Method for analyzing purity and related substances of fluorenylmethoxycarbonyl-L-valine

Abstract

The invention relates to the technical field of drug detection, in particular to a method for analyzing the purity of fluorenylmethoxycarbonyl-L-valine and related substances. The invention provides a method for analyzing the purity of fluorenylmethoxycarbonyl-L-valine and related substances, which comprises the following steps: high performance liquid chromatography is adopted, and the chromatographic conditions are as follows: a filler of a chromatographic column is octaalkylsilane bonded silica gel; the mobile phase A is an aqueous solution containing 0.08-0.12% of phosphoric acid; the mobile phase B is acetonitrile; a gradient elution procedure; according to the method, the Fmoc-Val-OH and the related substances as well as the related substances can be effectively separated, the separation degree is greater than or equal to 1.5, the peak shape symmetry is better, the detection sensitivity of each related substance is high, the content (purity) detection result of each component is accurate and effective, and the quality of the Fmoc-L-Val-OH can be correctly reflected.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to an analysis method for the purity of Fmoc-L-valine and related substances. Background Art

[0002] With the rapid development of peptide synthesis technology, the R&D cost and production cost of peptide drugs have been greatly reduced, which has promoted the development of peptide drugs. Peptide drugs are suitable for solving complex diseases that are difficult to solve with small molecule chemical drugs, including metabolic diseases, tumors, chronic diseases and rare diseases. The synthesis of large molecular peptides generally adopts solid phase synthesis method, and the starting materials used are mainly various Fmoc-protected amino acids, and fluorenylmethoxycarbonyl-L-valine (Fmoc-Val-OH) is one of them. For example, the solid phase synthesis method of the popular glucose-lowering and weight-loss drugs such as telpotide and semaglutide on the market will use this compound.

[0003] Fmoc-Val-OH is generally prepared from L-valine as the starting material through Fmoc protection. During the synthesis process, many impurities such as Fmoc-Gly-OH, Fmoc-β-Ala-OH, Fmoc-OH, Fmoc-β-Ala-Val-OH, Fmoc-Val-Val-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, etc. will be introduced due to raw material and process problems. If these impurities are not effectively controlled, they will be introduced into the final product, making it difficult to purify the product, thereby affecting the yield and quality.

[0004] At present, there are analytical methods for some Fmoc-protected amino acid related substances, such as Chinese patent CN 111505160B. However, this method cannot completely separate all the related impurities of Fmoc-Val-OH, so it cannot accurately control its corresponding impurities and is not suitable for effective control of Fmoc-Val-OH related substances. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is a method for analyzing the purity of Fmoc-L-Valine and related substances. The method can effectively separate Fmoc-Val-OH and related substances, the peak shape has good symmetry, the detection sensitivity of related substances is high, the detection results of the content (purity) of each component are accurate and effective, and the quality of Fmoc-L-Val-OH can be correctly reflected.

[0006] To this end, the present invention provides the following technical solutions:

[0007] The embodiment of the present invention discloses a method for analyzing the purity of Fmoc-L-valine and related substances, comprising: using high performance liquid chromatography, the chromatographic conditions comprising:

[0008] The filler of the chromatographic column is octaalkylsilane bonded silica gel; the mobile phase A is an aqueous solution containing 0.08-0.12 wt % phosphoric acid; the mobile phase B is acetonitrile; the gradient elution procedure is as follows:

[0009]

[0010] In some embodiments, the mobile phase A is an aqueous solution of 0.1 wt % phosphoric acid.

[0011] In some embodiments, in the gradient elution program, at 0 min, the volume ratio of mobile phase A to mobile phase B is 60%:40%.

[0012] In some embodiments, in the gradient elution program, at 0 min, the volume ratio of mobile phase A to mobile phase B is 60%:40%.

[0013] In some embodiments, the chromatographic conditions are 0.9 to 1.1 ml / min, optionally, 1 ml / min.

[0014] In some embodiments, in the chromatographic conditions, the column temperature is 38-42°C, optionally 40°C.

[0015] In some embodiments, in the chromatographic conditions, the detection wavelength is 260-264 nm, optionally 262 nm.

