Analysis and Detection Method of PEG3 and PEG5 Impurities in Fmoc-NH-PEG4-Propionic Acid

Abstract

The present invention discloses Fmoc-NH-PEG 4 The analytical detection method for PEG3 impurities and PEG5 impurities in propionic acid adopts a chromatographic column with octadecyl bonded silica gel as filler; an acidic buffer solution and acetonitrile volume ratio of 70-80:20-30 is used as mobile phase A; an acidic buffer solution and acetonitrile volume ratio of 50-60:40-50 is used as mobile phase B, and gradient elution is performed. The method of the present invention is durable to multiple chromatographic columns, the mobile phase used is relatively safe, the baseline is smooth at the detection wavelength, the solvent does not interfere with the detection of various impurities in the sample, the method has high sensitivity, good stability, and is more durable, and solves the problem of Fmoc-NH-PEG 4 The difficulty in separating the main peak of propionic acid from the impurity peaks of PEG3 and PEG5.

Description

Technical Field

[0001] The present invention belongs to the field of chemical analysis technology, and particularly relates to Fmoc-NH-PEG 4 -Analysis and detection method of PEG3 impurities and PEG5 impurities in propionic acid. Background Art

[0002] Fmoc-NH-PEG 4 In -propionic acid, Fmoc is used as a protective group and is often used to protect the amino group of amino acids. PEG is a polymer compound with good water solubility and biocompatibility. NH is an activating group and is used for coupling reactions of biomolecules. 4 -Propionic acid is mainly used in ligand research, peptide synthesis support, grafted polymer compounds, new materials and polyethylene glycol-modified functional coatings and other active compounds.

[0003] Fmoc-NH-PEG 4 The CAS number of -propionic acid is 557756-85-1, the chemical name is 5,8,11,14-tetraoxa-2-azaheptadecanedioic acid 1-(9H-fluoren-9-ylmethyl) ester, and the molecular formula is C 26 H 33 NO 8 , the chemical structure is:

[0004]

[0005] Its main preparation reaction equation is as follows:

[0006]

[0007] In Fmoc-NH-PEG 4 - If the reaction is incomplete or excessive during the propionic acid production process, Fmoc-NH-PEG is easily generated 4 -Propionic acid homologues, mainly Fmoc-NH-PEG 3 -Propionic acid (PEG3 impurity) and Fmoc-NH-PEG 5 -Propionic acid (PEG5 impurity).

[0008] In the presence of Fmoc-NH-PEG 4 In the reaction of Fmoc-NH-PEG as the reaction material, PEG3 impurities and PEG5 impurities as reaction material impurities will introduce new impurities into the reaction and affect the quality of subsequent products; therefore, the material Fmoc-NH-PEG 4 -Propionic acid, the content of PEG3 impurities and PEG5 impurities therein needs to be controlled.

[0009] PEG3 impurities and PEG5 impurities and Fmoc-NH-PEG 4 -Propionic acid contains some similar structures, making Fmoc-NH-PEG 4 -Propionic acid is difficult to separate from the above two impurities, and there are few studies on the separation of PEG3 impurities and PEG5 impurities from Fmoc-NH-PEG 4 -Research report on the separation of propionic acid.

[0010] The Chinese patent application with patent publication number CN 115236231 A discloses the Fmoc-NH-PEG n - Separation method of propionic acid homologues, separation of Fmoc-NH-PEG with n=1 to 12 n -Propionic acid homologues, perchloric acid is used in the mobile phase, and a specific chromatographic column Agilent ZORBAXSB-C18 (250mm*4.6mm, 5μm) is required to achieve a separation degree of more than 1.0. Perchloric acid is a controlled explosive hazardous chemical with strong corrosiveness and irritation, and has low safety. At the same time, the chromatographic column has a general effect on separating homologues and poor durability. In the mixed solution spectrum, the separation effect is not ideal, especially for the PEG5 impurity, which does not meet the requirement of complete separation. Summary of the invention

[0011] The purpose of the present invention is to provide a method capable of accurately analyzing and detecting PEG3 impurities and PEG5 impurities in Fmoc-NH-PEG4-propionic acid.

