phe-OH are obtained respectively. R ;

[0054] (4) Measurement results:

[0055] The area normalization method is used to calculate the content of each impurity in the test solution. The calculation formula is:

[0056]

[0057] A 杂质峰 : impurity peak area;

[0058] A 总峰 : The sum of all peak areas in the test sample;

[0059] Detection retention time t of mixed sample R The separation degree is shown in Table 2 below:

[0060] Table 2

[0061] name Retention time / min Separation HOSU 2.310 —— Fmoc-β-Ala-OH 18.069 84.38 Fmoc-OH 18.818 2.52 Fmoc-β-Ala-Phe-OH 24.403 18.46 Fmoc-Osu 27.908 11.30 Fmoc-Phe-OH 30.273 7.26 Fmoc-Phe-Phe-OH 34.954 14.54

[0062] The test results of the test solution content are shown in Table 3 below:

[0063] Calculation formula:

[0064]

[0065] A 杂质峰 : impurity peak area;

[0066] A 总峰 : The sum of all peak areas in the test sample.

[0067] Measurement results:

[0068] Table 3

[0069]

[0070] This method can effectively separate the known impurities from the main component Fmoc-Phe-OH, and the separation degrees between the impurities are greater than 1.5, indicating that this method can effectively detect related substances in Fmoc-Phe-OH.

[0071] Example 2

[0072] A method for detecting and analyzing Fmoc-Phe-OH related substances, comprising:

[0073] (1) Chromatographic conditions:

[0074] a. Chromatographic column: Octadecylsilane bonded silica gel is used as filler. The specific chromatographic column adopts C 18 Chromatographic column, specification: 4.6*250mm, inner diameter 5μm;

[0075] b. Mobile phase: Mobile phase A is purified water, and 85% v / v phosphoric acid is added to adjust the pH to 2.1-2.5;

[0076] Mobile phase B was acetonitrile; the mobile phase flow rate was 1.0 ml / min;

[0077] c. Elution program: The elution time is 50 min, using the gradient elution program shown in Table 1:

[0078] Table 1

[0079] Time / min Mobile phase A%, v / v Mobile phase B%, v / v 0 75 25 50 20 80

[0080] d. Detector: The detector is a photodiode array (i.e., DAD) detector with a detection wavelength of 220 nm;

[0081] e. Column temperature: 30℃;

[0082] (2) Mix the standard solution and the test solution:

[0083] a. Prepare standard solution: accurately weigh appropriate amount of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Phe-Phe-OH, and Fmoc-Phe-OH reference substances, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed standard solution containing 0.001 mg of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, and Fmoc-Phe-Phe-OH per ml, and 0.5 mg of Fmoc-Phe-OH;

[0084] b. Prepare the test solution: accurately weigh about 25 mg of the test sample, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain;

[0085] (3) Determination method: Take the diluent acetonitrile, the mixed standard solution and the test solution, and inject them into the high performance liquid chromatograph respectively. The injection volume is 10 μl. After injection, the retention time t of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Phe-Phe-OH and Fmoc-Phe-OH are obtained respectively. R ;

[0086] (4) Measurement results:

[0087] The area normalization method is used to calculate the content of each impurity in the test solution. The calculation formula is:

[0088]

[0089] A 杂质峰 : impurity peak area;

[0090] A 总峰 : The sum of all peak areas in the test sample;

[0091] Detection retention time t of mixed sample R The separation degree is shown in Table 4 below:

[0092] Table 4

[0093]

[0094]

[0095] The test results of the test solution are shown in Table 5 below:

[0096] Table 5

[0097] name result(%) HOSU Not detected Fmoc-β-Ala-OH Not detected Fmoc-OH 0.035 Fmoc-β-Ala-Phe-OH 0.033 Fmoc-Osu Not detected Fmoc-Phe-OH 99.66 Fmoc-Phe-Phe-OH 0.11 Maximum unknown single impurity 0.07 Total impurities 0.34

[0098] This method can effectively separate the known impurities from the main component Fmoc-Phe-OH, and the separation degrees between the impurities are greater than 1.5, indicating that this method can effectively detect related substances in Fmoc-Phe-OH.