[0016] In some embodiments, in the chromatographic conditions, the column length of the chromatographic column is 4.6 mm, the inner diameter is 250 mm, and the particle size is 5.0 μm.

[0017] In some embodiments, in the chromatographic conditions, the chromatographic column is Nano Chrom Core C8.

[0018] In some embodiments, the method further includes preparing a test sample solution, wherein the test sample is dissolved to prepare a solution containing about 0.8 to 1.2 mg per 1 ml.

[0019] In some embodiments, the method further includes preparing a test sample solution, wherein the test sample is dissolved to prepare a solution containing about 1 mg per 1 ml.

[0020] In some embodiments, the injection volume is 8 to 12 μl, optionally 10 μl.

[0021] The technical solution of the present invention has the following advantages:

[0022] 1. The present invention provides a method for analyzing the purity of Fmoc-L-valine and related substances, comprising: using high performance liquid chromatography, the chromatographic conditions comprising: the filler of the chromatographic column is octaalkylsilane bonded silica gel; the mobile phase A is an aqueous solution containing 0.08-0.12% phosphoric acid; the mobile phase B is acetonitrile; the gradient elution program is as follows: 0min→5min, the volume ratio of mobile phase A: mobile phase B is 58-62%:42-38%→60%:40%; 5min→15min, the volume ratio of mobile phase A: mobile phase B is 60%:40%→30%:70%; 15min→25min, the volume ratio of mobile phase A: mobile phase B is 30%:70%→30%:70%; 25min→35min, mobile phase A: The volume ratio of mobile phase B is 30%:70%→10%:90%; 35min→40min, the volume ratio of mobile phase A: mobile phase B is 10%:90%→10%:90%; 40min→42min, the volume ratio of mobile phase A: mobile phase B is 10%:90%→60%:40%; 42min→52min, the volume ratio of mobile phase A: mobile phase B is 60%:40%→60%:40%. The method can effectively separate Fmoc-Val-OH and related substances and related substances, with a separation degree of ≥1.5, good peak symmetry, high detection sensitivity of related substances, accurate and effective detection results of the content (purity) of each component, and can correctly reflect the quality of Fmoc-L-Val-OH.

[0023] Furthermore, through methodological verification, the method of the present invention has no interference from blank solvent, good specificity, good linearity, linear correlation coefficients greater than 0.99, and the detection limit of Fmoc-Val-OH and related substances can reach 0.003%, with high sensitivity and strong durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 It is the chromatogram in Comparative Example 1 of the present invention;

[0026] Figure 2 It is the chromatogram in Comparative Example 2 of the present invention;

[0027] Figure 3 It is the chromatogram in Comparative Example 3 of the present invention;

[0028] Figure 4 It is a chromatogram of the specific solution (system suitability solution) in the specificity test in the experimental example of the present invention. DETAILED DESCRIPTION

[0029] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0030] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0031] The instruments, reagents and reference substances used in the following examples are shown in the table below:

[0032] Table 1. Experimental instruments

[0033]

[0034] Table 2 Experimental reagents and reference substances

[0035]

[0036]

[0037] In the following examples, the separation degree between the chromatographic peak of the substance to be tested and the adjacent chromatographic peak is required to be no less than 1.5. The calculation formula of the separation degree is:

[0038] or

[0039] Where t R2 is the retention time of the latter of two adjacent chromatographic peaks;

[0040] t R1 is the retention time of the previous peak between two adjacent chromatographic peaks;

[0041] W1, W2 and W1,h / 2, W2,h / 2 are the peak width and half-peak width of the two adjacent chromatographic peaks respectively.

[0042] Example 1

[0043] This embodiment provides a method for separating Fmoc-L-valine related substances, comprising: using high performance liquid chromatography, the chromatographic conditions comprising:

[0044] The filler of the chromatographic column is octaalkylsilane bonded silica gel (the chromatographic column is Nano Chrom Core C8, 4.6×250 mm, 5.0 μm); 0.1% phosphoric acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, the flow rate is 1.0 ml per minute, and the column temperature is 40° C.; the ultraviolet detector has a detection wavelength of 262 nm; and gradient elution is performed according to Table 3.