[0012] The present invention is achieved through the following technical solutions:

[0013] A Fmoc-NH-PEG 4 -Analysis and detection method of PEG3 impurities and PEG5 impurities in propionic acid, using high performance liquid chromatography, the detection conditions are as follows:

[0014] Chromatographic conditions:

[0015] A chromatographic column with octadecyl bonded silica as filler was used; the mobile phase A was an acidic buffer solution-acetonitrile volume ratio of 70-80:20-30; the mobile phase B was an acidic buffer solution-acetonitrile volume ratio of 50-60:40-50, and gradient elution was performed; the gradient elution procedure was as follows:

[0016] 0 min → 3 min, 100% mobile phase A, 0% mobile phase B;

[0017] 3min→25min, 100% mobile phase A, 0% mobile phase B linearly changed to 0% mobile phase A, 100% mobile phase B; 25min→35min, 0% mobile phase A, 100% mobile phase B;

[0018] 35min→37min, 0% mobile phase A, 100% mobile phase B linearly changed to 100% mobile phase A, 0% mobile phase B; 37min→55min, 100% mobile phase A, 0% mobile phase B;

[0019] Preparation of test solution:

[0020] Use acetonitrile-water in a volume ratio of 45~55:45~55 as solvent to dissolve and quantitatively dilute.

[0021] In order to optimize the above technical solutions, the specific limitations adopted also include:

[0022] The chromatographic column adopts one of the following: SUPERIOREX Ⅱ ODS; 250mm×4.6mm, 5µm; XBridge RC18, 250mm*4.6mm, 5μm; YMC-Triart C18, 150×4.6mm, 3μm.

[0023] The acidic buffer in mobile phase A and mobile phase B is a phosphate buffer or a trifluoroacetic acid buffer, and the pH value is adjusted to 6.8-7.2 with alkali; the concentration volume percentage of the phosphate buffer is 0.1%-0.5%, and the concentration volume percentage of the trifluoroacetic acid buffer is 0.09%-0.11%; preferably, the concentration volume percentage of the phosphate buffer is 0.5%, and the pH value is 7.0; preferably, the concentration volume percentage of the trifluoroacetic acid buffer is 0.1%, and the pH value is 7.0.

[0024] The test solution was prepared by weighing Fmoc-NH-PEG 4 -propionic acid, dissolved in a solvent and quantitatively diluted to prepare a solution of 0.65~0.79mg / ml; the control solution is prepared by taking the test solution, dissolving it in a solvent and quantitatively diluting it to prepare a solution of 6.5~7.9μg / ml; preferably, the test solution is 0.72mgmg / ml and the control solution is 7.2μg / ml.

[0025] The detection wavelength is selected as 265±5 nm or 220±5 nm; preferably, the detection wavelength is selected as 265 nm.

[0026] The column temperature is 23-45°C; preferably, the column temperature is 40°C.

[0027] The detection flow rate is 0.9~1.1ml / min; preferably, the detection flow rate is 1.0ml / min.

[0028] The injection volume is 5-10 μl; preferably, the injection volume is 5 μl.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] Fmoc-NH-PEG 4 -In the process of propionic acid production, the main impurity Fmoc-NH-PEG 3 -Propionic acid (PEG3 impurity) and Fmoc-NH-PEG 5 -propionic acid (PEG5 impurity) content was controlled, while Fmoc-NH-PEG 4 -Propionic acid and PEG3 impurities, PEG5 impurities are difficult to separate. The present invention develops and optimizes a high performance liquid chromatography method for simultaneous analysis and detection of PEG3 impurities and PEG5 impurities that are difficult to separate in Fmoc-NH-PEG4-propionic acid, which has better chromatographic analysis conditions and can obtain better separation effects.

[0031] The analytical detection method of the present invention has low requirements on the selection of chromatographic columns and can be applied to a variety of C18 chromatographic columns. The baseline is smooth at the detection wavelength, and the solvent does not interfere with the detection of various impurities in the sample. The specificity, separation degree, durability, etc. of the analytical detection method are good after testing and can fully meet the analysis requirements.

[0032] The mobile phase adopted in the analysis and detection method of the present invention is relatively safe, and the analysis and determination can be completed in a better experimental environment, thereby avoiding potential safety hazards and being suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The HPLC spectrum in Example 1 for chromatographic condition screening and optimization in Example 1.

[0034] Figure 2 The HPLC spectrum in Example 2 for chromatographic condition screening optimization in Example 1.

[0035] Figure 3 This is the HPLC spectrum in Example 3 for chromatographic condition screening and optimization in Example 1.

[0036] Figure 4 This is the HPLC spectrum with a detection wavelength of 265 nm in Example 4 for chromatographic condition screening and optimization in Example 1.

[0037] Figure 5 This is the HPLC spectrum with a detection wavelength of 220 nm in Example 4 for chromatographic condition screening and optimization in Example 1.

[0038] Figure 6 This is the overall HPLC spectrum in Example 5 for chromatographic condition screening and optimization in Example 1.

[0039] Figure 7 This is a partial enlarged HPLC spectrum in Example 5 for chromatographic condition screening and optimization in Example 1.

[0040] Figure 8This is the overall HPLC spectrum in Example 6 for chromatographic condition screening and optimization in Example 1.

