Method for detecting related substances of 5 '(E)-VPA phosphoramidite monomer
By optimizing the detection conditions using liquid chromatography, the gap in the detection of related substances of 5'(E)-VPA phosphoridamide monomer was filled, enabling quality control of small nucleic acid drugs and improving detection efficiency and accuracy.
Patent Information
- Application Number
- CN202511069596.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-31
- Publication Date
- 2025-11-28
AI Technical Summary
The lack of effective methods in the current technology for detecting relevant substances in 5'(E)-VPA phosphorous amide monomers increases the difficulty of purifying small nucleic acid drugs and affects drug quality control.
The detection was performed using liquid chromatography with an octadecyl-bonded silica column, ammonium acetate solution and acetonitrile as the mobile phase, gradient elution, and a UV detector. The detection conditions were optimized to avoid sample degradation, thus achieving accurate separation and detection of related substances of 5'(E)-VPA phosphorous amide monomer.
It achieves effective separation and detection of related substances of 5'(E)-VPA phosphorous amide monomer, meets pharmacopoeia standards, reduces analysis costs, can complete the detection of multiple impurities in a short time, truly reflects sample quality, and is suitable for quality control of small nucleic acid drugs.
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Figure CN121027346A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of liquid chromatography detection, and in particular to a method for detecting related substances of 5'(E)-VPA phosphoramidite monomer. BACKGROUND
[0002] The mechanism of action of small nucleic acid drugs is to fundamentally regulate gene expression by interfering with gene transfer and translation process to achieve therapeutic effect. Compared with existing small molecule and antibody drugs, small nucleic acid drugs are widely concerned due to their advantages of fast target screening, wider treatment field, higher safety, longer treatment effect, higher success rate of research and development, etc.
[0003] The phosphate group at the end of the nucleic acid chain of small nucleic acid drugs is prone to degradation by exonuclease, but end modification can increase its nuclease resistance. At the same time, the antisense strand in the double-stranded small interfering RNA (siRNA) must have a 5' phosphate group to specifically bind to the side chain residues of the MID domain in Ago2, and the 5'(E)-VP phosphoramidite monomer is synthesized by chemical synthesis to make the 5' phosphorylation in advance, so as to realize the 5' phosphorylation of the antisense strand.
[0004] From the synthesis process of small nucleic acids, it is not difficult to find that the introduction of 5'(E)-VP phosphoramidite monomer impurities makes the impurities of small nucleic acid finished products more complex, which brings higher difficulty to purification. Therefore, as a key material of small nucleic acids, the research and control of related substances of 5'(E)-VP phosphoramidite monomer are crucial. However, there is no relevant research report on the analysis method of related substances of 5'(E)-VP phosphoramidite monomer. SUMMARY
[0005] In view of the technical problems in the background art, the present application provides a method for determining the related substances of 5'(E)-VPA phosphoramidite monomer based on liquid chromatography. The method is verified to be effective for detecting related substances in 5'(E)-VPA phosphoramidite monomer through system adaptability, specificity, linearity and range, detection limit and quantitative limit, and accuracy verification, which provides a reliable means for quality control of 5'(E)-VPA phosphoramidite monomer.
[0006] The technical scheme of the present application is as follows: The method for detecting the related substances of 5'(E)-VPA phosphoramidite monomer comprises the following steps: a 5'(E)-VPA phosphoramidite monomer sample is precisely weighed, and a test solution is prepared by using acetonitrile as a solvent; and the test solution is detected by using a high performance liquid chromatography method.
[0007] The 5'(E)-VPA solution is unstable under high-temperature, acidic and alkaline conditions, and the present application uses volatile ammonium acetate as the water phase and reduces the column temperature, thereby avoiding the misjudgment phenomenon that the impurity 5'(E)-VPA-OX generated by degradation in the chromatographic column is attributed to the sample impurity due to the limitation of the analysis and detection conditions, and the actual quality condition of the sample can be more truly reflected.
[0008] Further, the mobile phase A is 10 mmol / L ammonium acetate solution. Based on the mobile phase A and the mobile phase B, the gradient elution conditions are preferably as follows: at 0 min, the mobile phase A accounts for 50%, and the mobile phase B accounts for 50%; at 21 min, the mobile phase A accounts for 26%, and the mobile phase B accounts for 74%; at 21.5 min, the mobile phase A accounts for 10%, and the mobile phase B accounts for 90%; at 27 min, the mobile phase A accounts for 10%, and the mobile phase B accounts for 50%; at 27.5 min, the mobile phase A accounts for 50%, and the mobile phase B accounts for 50%; and at 32 min, the mobile phase A accounts for 50%, and the mobile phase B accounts for 50%.