[0099] Example 3

[0100] A method for detecting and analyzing Fmoc-Phe-OH related substances, comprising:

[0101] (1) Chromatographic conditions:

[0102] a. Chromatographic column: Octadecylsilane bonded silica gel is used as filler. The specific chromatographic column adopts C 18 Chromatographic column, specification: 4.6*250mm, inner diameter 5μm;

[0103] b. Mobile phase: Mobile phase A is purified water, and 85% v / v phosphoric acid is added to adjust the pH to 2.1-2.5;

[0104] Mobile phase B was acetonitrile; the mobile phase flow rate was 1.0 ml / min;

[0105] c. Elution program: The elution time is 50 min, using the gradient elution program shown in Table 1:

[0106] Table 1

[0107] Time / min Mobile phase A%, v / v Mobile phase B%, v / v 0 75 25 50 20 80

[0108] d. Detector: The detector is a photodiode array (i.e., DAD) detector with a detection wavelength of 220 nm;

[0109] e. Column temperature: 40℃;

[0110] (2) Mix the standard solution and the test solution:

[0111] a. Prepare standard solution: accurately weigh appropriate amount of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Phe-Phe-OH, and Fmoc-Phe-OH reference substances, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed standard solution containing 0.001 mg of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, and Fmoc-Phe-Phe-OH per ml, and 0.5 mg of Fmoc-Phe-OH;

[0112] b. Prepare the test solution: accurately weigh an appropriate amount of the test sample, about 25 mg, and place it in a 50 ml volumetric flask. Dissolve it in acetonitrile and quantitatively dilute it to make a solution containing 0.5 mg / mL of the test sample per 1 ml;

[0113] (3) Determination method: Take the diluent acetonitrile, the mixed standard solution and the test solution, and inject them into the high performance liquid chromatograph respectively. The injection volume is 10 μl. After injection, the retention time t of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Phe-Phe-OH and Fmoc-Phe-OH are obtained respectively. R ;

[0114] (4) Measurement results:

[0115] The area normalization method is used to calculate the content of each impurity in the test solution. The calculation formula is:

[0116]

[0117] A 杂质峰 : impurity peak area;

[0118] A 总峰 : The sum of all peak areas in the test sample;

[0119] Detection retention time t of mixed sample R The separation degree is shown in Table 6 below:

[0120] Table 6

[0121]

[0122]

[0123] The test results of the test solution are shown in Table 7 below:

[0124] Table 7

[0125] name result(%) HOSU Not detected Fmoc-β-Ala-OH Not detected Fmoc-OH 0.047 Fmoc-β-Ala-Phe-OH 0.033 Fmoc-Osu Not detected Fmoc-Phe-OH 99.64 Fmoc-Phe-Phe-OH 0.12 Maximum unknown single impurity 0.10 Total impurities 0.36

[0126] This method can effectively separate the known impurities from the main component Fmoc-Phe-OH, and the separation degrees between the impurities are greater than 1.5, indicating that this method can effectively detect related substances in Fmoc-Phe-OH.

[0127] Example 4

[0128] A method for detecting and analyzing Fmoc-Phe-OH related substances, comprising:

[0129] (1) Chromatographic conditions:

[0130] a. Chromatographic column: Octadecylsilane bonded silica gel is used as filler. The specific chromatographic column adopts C 18 Chromatographic column, specification: 4.6*250mm, inner diameter 5μm;

[0131] b. Mobile phase: Mobile phase A is purified water, and 85% v / v phosphoric acid is added to adjust the pH to 2.1-2.5;

[0132] Mobile phase B was acetonitrile; the flow rate of the mobile phase was 0.9 ml / min;

[0133] c. Elution program: The elution time is 50 min, using the gradient elution program shown in Table 1:

[0134] Table 1

[0135]

[0136]

[0137] d. Detector: The detector is a photodiode array (i.e., DAD) detector with a detection wavelength of 220 nm;

[0138] e. Column temperature: 35℃;

[0139] (2) Mix the standard solution and the test solution:

[0140] a. Prepare standard solution: accurately weigh appropriate amount of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Phe-Phe-OH, and Fmoc-Phe-OH reference substances, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed standard solution containing 0.001 mg of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, and Fmoc-Phe-Phe-OH per ml, and 0.5 mg of Fmoc-Phe-OH;

[0141] b. Prepare the test solution: accurately weigh an appropriate amount of the test sample, about 25 mg, and place it in a 50 ml volumetric flask. Dissolve it in acetonitrile and quantitatively dilute it to make a solution containing 0.5 mg / mL of the test sample per 1 ml;