[0041] Fig. 9 This is a partial enlarged HPLC spectrum in Example 6 for chromatographic condition screening and optimization in Example 1.

[0042] Fig.10 This is the specific overlay diagram in Example 2.

[0043] Fig.11 This is the linear relationship diagram of Fmoc-NH-PEG4-propionic acid in Example 2.

[0044] Fig.12 This is the linear relationship diagram of PEG3 impurities in Example 2.

[0045] Fig.13 This is the linear relationship diagram of PEG5 impurities in Example 2.

[0046] Fig.14 It is the HPLC spectrum in the chromatographic conditions of Comparative Example 1.

[0047] Fig.15 It is the HPLC spectrum of the system suitability solution of the present invention.

[0048] Fig.16 This is the solution HPLC spectrum of the system suitability of the comparative method of Comparative Example 2. DETAILED DESCRIPTION

[0049] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0050] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0051] Example 1 HPLC detection method

[0052] A method for detecting the content of PEG impurities in Fmoc-NH-PEG4-propionic acid specifically comprises the following steps:

[0053] The structures of PEG3 impurity and PEG5 impurity are shown in Table 1 below:

[0054] Table 1 Structures of PEG3 impurities and PEG5 impurities

[0055]

[0056] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0057] Solvent: acetonitrile-water (50:50).

[0058] Test solution: Take an appropriate amount of the product, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing approximately 0.72 mg per 1 ml.

[0059] Control solution: Take an appropriate amount of the test sample solution, dissolve it in solvent and quantitatively dilute it to make a solution containing approximately 7.2 μg per 1 ml.

[0060] System suitability solution: Take Fmoc-NH-PEG 4 -Propionic acid, Fmoc-NH-PEG 3 -Propionic acid (referred to as PEG3 impurity) and Fmoc-NH-PEG 5 -propionic acid (PEG5 impurity for short) is added with solvent to dissolve and dilute to about 7.2 μg of PEG3 impurity and PEG5 impurity per 1 ml, containing Fmoc-NH-PEG 4 -Solution of about 0.72 mg of propionic acid.

[0061] The high performance chromatography conditions are:

[0062] Chromatographic column: SUPERIOREX Ⅱ ODS, 250mm×4.6mm, 5µm;

[0063] Mobile phase A: phosphate buffer (5.0 ml of phosphoric acid was added to 1000 ml of water, mixed and adjusted to pH 7.0 with 10 mol / L potassium hydroxide solution)-acetonitrile (75:25);

[0064] Mobile phase B: phosphate buffer (5.0 ml of phosphoric acid was added to 1000 ml of water, and the pH value was adjusted to about 7.0 with 10 mol / L potassium hydroxide solution)-acetonitrile (55:45);

[0065] Gradient elution: see Table 2;

[0066] Table 2 Fmoc-NH-PEG 4 -Analysis and detection method of PEG3 impurities and PEG5 impurities in propionic acid Gradient elution conditions

[0067]

[0068] Flow rate: 1.0 ml per minute;

[0069] Column temperature: 40°C;

[0070] Detection wavelength: 265nm;

[0071] Injection volume: 5 μl;

[0072] Determination method: Accurately measure 5 μl of system suitability solution, control solution and test solution, respectively, inject them into liquid chromatograph, and record the chromatogram.

[0073] System suitability requirements: The peak order in the system suitability solution chromatogram is PEG3 impurity, Fmoc-NH-PEG 4 -Propionic acid and PEG5 impurities. Fmoc-NH-PEG 4 -The separation degree between the propionic acid peak and the PEG3 impurity peak and the PEG5 impurity peak is no less than 1.5, achieving complete separation.

[0074] Example of chromatographic condition screening and optimization:

[0075] The present application has tested a variety of chromatographic condition schemes, such as the studies of Comparative Example 1 and Comparative Example 2 below, investigated the separation effects of various detection conditions, developed and obtained an analytical detection method suitable for PEG3 impurities and PEG5 impurities in Fmoc-NH-PEG4-propionic acid, and gradually optimized it during the method development process, as shown in the following chromatographic condition screening optimization examples 1 to 6.

[0076] In the following chromatographic condition screening and optimization example, a mixed solution was selected for injection. The mixed solution was prepared as follows: Fmoc-NH-PEG 4 -Propionic acid, Fmoc-NH-PEG 3 -Propionic acid (PEG3 impurity) and Fmoc-NH-PEG 5 -propionic acid (PEG5 impurity) in appropriate amount, add solvent to dissolve and dilute to contain about 7.2 μg of PEG3 impurity and PEG5 impurity per 1 ml, containing Fmoc-NH-PEG 4 -Solution of about 0.72 mg of propionic acid.