[0009] Further, the chromatographic column is a Shim-pack Scepter C18 column with a specification of 4.6*150 mm, 3.0 μm.
[0010] Further, the injection volume is 10 µL, and the flow rate is 0.8-1.0 ml / min.
[0011] Further, an ultraviolet detector is used, and the detection wavelength is 260 nm.
[0012] Specifically, the detection method comprises the following steps: A 5'(E)-VPA sample to be detected is precisely weighed, and a test solution with a concentration of 2 mg / ml is prepared by using acetonitrile as a solvent; Precisely take 5'(E)-VPA 20 mg, and add mixed impurity solution 1 ml, and then dilute to 10 ml with acetonitrile, and mix to obtain a system suitability solution; in the mixed impurity solution, acetonitrile is the solvent, the concentration of impurity 5'-(E)-VPA-OX is 60 μg / ml, and the concentrations of impurities 5'-(E)-VP-2'-OMe-A(Bz), 5'(Z)-VPA and 5'-(E)-VP-2'-OMe-A-CE-P are all 40 μg / ml; Precisely take 5'(E)-VPA control, and prepare 5'(E)-VPA control solution with a concentration of 2 mg / ml by taking acetonitrile as a solvent; Take the system suitability solution, 5'(E)-VPA control solution and sample solution in turn for analysis, record the chromatogram, and calculate the detection amount of each related substance based on the chromatogram according to the main component self-control method.
[0013] Further, the liquid chromatography conditions include: taking Shim-pack Scepter C18 (4.6*150 mm, 3.0 μm) as a chromatographic column, taking 10 mmol / L ammonium acetate solution as a mobile phase A, acetonitrile as a mobile phase B, gradient elution, an ultraviolet detector and a detection wavelength of 260 nm, a flow rate of 1.0 mL / min, a column temperature of 25 DEG C, an injection amount of 10 μL, and an operation time of 32 min.
[0014] Compared with the prior art, the present application has the following beneficial effects: The method of the present application effectively separates four related substances in 5'(E)-VPA phosphoramidite monomer by adopting specific liquid chromatography conditions. The system adaptability, specificity, linearity and range, detection limit and quantitative limit and accuracy verification of the method all meet the acceptable standards of the pharmacopoeia, and the detection of multiple related substances can be completed in a short time, thereby reducing the analysis cost. Meanwhile, the method is compatible with mass spectrometry, and can meet the qualitative and impurity analysis requirements. In addition, the method can also reduce the degradation degree of 5'(E)-VPA in the detection process, so that the actual quality status of the sample can be more truly reflected. The present application fills the blank of 5'(E)-VP phosphoramidite monomer related substance detection, and has a positive significance for small nucleic acid drug development. BRIEF DESCRIPTION OF DRAWINGS
[0015] In order to more clearly illustrate the technical solutions of the present application, the drawings used in the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creating laborious work.
[0016] Figure 1Typical chromatogram of the test solution in the example; Figure 2 Chromatogram of the system suitability solution in the example; Figure 3 Typical chromatogram of the limit of detection solution in the example; Figure 4 Typical chromatogram of the limit of quantitation solution in the example; Legend: N-1 represents 5'-(E)-VP-2'-OMe-A(Bz) peak, N-OX, N-OX-1 and N-OX-2 represent 5'-(E)-VPA-OX peak, N-Bz-1 and N-Bz-2 represent 5'-(E)-VP-2'-OMe-A-CE-P peak, 5'(E)-VPA-1 and 5'(E)-VPA-2 represent 5'(E)-VPA peak, N-Z-1 and N-Z-2 represent 5'-(Z)-VPA peak. DETAILED DESCRIPTION
[0017] The technical solutions of the present application will be described in detail below in combination with the drawings and examples.
[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application; the terms "comprising" and "having," and any variations thereof, are intended to cover not exclusively including.