[0045] Table 3 Gradient elution program

[0046] Time (min) Mobile phase A (v / v%) Mobile phase B (v / v%) 0 60 40 5 60 40 15 30 70 25 30 70 35 10 90 40 10 90 42 60 40 52 60 40

[0047] Preparation of test solution: Take appropriate amount of three batches of Fmoc-Val-OH produced by Changzhou Gene Pharmaceutical Co., Ltd., dissolve them in acetonitrile and quantitatively dilute them to make solutions containing about 1 mg per 1 ml.

[0048] Preparation of system suitability solution: Take appropriate amount of Fmoc-β-Ala-OH, Fmoc-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Gly-OH, Fmoc-β-Ala-Val-OH, Fmoc-Val-Val-OH, and Fmoc-Val-OH reference substances, dissolve them in acetonitrile and dilute them to make a mixed solution containing 1 μg, 1 μg, 1 μg, 1 μg, 1 μg, 1 μg, 1 μg, 1 μg, and 1 mg of each substance per 1 ml.

[0049] System suitability requirements: Take 10 μl of the system suitability solution and inject it into the liquid chromatograph. Record the chromatogram. Fmoc-Gly-OH, Fmoc-β-Ala-OH, Fmoc-OH, Fmoc-β-Ala-Val-OH, Fmoc-Val-OH, Fmoc-Val-Val-OH, Fmoc-Ile-OH, and Fmoc-Leu-OH appear in sequence. The separation degree between adjacent peaks should be no less than 1.5.

[0050] Determination method: Take 10 μl of the test solution, inject it into the liquid chromatograph respectively, and record the chromatogram.

[0051] In the chromatogram of the test solution, except for the solvent peak, the content of each related substance was calculated according to the area normalization method with correction factors. The correction factor of Fmoc-Gly-OH was 0.8, the correction factor of Fmoc-β-Ala-OH was 0.9, the correction factor of Fmoc-OH was 0.6, the correction factor of Fmoc-β-Ala-Val-OH was 1.3, the correction factor of Fmoc-Val-Val-OH was 1.3, the correction factor of Fmoc-Ile-OH was 1.1, and the correction factor of Fmoc-Leu-OH was 1.1.

[0052] The test results of the test solution are shown in the following table:

[0053] Table 4 Example 1 test results

[0054]

[0055] As shown in the table above, the results of multiple batches of testing can accurately reflect the purity of Fmoc-Val-OH and related substances in each batch, indicating that this method is suitable for the detection of Fmoc-Val-OH purity and related substances.

[0056] Comparative Example 1

[0057] The difference between this comparative example and Example 1 is that the only difference is the chromatographic column, which is a C18 chromatographic column (4.6×250 mm, 5.0 μm). After the system is stable, 10 μl of the system suitability solution is injected into the liquid chromatograph and the chromatogram is recorded. The test results are shown in Tables 5 and Figure 1 .

[0058] Table 5. Test results of comparative example 1

[0059]

[0060]

[0061] Comparative Example 2

[0062] The difference between this comparative example and Example 1 is that the elution conditions in the liquid phase conditions are different. The elution conditions in this comparative example are isocratic elution, and the volume percentage of mobile phase A to mobile phase B is 40%:60%. After the system is stable, 10 μl of the system suitability solution is injected into the liquid chromatograph, and the chromatogram is recorded. The test results are shown in Tables 6 and Figure 2 .

[0063] Table 6. Test results of comparative example 2

[0064]

[0065] Comparative Example 3

[0066] The difference between this comparative example and Example 1 is that the mobile phase B is different. The mobile phase B in this comparative example is methanol. After the system is stable, 10 μl of the system suitability solution is injected into the liquid chromatograph and the chromatogram is recorded. The test results are shown in Tables 7 and Figure 3 .

[0067] Table 7. Test results of comparative example 3

[0068]

[0069] Experimental example method validation

[0070] 1) Exclusivity

[0071] Preparation of specific solution: the same as the system suitability solution in Example 1.

[0072] Preparation of Fmoc-β-Ala-OH positioning solution: Accurately weigh about 10 mg of Fmoc-β-Ala-OH reference substance into a 10 ml volumetric flask, dissolve it in acetonitrile and dilute to the mark, mix well; then accurately measure 0.1 ml into a 10 ml volumetric flask, dilute to the mark with acetonitrile, and mix well.