[0077] Chromatographic condition screening and optimization example 1: trifluoroacetic acid solution-acetonitrile system, see Table 3 for details.

[0078] Table 3 Chromatographic Condition Screening Optimization Example 1

[0079]

[0080] Conclusion: If Figure 1 As shown, PEG3 impurities and Fmoc-NH-PEG 4 -Propionic acid and PEG5 impurities emerge one after another, and the main peak (Fmoc-NH-PEG 4 -propionic acid) and the two adjacent impurity peaks are separated by 1.239 and 2.538, respectively, and the PEG3 impurity and the main peak are basically separated.

[0081] Chromatographic condition screening and optimization example 2: trifluoroacetic acid solution-acetonitrile system, reducing the injection volume, see Table 4 for details.

[0082] Table 4 Chromatographic Condition Screening Optimization Example 2

[0083]

[0084] In order to improve the separation of PEG3 impurities and main peak and reduce the injection volume, PEG3 impurities, Fmoc-NH-PEG 4 -The resolution between propionic acid and PEG5 impurities is 1.836 and 2.858, respectively, which is good. Figure 2 .

[0085] Chromatographic condition screening and optimization example 3: trifluoroacetic acid solution-acetonitrile system, increasing the temperature of the chromatographic column, see Table 5 for details.

[0086] Table 5 Chromatographic Condition Screening Optimization Example 3

[0087]

[0088] Conclusion: Increasing the column temperature can improve the PEG3 impurity and Fmoc-NH-PEG 4 - The separation between propionic acid and PEG5 impurities has a resolution of 2.120 and 3.255 respectively, and the separation effect has been further improved. Figure 3 .

[0089] Chromatographic Condition Screening and Optimization Example 4: Different chromatographic columns and different wavelengths for trifluoroacetic acid solution-acetonitrile system, see Table 6 for details.

[0090] Table 6 Chromatographic Condition Screening Optimization Example 4

[0091]

[0092] Conclusion: The separation effects of chromatographic columns of different brands, different particle sizes and different lengths are comparable. YMC-Triart C18, 150×4.6mm, 3μm and SUPERIOREX ODS, 250×4.6mm, 5μm are both preferred chromatographic columns. Comparing the detection wavelengths of 220nm and 265nm, both can effectively separate PEG3 impurities and Fmoc-NH-PEG in the mixed solution. 4 -Propionic acid and PEG5 impurities, see Figure 4 , Figure 5 Among them, the 265nm wavelength is selected as the preferred detection wavelength because of its smooth gradient baseline and high response value.

[0093] Chromatographic Condition Screening and Optimization Example 5 The preferred phosphoric acid concentration in the phosphoric acid solution-acetonitrile system (0.1% phosphoric acid) is shown in Table 7 for details.

[0094] Table 7 Chromatographic Condition Screening Optimization Example 5

[0095]

[0096] Conclusion: Fmoc-NH-PEG in the mixed solution in the mobile phase system of phosphoric acid solution-acetonitrile 4 - The resolution between propionic acid and PEG3 and PEG5 impurities is 2.711 and 3.379, respectively, which can achieve effective separation. Figure 6 , Figure 7 It can be seen that the effect of using phosphoric acid solution system is better.

[0097] Chromatographic Condition Screening and Optimization Example 6 Optimization of Phosphoric Acid Concentration in Phosphoric Acid Solution-Acetonitrile System

[0098] The concentration of the phosphoric acid solution system was adjusted to 0.5% phosphoric acid to further investigate the detection effect. See Table 8 for details.

[0099] Table 8 Chromatographic Condition Screening Optimization Example 6

[0100]

[0101] Conclusion: Increasing the concentration of phosphoric acid solution in the phosphoric acid solution-acetonitrile mobile phase system can effectively improve the separation of Fmoc-NH-PEG4-propionic acid and PEG3 impurities and PEG5 impurities in the mixed solution, and the resolution is 3.094 and 3.413, respectively. Figure 8 , Fig. 9 The use of 0.5% phosphate buffer in the mobile phase has a good separation effect on the separation of PEG3 impurities and PEG5 impurities in the Fmoc-NH-PEG4-propionic acid of the present application.

[0102] Since it was found through experimental research that the phosphoric acid solution system has a better separation effect on the substance system, in order to obtain better separation conditions, the present application replaced the acetonitrile in the mobile phase B with a certain proportion of 0.5% phosphoric acid-acetonitrile, and adjusted the gradient elution program accordingly according to the change of the mobile phase. Through comparative tests of various proportion conditions, the separation and detection effects were investigated, and the final preferred chromatographic condition method was obtained. For details, see the chromatographic condition screening optimization example 7 in Table 9. Under this condition, Fmoc-NH-PEG 4 -The separation and detection effect of PEG3 impurities and PEG5 impurities in propionic acid is outstanding.