[0019] 5'(E)-VP phosphoramidite monomer is a key material for the synthesis of small nucleic acid drugs, and its quality is extremely important for small nucleic acid drugs. From the preparation process and properties of 5'(E)-VP phosphoramidite monomer, its related substances mainly include two types of process impurities and degradation impurities. Specifically, for 5'(E)-VPA, its process impurities include starting material 5'-(E)-VP-2'-OMe-A(Bz), isomer impurity 5'(Z)-VPA, and key impurity base protection group loss 5'-(E)-VP-2'-OMe-A-CE-P, and its degradation impurities mainly include oxidation impurity 5'(E)-VPA-OX. The structures of 5'(E)-VPA and its impurities are shown in Table 1.
[0020] Table 1 Structures of 5'(E)-VPA and its related substances
[0021] In order to solve the technical problem of detecting the above-mentioned related substances in 5'(E)-VPA phosphoramidite monomer, the embodiments of the present application provide a liquid chromatography method for detecting related substances of 5'(E)-VPA phosphoramidite monomer, wherein the chromatography conditions include the following: Chromatographic column, stationary phase is octadecyl bonded silica gel; Column temperature 23-27℃; The mobile phase A is 10 mmol / L ammonium acetate solution, the mobile phase B is acetonitrile, gradient elution; The injection volume is 10 μL; The flow rate is 0.8-1.0 ml / min; The detection wavelength is 260 nm.
[0022] Specifically, the detection method comprises the following steps: Precisely weigh the 5'(E)-VPA sample to be tested, and prepare a test solution with a concentration of 2 mg / ml with acetonitrile as the solvent; Precisely weigh 5'(E)-VPA 20 mg, and add a mixed impurity solution 1 ml, then dilute to 10 ml with acetonitrile, mix well to obtain a system suitability solution; in the mixed impurity solution, acetonitrile is the solvent, the concentration of impurity 5'(E)-VPA-OX is 60 μg / ml, and the concentrations of impurities 5'(E)-VP-2'-OMe-A(Bz), 5'(Z)-VPA and 5'(E)-VP-2'-OMe-A-CE-P are all 40 μg / ml; Precisely weigh the 5'(E)-VPA control, and prepare a 5'(E)-VPA control solution with a concentration of 2 mg / ml with acetonitrile as the solvent; Take the system suitability solution, 5'(E)-VPA control solution and test solution in turn, analyze and record the chromatogram, and calculate the detection amount of each related substance based on the chromatogram according to the main component self-control method.
[0023] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application. If the specific technology or condition is not specified in the examples, it is carried out according to the technology or condition described in the literature in the art or according to the product instruction. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.
[0024] In the following examples, 5'(E)-VPA control, 5'(E)-VPA-OX control, 5'(E)-VP-2'-OMe-A(Bz) control, 5'(Z)-VPA control and 5'(E)-VP-2'-OMe-A-CE-P control are all purchased from Qingdao Sugar Intelligence; acetonitrile and ammonium acetate are HPLC grade; the liquid chromatograph used is Agilent 1260 liquid chromatograph.
[0025] Example 1 The example provides a liquid chromatography detection method for 5' (E)-VPA phosphoramidite monomer related substances, comprising the following steps: (1) Preparation of solution.
[0026] Mobile phase A (10 mmol / L ammonium acetate solution): 0.77 g of ammonium acetate was weighed into 1000 ml of purified water, stirred and dissolved, shaken uniformly, filtered with a 0.22 μm MCE water system filter, and ultrasonically degassed to obtain.
[0027] Test sample solution: 20 mg of the 5' (E)-VPA sample to be tested was accurately weighed into a 10 ml volumetric flask, dissolved with acetonitrile, and then diluted to the mark, shaken uniformly to obtain the test sample solution (2 mg / ml).
[0028] Impurity 5'-(E)-VPA-OX solution: 20 mg of impurity 5'-(E)-VPA-OX was accurately weighed into a 10 ml volumetric flask, dissolved with acetonitrile, and then diluted to the mark, shaken uniformly to obtain the impurity 5'-(E)-VPA-OX solution (2 mg / ml).
[0029] Impurity 5'-(E)-VP-2'-OMe-A(Bz) solution: 20 mg of impurity 5'-(E)-VP-2'-OMe-A(Bz) was accurately weighed into a 10 ml volumetric flask, dissolved with acetonitrile, and then diluted to the mark, shaken uniformly to obtain the impurity 5'-(E)-VP-2'-OMe-A(Bz) solution (2 mg / ml).