[0073] Preparation of Fmoc-OH positioning solution: Accurately weigh about 10 mg of Fmoc-OH reference substance into a 10 ml volumetric flask, dissolve it with acetonitrile and dilute to the scale, mix well; then accurately measure 0.1 ml into a 10 ml volumetric flask, dilute to the scale with acetonitrile, and mix well.

[0074] Preparation of Fmoc-Leu-OH localization solution: Accurately weigh about 10 mg of Fmoc-Leu-OH reference substance into a 10 ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, mix well; then accurately measure 0.1 ml into a 10 ml volumetric flask, dilute to the mark with acetonitrile, and mix well.

[0075] Preparation of Fmoc-Ile-OH positioning solution: Accurately weigh about 10 mg of Fmoc-Ile-OH reference substance into a 10 ml volumetric flask, dissolve it with acetonitrile and dilute to the scale, mix well; then accurately measure 0.1 ml into a 10 ml volumetric flask, dilute to the scale with acetonitrile, and mix well.

[0076] Preparation of Fmoc-Gly-OH positioning solution: Accurately weigh about 10 mg of Fmoc-Gly-OH reference substance into a 10 ml volumetric flask, dissolve it with acetonitrile and dilute to the scale, mix well; then accurately measure 0.1 ml into a 10 ml volumetric flask, dilute to the scale with acetonitrile, and mix well.

[0077] Preparation of Fmoc-β-Ala-Val-OH positioning solution: Accurately weigh about 10 mg of Fmoc-β-Ala-Val-OH reference substance into a 10 ml volumetric flask, dissolve it with acetonitrile and dilute to the scale, mix well; then accurately measure 0.1 ml into a 10 ml volumetric flask, dilute to the scale with acetonitrile, and mix well.

[0078] Preparation of Fmoc-Val-Val-OH positioning solution: Accurately weigh about 10 mg of Fmoc-Val-Val-OH reference substance into a 10 ml volumetric flask, dissolve it in acetonitrile and dilute to the scale, mix well; then accurately measure 0.1 ml into a 10 ml volumetric flask, dilute to the scale with acetonitrile, and mix well.

[0079] Preparation of Fmoc-Val-OH positioning solution: Accurately weigh about 10 mg of Fmoc-Val-OH reference substance into a 10 ml volumetric flask, dissolve it in acetonitrile and make up to the mark, and mix well.

[0080] After the system is balanced, 10 μl of each solution is injected into the liquid chromatograph, and the detection is carried out according to the method of Example 1, and the chromatogram is recorded. The measurement results are shown in Table 8 and Figure 4 .

[0081] Table 8. Specificity test results

[0082]

[0083]

[0084] Table 8, continued, specificity test results

[0085]

[0086] From the above results, it can be seen that the blank solvent has no interference, the separation degree between the Fmoc-Ile-OH peak and the Fmoc-Leu-OH peak is the smallest, which is 1.5, and the others are all greater than 1.5, which meets the requirements.

[0087] 2) Linearity and range

[0088] Linear stock solution: Accurately weigh appropriate amount of Fmoc-β-Ala-OH, Fmoc-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Gly-OH, Fmoc-β-Ala-Val-OH, Fmoc-Val-Val-OH, and Fmoc-Val-OH reference substances, dissolve them in acetonitrile and dilute them to make a mixed solution containing 10μg, 10μg, 10μg, 10μg, 10μg, 10μg, 10μg, 20μg of each substance per 1 ml.

[0089] 80% linear solution: Take 0.8 ml of the linear stock solution and transfer it to a 10 ml volumetric flask, dilute to the mark with acetonitrile, and mix well.

[0090] 90% linear solution: Take 0.9 ml of the linear stock solution and transfer it to a 10 ml volumetric flask, dilute to the mark with acetonitrile, and mix well.

[0091] 100% linear solution: Take 1.0 ml of the linear stock solution and transfer it to a 10 ml volumetric flask, dilute to the mark with acetonitrile, and mix well.

[0092] 110% linear solution: Take 1.1 ml of the linear stock solution and transfer it to a 10 ml volumetric flask, dilute to the mark with acetonitrile, and mix well.