[0103] Table 9 Chromatographic Condition Screening Optimization Example 7

[0104]

[0105] The following examples verify the final preferred chromatographic conditions:

[0106] Example 2: Method Validation

[0107] The present invention investigates the specificity, system applicability, linear range, limit of quantitation and limit of detection, accuracy, precision, solution stability and method durability of the method, and proves that the method can be used for Fmoc-NH-PEG 3 -Propionic acid (PEG3 impurity) and Fmoc-NH-PEG5-propionic acid (PEG5 impurity) controls.

[0108] 1) Exclusivity

[0109] Solvent: acetonitrile-water (50:50).

[0110] Test solution: Take Fmoc-NH-PEG 4 -Propionic acid (about 18 mg), weigh accurately, place in a 25 ml volumetric flask, add solvent to dissolve and dilute to scale, mix well.

[0111] PEG3 impurity-2% dilution: Take Fmoc-NH-PEG 3 About 18 mg, weigh accurately, put into a 25 ml volumetric flask, add solvent to dissolve and dilute to scale, shake well, and it is PEG3 impurity solution. Pipette 20 μl into the injection vial, add 980 μl of solvent, and mix well.

[0112] PEG5 impurity-2% dilution: Take Fmoc-NH-PEG 5 About 18 mg, weigh accurately, put into a 25 ml volumetric flask, add solvent to dissolve and dilute to scale, shake well, and it is PEG5 impurity solution. Pipette 20 μl into the injection vial, add 980 μl of solvent, and mix well.

[0113] Mixed solution (system suitability solution): Accurately pipette 0.5 ml of PEG3 impurity solution and PEG5 impurity solution into the same 50 ml volumetric flask as the stock solution; take Fmoc-NH-PEG 4 -Propionic acid (about 18 mg), accurately weighed, placed in a 25 ml volumetric flask, added with stock solution to dissolve and dilute to scale and mix well.

[0114] Determination: Accurately measure 5 μl of each of the above solutions, inject into the liquid chromatograph, record the chromatogram, and the results are shown in Fig.10 , blank solvent in Fmoc-NH-PEG 4 - There is no interference at the retention time of the chromatographic peaks of propionic acid, PEG3 impurities and PEG5 impurities, that is, the blank solvent does not interfere with Fmoc-NH-PEG 4-Detection of PEG3 and PEG5 impurities in propionic acid; PEG3 impurities were detected in the test solution, but PEG5 impurities were not detected. The separation between PEG3 impurities and adjacent peaks was 20.579 and 4.738; PEG3 impurities, Fmoc-NH-PEG 4 -Propionic acid and PEG5 impurities appear in turn, Fmoc-NH-PEG 4 The resolution between -propionic acid and PEG3 and PEG5 impurity peaks is 2.982 and 3.396, both greater than 1.5.

[0115] 2) Limit of quantification and limit of detection

[0116] Accurately weigh about 18 mg of PEG3 impurity and PEG5 impurity respectively, place them in different 25 ml volumetric flasks, add solvent to dissolve and dilute to the scale, and shake well; accurately measure 0.5 ml of each and place them in the same 50 ml volumetric flask, dilute to the scale with solvent, and shake well; dilute step by step to the appropriate concentration. The concentration when the signal-to-noise ratio is about 10:1 is the quantitative limit concentration; the concentration when the signal-to-noise ratio is about 3:1 is the detection limit concentration. The results are shown in Tables 10 and 11.

[0117] Table 10 Results of quantification limit and detection limit of PEG3 impurities

[0118]

[0119] Table 11 Results of quantification limit and detection limit of PEG5 impurities

[0120]

[0121] It can be seen from Table 10 and Table 11 that the detection limit of PEG3 impurity is equivalent to 0.03% of the test sample concentration, the quantification limit is equivalent to 0.05% of the test sample concentration; the precision RSD of the quantification limit is 2.0% (n=6). The detection limit of PEG5 impurity is 0.2041μg / ml, equivalent to 0.03% of the test sample concentration; the quantification limit of PEG5 impurity is 0.4082μg / ml, equivalent to 0.06% of the test sample concentration; the precision RSD of the quantification limit is 2.7% (n=6). It proves that this method can be accurately quantified with high sensitivity.

[0122] 3) Linearity and range

[0123] Fmoc-NH-PEG 4 -Propionic acid linear: accurately weigh Fmoc-NH-PEG 4 -propionic acid, add solvent to dissolve and quantitatively dilute to make each ml contain about Fmoc-NH-PEG 4-Propionic acid 0.3μg, 0.6μg, 1.2μg, 3.0μg, 6.0μg, 9.0μg, 12.0μg solution as Fmoc-NH-PEG 4 -Propionic acid linear solution.