[0030] Impurity 5'-(E)-VP-2'-OMe-A(Bz) solution: 20 mg of impurity 5'-(E)-VP-2'-OMe-A(Bz) was accurately weighed into a 10 ml volumetric flask, dissolved with acetonitrile, and then diluted to the mark, shaken uniformly to obtain the impurity 5'-(E)-VP-2'-OMe-A(Bz) solution (2 mg / ml).
[0031] Impurity 5'-(E)-VP-2'-OMe-A(Bz) solution: 20 mg of impurity 5'-(E)-VP-2'-OMe-A(Bz) was accurately weighed into a 10 ml volumetric flask, dissolved with acetonitrile, and then diluted to the mark, shaken uniformly to obtain the impurity 5'-(E)-VP-2'-OMe-A(Bz) solution (2 mg / ml).
[0032] Mixed impurity stock solution: precisely pipette 5'- (E) -VPA-OX solution (2 mg / ml) 1.5 ml, impurity 5'- (E) -VP-2'-OMe-A (Bz) solution (2 mg / ml) 1.0 ml, impurity 5' (Z) -VPA solution (2 mg / ml) 1.0 ml, 5'- (E) -VP-2'-OMe-A-CE-P solution (2 mg / ml) 1.0 ml into a 50 ml volumetric flask, dilute to the mark with acetonitrile, shake well to obtain the mixed impurity stock solution. In the impurity solution, the final concentration of impurity 5'- (E) -VPA-OX is 60 μg / ml, the concentration of impurity 5'- (E) -VP-2'-OMe-A (Bz) is 40 μg / ml, the concentration of impurity 5' (Z) -VPA is 40 μg / ml, and the concentration of impurity 5'- (E) -VP-2'-OMe-A-CE-P is 40 μg / ml.
[0033] System suitability solution: precisely weigh 5' (E) -VPA reference substance 20 mg into a 10 ml volumetric flask, add 1 ml of mixed impurity stock solution, dissolve with acetonitrile, and then dilute to the mark, shake well to obtain.
[0034] 5' (E) -VPA reference substance solution: precisely weigh 5' (E) -VPA reference substance 20 mg into a 50 ml volumetric flask, dissolve with acetonitrile, and then dilute to the mark, shake well to obtain the reference substance solution.
[0035] 0.2% 5' (E) -VPA reference solution: take 5' (E) -VPA reference substance solution 100 μl into a 20 ml volumetric flask, dissolve with acetonitrile, and then dilute to the mark, shake well to obtain 0.2% 5' (E) -VPA reference solution.
[0036] (2) Detection.
[0037] The liquid chromatograph is equipped with a UV detector (UV), the Column chromatographic column is Shim-pack Scepter C18 (4.6 x 150 mm, 3.0 μm), the injection volume is 10 μL, the column temperature is 25 ℃, the flow rate is 1.0 ml / min, and the elution gradient is shown in Table 2.
[0038] Table 2 Elution mode
[0039] Determination method: under the above detection conditions, the system suitability solution, 0.2% 5'(E)-VPA control solution and test sample solution were successively injected for analysis, and the chromatogram was recorded: then, based on the chromatogram of the test sample solution, the detection amount of the related substances 5'-(E)-VPA-OX, 5'-(E)-VP-2'-OMe-A(Bz), 5'-(E)-VP-2'-OMe-A-CE-P and 5'(Z)-VPA was calculated according to the main component self-control method. Among them, the limit (i.e. the impurity content) standard is: 5'-(E)-VPA-OX≤0.3%, 5'-(E)-VP-2'-OMe-A(Bz)≤0.2%, 5'-(E)-VP-2'-OMe-A-CE-P≤0.2% and 5'(Z)-VPA≤0.2%. Figure 1 Typical chromatogram of the test sample solution.
[0040] Example 2 For the detection method provided in Example 1, this example is evaluated from the following aspects: (1) System suitability.
[0041] The system suitability of liquid chromatography is a key index for verifying whether the chromatography system meets the analysis requirements. Specifically, the system suitability requirements are: the theoretical plate number of the main peak is not less than 5000, the separation degree between the main peak and the adjacent impurity peak is not less than 1.5, the separation degree between the impurities is not less than 1.2, and the main peak tailing factor is ≤1.8.