[0142] (3) Determination method: Take the diluent acetonitrile, the mixed standard solution and the test solution, and inject them into the high performance liquid chromatograph respectively. The injection volume is 10 μl. After injection, the retention time t of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Phe-Phe-OH and Fmoc-Phe-OH are obtained respectively. R ;

[0143] (4) Measurement results:

[0144] The area normalization method is used to calculate the content of each impurity in the test solution. The calculation formula is:

[0145]

[0146] A 杂质峰 : impurity peak area;

[0147] A 总峰 : The sum of all peak areas in the test sample;

[0148] Detection retention time t of mixed sample R The separation degree is shown in Table 8 below:

[0149] Table 8

[0150]

[0151]

[0152] The test results of the test solution are shown in Table 9 below:

[0153] Table 9

[0154] name result(%) HOSU Not detected Fmoc-β-Ala-OH Not detected Fmoc-OH 0.046 Fmoc-β-Ala-Phe-OH 0.041 Fmoc-Osu Not detected Fmoc-Phe-OH 99.66 Fmoc-Phe-Phe-OH 0.12 Maximum unknown single impurity 0.075 Total impurities 0.34

[0155] This method can effectively separate the known impurities from the main component Fmoc-Phe-OH, and the separation degrees between the impurities are greater than 1.5, indicating that this method can effectively detect related substances in Fmoc-Phe-OH.

[0156] Example 5

[0157] A method for detecting and analyzing Fmoc-Phe-OH related substances, comprising:

[0158] (1) Chromatographic conditions:

[0159] a. Chromatographic column: Octadecylsilane bonded silica gel is used as filler. The specific chromatographic column adopts C 18 Chromatographic column, specification: 4.6*250mm, inner diameter 5μm;

[0160] b. Mobile phase: Mobile phase A is purified water, and 85% v / v phosphoric acid is added to adjust the pH to 2.1-2.5;

[0161] Mobile phase B was acetonitrile; the mobile phase flow rate was 1.1 ml / min;

[0162] c. Elution program: The elution time is 50 min, using the gradient elution program shown in Table 1:

[0163] Table 1

[0164]

[0165]

[0166] d. Detector: The detector is a photodiode array (i.e., DAD) detector with a detection wavelength of 220 nm;

[0167] e. Column temperature: 40℃;

[0168] (2) Mix the standard solution and the test solution:

[0169] a. Prepare standard solution: accurately weigh appropriate amount of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Phe-Phe-OH, and Fmoc-Phe-OH reference substances, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed standard solution containing 0.001 mg of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, and Fmoc-Phe-Phe-OH per ml, and 0.5 mg of Fmoc-Phe-OH;

[0170] b. Prepare the test solution: accurately weigh an appropriate amount of the test sample, about 25 mg, and place it in a 50 ml volumetric flask. Dissolve it in acetonitrile and quantitatively dilute it to make a solution containing 0.5 mg / mL of the test sample per 1 ml;

[0171] (3) Determination method: Take the diluent acetonitrile, the mixed standard solution and the test solution, and inject them into the high performance liquid chromatograph respectively. The injection volume is 10 μl. After injection, the retention time t of HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, Fmoc-Phe-Phe-OH and Fmoc-Phe-OH are obtained respectively. R ;

[0172] (4) Measurement results:

[0173] The area normalization method is used to calculate the content of each impurity in the test solution. The calculation formula is:

[0174]

[0175] A 杂质峰 : impurity peak area;

[0176] A 总峰 : The sum of all peak areas in the test sample;

[0177] Detection retention time t of mixed sample R The separation degree is shown in Table 10 below:

[0178] Table 10

[0179]

[0180]

[0181] The test results of the test solution are shown in Table 11 below:

[0182] Table 11

[0183] name result(%) HOSU Not detected Fmoc-β-Ala-OH Not detected Fmoc-OH 0.047 Fmoc-β-Ala-Phe-OH 0.040 Fmoc-Osu Not detected Fmoc-Phe-OH 99.65 Fmoc-Phe-Phe-OH 0.11 Maximum unknown single impurity 0.079 Total impurities 0.35

[0184] This method can effectively separate the known impurities from the main component Fmoc-Phe-OH, and the separation degrees between the impurities are greater than 1.5, indicating that this method can effectively detect related substances in Fmoc-Phe-OH.