[0124] Linearity of PEG3 impurities and PEG5 impurities: Accurately weigh appropriate amounts of PEG3 impurities and PEG5 impurities, dissolve them in solvent and quantitatively dilute them to make mixed solutions containing approximately 0.3μg, 0.6μg, 1.2μg, 3μg, 6μg, 9μg and 12μg of PEG3 impurities and PEG5 impurities per 1ml, as linear solutions of PEG3 impurities and PEG5 impurities.

[0125] 5 μl was accurately measured and injected into the liquid chromatograph to record the chromatogram. Linear regression was performed with concentration C (μg / ml) as the horizontal axis and peak area A as the vertical axis. The results are shown in Tables 12, 13, 14 and Fig.11 , Fig.12 , Fig.13 .

[0126] Table 12 Fmoc-NH-PEG 4 -Propionic acid linear relationship results

[0127]

[0128] Table 13 PEG3 impurity linear relationship results

[0129]

[0130] Table 14 PEG5 impurity linear relationship results

[0131]

[0132] From Table 12~Table 14 and Figure 11~Figure 13 It can be seen that Fmoc-NH-PEG 4 -When the propionic acid concentration is in the range of 0.3193~12.7709μg / ml, the linear regression equation is y=11145.3017x+205.1219, and the correlation coefficient r is 1.0000, greater than 0.990, the ratio of the Y-axis intercept to the 100% limit response value is 0.25%, less than 15.0%; when the PEG3 impurity concentration is in the range of 0.3126~12.5048μg / ml, the linear regression equation is y=12326.6686x+195.0112, and the correlation coefficient ris 1.0000, greater than 0.990, the ratio of the Y-axis intercept to the 100% limit response value is 0.22%, less than 15.0%, and the correction factor is 0.9; when the PEG5 impurity concentration is in the range of 0.3284~13.1384μg / ml, the linear regression equation is y=10055.6097x+182.4413, and the correlation coefficient r It is 1.0000, which is greater than 0.990. The ratio of the Y-axis intercept to the 100% limit response value is 0.25%, which is less than 15.0%. The correction factor is 1.1, and the linear relationship is good.

[0133] 4) Accuracy

[0134] 50% stock solution: Accurately weigh appropriate amounts of PEG3 impurity and PEG5 impurity, dissolve them in solvent and dilute them to make a solution containing approximately 3.6 μg of PEG3 impurity and PEG5 impurity per 1 ml.

[0135] 100% stock solution: Accurately weigh appropriate amounts of PEG3 impurity and PEG5 impurity, dissolve them in solvent and dilute them to make a solution containing approximately 7.2 μg of PEG3 impurity and PEG5 impurity per 1 ml.

[0136] 120% stock solution: Accurately weigh appropriate amounts of PEG3 impurity and PEG5 impurity, dissolve them in solvent and dilute them to make a solution containing approximately 8.6 μg of PEG3 impurity and PEG5 impurity per 1 ml.

[0137] Test solution: Take about 18 mg of the product, weigh accurately, place in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0138] Test sample control solution: Accurately measure 0.5 ml of the test sample solution, place it in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0139] 50% recovery spiked test solution: Take about 18 mg of the product, accurately weigh it, put it in a 25 ml volumetric flask, add 50% of the stock solution to dissolve and dilute to the scale, shake well; prepare 3 parallel copies.

[0140] 100% recovery spiked test solution: Take about 18 mg of the product, accurately weigh it, place it in a 25 ml volumetric flask, add 100% stock solution to dissolve and dilute to the scale, shake well; prepare 3 copies in parallel.

[0141] 120% recovery spiked test solution: Take about 18 mg of the product, accurately weigh it, put it in a 25 ml volumetric flask, add 120% stock solution to dissolve and dilute to the scale, shake well; prepare 3 parallel copies.

[0142] Spiked test sample control solution: Accurately measure 0.5 ml of spiked test sample solution, place in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0143] Accurately measure 5 μl of the test solution and each recovery solution and inject them into the liquid chromatograph to record the chromatogram. Calculate the recovery of PEG3 impurity and PEG5 impurity. The results are shown in Tables 15 and 16.

[0144] Table 15 Accuracy Results - PEG3 Impurities

[0145]

[0146] Table 16 Accuracy Results - PEG5 Impurity

[0147]

[0148] Note: No PEG5 impurity was detected in the test solution.

[0149] It can be seen from Tables 15 and 16 that at three concentrations, the minimum PEG3 impurity recovery rate is 93.81% and the maximum is 98.02%, both between 92.0% and 105.0%; the RSD is 1.3% (n=9), which is less than 4.0%; the minimum PEG5 impurity recovery rate is 97.65% and the maximum is 102.33%, both between 92.0% and 105.0%; the RSD is 1.4% (n=9), which is less than 4.0%, which confirms that the method has good accuracy.