[0042] Figure 2 Typical chromatogram of the system suitability solution, from Figure 2 It can be known that the method of the present application meets the system suitability requirements.
[0043] (2) Specificity.
[0044] The specificity of the method was investigated by taking the blank solvent (10 mm ammonium acetate and acetonitrile), 5'(E)-VPA control solution, each impurity solution and sample impurity mixed solution (i.e. system suitability solution) as the specificity solution, it was determined that the blank solvent had no interference with the main peak, the purity of the main peak of the test sample solution was greater than or equal to 990, and the separation degree between the main peak and the adjacent impurity was greater than 1.5. The specificity analysis results are shown in Table 3, and it can be known from Table 3 that the method has good specificity.
[0045] Table 3 5'(E)-VPA specificity results
[0046] Note: " / " in the table indicates that there is no corresponding data or index.
[0047] (3) Detection limit and quantification limit.
[0048] Respectively, precision 5 '-(E)-VP-2 '-OMe-A (Bz) solution, 5 '-(E)-VPA-OX solution, 5 '-(E)-VP-2 '-OMe-A-CE-P solution, 5 '(E)-VPA control solution, 5 '(Z)-VPA solution 100 μl to 10 ml capacity bottle, with acetonitrile constant volume to the scale, take the above mixture 200 μl to 10 ml capacity bottle and acetonitrile constant volume to the scale, namely the limit of quantification solution (concentration is 0.40 μg / ml, equivalent to 0.02% of the concentration of test solution); The limit of quantification solution is diluted 3 times to obtain the detection limit solution (concentration is 0.13 μg / ml, equivalent to 0.0065% of the concentration of test solution).
[0049] The signal-to-noise ratio S / N of the limit of quantification solution is 12.0, 21.0, 56.1, 12.9, and 22.9, respectively. The signal-to-noise ratio S / N of the detection limit solution is 3.9, 7.7, 20.5, 4.5, and 7.2, respectively. The typical chromatograms of the detection limit solution and the limit of quantification solution are shown in Figure 3 and Figure 4 The experimental data show that only a conventional UV detector is needed to complete the inspection of 5 '(E)-VPA related substances within 32 min, and the concentration of the main component on the machine is 0.13 μg / ml, which is 0.006% of the main component content; The limit of quantification is 0.4 μg / ml, which is 0.02% of the main component content. It can be seen that the method has high sensitivity and meets the control requirements of related substances.
[0050] (4) Linearity and range.
[0051] According to the preparation method of the limit of quantification solution, 0.5 ml of the mixed impurity stock solution is precisely measured and placed in a 10 ml capacity bottle, acetonitrile is added to constant volume to the scale, and then shaken to obtain (equivalent to 50% impurity limit); 1.0 ml of the mixed impurity stock solution is precisely measured and placed in a 10 ml capacity bottle, acetonitrile is added to constant volume to the scale, and then shaken to obtain (equivalent to 100% impurity limit); 1.5 ml of the mixed impurity stock solution is precisely measured and placed in a 10 ml capacity bottle, acetonitrile is added to constant volume to the scale, and then shaken to obtain (equivalent to 150% impurity limit); 2.0 ml of the mixed impurity stock solution is precisely measured and placed in a 10 ml capacity bottle, acetonitrile is added to constant volume to the scale, and then shaken to obtain (equivalent to 200% impurity limit). The results of the above solutions are shown in Tables 4-7, which show that they have good linearity.
[0052] Table 4 Linearity results of impurity 5 '-(E)-VP-2 '-OMe-A (Bz) (control limit requirement ≤0.2%)
[0053] Table 5 Linear results of impurity 5'-(E)-VPA-OX (control limit requirement ≤0.3%)
[0054] Table 6 Linear results of impurity 5'-(E)-VP-2'-OMe-A-P (control limit requirement ≤0.2%)
[0055] Table 7 Linear results of impurity 5'-(Z)-VPA (control limit requirement ≤0.2%)
[0056] (5) Accuracy.
[0057] Prepare 50%, 100%, 150% limit level of test sample spiked solution, and prepare three replicates for each concentration.