[0185] According to the experimental results of Examples 1 to 5, the relative retention time of each impurity is calculated as shown in Table 12 below. The peak positions of each known impurity are shown in the attached table. Figure 1 shown.

[0186] Table 12

[0187]

[0188]

[0189] As can be seen from the above table, the Fmoc-Phe-OH related substance analysis method provided by the present invention can accurately locate and separate the known impurities HOSU, Fmoc-OH, Fmoc-Osu, Fmoc-β-Ala-OH, Fmoc-β-Ala-Phe-OH, and Fmoc-Phe-Phe-OH in Fmoc-Phe-OH, and can accurately determine the impurity content of Fmoc-Phe-OH.

Claims

1. A method for detecting related substances in N-Fmoc-L-phenylalanine, characterized in that: The related substances are HOSU, Fmoc-OH, Fmoc-Osu, Fmoc- β -Ala-OH, Fmoc- β -Ala-Phe-OH and Fmoc-Phe-Phe-OH; The detection method comprises: (1) Chromatographic conditions: a. Chromatographic column: octadecylsilane bonded silica gel is used as filler; b. Mobile phase: Mobile phase A is purified water, and 85% v / v phosphoric acid is added to adjust the pH to 2.1-2.5; Mobile phase B was acetonitrile; c. Elution program: The elution time is 50 min, using the gradient elution program shown in Table 1: Table 1 d. Detector: The detector is a photodiode array detector; e. Column temperature: 30℃~40℃; (2) Mix standard solution and test solution: a. Prepare standard solution: accurately weigh HOSU, Fmoc-OH, Fmoc-Osu, Fmoc- β -Ala-OH, Fmoc- β -Ala-Phe-OH, Fmoc-Phe-Phe-OH, Fmoc-Phe-OH reference substances are dissolved in a diluent and quantitatively diluted to make each ml contain 0.001 mg HOSU, Fmoc-OH, Fmoc-Osu, Fmoc- β -Ala-OH, Fmoc- β - Ala-Phe-OH, Fmoc-Phe-Phe-OH and a mixed standard solution containing 0.5 mg of Fmoc-Phe-OH; b. Prepare the test solution: accurately weigh an appropriate amount of the test sample, dissolve it in a diluent and quantitatively dilute it to make a solution containing 0.5 mg / mL of the test sample per 1 ml; (3) Determination method: Take the diluent, mixed standard solution and test solution and inject them into the high performance liquid chromatography. After injection, HOSU, Fmoc-OH, Fmoc-Osu, Fmoc- β -Ala-OH, Fmoc- β -Retention time t of Ala-Phe-OH, Fmoc-Phe-Phe-OH and Fmoc-Phe-OH R ; (4) Content calculation results: The area normalization method is used to calculate the content of each impurity in the test solution. The calculation formula is: ； A 杂质峰 : impurity peak area; A 总峰 : The sum of all peak areas in the test sample.

2. The method for detecting related substances in N-Fmoc-L-phenylalanine according to claim 1, characterized in that: The detection wavelength of the DAD detector was 220 nm.

3. The method for detecting related substances in N-Fmoc-L-phenylalanine according to claim 1, characterized in that: The flow rate of the mobile phase is 0.8-1.2 ml / min.

4. The method for detecting related substances in N-Fmoc-L-phenylalanine according to claim 3, characterized in that: The flow rate of the mobile phase was 1.0 ml / min.

5. The method for detecting related substances in N-Fmoc-L-phenylalanine according to claim 1, characterized in that: The injection volume is 5 to 20 μ l.

6. The method for detecting related substances in N-Fmoc-L-phenylalanine according to claim 5, characterized in that: The injection volume is 10 μ l.

7. The method for detecting related substances in N-Fmoc-L-phenylalanine according to claim 1, characterized in that: The diluent is acetonitrile.

8. The method for detecting related substances in N-Fmoc-L-phenylalanine according to claim 1, characterized in that: The HOSU, Fmoc- β -Ala-OH, Fmoc-OH, Fmoc- β -Retention time t of Ala-Phe-OH, Fmoc-Osu, Fmoc-Phe-Phe-OH and Fmoc-Phe-OH R They are 2.3~2.4 min, 18.0~18.5 min, 18.5~19.0 min, 24.0~25.0 min, 27.5~28.0 min, 34.5~36.0 min and 30.2~31.0 min respectively.

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