[0150] 5) Precision

[0151] Different personnel used different instruments to perform 100% spike recovery tests on different dates. Stock solutions, test solutions, test control solutions, spiked test solutions and spiked test control solutions were prepared. The recoveries of PEG3 impurities and PEG5 impurities in the spiked test solutions were determined. The results are shown in Table 17.

[0152] Table 17 Intermediate precision results

[0153]

[0154] The results showed that the RSD of the recovery rate of PEG3 impurity in 12 solutions was 1.1%, and the RSD of the recovery rate of PEG5 impurity was 2.7%, both less than 5.0%, confirming that the method has good precision.

[0155] 6) Solution stability

[0156] Prepare the test solution, spiked test solution and spiked test control solution, place them at room temperature and in the dark, take the test solution and inject it at 0h, 7.3h, 15.6h, 23.8h and 32.1h, take the spiked test solution and spiked test control solution and inject them at 0h, 6.4h, 14.7h, 22.9h and 31.2h, record the chromatogram and examine the solution storage stability. Compared with 0 hour, the relative difference of the peak area of ​​PEG3 impurity and PEG5 impurity in the test solution, spiked test solution and spiked test control solution should not be greater than 10.0%, the results are shown in Table 18.

[0157] Table 18 Solution stability results

[0158]

[0159] Table 18 shows that the reference solution has good stability when placed at room temperature and protected from light for 32.1 hours; the spiked test solution and the spiked test control solution have good stability when placed at room temperature and protected from light for 31.2 hours.

[0160] 7) Durability

[0161] The proportion of organic phase in mobile phase A, pH value of mobile phase water, column temperature and flow rate were changed, and the system applicability and detection and recovery of impurities in samples were investigated under different experimental conditions. The results are shown in Table 19.

[0162] Table 19 Durability results

[0163]

[0164] Note: Verification column: SUPERIOREX Ⅱ ODS 250mm*4.6mm 5μm Different brand column: XBridge RC18 250mm*4.6mm 5μm. Separation 1 is PEG3 impurity and Fmoc-NH-PEG 4 -propionic acid; resolution 2 is Fmoc-NH-PEG 4 - Resolution between propionic acid and PEG5 impurities.

[0165] It can be seen from Table 19 that by changing the proportion of organic phase in mobile phase A (20%~30%), the pH value of the mobile phase water phase (7.0±0.2), the column temperature (40±5℃), and the flow rate (1.0±0.1ml / min), the system suitability met the requirements. Only PEG3 impurity was detected in the test solution, with an RSD value of 0.9% (n=10). The RSD value of PEG3 impurity in the spiked test solution was 1.2% (n=10), and the RSD value of PEG5 impurity was 1.2% (n=10). The recoveries under each condition were between 94.37% and 101.31%, and were all between 92.0% and 105.0%. The method had good durability.

[0166] Comparative Example 1 Comparison with other conditions

[0167] The aqueous phase of the mobile phase was 0.1% trifluoroacetic acid, and the chromatographic conditions were shown in Table 20.

[0168] Table 20 Chromatographic conditions of Comparative Example 1

[0169]

[0170] Results Fig.14 . Fmoc-NH-PEG in the mixed solution 4 The peak retention times of -propionic acid, PEG3 impurity and PEG5 impurity are basically the same and cannot be separated.

[0171] Comparative Example 2 Comparison with the solution of patent publication number CN 115236231 A

[0172] The patent application with patent publication number CN 115236231 A discloses a Fmoc-NH-PEG n -Propionic acid homologue separation method, this comparative example cites the patent publication number CN 115236231 A scheme Fmoc-NH-PEG n -The chromatographic conditions and the results of the impurity mixed solution (system suitability solution) in the separation method of propionic acid homologues are compared with the chromatographic conditions and the mixed solution (system suitability solution) results of the present invention. The specific description is shown in Table 21.

[0173] Table 21 Comparison with Chinese Patent CN 115236231 A Method

[0174]

[0175] By comparison with Table 21, it can be seen that Fmoc-NH-PEG 4-The separation degree between propionic acid and the adjacent PEG3 impurity and PEG5 impurity peaks is not less than 2.5, that is, the separation effect is better; this application uses two chromatographic columns (SUPERIOREX Ⅱ ODS 250mm*4.6mm 5μm and XBridge RC18 250mm*4.6mm 5μm) for detection, and the system applicability meets the requirements and the recovery rate is within the range, and the durability is better; this application uses 265m wavelength detection, the baseline is smoother, the sensitivity of the method is improved, and the interference of low-wavelength absorption substances is also reduced; this application uses phosphoric acid to prepare the mobile phase, which is safer than perchloric acid.