[0058] The detection results show that the recovery rate of impurity 5'-(E)-VPA-OX is 90.1% to 101.7%, the average recovery rate is 97.5%, and the recovery rate RSD is 5.1%; the recovery rate of impurity 5'-(E)-VP-2'-OMe-A(Bz) is 95.5% to 103.0%, the average recovery rate is 100.3%, and the recovery rate RSD is 3.3%, less than 15.0%; the recovery rate of impurity 5'-(Z)-VPA is 103.8% to 108.0%, the average recovery rate is 105.7%, and the recovery rate RSD is 1.5%, less than 15.0%; the recovery rate of impurity 5'-(E)-VP-2'-OMe-A-P is 100.3% to 103.5%, the average recovery rate is 102.5%, and the recovery rate RSD is 1.3%, less than 15.0%. The above results all meet the acceptable standard of pharmacopoeia, and the method has good accuracy.
[0059] In summary, the method has the characteristics of good separation degree between impurities, good system applicability, good sensitivity, good linear relationship and good accuracy, and can realize rapid and accurate detection and monitoring of the quality of 5'(E)-VPA phosphoramidite monomer.
[0060] Note that the present application is not limited to the above-described embodiments. The above-described embodiments are merely examples, and embodiments having substantially the same configuration as the technical idea and achieving the same effects within the scope of the technical idea of the present application are included in the technical scope of the present application. Furthermore, other modes constructed by applying various modifications that can be thought of by those skilled in the art to the embodiments or by combining part of the constituent elements of the embodiments are also included in the scope of the present application without departing from the spirit of the present application.
Claims
1. A method for detecting related substances of 5'(E)-VPA phosphorous amide monomer, characterized in that, Liquid chromatography was used, and the chromatographic conditions included: column temperature 23-27℃, stationary phase of octadecyl bonded silica gel, mobile phase A being ammonium acetate solution, mobile phase B being acetonitrile, and gradient elution; the related substances included 5'-(E)-VP-2'-OMe-A(Bz), 5'(Z)-VPA, 5'-(E)-VP-2'-OMe-A-CE-P, and 5'(E)-VPA-OX.
2. The detection method according to claim 1, characterized in that, The mobile phase A is a 10 mmol / L ammonium acetate solution.
3. The detection method according to claim 2, characterized in that, The gradient elution conditions are as follows: 0 min, mobile phase A accounts for 50% and mobile phase B accounts for 50%; 21 min, mobile phase A accounts for 26% and mobile phase B accounts for 74%. At 21.5 min, liquid phase A accounts for 10% and mobile phase B accounts for 90%; at 27 min, liquid phase A accounts for 10% and mobile phase B accounts for 50%. At 27.5 min, the proportion of liquid phase A was 50% and that of liquid phase B was 50%; at 32 min, the proportion of liquid phase A was 50% and that of liquid phase B was 50%.
4. The detection method according to claim 1, characterized in that, The chromatographic column was a Shim-pack Scepter C18 with dimensions of 4.6 × 150 mm and a diameter of 3.0 μm.
5. The detection method according to claim 1, characterized in that, The injection volume was 10 µL.
6. The detection method according to claim 1, characterized in that, The flow rate is 0.8-1.0 ml / min.
7. The detection method according to claim 1, characterized in that, The column temperature is 25°C.
8. The detection method according to claim 1, characterized in that, The detection wavelength is 260 nm.
9. The detection method according to claim 1, characterized in that, Includes the following steps: Accurately weigh the 5'(E)-VPA sample to be tested and prepare a test solution with a concentration of 2 mg / ml using acetonitrile as solvent; Accurately weigh 20 mg of 5'(E)-VPA and add 1 ml of mixed impurity solution. Dilute to 10 ml with acetonitrile and mix well to obtain the system suitability solution. In the mixed impurity solution, acetonitrile is used as the solvent, the concentration of impurity 5'-(E)-VPA-OX is 60 μg / ml, and the concentrations of impurities 5'-(E)-VP-2'-OMe-A(Bz), 5'(Z)-VPA, and 5'-(E)-VP-2'-OMe-A-CE-P are all 40 μg / ml. Accurately weigh 5'(E)-VPA reference standard and prepare a 5'(E)-VPA reference standard solution with a concentration of 2 mg / ml using acetonitrile as solvent; Inject the system suitability solution, 5'(E)-VPA reference solution, and test solution sequentially for analysis, record the chromatograms, and calculate the detection limits of each relevant substance based on the chromatograms.
10. The detection method according to claim 9, characterized in that, Based on the chromatogram, the detection amounts of each related substance were calculated using the principal component self-comparison method.