[0093] 120% linear solution: Take 1.2 ml of the linear stock solution and transfer it to a 10 ml volumetric flask, dilute to the mark with acetonitrile, and mix well.

[0094] After the system was balanced, 10 μl of each solution was injected into the liquid chromatograph, and the test was performed according to the method of Example 1. The chromatogram was recorded. The test results are shown in Table 9.

[0095] Table 9. Linearity and range test results

[0096]

[0097] 3) Detection limit

[0098] Take each positioning solution, dilute it step by step with acetonitrile to a suitable multiple, accurately measure 10 μl and inject it into the liquid chromatograph, detect it according to the method of Example 1, and record the chromatogram. When the signal-to-noise ratio is about 3:1, the concentration of the solution at this time is the detection limit of each impurity. The measurement results are shown in Table 10.

[0099] Table 10. Detection limit test results

[0100]

[0101] 4) Solution stability

[0102] After the system is balanced, accurately measure the test solution (prepared according to Example 1 (select batch C67231005003) stored at room temperature at 0h, 4h, 8h, and 12h. F moc - V a l -OH as the test sample) 10 μl was injected into the liquid chromatograph, and the detection was carried out according to the method of Example 1. The chromatogram was recorded, and the measurement results are shown in Table 11.

[0103] Table 11. Solution stability test results

[0104]

[0105]

[0106] 5) Durability

[0107] The chromatographic conditions were changed according to the single factor in Table 12, and the rest were tested according to the method in Example 1. 10 μl of the system suitability solution was measured and injected into the liquid chromatograph, and the chromatogram was recorded. The measurement results are shown in Table 13.

[0108] Table 12. Range of variation of chromatographic condition parameters in durability test

[0109]

[0110] Table 13. Durability test results

[0111]

[0112]

[0113] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for analyzing the purity of Fmoc-L-valine and related substances, characterized in that: include: High performance liquid chromatography was used, and the chromatographic conditions included: The filler of the chromatographic column is octaalkylsilane bonded silica gel; the mobile phase A is an aqueous solution containing 0.08-0.12 wt % phosphoric acid; the mobile phase B is acetonitrile; the gradient elution procedure is as follows:

2. The method for analyzing the purity and related substances of Fmoc-L-valine according to claim 1, characterized in that: The mobile phase A is an aqueous solution of 0.1 wt % phosphoric acid.

3. The method for analyzing the purity and related substances of Fmoc-L-valine according to claim 1 or 2, characterized in that: In the gradient elution program, at 0 min, the volume ratio of mobile phase A to mobile phase B was 60%:40%.

4. The method for analyzing the purity of Fmoc-L-valine and related substances according to any one of claims 1 to 3, characterized in that: In the chromatographic conditions, the flow rate is 0.9 to 1.1 ml / min, optionally 1 ml / min.

5. The method for analyzing the purity of Fmoc-L-valine and related substances according to any one of claims 1 to 4, characterized in that: In the chromatographic conditions, the column temperature is 38-42°C, optionally 40°C.

6. The method for analyzing the purity of Fmoc-L-valine and related substances according to any one of claims 1 to 7, characterized in that: In the chromatographic conditions, the detection wavelength is 260-264 nm, and optionally 262 nm.

7. The method for analyzing the purity of Fmoc-L-valine and related substances according to any one of claims 1 to 6, characterized in that: In the chromatographic conditions, the chromatographic column has a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5.0 μm.

8. The method for analyzing the purity of Fmoc-L-valine and related substances according to any one of claims 1 to 7, characterized in that: In the chromatographic conditions, the chromatographic column is a C8 bonded silica gel column.

9. The method for analyzing the purity of Fmoc-L-valine and related substances according to any one of claims 1 to 8, characterized in that: The method also includes preparing a test sample solution, dissolving the test sample to prepare a solution containing about 0.8 to 1.2 mg per ml, and optionally, preparing a solution containing about 1 mg per ml.

10. The method for analyzing the purity of Fmoc-L-valine and related substances according to any one of claims 1 to 9, characterized in that: The injection volume is 8 to 12 μl, and 10 μl is optional.

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