[0176] It can be seen from the above embodiments and comparative examples that the detection and analysis method of the present application, after examining the key conditions such as the chromatographic column and the mobile phase in the high performance liquid chromatography analysis, obtains a better mobile phase system, establishes an HPLC gradient elution method suitable for the detection and analysis, and can ensure that the solvent does not interfere with the detection of various impurities in the sample. 4 -The separation effect between propionic acid and PEG3 impurity and PEG5 impurity peaks is significant, achieving the separation of Fmoc-NH-PEG 4 - Accurate detection and control of PEG3 and PEG5 impurities in propionic acid.

[0177] In summary, the method of the present application is durable to multiple chromatographic columns, the mobile phase used is safe, the baseline is smooth at the detection wavelength, the solvent does not interfere with the detection of various impurities in the sample, the method has high sensitivity, good stability, and is more durable, which solves the problem of Fmoc-NH-PEG 4 -The main peak of propionic acid and the main impurity peaks of PEG3 and PEG5 are difficult to separate.

[0178] The above are only preferred embodiments of the present invention and are not intended to limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention and based on the technical essence of the present invention, may make any simple modifications, equivalent substitutions and improvements to the above embodiments, which are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for analyzing and detecting PEG3 impurities and PEG5 impurities in Fmoc-NH-PEG4-propionic acid, using high performance liquid chromatography, characterized in that: The test conditions are as follows: Chromatographic conditions: A chromatographic column with octadecyl bonded silica as filler was used; the mobile phase A was an acidic buffer-acetonitrile volume ratio of 70-80:20-30; the mobile phase B was an acidic buffer-acetonitrile volume ratio of 50-60:40-50, and gradient elution was performed. The acidic buffer in mobile phase A and mobile phase B was a phosphate buffer or a trifluoroacetic acid buffer, and the pH value was adjusted to 6.8-7.2 with alkali; the gradient elution program was as follows: 0 min → 3 min, 100% mobile phase A, 0% mobile phase B; 3min→25min, 100% mobile phase A, 0% mobile phase B linearly changed to 0% mobile phase A, 100% mobile phase B; 25 min → 35 min, 0% mobile phase A, 100% mobile phase B; 35 min → 37 min, 0% mobile phase A, 100% mobile phase B linearly changed to 100% mobile phase A, 0% mobile phase B; 37 min → 55 min, 100% mobile phase A, 0% mobile phase B; Preparation of test solution: Use acetonitrile-water in a volume ratio of 45~55:45~55 as solvent to dissolve and quantitatively dilute.

2. The method for analyzing and detecting PEG3 impurities and PEG5 impurities in Fmoc-NH-PEG4-propionic acid according to claim 1, characterized in that: The chromatographic column is one of: SUPERIOREX Ⅱ ODS, 250mm×4.6mm, 5µm; XBridge RC18, 250mm*4.6mm, 5μm; YMC-Triart C18, 150×4.6mm, 3μm.

3. The method for analyzing and detecting PEG3 impurities and PEG5 impurities in Fmoc-NH-PEG4-propionic acid according to claim 1, characterized in that: The volume percentage of the phosphate buffer solution is 0.1% to 0.5%, and the volume percentage of the trifluoroacetic acid buffer solution is 0.09% to 0.11%.

4. The method for analyzing and detecting PEG3 impurities and PEG5 impurities in Fmoc-NH-PEG4-propionic acid according to claim 1, characterized in that: The test solution was prepared by weighing Fmoc-NH-PEG4-propionic acid, dissolving it in a solvent, and quantitatively diluting it to make a solution of 0.65~0.79 mg / ml; the control solution was prepared by taking the test solution, dissolving it in a solvent, and quantitatively diluting it to make a solution of 6.5~7.9 μg / ml.

5. The method for analyzing and detecting PEG3 impurities and PEG5 impurities in Fmoc-NH-PEG4-propionic acid according to claim 1, characterized in that: The detection wavelength is selected as 265±5nm or 220±5nm.

6. The method for analyzing and detecting PEG3 impurities and PEG5 impurities in Fmoc-NH-PEG4-propionic acid according to claim 1, characterized in that: The column temperature is 23~45°C.

7. The method for analyzing and detecting PEG3 impurities and PEG5 impurities in Fmoc-NH-PEG4-propionic acid according to claim 1, characterized in that: The detection flow rate is 0.9~1.1ml / min.

8. The method for analyzing and detecting PEG3 impurities and PEG5 impurities in Fmoc-NH-PEG4-propionic acid according to claim 1, characterized in that: The injection volume is 5~10μl.

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